Dynamic Approaches to Improve Glycemic Control and Primary Diabetes Care 1‑Or 2‑Or 3‑Or

Home , Dasiglucagon, Diabetes medication, Diabetic foot ulcer, Dulaglutide, Insulin analog, Insulin detemir

DYNAMIC APPROACHES TO IMPROVE GLYCEMIC CONTROL AND PRIMARY DIABETES CARE

DYNAMIC APPROACHES TO IMPROVE GLYCEMIC Figure. CONTROL AND PRIMARY DIABETES CARE

1‑OR Characterizing Clinical Inertia in a Large, National Database CORI R. RATTELMAN, ANUPAMA ARORA, JOHN K. CUDDEBACK, ELIZABETH L. CIEMINS, Alexandria, VA Objective: To characterize clinical inertia in the treatment of diabetes using a large, geographically diverse clinical database. Study Design: A retrospective descriptive analysis was conducted in a clinical database containing 22 million patient records across 22 health care organizations (HCOs). Population Studied: A total of 281,000 patients aged 18-75 were included during the 5.5-year study period (1/2012-6/2017). Patients had an outpatient visit in the last 12 months of the study period, an HbA1c in the last 24-30 months (index A1c), and a diagnosis of type 2 DM on a claim or EHR problem list at least 6 months prior to index A1c. A subset of 47,693 patients with an index A1c ≥8 and a prior A1c ≥8 or lack thereof, was observed for Supported By: National Institute of Diabetes and Digestive and Kidney Diseases four 6-month follow-up periods for actions including a new class of diabetes (T35DK104689); Yale University medication prescribed or an A1c <8. The absence of observable action following index A1c suggests potential “clinical inertia.” Principal Findings: Six months following an index A1c≥8, 55% of patients 3‑OR received no observable clinical action ranging from 45-65% across HCOs Influence of Diabetes Complications on the Cost-Effectiveness of and 18-96% across individual providers. A new diabetes prescription was A1c Treatment Goals in Older U.S. Adults observed in 35% of patients (7.5% moved into glycemic control, A1c<8) HUI SHAO, DEBORAH B. ROLKA, EDWARD W. GREGG, PING ZHANG, Atlanta, GA and 10% moved into glycemic control without a new prescription. Patient The American Diabetes Association (ADA) recommends glycemic man- characteristics associated with increased clinical inertia, i.e., no observable agement goals for older diabetes patients take into account the complexity action, during the 6- and 24-month follow-up periods included black race, of complications. Little information is available on how cost-effectiveness low-income insurance, normal body mass index, and being on bolus insulin (CE) of glycemic control goals relates to a patient’s level of complications. (all P<.01). Within 24 months, clinical inertia was reduced to 19%, ranging We examined the CE of intensive glycemic control (A1c <7.5%) compared to from 13-28% across HCOs. standard treatment (A1c <8.5%) among adults aged 65 years with type 2 Conclusions: Lack of clinical action in the 6 months following an A1c ≥8 diabetes having various complications. 2011-2016 National Health and Nutri- suggests clinical inertia vs. ADA guidelines. Greater rates in low-income tion Examination Survey data were used to generate nationally representa- insurance and race/ethnic minority adults suggests potential populations tive samples with various complications. The CDC-RTI diabetes simulation to target to ensure adequate treatment for diabetes. The decline in clinical model was used to project the long-term health and cost consequences of inertia within 24 months suggests either actions not seen in the data, or intensive/standard glycemic control. The CE of the intensive glycemic con- later interventions that were ultimately effective. trol was measured in costs (2017 USD) per quality adjusted life year (QALY). Supported By: Novo Nordisk Based on a $50,000/QALY threshold, intensive glycemic control was only marginally cost effective for patients with no complications or any microvascular complication (excluding renal failure) but was not cost-effective 2‑OR for patients with one or more macrovascular complications. Current ADA Cost-Related Insulin Underuse Is Common and Associated with standards recommend intensive glycemic control among older adults with Poor Glycemic Control fewer than 3 complications. From a CE perspective, our results only support DARBY M. HERKERT, PAVITHRA VIJAYAKUMAR, JING LUO, JEREMY SCHWARTZ, the intervention among those with no complications or only microvascular TRACY L. RABIN, EUNICE M. DEFILIPPO, KASIA J. LIPSKA, New Haven, CT, Boston, complication. MA, West Haven, CT Insulin is an essential medicine for people with diabetes (DM), but increasing prices have threatened its affordability. We examined the prevalence of and factors associated with cost-related insulin underuse. We administered a cross-sectional survey to patients with DM prescribed insulin at Yale Diabetes Center (YDC). Our primary outcome was cost-related underuse in the past 12 months, defined by a positive response to any 1 of

6 questions: Did you … 1.) Use less insulin than prescribed 2.) Try to stretch ORALS out your insulin 3.) Take smaller doses of insulin than prescribed 4.) Stop insulin 5.) Not fill an insulin prescription 6.) Not start insulin … because of cost? We examined the association of cost-related underuse with HbA1c >9% using logistic regression controlling for age, sex, age, DM duration, and income. Out of 354 patients prescribed insulin who had YDC visit in July 2017, 199 (56.2%) completed the survey (50.8% female, 60.8% white, 41.7% type 1). Of these patients, 51 (25.5%) reported cost-related insulin underuse. Patients with cost-related underuse had lower income levels, variable drug coverage and employment (Figure), and 3-fold higher odds of HbA1c >9% (p = 0.03) than patients who did not report underuse. One in four patients at our urban diabetes center reported cost-related insulin underuse, and this was associated with poor glycemic control. These results highlight an urgent need to address high insulin prices in the U.S.

ADA-Supported Research

A1 DYNAMIC APPROACHES TO IMPROVE GLYCEMIC CONTROL AND PRIMARY DIABETES CARE

4‑OR 6‑OR The Clinical and Economic Effects of Switching Medicare Benefi‑ Management of Patients with Type 2 Diabetes Shared between a ciaries with Type 2 Diabetes from Analog to Human Insulin Specialized Outpatient Clinic and General Practice Was Noninfe‑ JING LUO, NAZLEEN F. KHAN, THOMAS MANETTI, JAMES J. ROSE, ANI rior to Mono Management in a Specialized Outpatient Clinic—A KALOGHLIAN, BALU GADHE, SACHIN H. JAIN, JOSHUA GAGNE, AARON KES- Prospective, Randomized, Noninferiority Trial SELHEIM, Boston, MA, Cerritos, CA LENE MUNCH, BIRGITTE B. BENNICH, DORTHE OVERGAARD, HANNE KONRAD- Objective: To evaluate the clinical and economic effects of a managed SEN, HELLE V. MIDDELFART, NIELS KAARSBERG, FILIP K. KNOP, TINA VILSBØLL, care organization’s conversion program switching Medicare beneficiaries MICHAEL RØDER, Gentofte, Denmark, Hellerup, Denmark, Copenhagen, Denmark, with type 2 diabetes mellitus from analog to human insulin. Huddinge, Sweden, Holte, Denmark Methods: A consecutive sample of 14,635 participants who filled insulin To ensure high-quality care for patients with type 2 diabetes (T2D), it is prescriptions between January 2014 and December 2016 were identified necessary to consider how clinical management is organized. We investi- from a Medicare Advantage and prescription drug plan operating in 4 U.S. gated if management of patients with T2D shared between a specialized states (CA, AZ, NV, VA). Outcomes were mean hemoglobin A1c (%), rates of outpatient clinic and primary health care has a non-inferior outcome of severe hypoglycemia or hyperglycemia events and risk of reaching the Part HbA1c compared to ‘mono’ management in a specialized outpatient clinic. D coverage gap. Data were analyzed using interrupted time series and seg- The study was a 12-month randomized controlled, non-inferiority trial. Non- mented regression with cut points at the beginning and end of 2015. inferiority margin for HbA1c was 4.4 mmol/mol. Patients with T2D and incipi- Results: 14,635 members (mean age=72.5) filled 221,866 insulin prescrip- ent complications were eligible for the study. The shared care intervention tions. Insulin conversion decreased the proportion of scripts filled by analog consisted of one annual comprehensive check-up at the outpatient clinic and insulins from 90% to 30%. The baseline mean hemoglobin A1c was 8.46% three quarterly visits at the primary health care physician. The control group and decreased at a rate of -0.02% per month during 2014. The beginning was offered four quarterly visits at the outpatient clinic, including an annual of the conversion program in 2015 was associated with a level change of comprehensive check-up. We randomized 140 patients (age: 65.0±0.9 years +0.14% (p<0.01) and a slope change of 0.02% (p<0.01). The baseline rate of (mean±SEM), BMI: 30.8±0.5 kg/m2, diabetes duration: 9.1±0.5 years, HbA1c: severe hypoglycemia was 4.21 per 1000-person years (py) and increased at a 51.9±0.8 mmol/mol, systolic blood pressure: 135.6±1.1 mmHg) with no sig- rate of 0.36/1000py during 2014. The baseline rate of severe hyperglycemia nificant baseline differences between the groups. Ninety-five (68%) patients was 22.33/1000py and increased at a rate of 0.30/1000py. For both hypogly- had peripheral neuropathy, 26 (19%) microalbuminuria, and 21 (15%) previ- cemia and hyperglycemia, the level and slope changes in the 12-month inter- ous major cardiovascular event. At end-of-trial, mean HbA1c change from vention and post-intervention segments were not statistically significant. baseline was 2.0 mmol/mol in the intervention group and 0.9 mmol/mol in The intervention reduced the risk of reaching the coverage gap by 55% (HR the control group. The between-group difference of 1.1 mmol/mol (95% con- 0.45, nominal 95% CI: 0.43-0.48, p<0.001). fidence interval: -2.0, 4.1) met the pre-specified non-inferiority criterion. Our Conclusions: Switching Medicare beneficiaries with diabetes from analog study shows that a shared care program is non-inferior to an established to human insulin did not change the rates of hospitalization for hypoglyce- program in a specialized outpatient clinic in maintenance of glycemic control mia or hyperglycemia, slightly increased mean A1c, and reduced the risk of of T2D patients with incipient complications. Shared care could be part of reaching the Part D coverage gap. future T2D management, as more patients will benefit from the specialized diabetes team while keeping close contact with the primary care physician. 5‑OR Supported By: Jascha Foundation; Capital Region of Denmark; Lilly and Herbert Dietary Intervention for Inpatient Glycemic Control Hansen Foundation SARA CROMER, MAGDALENA M. BOGUN, New York, NY While recommendations for inpatient glycemic control suggest a conser- 7‑OR vative target of 140-180mg/dL, many struggle to achieve this goal. Glycemic Diabetes Inside—Following the Long-Term Impact of a Diabetes control begins with a carbohydrate-controlled diet, but many inpatient diets Quality Improvement (QI) Initiative in Primary Care continue to be suboptimal and can contribute to hyperglycemia. At our insti- UMA GUNASEKARAN, ROY E. FURMAN, KELLIE M. RODRIGUEZ, E. ELIZA- tution, the carbohydrate-controlled breakfast diet was changed to better BETH OBIALO, SENTAYEHU KASSA, NOEL O. SANTINI, ELISE FURMAN, LUIGI reflect carbohydrate goals (e.g., replacing french toast on the “bad diet” with MENEGHINI, Dallas, TX, Bala Cynwyd, PA vegetable frittatas on the “good diet”). Pre- and post-prandial glucose values In collaboration with the ADA, we implemented a QI project to improve for patients with types 1 and 2 diabetes receiving a carbohydrate-controlled glycemia in patients with type 2 diabetes (T2D) and poor glycemic control diet were collected by retrospective chart review during the weeks before (A1C > 9%) receiving care in a large urban safety net health system. The and after these changes took effect. Patients on steroids were analyzed 18-month initiative focused on developing shared medical appointments for separately. Individual post-prandial glucose values were recorded in a binary insulin initiation and management, supported by a primary care QI commit- fashion as meeting or exceeding predefined glucose targets (<180 mg/dL, tee, educational interventions and development of EMR tools to facilitate <250mg/dL, and <350mg/dL). During this period, 369 patients not receiving diabetes management and documentation. We report 2-year data following steroids and 39 patients receiving steroids had at least one relevant glu- implementation of the QI intervention. cose value. In the non-steroid group, there was no difference in proportion Of 23,611 individuals (mean age 58 years) with 73,849 A1C data points over ORALS of patients who exceeded any of the predefined thresholds. However, in the a period of 3 years 60% were female; 57% Hispanic white, 27% non-Hispanic steroid group, there was a trend toward reduction in proportion of patients black, 9% non-Hispanic white; 62% received charity care, 30% Medicare/ exceeding blood glucose of 180mg/dL (absolute risk reduction 19%, 95% Medicaid, 5% commercial insurance. Control charts first demonstrated sta- confidence interval -4% to 42%, p = 0.12) and 250mg/dL (absolute risk reduc- tistically significant increases in insulin use in poorly controlled patients fol- tion 17%, 95% confidence interval -2% to 35%, p = 0.08) on the days after lowed by statistically significant improvement in population glycemic control the dietary change. Although all patients with diabetes may experience (A1C average) and proportion of patients with A1c>9% (Figure). hyperglycemia proportionate to their carbohydrate intake, those on steroids A primary care-based QI initiative targeting patients with T2D and A1C experience induced insulin resistance leading to especially diet-sensitive > 9% led to appropriate increases in insulin prescriptions, which was fol- glucose excursions. Our data suggests that patients receiving steroids can lowed by a system-wide improvement in diabetes control. likely achieve improved inpatient glucose control through dietary modification. Physician and administration attention to carbohydrate-controlled menus represents an opportunity for improved quality of care for inpatients with diabetes.

ADA-Supported Research

A2 GLUCOSE MONITORING—ADVANCES, PITFALLS, AND CLINICAL RELEVANCE

Figure. curacy can lead to reduced glycemic control and insulin dosing errors. In order to assess the accuracy of these BGMs and identify trends that might explain these results, we performed a literature review of publications between 2010 and 2017 that present data about the accuracy of BGMs using ISO 15197 2003 and/or ISO 15197 2013 as target standards. We found 58 publications describing the performance of 143 unique BGM systems, 59 of which were FDA-cleared. When compared with non-FDA cleared BGMs, FDA-cleared BGMs were significantly more likely to pass both the ISO 15197 2003 (OR= 2.39, CI: 1.45-3.92, p<0.01) and ISO 15197 2013 standards (OR= 2.20, CI: 1.51-3.27, p<0.01). We also found that newer meters were more likely to pass each ISO standard for both ISO 15197 2003 (OR: 1.06, CI: 1.06-1.07, p<0.001, odds ratio reported for a one-year period) and ISO 15197 2013 standards (OR=1.06, CI: 1.03-1.06, p<0.01, odds ratio reported for a one year period). Newer meters tend to favor using the test strip enzyme glucose dehydrogenase (GDH) compared to glucose oxidase (GOX), which correlates to an increased likelihood of passing ISO 15197 2003 (OR=0.23, CI: 0.13-0.38, p<0.001) and ISO 15197 2013 (OR=0.24, CI: 0.15-0.35, p<0.001). We note that many of these studies were supported by BGM manufactur- ers, and when compared with independent studies, an FDA-cleared BGM was significantly more likely to pass in a manufacturer-supported study for both ISO 15197 2003 (OR=22.4, CI: 8.73-21.57, p < 0.001) and ISO 15197 2013 (OR=23.08, CI: 10.16-60.03, p < 0.001). We conclude that BGM accuracy should be assessed independently, both prior to FDA clearance and again Supported By: Eli Lilly and Company; Novo Nordisk; Sanofi during additional surveillance studies, to ensure accurate performance over time in real-world settings. Failure to do so can result in deleterious clinical 8‑OR and economic effects. Economic Evaluation of Group Medical Visits for Adults with Diabe‑ tes in Community Health Centers 10‑OR WEN WAN, ERIN M. STAAB, SANDRA A. HAM, AMANDA CAMPBELL, CYNTHIA Analysis of Prevalence, Magnitude, and Timing of the Dawn Phe‑ T. SCHAEFER, MICHAEL T. QUINN, ARSHIYA BAIG, Chicago, IL, East Lansing, MI, nomenon in Type 1 Diabetes—Descriptive Analysis of Two Insulin Evansville, IN Pump Trials Background: Diabetes group visits (GVs)—shared appointments that ILIA OSTROVSKI, LEIF ERIK LOVBLOM, DANIEL SCARR, ALANNA WEISMAN, incorporate medical care, diabetes self-management education, social ANDREJ ORSZAG, EMILIE D’AOUST, AHMAD HAIDAR, RÉMI RABASA-LHORET, support, and goal-setting—were recently examined in a network of U.S. LAURENT LEGAULT, BRUCE A. PERKINS, Toronto, ON, Canada, Montreal, QC, community health centers (HCs) and found to improve glucose control and Canada diabetes-related quality of life. To better understand the dawn phenomenon in type 1 diabetes, we sought Objective: To evaluate the economic impact of group visits (GV) in adults to determine its prevalence, timing and magnitude in studies specifically with diabetes in HCs. designed to assess insulin pump basal requirements. Thirty-three partici- Methods: In a pilot prospective matched case-control trial, we imple- pants from two insulin pump studies were analyzed. Twenty were obtained mented 6 monthly GV sessions in 5 HCs and compared intervention patients from a methodologically-ideal semi-automated basal analysis trial in which (n=49) to control patients receiving usual care within the same HCs (n=72). basal rates were determined from repeated fasting tests (the derivation set) We conducted patient chart reviews to obtain healthcare utilization data and 13 from an artificial pancreas trial in which duration of fasting was vari- for the prior 6 months, at baseline, 6, and 12 months and collected monthly able (the validation set). Prevalence was determined for the total cohort and logs of HC expenses and staff time spent on activities related to GVs, includ- the individual trials using the standard definition of an increase in insulin ing training, planning, implementation, and evaluation. Total costs included exceeding 20% and lasting 90 minutes or more. Among cases, time of onset HCs’ expenses and activities and patients’ healthcare use over one year. and percent change in the magnitude of basal delivery was determined. Wilcoxon rank-sum tests, generalized linear models, and modified Park tests Seventeen of the 33 (52%) participants experienced the dawn phenomenon were used to compare the healthcare use and cost data. [11 of 20 (55%) in the derivation set, 6 of 13 (46%) in the validation set]. Results: Despite each HC spending $17,342 on average (or $1,770 per- Time of onset was 3:00 am [IQR; 3:00, 4:15 am] in the derivation set and patient) for the GV preparation and implementation, the per-patient annual 3:00 am [3:00, 4:00 am] in the validation set. The magnitude of the dawn total costs were not different between the groups (GV: $3,982 vs. control: phenomenon was a 58.1% [28.8, 110.6%] increase in insulin requirements in $4,287, p=0.73). Most of the benefits were due to GV patients having fewer the derivation set and 65.5% [45.6%, 87.4%] in the validation set. The dawn hospitalizations (p=0.08) and fewer primary care visits that occurred outside phenomenon occurs in approximately half of patients with type 1 diabetes, ORALS the trial (p=0.01). After excluding the staff training costs (mean $7,962 per when present it has predictable timing of onset (generally 3:00 am), and has HC), the GV group had significantly lower total costs ($3,169) than the control substantial but highly variable magnitude. These findings imply that opti- group (p <0.01). mization of glycemic control requires clinical emphasis on fasted overnight Conclusions: The diabetes group visits in health centers improved basal insulin assessment. patients’ healthcare quality without increasing overall healthcare costs. In Supported By: JDRF; Diabetes Canada subsequent GV sessions, excluding the need for staff training, group visits may be cost saving. Future studies should assess the cost-effectiveness of 11‑OR group visits. Distinct Patterns of Daily Glucose Variation by Pubertal Stage in Supported By: Chicago Center for Diabetes Translation Research Youth with Type 1 Diabetes (T1D) JIA ZHU, LISA VOLKENING, MICHELLE KATZ, LORI M. LAFFEL, Boston, MA Background: Glucose variability is an emerging risk factor for diabetes GLUCOSE MONITORING—ADVANCES, PITFALLS, complications. As basal insulin requirements vary in youth with T1D by age AND CLINICAL RELEVANCE and time of day, we analyzed daily patterns of glucose variation by pubertal stage in youth with T1D. 9‑OR Methods: Glucose patterns were assessed using 4 weeks of continuous Performance of Cleared Blood Glucose Monitor Systems glucose monitoring (CGM) data from 107 youth ages 8-17 years with T1D for FRAYA G. KING, DAVID T. AHN, VICTORIA HSIAO, TRAVIS PORCO, DAVID C. >1 year. Glucose values per hour were expressed as percentages relative to KLONOFF, New Orleans, LA, Los Angeles, CA, San Francisco, CA, San Mateo, CA the mean glucose over a 24-hour period. Glucose patterns were compared by Many blood glucose monitor systems (BGMs) do not pass international pubertal stage (pre-pubertal: Tanner (T) 1, pubertal: T2-4, post-pubertal: T5). standards of accuracy, even when they have been previously cleared by Results: The sample had a mean (±SD) age of 13.1±2.6 years, T1D duration the FDA. Patients rely on BGM data to make treatment decisions, and inac- of 6.3±3.5 years, A1c of 7.8±0.8; 88% were pump-treated. Pre-pubertal youth

ADA-Supported Research

A3 GLUCOSE MONITORING—ADVANCES, PITFALLS, AND CLINICAL RELEVANCE

had greater mean standard deviation (86.4±12.0, p=.008) and coefficient of performance of the Eversense XL through 180 days in a primarily adolescent variation (0.43±.05, p=.04) compared to pubertal (79.8±13.4; 0.41±.05) and population with type 1 diabetes (T1D). post-pubertal youth (75.8±13.3; 0.40±.05). There was no difference in mean Methods: This study was a prospective, single-center, single-arm, 180- glucose or A1c by pubertal stage (p >.4). Patterns of glucose variation appear day evaluation of the effectiveness and safety of the implantable CGM sys- in Figure. tem among Canadian adolescent and adult participants with T1D. Effective- Conclusion: Pre-pubertal compared with pubertal and post-pubertal ness measures included mean absolute relative difference (MARD), system youth appear to have greater glucose variability independent of A1c, with agreement with Yellow Springs Instrument (YSI) glucose values, and Clarke a notable dawn-dusk pattern. Thus, A1c alone does not seem to capture the Error Grid analysis using paired CGM and reference YSI glucose analyzer full range of glycemic parameters in youth with T1D, highlighting the added values. Adult participants were inserted with two sensors and adolescent value of CGM data when determining optimal insulin doses. participants were inserted with one sensor in the upper arm. CGM system accuracy studies were performed every 30 days. The safety assessment included the incidence of insertion or removal procedure-related and device- related serious adverse events (SAEs) through 180 days post-insertion. Results: Thirty-Six participants (30 adolescent/6 adult, 13 female/23 male, mean age 17±9.2 years, mean BMI 22±4 kg/m2) received the CGM system. One subject withdrew at Day 1 due to intravenous access issues. CGM system agreement with YSI glucose within 15 mg/dL or 15% of YSI glucose values (N = 7163) through 60, 120 and 180 days was 82.9% (95% CI: 78.4%- 86.1%), 83.6% (95% CI: 80.4%-85.7%) and 83.4% (95% CI: 79.7%-85.5%), respectively. Overall MARD was 9.4% (95% CI: 8.6%-10.5%). Clarke Error Grid analysis showed 99% of paired values in clinically acceptable error zones A and B. No insertion/removal or device-related SAEs were reported. Conclusions: The Eversense XL CGM system is safe and accurate through 180 days of Sensor wear in a primarily adolescent population. Supported By: Senseonics, Inc.

Supported By: National Institutes of Health (R01DK089349, K12DK094721, 14‑OR K23HL125976, P30DK036836) A Three-Way Accuracy Comparison of the Dexcom G5, Abbott Free‑ style Libre Pro, and Senseonics Eversense CGM Devices in an Out‑ 12‑OR patient Study of Subjects with Type 1 Diabetes What Is the Role of A1C in Comorbidities? RABAB Z. JAFRI, COURTNEY A. BALLIRO, FIRAS EL-KHATIB, MICHELE MAHENO, JORDAN E. PERLMAN, JENNIFER L. ROSENBAUM, BRIDGET K. MCNULTY, IRL B. MALLORY A. HILLARD, ALEXANDER J. O’DONOVAN, RAJENDRANATH SELA- HIRSCH, Seattle, WA GAMSETTY, HUI ZHENG, EDWARD DAMIANO, STEVEN J. RUSSELL, Boston, MA The ADAG Study Group defined the relationship between average blood There are no published studies directly comparing the accuracy of con- glucose (AG) and A1C. They derived an equation valid across diabetes types tinuous glucose monitoring (CGM) devices in the outpatient setting. We but excluded comorbidities known to impact A1C. The goal of this study is tested the performance of the Dexcom G5, Abbot Freestyle Libre Pro, and to examine the relationship between AG and A1C in patients omitted from Senseonics Eversense (an implantable CGM approved in Europe) during a prior analyses. A retrospective chart review was performed of downloaded 6-week, free-living, outpatient bionic pancreas study involving 23 subjects glucose data from patients prescribed insulin at the UW Diabetes Center with type 1 diabetes who wore all 3 devices concomitantly. The primary between 1/2011-10/2017. Patients were identified who had either 1.) docu- outcome was the mean absolute relative difference (MARD) vs. plasma mentation and/or laboratory confirmation of anemia; 2.) CKD; or 3.) NAFLD. glucose (PG) values measured with the Nova Biomedical StatStrip Xpress Eligible charts required: 1.) 2 weeks of finger stick glucose (SMBG) data with meter that was also used for calibrations according to the manufacturer’s >14 checks or 1 month of CGM with >14 complete days and, 2.) an A1C mea- instruction (except for Libre Pro that is not calibrated). We compared PG sured within 1 month of the encounter. The SMBG and CGM means were values with CGM readings when they were available from all 3 CGMS in the obtained from downloads. An AG was extrapolated from the A1c using the 5 minutes preceding the PG values (n=829 sets). Since the Libre Pro records ADAG equation. The downloaded data and A1C-derived AGs were compared. readings every 15 minutes, we also did a two-way comparison between the There was a linear correlation between AG and A1C (R2=0.48) in anemic G5 and the Eversense that allowed a higher number of comparisons (n=2277 patients regardless of etiology. Non-anemic patients (N=220) had better sets). Statistical significance was determined using a repeated measure- ADAG agreement (70% of AGs inside 15% of predicted) than anemic patients ments model fitted with the generalized estimating equation method. All 3 (55% of AGs, N=123, p=0.006). Normal GFR yielded 71% of AGs inside 15% CGM systems produced higher average MARDs than during in-clinic studies. of ADAG estimation (N=440). This decreased to 59% if limited to anemic However, since all three CGM systems were worn by the same individuals patients (N=83, p<0.001). ADAG agreement decreased as a function of renal and used the same meter for calibration and as comparator, we were able ORALS impairment. The impact of anemia was only significant in CKD stage 3A to directly compare their performance under real-world conditions. In the (N=49, p=0.02). In CKD stage 4/ESRD there was no correlation between AG 3-way comparison Eversense achieved the lowest nominal MARD (14.8%) and A1C (N=14, R2=0.0004). Abnormal LFTs did not affect ADAG agreement. followed by Dexcom G5 (16.3%) and Libre Pro (18.0%) (Eversense vs. Libre In NAFLD there was no correlation between AG and A1C (N=14, R2=0.03) Pro p=0.004, other comparisons p=NS). In the 2-way comparison the MARD but 64% of AGs were inside 15% of ADAG projection. For many, SMBG and difference between Eversense (15.1%) and G5 (16.9%) was statistically sig- CGM data provide an important confirmatory assessment of A1C. However, nificant (p=0.008). We found that the point accuracy of the Eversense was in certain conditions there is significant discordance between AG and A1C. significantly better than two other CGM systems. The Eversense CGM sys- There is a complete lack of correlation between AG and A1C in advanced tem may be useful to provide glucose values to artificial pancreas devices. renal dysfunction and NAFLD as measured by our lab. These findings suggest Supported By: The Leona M. and Harry B. Helmsley Charitable Trust glucose downloads are a better assessment of diabetes control in several common comorbidities. 15‑OR Efficacy of a Novel Interim Intervention Technique (IIT) with Ret‑ 13‑OR rospective Flash Glucose Monitoring to Improve Glycemic Control First Assessment of the Performance of an Implantable CGM Sys‑ AKSHAY B. JAIN, Mumbai, India tem through 180 Days in a Primarily Adolescent Population with Aim: Retrospective blinded flash glucose monitoring system (FGMS) is Type 1 Diabetes typically used for 14 days, prior to modification of therapy. We sought to RONNIE ARONSON, RAVI RASTOGI, COLLEEN MDINGI, XIAOXIAO CHEN, KATH- assess the efficacy of a novel approach wherein an interim analysis was ERINE TWEDEN, Toronto, ON, Canada, Germantown, MD done within a week of starting FGMS and utilized to implement therapeutic Background: An implantable continuous glucose monitoring (CGM) system modifications. The same sensor was reassessed within the following week (Eversense® XL, Senseonics, Maryland U.S.A) recently received CE Mark for to see the changes in glycemic control, thereby maximizing utility of a single 180-day duration in adults. The current study is the first investigation of the sensor.

ADA-Supported Research

A4 GENETICS, ADIPOSE TISSUE, OBESITY, AND TYPE 2 DIABETES

Methods: This is a retrospective analysis of 105 consecutive adults with other tissue-specific GHR knockouts, AdGHRKO was found to be maternally T2DM and HbA1c >7% on pharmacotherapy (oral agents and/or insulin) at a inheritable. Compared with female AdGHRKO mice born from Flox mothers, single centre. Patients started on blinded FGMS (Freestyle Libre Pro) were female KO mice born from KO mothers displayed resistance to diet-induced assessed within 1 week to get baseline estimate of glycemic trends. Glucose obesity from a high-fat diet for 8 weeks. and the difference in body weight target range was set at 70-180 mg/dL. Patients kept a food log while on was exclusively due to a significant reduction in white fat tissue, especially FGMS. Based on the glucose profile reports, dietary and pharmacotherapy from decreased subcutaneous deposition. Moreover, KO mice from KO changes were made and patients were re-evaluated in the next 7 days to mothers had better insulin sensitivity and glucose tolerance after high-fat assess the changes due to IIT, while wearing the same sensor. Analysis treatment for 8 weeks. Our data suggests that the phenotype of metabolic of pre and post: daily average glucose (DAG), time in target range (TITR), stability in progeny is strongly affected by the maternal genotype. To sup- time above target range (TATR) and time below target range (TBTR) was port this, we also found that Flox mice born from KO mothers presented performed. similar phenotypic traits (improved glucose tolerance and insulin resistance) Results: At baseline, patients had a DAG of 191.3 mg/dL. Average time for of protection from high fat induced diabetes, which was comparable to the interim analysis was 5 days after FGMS initiation. After IIT, the DAG dropped KO mice born from KO mothers. Our ongoing studies are to determine if this to 137.4 mg/dL, within 14 days (p <0.001). The TATR dropped from 52.1% to phenomenon is caused by alterations in maternal hormone levels or some 18.3% (p<0.001), with a concurrent decrease in TBTR from 5.7% to 1.5% unknown mitochondrial activities. Nonetheless, how adipose tissue specific (p<0.001). Recurrent hypoglycemias were detected in 27 (25%) patients, ablation of GHR exerts its effects on metabolic syndromes through maternal with average TBTR being 21.1% and TITR being 65.7% on interim analysis. cues is of high interest not only to pure genetic studies but also to medical Following IIT, the TBTR reduced to 1.9% (p<0.001) and TITR changed to practice. 86.8% (p<0.001), without increase in TATR in these patients. Supported By: National Natural Science Foundation of China (81471000, Conclusion: Using IIT, changes to patient’s lifestyle and/or pharmacother- 81600668) apy can be made within a few days of FGMS, with the ability to gauge subsequent changes while still utilizing the same sensor. Significant improvement 18‑OR in glycemic control was achieved using this technique. Shared Genetic Influence on Early Growth and Type 2 Diabetes in Adulthood 16‑OR FASIL TEKOLA-AYELE, ANTHONY LEE, TSEGASELASSIE WORKALEMAHU, DEEP- Real-World Assessment of Sugar.IQ with Watson—A Cognitive IKA SHRESTHA, Bethesda, MD, Rockville, MD Computing-Based Diabetes Management Solution Background: There is a strong evidence of association between birth- YUXIANG ZHONG, SIDDHARTH ARUNACHALAM, PRATIK AGRAWAL, HUZEFA weight (BW) and type 2 diabetes (T2D) in adulthood. Although shared genet- NEEMUCHWALA, TONI L. CORDERO, FRANCINE R. KAUFMAN, Northridge, CA ics has been suggested to be a significant contributor to these associations, Background: Cognitive computing technology providing personalized to date, we know very few shared genetic loci. We conducted a system- insights based on correlations between behavior patterns and glycemic atic genome-wide investigation of shared genetic effects (pleiotropy) and outcomes was leveraged to create an interactive mobile assistant for T1D enrichment of biologically functional loci implicated in BW-T2D associations. patients. Methods: We evaluated pleiotropy, enrichment of functional annotation, Method: Medtronic MiniMed™ Connect users (N=256) were invited to take and association of single nucleotide polymorphisms (SNPs) using genome- part in a 90-day Sugar.IQ app pilot program that began April 11, 2017. The per- wide summary statistics of 2,893,797 SNPs for BW from the Early Growth centage of time in target range (TIR, 70-180 mg/dL), <70mg/dL, >180mg/dL, Genetics Consortium, and T2D from the Diabetes Genetics Replication And and excursions (periods >20 minutes and <70mg/dL or >180mg/dL) Meta-analysis Consortium. Analyses were conducted using the Genetic were collected 30 days before Sugar.IQ onboarding, and compared to those analysis incorporating Pleiotropy and Annotation (GPA). Tests for enrichment 90 days later (August 2017). Insights delivered to users and user feedback of annotations from functional datasets were performed in loci associated were also analyzed. with pairs of traits. Results: There were 11,356 sensor-wear days; 10,761 unique Sugar.IQ Results: We found significant enrichment of genetic pleiotropy (P <10-30) usage sessions collected; and 4,688 insights delivered (1 every 3 days). and biologically functional loci jointly associated with BW and T2D (enrich- Insights included 655 and 699 identifying behaviors associated with more ment fold of shared vs. BW-specific vs. T2D-specific loci: 1.45 vs. 1.24 time <70mg/dL and >180mg/dL, respectively. The Sugar.IQ app was used 2.1 vs. 0.57; P=1.73x10-21). A total of 178 independent SNPs in genes NYAP2- times/day. Compared to baseline, TIR was 33 minutes longer per day (P<0.15) IRS1, ADCY5, ST6GAL1, CDKAL1, ANK1, HHEX-ECOC6, FAT3-MTNR1B, and and hypoglycemia events reduced by 1.0 per month (P<0.001). A week after MPHOSPH9 were associated with both BW and T2D at a false discovery receiving insights associated with hypoglycemia, 55% and 54% of users rate of 5%. The locus at NYAP2-IRS1 (rs1515098 C) associated with reduced had fewer hypoglycemia and hyperglycemia events, respectively. Hypergly- BW and increased T2D risk was novel. Functional analysis showed that cemia events >2 hours reduced by 1.3 per month (P<0.001). After receiving rs62188784, in strong LD with rs1515098 (r2=0.91), has potential regulatory insights about low glucose associated with boluses delivered at rapid rates (cis-eQTL) effect on expression of the IRS1 gene in adipose tissues. of change, users tended to take smaller boluses and consume less carb in the Conclusions: We found shared genetic loci that influence early growth following 7 days. Among insights that included user feedback, 86% of users and type 2 diabetes, including novel shared variant in the insulin receptor rated them as “Helpful” vs. “Not helpful.” substrate 1 gene. Findings suggest that genetic regulation of insulin signal- ORALS Conclusion: Timely and personalized insights, such as those provided during in adipose tissue potentially influences the associations of early growth ing the Sugar.IQ pilot, may advance patient understanding of glucose trends, and type 2 diabetes later in life. aid in behavioral change that improves therapy adherence, and lead to bet- Supported By: Eunice Kennedy Shriver National Institute of Child Health and ter outcomes. Human Development; National Institutes of Health

19‑OR GENETICS, ADIPOSE TISSUE, OBESITY, AND Genetically Regulated Mechanisms of Insulin Resistance in Adi‑ TYPE 2 DIABETES pose Tissue NEERAJ K. SHARMA, CHIA-CHI C. KEY, MARY E. COMEAU, CARL D. LANGEFELD, 17‑OR JOHN S. PARKS, SWAPAN K. DAS, Winston-Salem, NC The Maternal Impact of AdGHRKO Mice on Glucose Metabolism Insulin resistance (IR) is a harbinger of type 2 diabetes (T2D) and partly XIAOSHUANG WANG, AI MI, YANSHUANG LIU, DAN WANG, LIYUAN RAN, determined by genetic factors. However, genetically-regulated mechanisms YINGJIE WU, Dalian, China of IR remain poorly understood. Using gene expression, genotype, and insu- Genetically modified mouse models provide effective tools to explore lin sensitivity (SI and Matsuda index from FSIVGT and OGTT) data from an the occurrence development and therapy of various diseases, especially African American cohort (AAGMEx, N=256), we performed transcript-wide the metabolic diseases such as diabetes. In line with the reported maternal association and expression quantitative trait loci (eQTL) analyses to identify inheritance of glucose homeostasis via maternally expressed gene activity, IR-associated cis-regulated transcripts (cis-eGenes) in adipose tissue. Data we found that a tissue-specific knockout exhibited maternal influence on from a European ancestry cohort (METSIM, N=770) was used for testing rep- insulin sensitivity, resulting in a lower liability for diabetes in the offspring. lication of Matsuda index-associated cis-eGenes. In vitro genetic silencing Using the Cre/LoxP system, we constructed a mouse model of adipose tis- studies were performed in a human adipocyte model to define the molecular sue-specific growth hormone receptor (GHR) knockout (AdGHRKO). Unlike mechanism of selected genes. Expression level of 2724 transcripts in adi-

ADA-Supported Research

A5 GENETICS, ADIPOSE TISSUE, OBESITY, AND TYPE 2 DIABETES

pose were negatively (1338) or positively (1386) associated (p<0.001) with sizes. Diet was recorded using validated cohort-specific dietary assessment Matsuda index in both cohorts. In the AAGMEx cohort, we identified cis- tools. eQTLs (FDR<1%) for 786 Matsuda index-associated transcripts. Cis-eQTLs Findings: During a median follow-up of 12 years, 20,451 participants for 510 Matsuda index-associated genes were identified in both the AAG- developed T2D. The relative risk of T2D per increment of 10 risk alleles in the MEx and METSIM cohorts. These genes were enriched for mitochondrial GRS was 1.68 (95% confidence interval [CI] 1.62-1.74). Increasing monoun- dysfunction, phagosome maturation, and branched-chain amino acid degra- saturated fat intake in place of carbohydrates was associated with a higher dation pathways. The enoyl-CoA hydratase domain-containing 3 (ECHDC3) risk of T2D (HR per 5% of energy 1.08, 95% CI 1.02-1.15), while increasing was the top ranked Matsuda index-associated cis-eGene. ECHDC3 expres- polyunsaturated or total ω-3 fat intake in place of carbohydrates was asso- sion level was positively correlated with Matsuda index (r=0.47, p=5.3x10-15) ciated with a lower risk of T2D (HR per 5% of energy 0.92, 95% CI 0.85-1.00; and rs200943982 was the top ECHDC3 cis-eSNP (p=1.94x10-9) in AAGMEx. and HR per increment of 1g/d 0.95, 95% CI, 0.92-0.99, respectively). We did The shRNA-mediated silencing of ECHDC3 in SGBS adipocytes reduced not observe evidence of significant interactions between dietary fat sub- insulin-stimulated Akt phosphorylation. RNA-seq analysis identified 691 types and GRS on the risk of T2D. differentially-expressed genes in ECHDC3 knockdown cells, which were Interpretation: In the present long-term prospective study including 103,206 enriched in γ-linolenate and cholesterol biosynthesis, LXR/RXR activation participants, our results support that genetic risk profile and monounsatu- pathways, and known IR genes. rated fat intake were each associated with a higher risk of T2D, whereas In summary, variation-based genomic analysis with concurrent in vitro polyunsaturated fat intake was associated with a lower risk of T2D. Findings genetic silencing studies identified novel genetic regulatory mechanisms of IR. from this study suggest that dietary fat recommendations do not need to be Supported By: National Institutes of Health tailored to individual T2D genetic risk profile for the primary prevention of T2D, and that dietary fat subtypes associate with the risk of T2D across the 20‑OR spectrum of T2D genetic risk. Genome-Wide and Abdominal Imaging Data Characterizes Common Alleles Associated with Higher BMI and Subcutaneous Fat but Less 22‑OR Liver Fat and Lower Risk of Type 2 Diabetes MicroRNA Drivers of the Effects of High-Fat Diet and Bariatric Sur‑ HANIEH YAGHOOTKAR, YINGJIE JI, ANDRIANOS M. YIORKAS, FRANCESCA gery on the Intestine FRAU, DENNIS MOOK-KANAMORI, RENÉE DE MUTSERT, JESSICA TYRRELL, PRAVEEN SETHUPATHY, Ithaca, NY SAMUEL E. JONES, ROBERT BEAUMONT, ANDREW R. WOOD, LOUISE THOMAS, High-fat diet (HFD) leads to obesity and insulin resistance, which are key KARLA V. ALLEBRANDT, NORBERT STEFAN, HARALD STAIGER, EWAN PEAR- risk factors for type 2 diabetes (T2D). Many studies have investigated the SON, ALEX I. BLAKEMORE, JIMMY D. BELL, TIMOTHY M. FRAYLING, Exeter, effects of HFD on several metabolic tissues, but the intestine is highly under- United Kingdom, London, United Kingdom, Frankfurt, Germany, Leiden, Netherlands, explored. The small intestine is the primary site of the positive metabolic Tübingen, Germany, Dundee, United Kingdom, Uxbridge, United Kingdom effects of the most successful pharmacologic treatments (metformin) and Genetic studies have identified “favourable adiposity” variants, where surgical interventions (bariatric surgery) for T2D. The innermost cell layer the allele associated with higher adiposity is associated with lower risk of of the small intestine is the epithelium, which is the most rapidly renewing type 2 diabetes. We took a novel approach to find more of these alleles and tissue in the body and is comprised of several cell types that mediate nutri- find the underlying mechanisms. ent absorption and energy homeostasis. The high renewal rate for these We first performed a genome wide association study (GWAS) of body fat cells is driven by a small but highly proliferative population of intestinal stem percentage using 451000 individuals from UK Biobank. Second, we used cells (ISCs). In this study, we showed that an obesogenic HFD significantly published genetic data in a multivariate test to find alleles associated with increases proliferation of ISCs, and alters allocation of ISCs to different higher adiposity but a “favourable” metabolic phenotype: higher HDL-C, lineages in mice. To define the molecular mechanisms that underpin these adiponectin, sex hormone binding globulin, but lower triglycerides, fasting effects, we performed whole transcriptome analyses (both small RNA-seq insulin and alanine transaminase. Third, we used abdominal imaging data to profile microRNAs (miRNAs) and RNA-seq for long, non-coding RNAs and from 4 studies to define the adiposity phenotype in more detail. mRNAs) in Lgr5+ ISCs. We demonstrated that HFD shifts the miRNA profile We identified 620 variants associated with body fat percentage (p<5x10-8). of ISCs toward that of enterocytes, the cells involved in nutrient absorption. Fourteen alleles had a “favourable adiposity” phenotype, including 7 previ- Further bioinformatics analysis revealed that this shift is driven primarily by ously known variants (in/near PPARG, LYPLAL1, GRB14, IRS1, PEPD, FAM13A changes in two miRNAs, miR-194 and miR-375. Specifically, HFD suppresses and ANKRD55), 5 known to be associated with a relevant trait (TRIB1, KLF14/ miR-375 by over five-fold (P < 0.05). Notably, follow-up studies showed that MKLN1, DNAH10, VEGFA/C6orf223 and AEBP2/PDE3A) and 2 novel variants vertical sleeve gastrectomy, which reverses the effects of HFD, significantly (MAFF and CITED2). Carrying 10 additional “favourable adiposity” allele was rescues the levels of miR-375 in Lgr5+ ISCs. We hypothesized that suppres- associated with higher BMI (0.63 kg/m2, p = 4x10-45) but lower risk of type 2 sion of miR-375 promotes ISC proliferation and allocation to enterocytes. diabetes (odds ratio: 0.60, p = 4x10-44, 14371 cases), lower risk of heart dis- To test this hypothesis, we generated miR-375-/- mice and showed that loss ease (0.82, p = 3x10-14, 37741 cases) and lower risk of hypertension (0.82, of miR-375 in vivo leads to a marked increase in ISC proliferation and vil- p = 1x10-33, 241691 cases). Using MRI/CT scans of abdominal fat from UK lus height, mimicking the effects of HFD. Currently we are investigating Biobank (N = 5000), NEO (2236), TÜF (900), and a published GWAS (10557), whether miR-375 is essential for the effects of bariatric surgery. ORALS carrying more “favourable adiposity” alleles was associated with higher Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-16- subcutaneous fat (p = 1x10-12) but lower liver fat (p = 1x10-7). These effects ACE-47) were similar when we removed the 7 known “favourable adiposity” variants. “Favourable adiposity” alleles associated with higher BMI but lower risk 23‑OR of type 2 diabetes are characterized by higher subcutaneous but lower liver Physical Inactivity and Sleep Inefficiency Accentuate the Genetic fat. Risk of Obesity Supported By: Diabetes UK; European Foundation for the Study of Diabetes; ANDREW R. WOOD, SAMUEL E. JONES, REBECCA RICHMOND, SHAFQAT Novo Nordisk AHMAD, HANIEH YAGHOOTKAR, ROBERT BEAUMONT, KATHERINE S. RUTH, MARCUS TUKE, ANNA MURRAY, RACHEL M. FREATHY, GEORGE DAVEY SMITH, 21‑OR I-MIN LEE, NIC TIMPSON, DANIEL CHASMAN, ZOLTAN KUTALIK, MICHAEL N. Dietary Fat Quality and Genetic Risk of Type 2 Diabetes WEEDON, JESSICA TYRRELL, TIMOTHY M. FRAYLING, Exeter, United Kingdom, JORDI MERINO, HASSAN S. DASHTI, MARTA GUASCH, CHRISTINA ELLERVIK, Bristol, United Kingdom, Boston, MA, Lausanne, Switzerland CAREN SMITH, TUOMAS O. KILPELAINEN, DANIEL CHASMAN, JOSE C. FLOREZ, Gene-lifestyle interactions of relevance to type 2 diabetes have been dif- Boston, MA, Copenhagen, Denmark ficult to identify but may help stratify groups of people at especially high risk. Background: Type 2 diabetes (T2D) is a complex disease driven by lifestyle In large genetic studies, measures of diabetes risk factors such as diet and and genetic factors. The extent to which dietary fat quality may modify T2D activity are usually limited to self-reported data. We used genetic and accel- genetic burden on the incidence of T2D is unknown. erometer data from 97,806 individuals to test for genetic interactions with Methods: We used Cox proportional-hazards models to calculate adjusted physical activity and sleep lifestyles. We tested the hypothesis that lower hazard ratios (HRs) for T2D among 103,206 participants of European descent levels of physical activity and abnormal sleep patterns accentuate genetic from 15 prospective cohort studies. T2D genetic risk profile was character- susceptibility to obesity. We used 82,322 individuals from the UK Biobank, ized by a 68-variant genetic risk score (GRS) weighted by published effect 10,885 from the Women’s Genome Health Study (WGHS) and 4,599 individuals from the Avon Longitudinal Study of Parents and Children (ALSPAC). We

ADA-Supported Research

A6 NOVEL APPROACHES TO ADVANCING ISLET TRANSPLANTATION—BASIC SCIENCE derived measures for physical activity and sleep, including total physical thicker and looser membranes. We observed intra-AgMc IgM retention and activity, time spent undertaking sedentary, light, moderate and vigorous their progressive release into the culture medium in vitro. Prediabetic NOD activity, and sleep duration and sleep efficiency. We used BMI as the out- mice were divided into two groups: (1) n=20, received graft (TX) of G3C con- come and tested for interactions between a BMI genetic risk score (GRS) taining microcapsules; (2) n=10 received TX of empty microcapsules. Both and the accelerometer derived measures of activity and sleep. We also per- (1) and (2) were monitored for the onset of DM. BG in basal and after IPGTT, formed several negative control experiments to test for residual confound- morphological, immuno-molecular, and cell immuno-phenotyping testing, as ing. We found evidence of gene-activity interactions. For example, in the well as G3C plasma levels assay in (1) were performed. In (2): 5/10 animals UK Biobank, when analysing non-sedentary physical activity, the 10% of developed DM within 70d; in (1) no animals developed DM at 180 of TX. individuals carrying the most BMI-raising alleles were 5.9kg heavier if they In the spleen of G3C-AgMc-treated mice either CD4+FoxP3+GITRbright, or were inactive compared to active (for height 1.73m). In contrast, the 10% of CD4+CD25-GITR(sp), or CD8+CD25+GITRlowFoxP3+, or CD8+CD25-GITRint- individuals carrying the fewest BMI-raising alleles were 4.1kg heavier if they FoxP3+ Tregs significantly increased from 4 week of TX as compared to con- -4 were inactive compared to active (Pinteraction=4x10 ). This observation was trols. Pancreatic histology at 26 week of TX was associated with either com- consistent in the WGHS and ALSPAC data although not all tests reached pletely destroyed islets (1) or intact islets with minor insulitis (2). Significant P<0.05. The results were robust to negative control tests, including the test- diabetes-free time of (1) vs. (2) may relate to increase of islet B-cell specific ing of interactions using random variables simulating the BMI-activity cor- Tregs, expanded by G3C-released MAb, and antigenic stimulation, at early relations. Our results are consistent with previous studies suggesting that stage of the autoimmune disease process. low levels of physical activity accentuate the genetic risk of obesity. In conclusion, the newly engineered AgMc, by permitting GITR triggering Supported By: European Research Council; British Heart Foundation by anti-GITR IgM, may represent a new approach to expand Tregs and inhibit Teff, and to avert the onset of autoimmune DM in NOD mice. 24‑OR Whole Genome Sequence Association Analysis and Diabetes-cen‑ 26‑OR tric Functional Annotation of Type 2 Diabetes Subcutaneous Transplantation of MIN6 Beta Cells Embedded in JENNIFER WESSEL, JENNIFER A. BRODY, ALISA MANNING, TOPMED DIABETES mPEG-Ala Hydrogel WORKING GROUP, Indianapolis, IN, Seattle, WA, Cambridge, MA JYUHN-HUARNG JUANG, HSIU-CHAO LIN, CHEN-YI CHEN, CHEN-WEI KAO, Our understanding of the genetic basis of T2D has been significantly SHU-TING WU, SUNG-HAN LIN, CHIA-RUI SHEN, JIUN-JIE WANG, I-MING CHU, advanced by identification of T2D loci through analysis of common variation; Taoyuan, Taiwan, Hsinchu, Taiwan however, evidence suggests a role for rare regulatory variation influencing The liver is the current site of choice for clinical islet transplantation, but T2D. We leveraged the largest available whole genome sequence (WGS) many intraportal grafts were lost in the early stage of transplantation. Other data from NHLBI’s Trans-Omics for Precision Medicine (TOPMed) initiative disadvantages include the difficulty of monitoring the islets and procedure- and diabetes-centric genomic function data to the interpretation of results. associated complications. In contrast, the subcutaneous space offers the We performed single-variant pooled-ancestry association analyses with advantages of a large space, a less invasive procedure and easy graft moni- (>30x deep sequence) WGS in 13,200 individuals (2,601 T2D and 10,599 toring and removal. However, islet transplantation into an unmodified subcu- controls) from 6 studies. The association analyses were performed in the taneous site has poor efficacy; possibly due to poor oxygenation and inade- Analysis Commons hosted on DNANexus. We demonstrated the utility of quate vascularization of the transplanted tissue. In this study, we tested the the OASIS (Omics Analysis, Search and Information System) platform and feasibility of subcutaneous transplantation of MIN6 beta cells embedded in integrating diabetes-specific functional annotation in a recently discovered temperature-sensitive poly(ethylene glycol) methyl ether (mPEG)-Ala hydro- locus (GIP-IGF2BP1) to fine-map to the likely causal variants. Our results gels as scaffolds. After culturing for 14 days, the viability of MIN6 beta cells identified common (minor allele frequency [MAF] > 0.05) variant associations in mPEG-Ala hydrogel was comparable with those in medium, either assayed (P < 5 x 10-8) at a known locus with T2D: TCF7L2 (rs7903146, P = 3.8 x 10-12). by MTT or LIVE/DEAD. Static incubation showed that both had comparable We also identified a potentially novel association on Chr15q21 with 27 rare insulin secretion. For in vivo experiments, we infected MIN6 beta cells with variants (MAF = 0.2%, P =1.3 x 10-8, r2=1) spanning a 7.5 MB region. In the luciferase by AAV and then transplanted 5*106 cells embedded in mPEG-Ala Old Order Amish study, 53 individuals carry the haplotype tagged by these hydrogel into the subcutaneous space of each nude mouse’s upper back. 27 variants; 17% of carriers are diagnosed with T2D compared to 3% of Positive In Vivo Imaging System (IVIS) images and positive insulin staining of the non-carriers. Interrogation of the GIP-IGF2BP1 locus, utilizing diabetes- tissue histology were observed up to 41 days after transplantation. Mean- specific annotation and association evidence of both common and rare non- while, we incubated MIN6 beta cells with chitosan-coated superparamag- coding variants, within the OASIS browser implicated nearby genes in the netic iron oxide (CSPIO) overnight and then transplanted 5*106 MIN6 beta region as being the likely biologic candidates. cells into the subcutaneous tissue of each nude mouse’s left flank. The graft In conclusion, preliminary results suggest WGS analysis can discover of CSPIO-labeled MIN6 beta cells was visualized on Magnetic Resonance novel loci for complex traits. Work is ongoing to refine annotation for rare (MR) scans as distinct hypointense spots on T2-weighted images located at variant association tests and perform functional fine-mapping across the the transplantation site at day 6 and 20. These results indicate that subcuta- genome. We soon will extend our analysis into the next release of WGS data neous transplantation of MIN6 beta cells embedded in temperature-sensi- from TOPMed (N~69,000 individuals). tive mPEG-Ala hydrogels is feasible. Moreover, the IVIS and MR imaging are Supported By: National Institutes of Health (5U01DK078616, 5K01DK107836); useful tools for detecting and monitoring beta cells at the subcutaneous site. ORALS National Institute of Diabetes and Digestive and Kidney Diseases; National Heart, Lung, and Blood Institute 27‑OR Predicting Insulin Secretion Profiles for Immunoisolating Devices with Transplanted Islets NOVEL APPROACHES TO ADVANCING ISLET PETER BUCHWALD, ALICE A. TOMEI, CHERIE L. STABLER, Miami, FL, Gainesville, FL TRANSPLANTATION—BASIC SCIENCE Avascular islets or stem cells transplanted under immunoisolating conditions offer considerable hope in overcoming the challenges of islet trans- 25‑OR plantation; however, because of diffusional limitations, implants face Newly Designed Alginate-Based Microcapsules (AgMc) for the long-term viability and efficiency-of-response problems. To quantitatively Molecular Therapy of Type 1 Diabetes characterize the glucose-stimulated insulin release of pancreatic islets, we RICCARDO CALAFIORE, PIA MONTANUCCI, GIUSEPPE P. BASTA, GIUSEPPE conducted parallel dynamic perifusion experiments with free and hydro- NOCENTINI, TERESA PESCARA, CARLO RICCARDI, Perugia, Italy gel encapsulated islets. Perifused human islets secreted less insulin than Glucocorticoid-induced tumor necrosis factor receptor-related protein murine islets per unit mass (IEQ) and had a less pronounced first-phase peak. (GITR) plays a crucial role in either thymic t-Treg differentiation and expan- The data was used to develop a computational model of insulin secretion. sion, or peripheral p-Treg expansion, thus representing a potential tool to Our model couples hormone secretion and nutrient consumption kinetics contrast the autoimmune process. AgMc are usually intended to constitute with diffusive and convective transport, fits well the experimental data, and an immunobarrier to prevent rejection of the embodied islet/insulin produc- can be used to predict insulin release profiles for arbitrary geometries and ing cell grafts in T1D. We aimed to reverse this principle, and engineered glucose challenges. We found that larger diffusion distances (larger cap- the AgMc membranes, in order to make them permeable to outflowing rat sules) unavoidably dampen the first-phase insulin response, resulting in a anti-mouse GITR IgM MAb, derived from microencapsulated rat G3C hybrid- sustained-release type insulin secretion that sluggishly responds to changes oma cells. To this end, we changed our AgMc original formulation to obtain in glucose concentration. Hence, bioartificial pancreas devices can provide

ADA-Supported Research

A7 NOVEL APPROACHES TO ADVANCING ISLET TRANSPLANTATION—BASIC SCIENCE

long-term and physiologically desirable solutions only if immunoisolation We hypothesize that islet encapsulation with a nanothin multilayer coat- and biocompatibility considerations are considered together with optimized ing consisting of tannic acid (TA), an immunomodulatory antioxidant, and nutrient diffusion and insulin release characteristics. poly(N-vinylpyrrolidone) (PVPON), will provide an immunoprotective barrier Figure. and maintain β-cell function after transplantation. In support of our hypothesis, we demonstrate that (PVPON/TA)-encapsulated syngeneic NOD.Rag islets restored euglycemia after transplantation under the kidney capsule of streptozotocin-treated diabetic NOD mice by more than 30 days in comparison to non-encapsulated islets. Encapsulated allogeneic B6 islets also restored euglycemia in diabetic NOD mice and delayed rejection by 7.5 days in contrast to non-encapsulated islets. Gene expression of allogeneic B6 islets at 5 days post-transplant displayed an increase in Ins2 (6-fold), Gcg (6-fold), Pdx1 (5-fold), Ccl22 (3-fold), and Arg1 (2.5-fold) mRNA accumulation with (PVPON/TA)-encapsulated islet grafts, suggesting a potential shift toward an alternatively-activated M2 macrophage phenotype. To address limited islet availability, the use of (PVPON/TA)-encapsulated differentiated neonatal porcine islets restored euglycemia and maintained β-cell function for more than 200 days after transplantation into diabetic NOD.scid mice. Our results support the hypothesis that islet encapsulation with (PVPON/ TA) coatings maintain function and may elicit protection following islet allo- and xenotransplantation. Future studies will determine the immunological mechanism of protection following (PVPON/TA)-encapsulation of islets. Supported By: American Diabetes Association (7-12-CD-11 to H.M.T.); JDRF (1-SRA-2015-42-A-N)

30‑OR Prolongation of Allograft Survival by Intrathymic Introduction of Allogeneic Thymic Epithelial Cells ISHA PRADHAN, STEPHANIE WONG-NOONAN, SUZANNE BERTERA, ROBERT J. LAKOMY, HENRY COHEN, DERRICK WELLS, MASSIMO TRUCCO, YONG FAN, Supported By: National Institutes of Health/National Institute of Diabetes and Pittsburgh, PA Digestive and Kidney Diseases (1UC4DK104208, 1R01DK109929); JDRF (3SRA Thymic epithelial cells (TECs), the primary stromal population in the thy- 2017-347MB) mus, are critical to establish the microenvironments required for generating self-tolerant, pathogen-responsive T cells. However, due to the inacces- 28‑OR sibility of the thoracic cavity and the fragility of TECs, manipulating the Comparison of Xenogeneic Encapsulated Islet Transplantation in thymus to modulate adaptive immune responses remains challenging. We Nonhuman Primates With and Without Immunosuppression developed a novel surgical procedure that allowed access to thymus glands EUN YOUNG LEE, HEON SEOK PARK, JIWON KIM, YEOREE YANG, JAYOUNG LIM, for intrathymic injection (ITI), without the need to cut the manubrium of the HUN-SUNG KIM, SEUNG-HWAN LEE, JAE HYOUNG CHO, BONG YUN CHA, KUN- sternum bone. Horizontal cuts of the muscle layers between the first and HO YOON, Seoul, Republic of Korea second bones of the rib cage expose the thymus with minimal side effects. Previously, we have shown the long-term efficacy and biocompatibil- This novel ITI procedure enabled us to implant 16.5-day embryonic TECs into ity of chitosan-coated alginate capsule in allogeneic islet transplantation an adult syngeneic recipients’ thymus. Long-term survival and colonization with canine models of diabetes. In this study, we compared the efficacy of the implanted TECs was observed via live imaging, flow cytometry, and and immune response of chitosan-coated alginate capsule in xenogeneic immunohistochemistry. We further investigated if introduction of allogeneic islet transplantation with non-human primates (NHPs). Porcine islets were TECs in the thymus promote negative selection against allo-reactive T cells and induce donor-specific immune tolerance. We engrafted B6 islets under encapsulated alginate crosslinked with BaCl2, followed by suspension in chitosan solution. Encapsulated porcine islets were transplanted intraperi- the kidney capsules of chemically induced diabetic Balb/C mice, precondi- toneally in NHPs with and without immunosuppressants. Anti-αGal IgM tioned with T-cell depletion sera and intrathymic injection of B6 16.5-day and IgG was measured from the day of transplantation. After transplanta- embryonic TECs. Prolonged survival of the transplanted islets was observed tion of encapsulated porcine islets, hyperglycemia and exogenous insulin compared to sham-operated controls. Mixed lymphocyte reaction experi- requirements were decreased in the NHP recipients. Porcine C-peptide was ments further showed dampened T cell proliferation responses against detected in plasma after transplantation, but decreased with time. Anti- donor B6 antigens. Moreover, increased levels of Foxp3+ T-regulatory cells were detected in Balb/C recipients with ITI of B6 TECs, suggesting that

ORALS αGal IgM and IgG began to increase a week after transplantation in NHP without immunosuppressants, while they showed little increase in NHP implanted TECs could also promote the generation of alloantigen-specific with immunosuppressants. Exogenous insulin began to be administered T regulatory cells in addition to facilitating negative selection of donor anti- to the NHP recipients from 3 to 5 days after transplantation regardless of gen-reactive T cells. Our results suggest that intrathymic transplantation of immunosuppressants. However, daily insulin requirement increased earlier TECs could be an effective way to induce donor-specific immune tolerance in NHP without immunosuppressants than those with immunosuppressants. with limited general immune suppression. Retrieved encapsulated porcine islets from showed fibrosis more than 70% Supported By: National Institutes of Health in NHP without immunosuppressants, whereas only 5% of retrieved capsules showed fibrosis in NPH with immunosuppressants. In addition, in NPH 31‑OR with immunosuppressants, dithizone positive islets were observed over a Immunosuppressive PLGA TGF-β1 Microparticles Induce Anti‑ month after transplantation. The use of immunosuppressants in xenoge- gen-Specific -T Cell Tolerance for Enhancing Islet Transplantation neic encapsulated islet transplantation in NHP may help reduce fibrosis and Outcomes improve islet function by modulating immune reaction. YING LI, ANTHONY FREI, STEVEN D. BARASH, CHERIE L. STABLER, Gainesville, FL Supported By: Korean Ministry of Health and Welfare (HI13C0954) Clinical islet transplantation (CIT) is a potential therapy for type 1 diabetes. Allorejection mediated by the host CD4 T cells is a major hurdle for successful 29‑OR CIT, despite potent systemic immunosuppression (SI). To address these issues, Tannic Acid-Encapsulated Islets Suppress Autoimmunity and we engineered PLGA microparticles (PLGA-MPs) that allow the localized deliv- Restore Euglycemia following Transplantation ery of TGF-β1 for regulatory T cells (TRegs) induction at the transplant site. JESSIE BARRA, HUBERT M. TSE, Birmingham, AL TGF-β1 is an immunoregulatory agent that promotes the TRegs differentiation Type 1 diabetes (T1D) is an autoimmune disease of pancreatic β-cell death from naïve CD4 cells by inducing FoxP3 expression while suppressing inflam- caused by islet-infiltrating leukocytes. Islet transplantation can restore matory Th1 and Th2 responses. To date, we have (i) fabricated TGF-β1 loaded β-cell function, however, limited islet availability, toxicity of immunosup- PLGA particles by double emulsion (PVA(aq)- CH2Cl2- PVA(aq)) with character- pressants, and poor graft survival are major hurdles for clinical application. ization in size (45±26 μm), TGF-β1 encapsulation efficiency (49.1±0.1%), and

ADA-Supported Research

A8 ADIPOSE DIFFERENTIATION AND FUNCTION release kinetics (durable biphasic release for 14 days); (ii) proved effective ADIPOSE DIFFERENTIATION AND FUNCTION TRegs conversion in OVA-OTII antigen-specific murine model (Fig.1); and (iii) shown efficacy for in vivo Treg conversion by MPs in rodent islet allografts, 33‑OR where accepted grafts showed improved insulin production and elevated Functional Roles of Proline/Arginine-Rich End Leucine-Rich Repeat TRegs. Future studies exploring the competency of PLGA-MPs to provide dura- Protein (PRELP) in Adipocytes ble allograft protection in rodent models is on-going. Based on these promising JUN EGUCHI, JUN WADA, Okayama, Japan preliminary results, TGF- 1 PLGA-MPs could provide a translational therapeu- β Adipose tissue fibrosis is recognized as a hallmark of adipose tissue dys- tic strategy to minimize islet graft rejection and the dependency on SI. function. We have previously identified PRELP as a novel regulator of adipogenesis by using lentiviral short hairpin RNA libraries targeting murine genomes. PRELP belongs to the family of leucine-rich repeat proteins, which are characterized by a series of adjacent leucine-rich motifs flanked by disul- fide-bounded domains. Functionally, PRELP anchors basement membranes to connective tissues. However, roles of PRELP in adipocytes remain unclear. In the present study, we show that PRELP expression is up-regulated both in adipocytes of high-fat diet induced obesity mice and ob/ob mice. PRELP reduction in 3T3-L1 adipocytes leads to enhanced insulin signaling and insulin-stimulated glucose uptake. To determine functions of PRELP in the development of metabolic phenotypes, we generated adipocyte-specific PRELP transgenic mice. Adipose-specific overexpression of PRELP results in the development of significant adipose tissue fibrosis and insulin resistance on high fat diet without difference of body weight and adiposity. This phenotype is associated with increased expression of inflammation genes and decreased insulin signaling in adipose tissue of PRELP transgenic mice. Fur- thermore, we find that global Prelp deficient mice are protected from adipose tissue fibrosis and insulin resistance associated with high fat feeding. These data suggest that PRELP might play a key role in the development of adipose tissue remodeling in obesity. Supported By: Japan Society for the Promotion of Science; Okayama Medical Foundation

34‑OR Adipocyte mTORC1 Suppresses Treg Cell Development and Brown‑ ing of White Adipose Tissue through CRTC2/COX-2/PGs Pathway XING ZHANG, YAN LUO, XIAOFENG DING, CHUNQING WANG, XUEXIAN YANG, MEILIAN LIU, Albuquerque, NM Adipose tissue (AT) contains abundant CD4+Foxp3+ regulatory T (Treg) cells; however, the physiological signals that control AT Treg cell and the role of AT Treg cell in thermogenesis have not been defined. Here we show that obesity activated mTORC1 signaling while decreased frequency of Treg cells Supported By: National Institutes of Health in white adipose tissue (WAT), and adipose-specific inhibition of mTORC1 by raptor deficiency promoted development of Treg cells and beige adipocytes 32‑OR and protected mice against diet-induced reduction of Treg cells and beige Acceptance of Murine Islet Allografts without Immunosuppression adipocytes in WAT and diet-induced obesity. Mechanistically, mTORC1 sig- in the Inguinal Subcutaneous White Adipose Tissue Pretreated naling phosphorylated CREB regulated transcription coactivator 2 (CRTC2) with bFGF and subsequently suppressed expression of cyclooxygenase 2 (COX-2) and YUKI NAKAFUSA, NAOYOSHI NITTA, MASAFUNI NAKAMURA, YOHICHI YASU- secretion of prostaglandins (PGs) including PGI2, PGD2 and PGE2 in vivo and NAMI, Fukuoka, Japan in adipocytes. Treatment of PGE2 but not PGI2 and PGD2 promoted differ- Prevention of immune rejection without immunosuppression is a goal for entiation and activation of Treg cells in vitro and in vivo. Pharmacological transplant immunology. Previous report demonstrated that rejection of rat inhibition of COX-2 or blocking Treg cell by neutralization of CD25 attenu- islet allografts in sc space of the back between low responder strains was ated mTORC1 inhibition-induced browning of white fat. Further, deficiency of prevented by pretreatment of the site with bFGF (AJT2014). However, it was Treg cell suppressed the inducing effect of PGE2 administration on thermo-

not in the high responder combination of mice (BALB/c to C57BL/6). Here, genic gene expression and oxygen consumption in inguinal and epididymal ORALS we determined whether islet allografts in the inguinal subcutaneous white WAT. Taken together, these results uncover that adipocyte mTORC1 is a key adipose tissue (ISWAT), a novel site of islet transplantation (tx), pretreated regulator of Treg cell and beige adipocyte development in adipose tissue via with bFGF are accepted without immunosuppression in mice. An agarose CRTC2/COX-2/PGs pathway-dependent mechanisms. rod containing bFGF was buried in the ISWAT of a C57BL/6 mouse, and 1-3 Supported By: American Diabetes Association (1-17-IBS-261 to M.L.); National weeks later 400 BALB/c islets (2 donors) were grafted in the space after the Institute of Diabetes and Digestive and Kidney Diseases (R01DK110439-01); removal of a rod. Recipients were made diabetic by STZ (180mg/kg, iv) at 3 National Institutes of Health (NIGMSP20, GM121176-01) days prior to tx. Eventually, we found that 10µg bFGF/50µl/rod with 2 week interval is the optimal, in which 11/14 recipient mice remained normoglycemic (<200mg/dl) for more than 60 days. Removal of the ISWAT with islets 35‑OR The Adipose-Derived Insulin Sensitizer Adiponectin Can Activate promptly made recipient mice hyperglycemic again. Histologically, intact Intracellular Signaling Independent of the Well Known AdipoR1 or islets were seen in the ISWAT. In contrast, islet allografts (n=7) in the ISWAT AdipoR2 Receptors pretreated with vehicle were rejected by 10 days. FACS revealed an expan- JOSEPH W. GUNNET, WENYU LI, YUANPING WANG, JOSE A. CHAVEZ, MATT sion of CD8 T cells in the ISWAT rejecting but not accepting islet allografts. In HUSOVSKY, RONG MENG, WENSHENG LANG, JEY R. JEYASEELAN, GARY W. the latter, IL-10+cells including Gr-1+ cells were seen. Re-tx of BALB/c islets CALDWELL, SUZANNE EDAVETTAL, Spring House, PA, San Diego, CA (n=3) to the contralateral ISWAT of normoglycemic mice induced rejection Adiponectin (ADN) is a crucial mediator of insulin sensitivity and vascu- of the initial BALB/c grafts and made recipient mice hyperglycemic again, lar health. The discoveries of the adiponectin receptors AdipoR1 and Adi- while that of C3H (n=3) did not. These findings indicate that acceptance of poR2 (R1 and R2) prompted efforts to identify ADN-mimetics. R1 and R2 are murine islet allografts without immunosuppression was achieved when the especially difficult drug targets due to the complex higher-order structures ISWAT pretreated with bFGF was the site and that the acceptance appeared of ADN as well as challenges in creating receptor knock-out cell lines. We mediated by local rather than systemic unresponsiveness. approached R1 and R2 as possible targets beginning with a rigorous valida- Supported By: Japan Ministry of Education, Culture, Sports, Science and tion of the ligand and receptors. ADN preparations from multiple internal Technology and external sources were characterized in physicochemical, cell binding

ADA-Supported Research

A9 ADIPOSE DIFFERENTIATION AND FUNCTION

and in vitro assays (pAMPK/pACC and ceramidase). It is important to note augment efficiency of energy storage. Further elucidation of the integration that ADN from only one source passed this evaluation. This ADN triggered of insulin and GIP signaling will be needed to support efforts to counteract in vitro activities associated with this protein, including stimulation of phos- dysregulated nutrient metabolism in obesity and diabetes. phorylation of ACC (EC50 = 9.3 ug/ml), calcium influx (EC50 = 2.2 ug/ml), glucose uptake, fatty acid oxidation and ceramidase activity. With a biologi- 38‑OR cally active ADN preparation identified, we strove to understand the inter- The Cell Junction Protein Afadin Negatively Regulates Insulin actions of ADN with R1 and R2. However, both R1 and R2 are ubiquitously Action and Modulates Adipose Tissue Function expressed, complicating the in vitro confirmation of receptor-dependent MORTEN LUNDH, MARIE S. ISIDOR, KAJA PLUCINSKA, FARNAZ SHAMSI, functional activity. We selected HAP1 cells, an ADN-responsive haploid PATRICIA PETERSEN, YU-HUA TSENG, BRICE EMANUELLI, Copenhagen, Den- CML derived line well-suited for gene deletion via CRISPR-Cas9 technology, mark, Boston, MA to generate R1, R2 and R1/R2 deficient cells. Surprisingly, the stimulatory Adipose tissue dysfunction is one of the main culprits triggering metabolic activity of adiponectin in pACC and ceramidase assays in HAP1 cells defi- imbalance associated with obesity. We identified the cell junction protein cient in R1, R2 or both R1 and R2 were indistinguishable from those in wild Afadin as one of the most highly phosphorylated proteins in response to type HAP1 cells. In addition, transient overexpression of R1 in HAP1 cells or insulin/IGF-1 stimulation using quantitative phospho-proteomic analysis pretreatment with antibodies to block R1 did not alter their pACC responses in pre-adipocytes, and investigated the role of Afadin in mediating insulin to ADN. These data indicate R1 and R2 are not responsible for all the in vitro action and in adipocyte biology. We found that phosphorylation of Afadin biological activity attributed to ADN and suggest that there must be other occurred in response to insulin in both pre- and mature adipocytes obtained yet unidentified receptors for ADN. from mice and human subjects. In addition, Afadin phosphorylation was observed in mouse adipose tissues following insulin injection, and after re- 36‑OR feeding, indicating physiological relevance. In obese mice, however, brown Silencing Mediator of Retinoid and Thyroid Hormone Receptors fat insulin-induced phosphorylation of Afadin was abolished. Surprisingly, (SMRT)—A Link betweenWITHDRAWN Adipocytes, Inflammation, and Glucose glucose and insulin tolerance were improved in Fat-specific Afadin knock-out Intolerance (KO) mice on a high fat diet, despite similar body weight gain and adipose JONATHAN KAHN, ANNA A. GODDI, RONALD N. COHEN, Chicago, IL mass as wild type controls. Furthermore, ablation of Afadin in vitro did not Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) affect adipogenesis. Yet, prolonged insulin-induced phosphorylation of Akt is a nuclear corepressor, which regulates the transcriptional activity of was observed in KO adipocytes, and was associated with increased insulin- metabolically essential transcription factors. While SMRT has been shown mediated fatty-acid uptake. Finally, phosphorylation of Afadin regulates to modulate adipocyte function, in vivo experiments utilizing knock-out (KO) the docking of several proteins, which may be involved in the regulation of models have led to conflicting results. To more rigorously define the role of insulin action and adipocyte metabolism. Thus, Afadin is a novel bona fide SMRT in the adipocyte, we generated adipocyte-specific SMRT KO (adSMRT substrate of the insulin signaling pathway, and is involved in the regulation KO) mice by crossing adiponectin-Cre mice with floxed SMRT mice on a C57/ of insulin action and metabolism. BL6 background. When adSMRT KO mice are challenged with a 45% high-fat Supported By: Novo Nordisk Foundation; Danish Council for Independent diet, we observe 20% increase in glucose intolerance (p=0.006) compared Research to wild type (WT) counterparts. Additionally, RNA-Seq data of adipose tissue from these mice indicate a dramatic up-regulation of inflammatory gene 39‑OR expression. To further characterize the pro-inflammatory phenotype, we uti- Insulin Sensitizing Effects of Vitamin D Mediated through Reduced lized flow cytometry to identify infiltration of specific populations of adipose Adipose Tissue Inflammation and Fibrosis tissue inflammatory cells. We found that overall macrophage infiltration ERIC LONTCHI-YIMAGOU, SONA KANG, KEHAO ZHANG, AKANKASHA GOYAL, in the adipose tissue of KO mice increased two-fold (p=0.026), with anti- JEE YOUNG YOU, PREETI KISHORE, EVAN ROSEN, MEREDITH HAWKINS, Bronx, inflammatory M2 macrophages infiltrating in significantly lower proportions NY, Berkeley, CA, New York, NY, Boston, MA (p=0.017), indicating a higher ratio of M1:M2 cells in the KO mice compared Adipose tissue inflammation and fibrosis appear to be mediated by adi- to WT. In contrast, body weight and total fat mass were not altered. These pocytes and contribute to insulin resistance. Since vitamin D (25(OH)D) has data suggest a role for SMRT in the cross-talk between adipocytes and pro- anti-inflammatory and anti-fibrotic effects, expression of its receptor in adi- inflammatory macrophages for the regulation of systemic glucose tolerance, pocytes and macrophages suggests that 25(OH)D signaling could mediate distinct from the development of obesity. SMRT therefore integrates meta- paracrine effects within adipose tissue and improve insulin resistance. We bolic and inflammatory signals to maintain physiological homeostasis. performed a randomized, double-blinded placebo-controlled trial to examine Supported By: American Diabetes Association (7-13-BS-033 to R.N.C.) the effects of repleting vitamin D in 25(OH)D-deficient (<20 ng/ml), insulin resistant, overweight-to-obese humans (n=19). 25(OH)D repletion to >30 ng/ml 37‑OR was associated with reductions in adipose tissue expression of inflamma- GIP Signaling Integrates with Insulin Signaling to Regulate Glucose tory (0.6-0.7-fold decreased expression of TNF-α, IL-6, iNOS and PAI-1) and and Lipid Metabolism in Human Adipocytes pro-fibrotic (0.4-0.8-fold decreased expression of TGF-β1, HiF1α, Collagen I,

ORALS AJIT REGMI, MELISSA K. THOMAS, M. DODSON MICHAEL, WILLIAM C. ROELL, V, VI and MMP7) genes, decreased collagen VI immunofluorescence (19% Indianapolis, IN reduction, p=0.02) and improved hepatic insulin sensitivity, assessed as sup- Recent studies suggest that activation of GIP (glucose-dependent insuli- pression of endogenous glucose production (EGP) during hyperinsulinemic notropic peptide) receptor in conjunction with GLP-1 and/or glucagon recep- clamp studies (1.28 ± 0.20 vs. 0.88 ± 0.18 mg/kg/min, p=0.03). tors reduces adiposity and body weight in mice. Incretin actions of GIP on To determine whether vitamin D’s effects are mediated through adipo- pancreatic beta cells are well characterized, but functions of GIP on adipose cytes, we studied an adipocyte-specific vitamin D receptor knockout mouse tissue are incompletely understood. Using differentiated human adipocytes model (Adiponectin-Cre+VDR+/ fl) after 12 weeks on high fat diet. Despite we investigated GIP regulation of carbohydrate and lipid metabolism in no differences in body weight or adiposity, VDR KO mice exhibited increased the context of insulin signaling. GIP dose-dependently stimulated lipolysis adipose tissue expression of several pro-inflammatory (Tnf-α, iNos, Pai-1, without affecting ATGL or HSL lipolytic enzyme mRNA expression. Insulin Mcp-1 and F4/80; 4-10 fold) and pro-fibrotic genes (Tgf-β1, Collagen VI, and dose-dependently suppressed lipolysis, and addition of GIP attenuated insu- Tsp1; 2-4 fold), in concert with hepatic insulin resistance during 2h hyperin- lin effects on lipolysis without affecting Akt phosphorylation at Thr308 or sulinemic (4 mU/kg/min)-euglycemic clamps (EGP 10 ± 3 vs. 3 ± 2 mg/kg/min Ser473. Insulin administration increased lipogenesis, but GIP, in the presence in WT, p = 0.021). or absence of insulin, had little effect on lipogenesis in [14C]glucose incorpo- These human and rodent studies establish a beneficial role of vitamin D ration assays. GIP increased glucose utilization in adipocytes, as measured in restraining adipose tissue inflammation and fibrosis as well as hepatic by depletion of glucose in culture media over a 24-hour period. Notably GIP insulin resistance, and suggest that normalizing 25(OH)D levels could have dose-dependently enhanced insulin-stimulated glucose uptake ([14C]-2-deox- metabolic benefits in targeted individuals. yglucose), but GIP alone did not appreciably regulate acute glucose uptake. Supported By: American Diabetes Association (Fellowship Award to P.K.); Dia- We propose a new mechanistic model in which GIP signaling integrates with betes Action Research and Education Foundation insulin signaling to regulate glucose and lipid metabolism. In the presence of low plasma insulin levels resembling a fasting state, GIP can increase lipolysis to mobilize nutrients from adipocytes. When insulin levels rise in a fed state, GIP can increase insulin-mediated glucose uptake in adipocytes to

ADA-Supported Research

A10 DIRECT LINKS BETWEEN HEPATIC METABOLISM, GLUCAGON ACTION, AND GLUCOSE TURNOVER

DIRECT LINKS BETWEEN HEPATIC METABOLISM, 42‑OR GLUCAGON ACTION, AND GLUCOSE TURNOVER Diabetes Remission Clinical Trial (DiRECT)—Changes in Hepatic VLDL1-TG Production and Intrapancreatic Fat during Weight Main‑ 40‑OR tenance Phase Pathophysiology of Remission in Type 2 Diabetes—The Diabetes AHMAD AL-MRABEH, SVIATLANA V. ZHYZHNEUSKAYA, CARL PETERS, ALISON Remission Clinical Trial (DiRECT) C. BARNES, BENJAMIN ARIBISALA, KIEREN G. HOLLINGSWORTH, NAVEED SATTAR, MICHAEL E. LEAN, ROY TAYLOR, Newcastle upon Tyne, United Kingdom, ROY TAYLOR, AHMAD AL-MRABEH, SVIATLANA V. ZHYZHNEUSKAYA, CARL Lagos, Nigeria, Glasgow, United Kingdom PETERS, ALISON C. BARNES, BENJAMIN ARIBISALA, KIEREN G. HOLLING- Type 2 diabetes is characterized by excess hepatic and intrapancreatic fat SWORTH, NAVEED SATTAR, MICHAEL E. LEAN, Newcastle upon Tyne, United deposition. Liver-derived Very Low Density Lipoprotein-Triglyceride (VLDL1-TG) Kingdom, Lagos, Nigeria, Glasgow, United Kingdom DiRECT is a prospective, randomised study of type 2 diabetes of <6 years delivers fat to all peripheral tissues. Relative changes in VLDL1-TG produc- duration. In the intervention group, weight loss was induced using a liquid tion and intrapancreatic fat were investigated in a sub-group of the prospec- diet replacement (825-853 kcal/day) for 3-5 months with withdrawal of all tive, randomized Diabetes Remission Clinical Trial (DiRECT). Individuals with antidiabetic drugs on day 1. A subgroup underwent 3-point Dixon MRI of complete datasets at 12 months (n=45) were included. Detailed metabolic liver and pancreas fat and measurement of hepatic very low density lipo- tests were carried out at baseline, 4 months, and 12 months after low calorie protein triglyceride (VLDL1-TG) metabolism and first phase insulin secre- diet (825-853 kcal/day). Intra-organ fat was quantified using 3-point Dixon tion, before and after 3-5 months. Those with HbA1c <48mmol/mol (<6.5%) MRI, VLDL1-TG production was quantified using a competitive blocking non- after weight loss (n=37; responders) were compared with those remaining isotopic method, and insulin secretion was measured by the Stepped Insulin >48mmol/mol (n=16; non-responders). At baseline, the two groups were sim- Secretion Test with Arginine (SISTA). Weight loss induced major changes in ilar (age 53.3±1.2 vs. 53.6±2.2 years, weight 100.3±2.8 vs. 98.2 ±3.9kg, BMI liver and intrapancreatic fat with 69% remission of diabetes (responders, 34.9±0.7 vs. 34.9±1.2kg/m2) but not duration of diabetes (2.7±0.3 vs. 3.8±0.4 defined as HbA1c <6.5%). This was followed by weight regain between 4-12 years, p=0.04) nor HbA1c 57.1±1.8 vs. 62.5±2.3 mmol/mol; p<0.07). Weight months in responders (82.3±2.8 to 85.4±3.2kg, p=0.001) and non-responders decreased similarly in responders (100.3±2.8 vs. 84.2±2.3kg; p<0.0001) and (84.5±3.2 to 89.6±3.3kg, p<0.0001). However, liver fat, VLDL1-TG production, in non-responders (98.2 ±3.2 vs. 84.3 ±3.6kg (p<0.0001). HbA1c changed and intrapancreatic fat remained stable in responders (2.5 ±1.9 to 2.9±0.6%, only in responders (57.1±1.8 to 41.2±0.6 mmol/mol, p<0.0001; 62.5±2.3 to p=0.20; 386.7±30.2 to 428.5±21.2 mg/kg/day, p=0.14; and 8.0±0.5 to 7.7 63.8±4.7 mmol/mol, p=0.77, respectively). Decreases occurred in liver fat ±0.4%, p=0.26, respectively). In contrast, these parameters increased in in responders and non-responders (16.7±1.6 to 3.4±0.7%, p<0.0001 and non-responders (2.7±0.5 to 6.2 ±1.8%, p=0.02; 460.2±39.8 to 596.2±36.6 15.0±2.7 to 2.4±0.5%, p<0.0001), plasma VLDL1-TG concentration (0.69±0.07 mg/kg/day, p=0.001; and 6.7±0.3 to 7.0±0.4%, p=0.18, respectively). The to 0.44±0.06 mmol/l, p=0.001 and 0.64±0.10 to 0.46±0.11 mmol/l, p=0.15), recovered first phase insulin secretion in responders continued to improve between 4-12 months (0.13±0.02 to 0.17±0.04 nmol/min/m2, p=0.17). There and intra-pancreatic fat (8.7±0.4 to 7.8±0.4%, p<0.0001 and from 7.6±0.5 2 to 6.7±0.4%, p=0.001). In contrast, first phase insulin secretion increased was no change in the non-responders (0.03±0.01 to 0.03±0.01 nmol/min/m , in responders only (0.05±0.01 to 0.12±0.02, p<0.0001 vs. 0.02± 0.01 to p=0.98). These data consistent with VLDL1-TG being the link between liver 0.02±0.01, p=0.98, respectively). Failure of first phase insulin secretion fat and intrapancreatic fat, and a major modulator determining remission of response but not differential lipid or hepatic responses characterises type 2 diabetes. non-responders. De-differentiation of beta cells in early type 2 diabetes Supported By: Diabetes UK is not reversible in all, despite removal of the metabolic stress. Such individuals are characterised by longer duration of diabetes even in early type 2 43‑OR diabetes. Amino Acid Metabolism Is Regulated by Glucagon Receptor Signal‑ Supported By: Diabetes UK ing in Mice MARIE WINTHER-SOERENSEN, KATRINE D. GALSGAARD, RUNE E. KUHRE, 41‑OR JENS PEDERSEN, NICOLAI J. WEWER ALBRECHTSEN, JENS J. HOLST, Copen- Mild Physiologic Hyperglycemia Induces Hepatic Insulin Resis‑ hagen, Denmark The pancreatic hormone glucagon contributes to diabetic hyperglycemia tance in Healthy Normal Glucose Tolerant Subjects—Role of Glu‑ and glucagon receptor antagonists lower blood glucose in patients with cagon and Gluconeogenic Substrates type 2 diabetes. A liver-alpha cell axis has recently been identified which DEVJIT TRIPATHY, AURORA MEROVCI, ENRIQUE R. MALDONADO CORCHADO, when disrupted leads to hypersecretion of glucagon and may contribute to RITA BASU, RALPH A. DEFRONZO, San Antonio, TX, Charlottesville, VA The aim of the present study was to evaluate the effect of a physiologic the development of diabetes. Here, we investigated the hepatic effects of increase in plasma glucose concentration on endogenous glucose produc- glucagon receptor signaling in rodents and hypothesized that intact gluca- tion (EGP) in healthy NGT individuals. 16 NGT subjects [8 with and 8 without gon receptor signaling is necessary for amino acid metabolism via non-tran- family history of T2DM (9M/7F, age = 44± 3 years, BMI = 26 ± 1 kg/m2)] scriptional changes leading to increased ureagenesis. Female C57BL/6JRj participated in a 3-step hyperinsulinemic (10, 20, 40 mU/m2·min) euglycemic mice (n=64) were given a glucagon receptor antagonist (GRA) or vehicle orally three hours prior to an intraperitoneal injection with amino acids clamp before and after a 48 hour glucose infusion to increase plasma glu- ORALS cose conc by ~45 mg/dl above baseline (89±4 to 136±4.9 mg/dl). EGP was (Vamin, 3.6 µmol/g body weight), glucagon (200 ng/g body weight), or both, measured with [3-3H] glucose and gluconeogenic rate was measured follow- and blood samples were obtained at 0, 4, 12, and 20 minutes after injection. ing administration of deuterated water. Plasma FFA, glucagon, lactate, glyc- GRA treated animals had a significant decrease in plasma urea (8.6±0.3 vs. erol and alanine concentrations also were measured. Following 48 hours of 11.1±0.4 mmol/L, P<0.0001) and blood glucose (8.1±0.2 vs. 9.0±0.2, P<0.001 mmol/L) and a concomitant increase in plasma amino acids (3.5±0.1 vs. glucose infusion basal EGP increased from 2.05±0.09 to 3.06±0. mg/kg⋅min (p=0.003) and hepatic insulin resistance index (EGP X Fasting Plasma Insulin) 2.7±0.1 mmol/L, P<0.0001) and glucagon (32.5±3.5 vs. 12.5±1.7 pmol/L, increased markedly and similarly (25.1±1.0 vs. 48.3± 3.2, p<0.005). The base- P<0.0001). In addition, amino acid-stimulated ureagenesis, reflected by line plasma glucagon concentration tended to be higher following 48 hours increased plasma urea, was inhibited in GRA treated mice compared to con- of glucose infusion (64±3 vs. 77± 8 pg/ml, p=0.2) while the product of the trols (incremental area under the curve: 27.6±3.3 vs. 41.2±5.3 min × mmol/L, plasma glucagon and insulin concentrations was markedly increased (540 ± P<0.05). Production of urea in perfused rat livers (n=6) increased rapidly 56 vs. 1336 ± 262, p=0.004). Although the baseline plasma FFA concentration upon administration of amino acids (1 mM) compared to baseline (683±254 was markedly reduced following 48 hours of glucose infusion (0.482 ±0.05 vs. 518±198 pmol/min/100 g body weight, P<0.05) and was further increased to 0.055± 0.01mM), there was no difference in plasma glycerol concentra- in response to amino acids + glucagon (10 nM) stimulation (846±229 vs. tion (13.4±1.2 vs. 13.3 ± 2.2 mg/L p=ns). There were small increases in both 518±198 pmol/min/100 g body weight, P<0.001). Glucagon therefore acutely the plasma lactate and alanine concentrations. Total body insulin sensitivity regulates hepatic amino acid metabolism through increased ureagenesis via declined from 8.75±2.1 to 6.92± 6 mg.kg/min (p<0.05). non-transcriptional mechanism(s), and disruption of this, due to e.g., nonal- Conclusion: Chronic (48-hr) physiologic hyperglycemia to levels seen in coholic liver disease, may therefore contribute to the diabetogenic effect of T2DM increases basal hepatic glucose production and induces hepatic and hyperglucagonemia. skeletal muscle insulin resistance in healthy NGT subjects. These results demonstrate, for the first time that glucotoxicity causes resistance to the suppressive effect of insulin on hepatic glucose production. Supported By: National Institutes of Health

ADA-Supported Research

A11 DIRECT LINKS BETWEEN HEPATIC METABOLISM, GLUCAGON ACTION, AND GLUCOSE TURNOVER

44‑OR to pre-E values when E stops. Taken together, euglycemia coupled with low Effect of Glucagon on Nocturnal Rates of Endogenous Glucose Pro‑ insulin appear to be the most favorable milieu for T1D during E. duction in Type 2 Diabetes Figure. ANANDA BASU, HUALING ZHAI, RICKEY CARTER, RITA BASU, Charlottesville, VA, Shanghai, China, Jacksonville, FL The role of nocturnal glucagon on regulation of overnight endogenous glucose production (EGP) in T2D is unknown. We studied 13 T2D and 13 age, BMI matched nondiabetic subjects (ND) on two occasions at the Clinical Research Unit at Mayo Clinic. Deuterated water was provided to measure gluconeogenesis (GNG) and glycogenolysis (GGL) using the HMT (hexameth- ylenetetramine) method. A somatostatin pancreatic clamp was initiated at 10 PM with 0.25 mU/kg/min insulin infusion until 7 AM along with [3-3H] glucose to measure turnover in the presence of glucagon infusions at 0.65 ng/ kg/min (basal) or 1.5 ng/kg/min (high) in random order. Blood was sampled periodically for the measurements of plasma glucose, [3-3H] glucose, hormones; EGP rates were measured at 7 AM. Results are presented in the Table. Fasting glucose, insulin and c-peptide concentrations were not different in either group during either visit. Glucose infusion rates (GIR) required to maintain euglycemia were lower during high glucagon in both groups and lower in T2D vs. ND regardless of visit indicating insulin resistance. Rates of EGP at 7AM were higher in both groups with the high vs. basal glucagon (matched data shown for calculation). Partial GNG data that are available indicate increased rates of GGL at 7 AM in the presence of high glucagon. In T2D higher glucagon overnight could contribute to higher rates of GGL and EGP. Therapies to reduce GGL may help lower overnight EGP. Table. Diabetes Basal High 7 AM Status Glucagon Glucagon p-value 7 AM 7 AM Basal vs. High Supported By: National Institutes of Health (R01DK090541, R01DK029953) Glucose ND 5.4 ± 0.6 5.2 ± 0.6 0.4 (mmol/L) T2D 8.2 ± 4.0 8.2 ± 4.0 0.9 (p < 0.001) (p < 0.001) 46‑OR Glucagon ND 82.0 ± 14.0 138.3 ± 15.4 p < 0.01 NREP Bridges TGF-β Signaling and Lipid Metabolism in the Epigen‑ (pg/ml) T2D 90.5 ± 12.5 151.5 ± 17.2 p < 0.01 etic Reprogramming of NAFLD in the Offspring of Insulin-Resistant (p = 0.56) (p = 0.20) Parents GIR ND 23.0 ± 12.8 21.0 ± 11.4 p < 0.01 DARIO F. DE JESUS, KAZUKI ORIME, CHIH-HAO WANG, JIANG HU, ERCUMENT (µmol/kgFFM/min) T2D 6.7 ± 8.4 4.0 ± 6.8 p < 0.01 DIRICE, AMÉLIA M. SILVA, YU-HUA TSENG, JUSSI PIHLAJAMAKI, ROHIT (p <0.001) (p <0.001) KULKARNI, Boston, MA, Vila Real, Portugal, Kuopio, Finland EGP ND n=13 8.0 ± 4.2 10.3 ± 6.6 Nonalcoholic fatty liver disease (NAFLD) prevalence is increasing world- (µmol/kgFFM/min) T2D n=13 13.3 ± 4.7 15.8 ± 4.3 wide and few studies have associated maternal diabetes and birth weights with increased risk for NAFLD. We used an unique non-dietary model, mani- HMT data ND n=4 10.3 ± 4.8 15.1 ± 8.7 (EGP) (µmol/kgFFM/min) T2D n=7 15.2 ± 5.6 22.0 ± 5.0 festing hyperglycemia and hyperinsulinemia-two hallmarks of gestational and type 2 diabetes. We aimed to determine the genetic and epigenetic Gluconeogenesis ND n=4 3.8 ± 1.3 4.7 ± 3.4 effects of paternal vs. maternal genetic insulin resistance on the develop- T2D n=7 5.6 ± 2.2 9.4 ± 1.6 mental programming in the offspring of the liver-specific insulin receptor Glycogenolysis ND n=4 6.5 ± 3.4 10.4 ± 1.2 knockout (LIRKO) mice. Male control F1 offspring from father LIRKO (FL), T2D n=7 9.6 ± 3.3 12.6 ± 3.5 mother LIRKO (ML) or control mothers and fathers (C) were weaned on a All data are Mean +/- SD. Statistical analyses was not performed on EGP and chow or high-fat-diet (HFD) and followed for 3 months. FL and ML showed HMT data as it is preliminary. impaired growth and body weight composition. FL and ML developed hepatic Supported By: National Institutes of Health (R01DK29953) steatosis compared to C when challenged with HFD and exhibited increased hepatic expression of lipogenesis-associated genes. Hepatic transcriptomic

ORALS 45‑OR and genome-wide DNA methylation analyses of FL and ML on chow diet presented enriched-pathways associated with TGF-β signaling and lipid syn- Effects of Hyperglycemia and Hyperinsulinemia on Glucose Turn‑ thesis. FL and ML hepatic NREP mRNA levels were decreased 50% (p<0.05) over during Exercise in Type 1 Diabetes on HFD compared to C. In-vivo and in-vitro modeling of hepatic steatosis in DAVIDE ROMERES, ANANDA BASU, MICHELE SCHIAVON, CLAUDIO COBELLI, HepG2 and human primary hepatocytes decreased NREP mRNA and protein CHIARA DALLA MAN, RITA BASU, Padova, Italy, Charlottesville, VA levels. Knock-down experiments performed in HepG2 cells revealed that Exercise (E) induced increase in glucose uptake (Rd) is offset by com- NREP acts by regulating triglyceride and cholesterol synthesis transcrip- pensatory increase in endogenous glucose production (EGP) in health. To tional network via regulation of ATP-citrate lyase (ACL) in a phospho-AKT determine the extent to which this occurs in type 1 diabetes (T1D) and to dependent manner. Finally, reduced NREP mRNA levels in patients with assess the effects of hyperglycemia and hyperinsulinemia, we studied six hepatic steatosis by 40% (p<0.05) and recent preclinical trials implicating T1D subjects (age 29±7 years, BMI 28.3±4.8 kg/m2, VO max 28.7±10 ml/kg/ 2 ACL in NAFLD highlight the translation relevance of our findings. These data min) on three visits (V : euglycemia, low insulin; V : euglycemia, high insulin; 1 2 suggest that prenatal insulin resistance epigenetically regulates a novel V : hyperglycemia, low insulin) in random order. Glucose fluxes were mea- 3 gene, NREP, that has detrimental effects on metabolic adaptation and tran- sured with tracer-tracee clamp using [6,6-2H ] glucose before, during and 2 scriptional regulation of hepatic metabolism in the development of NAFLD. after, 60 min of E at 65% VO max. By design, plasma glucose concentra- 2 Supported By: National Institutes of Health (R01DK67536, R01DK103215 to R.K.); tions were higher in V than V and V (9.7±0.5, 5.2±0.1, 5.2±.0.1 mM respec- 3 1 2 SFRH/BD/51699/2011 (to D.F.D.J.) tively, p<0.05), while plasma insulin concentrations were higher in V2 than V1 and V3 (324.3±19.4, 129.3±9.3, 111.6±5.9 pM, p<0.05). Rd increased with E in all visits (p<0.05) and returned to pre-E values thereafter. EGP during E increased (p<0.05) in V1 and to a lesser extent in V3, but was unchanged in V2. To summarize, hyperinsulinemia, but not hyperglycemia inhibits E induced compensatory increase in EGP; both hyperinsulinemia and hyperglycemia augments increase in Rd with E albeit to different extents; Rd returns

ADA-Supported Research

A12 BALANCING PROLIFERATION AND MATURATION IN THE ISLET

47‑OR 49‑OR Effects of NAFLD on Acetyl-CoA Partitioning and Ketone Kinetics in Identification of Pancreatic Elastase Inhibitors That Can Stimulate Response to a 24-Hour Fast Beta-Cell Proliferation JUSTIN A. FLETCHER, STANISLAW DEJA, SHAWN C. BURGESS, JEFFREY GIORGIO BASILE, AMEDEO VETERE, JIANG HU, BRIDGET WAGNER, ROHIT BROWNING, Dallas, TX KULKARNI, Boston, MA, Cambridge, MA During an overnight fast (12 hour), the liver provides glucose as fuel for The identification of novel small molecules that can target endogenous other tissues via glycogenolysis and the energetically expensive process factors to regulate β-cell proliferation is a desirable goal to counter all of gluconeogenesis. During this period, energy to support this process is forms of diabetes. Recently, we identified serpinB1 (SB1), an endogenous generated primarily by the tricarboxylic acid (TCA) cycle. As the duration protease inhibitor, as a protein that promotes human β-cell proliferation by of fasting prolongs, and glycogen stores are depleted (>24 hour), the liver inhibiting elastase activity, and demonstrated that the elastase inhibitor shifts to the production of ketone bodies that can be used as a secondary sivelestat also induced β-cell proliferation. Here, we report high-throughput fuel source by peripheral tissues. In healthy humans, this period of fasting is screening to identify novel elastase inhibitors and to evaluate their ability to generally associated with increased β-oxidation and partitioning of acetyl- induce human β-cell proliferation. We screened 16,320 compounds using a CoA away from the TCA cycle and toward ketone body production. We have fluorescence-based porcine pancreatic elastase (pPE) assay, and confirmed previously shown that subjects with nonalcoholic fatty liver disease (NAFLD) inhibition of human neutrophil (hNE) and pancreatic (hPE) elastases using an have increased rates of acetyl-CoA oxidation in the TCA cycle after a 12 absorbance-based assay. We observed that telaprevir and tebipenem each hour fast. To examine how NAFLD affects hepatic response to a 24 hour inhibited pPE with greater potency than sivelestat (IC50: telaprevir 33.9 nM, fast, we infused stable isotope tracers into BMI and age matched subjects tebipenem 1.1 µM, sivelestat 2.4 µM; n=3). The results were confirmed on with (n=12) and without (n=12) NAFLD. At enrollment, NAFLD subjects had hPE (IC50: telaprevir 15.7 nM, tebipenem 3.6, sivelestat 2.3 µM; n=3), while greater liver triglycerides (P<0.05) and hepatic insulin resistance (P<0.05), neither telaprevir nor tebipenem blocked hNE activity. Interestingly, telapre- as well as higher fasting plasma concentrations of cholesterol, triglycerides, vir and SB1 inhibited hPE and pPE at similar concentrations, enforcing the and glucose (P<0.05). After fasting for 24 hours, subjects with NAFLD failed hypothesis of a shared mechanism of action between the protein and the to suppress endogenous glucose production as compared to controls. This compound. We then assessed the mitogenic properties of the compounds was associated with a higher ratio of plasma insulin to glucagon as well as in in vitro cultures of mouse and human islets by immunofluorescence. Tela- a blunted increase in the concentration of plasma free fatty acids as fasting previr and tebipenem each increased proliferating β-cells in rodent (~6-fold duration prolonged. As previously shown in overnight fasted subjects, TCA and 1.5-fold increase, respectively; n=3) and human islets (~4-fold and 1.5- cycle activity was significantly higher in NAFLD subjects after a 24 hour fast. fold increase, respectively; n=3) compared to vehicle-treated islets. Taken However, despite elevated TCA cycle flux, NAFLD subjects failed to increase together, these data confirm the identification of two novel compounds with overall β-oxidation compared to controls due to reduced utilization of acetyl- the ability to inhibit pancreatic elastase and enhance mammalian β-cell pro- CoA for ketone synthesis. liferation. These results highlight the therapeutic potential of using elastase In conclusion, after a 24 hour fast, the liver of NAFLD subjects produced inhibitors to stimulate β-cell proliferation to treat diabetes. more glucose and disposed of less fat via β-oxidization by shuttling more acetyl-CoA to the TCA cycle but much less to ketogenesis. 50‑OR Supported By: National Institutes of Health (1R01DK087977-01A1 to J.B.), DNA Hydroxymethylation Regulates Beta-Cell Maturation and (R01DK078184 to S.C.B.), (5F32DK107124-03 to J.A.F.) Expansion TATYANA GURLO, SENTA GEORGIA, SANGEETA DHAWAN, Los Angeles, CA, Duarte, CA BALANCING PROLIFERATION AND MATURATION IN Epigenetic mechanisms play a central role in governing functional beta- THE ISLET cell mass. In particular, the methylation of DNA to generate 5-methylcyto- sine (5mC), directed by DNA methyltransferases, has been shown to regu- 48‑OR late the establishment and maintenance of beta-cell identity and function. CRISPR-cas9 Gene Editing Restores Beta-Cell Differentiation and While the contribution of DNA methylation to beta-cell homeostasis and Function in Patient-Specific iPSCs diabetes pathogenesis has been studied in detail, not much is known about KATELYN MILLETTE, KYLE R. VOGT, ANDREW SALAS, PISIT PITUKCHEEWANONT, the role of DNA demethylation in beta-cells. Oxidative conversion of 5mC to JULIANA AUSTIN, MARTIN MARTIN, SENTA GEORGIA, Los Angeles, CA 5-hydroxymethylcytosine (5hmC) by Ten Eleven Translocation (Tet) enzymes Genetic knockout studies in mice concluded that the differentiation of constitutes a key step towards active DNA demethylation. In the present endocrine lineages in the pancreas and intestine requires the transcription study, we provide evidence that the 5hmC landscape is dynamic, and under- factor neurogenin3 (NGN3). However, case reports of patients with NGN3 goes specific changes during embryonic and postnatal islet development, mutations show that most patients do not develop diabetes until later in islet adaptation, and in diabetes. We report that the Pdx1-positive pancre- childhood, suggesting that NGN3 may not be required for human beta cell atic progenitors are marked by very low 5hmC, and beta-cell differentia- differentiation in vivo. We have identified a patient with a previously unde- tion and functional maturation is accompanied by a progressive increase in 5hmC. However, under conditions during adaptive beta-cell expansion as scribed NGN3 loss-of-function mutation and generated induced pluripotent ORALS stem cells from patient fibroblasts to analyze the role of NGN3 in human well as in diabetes 5hmC levels decline in part due to the oxidative stress. beta cell differentiation. We used established protocols to differentiate These changes in 5hmC pattern are conserved in mouse and human pan- patient-specific iPSCs (PS-iPSCs) into pancreatic progenitor cells and beta- creas, and are accompanied by the changes in Tet2 levels. We show that like cells in parallel with the H1 hESC line. The PS- iPSCs displayed signifi- deletion of Tet2 in the pancreatic lineage results in improved glucose tol- cantly lower pancreatic progenitor cell differentiation with only 10.7% of the erance and beta-cell function. Together, our data show the importance of double positive PDX1+/NKX6.1+ cell population present, whereas the control Tet2-dependent 5hmC patterning in maintaining a differentiated beta-cell H1s hESCs produce 60% double positive cells. After further differentiation phenotype, and highlight the remodeling of beta-cell epigenetic landscape into beta-like cells, H1 cells produced multiple endocrine cell types, while as a key component of diabetes pathogenesis. the PS-iPSC line was not able to make significant numbers of endocrine cells. Supported By: Larry L. Hillblom Foundation; JDRF To assess if the loss of NGN3 was the primary cause for the loss of pancreatic progenitor cells and beta-like cell differentiation, we used CRISPR-cas9 51‑OR gene editing to correct the patient’s mutation in the PS-iPSCs (cPS-iPSCs). Serpin B13 Plays a Role in Beta-Cell Development and Progression Correcting the patient’s mutation restored the PDX1+/NKX6.1+ cell popula- to Insulin-Dependent Diabetes tion in the pancreatic progenitor stage. Beta-like cells differentiated from YURY KRYVALAP, MATTHEW L. JIANG, NADZEYA KRYVALAP, KYLE A. MUELLER, cPS- iPSCs were capable of glucose responsive insulin secretion at the end JAN CZYZYK, Rochester, NY, Webster, NY of the differentiation protocol. Identifying natural factors of the progression to type 1 diabetes (T1D) pro- In conclusion, our data suggests that NGN3 may have a previously uniden- vides a unique opportunity to develop novel preventative strategies for this tified role important for the differentiation of pancreatic progenitor cells and disease. Our laboratory has identified serpin B13 proteinase inhibitor as a that there may be a relationship between NKX6.1 and NGN3 that is critical potential player, activity of which is involved in modifying the timing of T1D for pancreatic development. development. In this study we examined whether blocking of serpin B13 with Supported By: California Institute for Regenerative Medicine a monoclonal antibody (mAb) in early life causes any changes in pancreatic islet development that may explain beneficial impact of this activity later

ADA-Supported Research

A13 BALANCING PROLIFERATION AND MATURATION IN THE ISLET

in life. First, our animal studies showed that blocking of serpin B13 during tions, I hypothesize that SAD-A promotes pancreatic β-cell proliferation by mid-pregnancy caused robust increase in the number of Ngn3+ progenitor regulating β-cell cycle as a downstream target of mTORC1 signaling. cells at day E16.5 and accelerated conversion of these cells to beta cells. Supported By: American Diabetes Association (1-14-BS-185 to J.N.); National Similar changes were observed in embryonic pancreatic buds in vitro. On Institutes of Health the other hand, the serpin B13 protein significantly downregulated the out- put of Ngn3+ cells in these in vitro cultures. Importantly, at 2 to 3 months 54‑OR after birth, the offspring of serpin B13 mAb-treated mothers demonstrated Interaction of Exocrine and Endocrine Pancreas in Obesity increased islet cell mass and responded to the diabetes-inducing agent, Ciliopathies streptozotocin, with markedly improved glucose tolerance and renal func- TIMOTHY L. HOSTELLEY, JR., JESSICA DUNLEAVEY, NORANN A. ZAGHLOUL, tion, compared with control animals. Second, our screening of 280 children, Baltimore, MD previously enrolled in a DPT-1 clinical trial, for serpin B13 autoantibody (AA) Ciliopathies are rare genetic disorders characterized by dysfunction in pri- revealed an inverse correlation of this AA with risk level for T1D, as well as a mary cilia. Two ciliopathies, Alström Syndrome and Bardet-Biedl Syndrome positive association with longer diabetes-free interval. These data demon- (BBS), are characterized by high rates of obesity. However, they have strik- strate the significant, long-term impact of serpin B13 activity on islet biology ingly different rates of diabetes. The rate is much higher in Alström patients and suggest that blocking this serpin has the potential to partially prevent, relative to BBS. To gain insight into genetic mechanisms for this discrepancy or at least slow down, the development of T1D both in the mouse and human. we performed whole transcriptome sequencing on zebrafish depleted of the Supported By: American Diabetes Association (1-17-ICTS-083 to J.C.); JDRF Alström gene, alms1, or the BBS gene, bbs1. We identified gene expression changes and found 8 genes that were differentially expressed in opposite 52‑OR directions between the two models. Among these, several exocrine pan- Generation of a Novel Mouse Model to Study Pancreatic Beta-Cell creas enzymes were downregulated in Alström and/or upregulated in BBS. Dedifferentiation Based on this, and our previous demonstration of β-cell mass changes in TRACY C.S. MAK, MATHIEU LATREILLE, London, United Kingdom models of both disorders, we hypothesized that the expression of exocrine Compromised β-cell identity triggered by metabolic stress has been pancreas enzymes affects endocrine pancreas function. This might sug- revealed as a novel mechanism of insulin insufficiency in type 2 diabetes gest that impaired β-cell function in Alström results from reduced expres- (T2D). Previous work from our group showed increased microRNA-7 (miR- sion of exocrine pancreas enzymes, while the opposite may be true in BBS. 7) expression in islets of mouse models of T2D and human islets exposed These findings also support a non-ciliary role for BBS and Alström genes to an obesogenic environment. Murine β-cell-specific overexpression of as we found them to be expressed in the non-ciliated acinar cells. To test miR-7 is sufficient to causes diabetes and is accompanied with decreased our hypothesis, we overexpressed each enzyme in transgenic zebrafish expression of β-cell identity markers, thus revealing miR-7 induction as a embryos, in which β-cells can be visualized. In control animals, overexpres- novel mechanism impairing β-cell identity. To address if insulin-producing sion of the exocrine pancreas enzymes significantly increased the number of cells can recover their function and identity in T2D, we engineered a novel β-cells, suggesting that these enzymes play a role in regulating β-cell mass mouse enabling us to temporally compromise the identity of β-cells through and function. Overexpression could also rescue the loss of β-cells observed doxycycline-mediated miR-7 overexpression in vivo. We report that mutant in animals depleted of alms1. We have also found that the inactive form of mice exposed to doxycycline (Dox) displayed a severe diabetic phenotype these enzymes are selectively taken up by β-cells in vitro and induce prolif- characterized by hyperglycemia, glucose intolerance and reduced circulating eration in cultured MIN6 β-cells. This suggests a direct interaction between insulin levels. Strikingly, four weeks after withdrawing Dox from the diet, we the exocrine and endocrine pancreas. Taken together, these data support the observed a complete normalization of glycemia, circulating insulin as well as possibility that exocrine pancreatic enzymes may play an important role in the number of bihormonal cells in mutant mice. RNA seq analysis revealed the modulation of β-cells in diabetes. that islets from miR-7-overexpressing mice exhibit a striking downregula- Supported By: National Institutes of Health (F31DK115179, T32DK098107, tion of β-cells identity markers and increased levels of α- and δ-cell specific R01DK102001) genes. Interestingly, we measured upregulation of several genes involved in EMT, Inflammation and Stemness, pointing to a dedifferentiation ofβ -cells 55‑OR following induction of miR-7 expression. We provide novel evidence that Intrauterine Growth Retardation Induced Long-Term Gene Dysregu‑ miR-7 triggers an EMT process by repressing components of ATP-dependent lation Is Associated with Altered Histone Modifications in Rat Islets chromatin remodeling complexes, altering nucleosome positioning at β-cell YU-CHIN LIEN, PAUL WANG, REBECCA A. SIMMONS, Philadelphia, PA specific loci and ultimately causing dedifferentiation ofβ -cells. Together, Intrauterine growth retardation (IUGR) is associated with the develop- our data reveal the mechanism underlying β-cell dedifferentiation by miR-7 ment of type 2 diabetes in adulthood. Although the underlying mechanisms and provide a proof of concept for the therapeutic efficacy of anti-miR-7 mol- remain unclear, permanent alterations in gene expression implicate epi- ecules in reversing β-cell dedifferentiation and improving β-cell function. genetics. Using a rat model of IUGR induced by bilateral uterine artery liga- Supported By: UK Medical Research Council tion at e18, we sought to determine if histone modifications are a potential mechanism for permanent IUGR-induced gene dysregulation. We assessed

ORALS 53‑OR genome-wide histone modifications in islets from 2 and 10 week control SAD-A Promotes Pancreatic β-Cell Proliferation by Regulating and IUGR rats using ChIP-Seq. IUGR induced significant changes in enrich- β-Cell Cycle as a Downstream Target of mTORC1 Signaling ment of H3K4me3, H3K27me3, and H3K27Ac marks in 2 week islets at 2181, JIA NIE, NICOLAS MUSI, YUGUANG SHI, San Antonio, TX 1482, and 4572 annotated genes, respectively. At 10 week there were far Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and more changes in enrichment of these marks (9988, 3489, and 4355 genes, β-cell dysfunction, which leads to the depletion of β-cell mass. Understand- respectively). We then integrated our RNA-Seq transcriptome data obtained ing the molecular mechanisms driving islet β-cell dysfunction is paramount at the same ages and found that the expression levels of multiple genes for developing therapies to increase β-cell function. The mammalian target in IUGR islets were correlated with changes in histone marks. In 10 week of rapamycin complex 1 (mTORC1) plays a key role in regulating β-cell func- islets, the expression of 639, 355, and 400 genes correlated with changes in tion by integrating growth factors and mitogens to direct cell cycle and sur- enrichment of H3K4me3, H3K27me3, and H3K27Ac, respectively; whereas vival; yet, mechanistically this process is not fully understood. Synapses of in 2 week islets, only 43, 64, and 149 genes were correlated with enrich- amphids defective protein kinase A (SAD-A) is a member of AMP-activated ment of these marks. Interestingly, 6 genes in 10-week islets correlated protein kinase (AMPK)-related family kinases, with high expression in the with changes in all 3 histone marks, strongly suggesting that changes in pancreas. My earlier work demonstrated that SAD-A is a β-cell specific gene expression were induced by histone modifications. Ingenuity pathway mediator of insulin secretion and islet β-cell size. Furthermore, glucose dra- analysis of differential expressed genes that correlated with histone modifi- matically stimulated SAD-A protein translation in islets that was potently cations revealed significant enrichment in pathways critical for normal islet inhibited by rapamycin, an inhibitor of mTORC1. My recent preliminary stud- function, such as cAMP-mediated signaling, immune function, axonal guid- ies showed that 1.) SAD-A overexpression increases β-cell proliferation in ance, and VEGF family ligand-receptor interaction. Collectively, the present the MIN6 β-cell line and islets; 2.) Ablation of SAD-A in pancreas of mice findings identify histone modification as a potential epigenetic mechanism (pSADKO) promotes β-cell apoptosis and attenuates compensatory islet that may contribute to long-term gene dysregulation and abnormal islet phe- growth in obese mice; 3.) SAD-A depletion in the pancreas increases mRNA notype in IUGR animals. expression of cyclin D2 in islets of pSADKO mice. Based on these observa- Supported By: National Institutes of Health (R01DK055704, R01DK114054-01)

ADA-Supported Research

A14 WHAT’S NEW IN DIABETIC NEUROPATHY? EQUAL PARTS CLINICAL AND BASIC SCIENCE

WHAT’S NEW IN DIABETIC NEUROPATHY? EQUAL proteins and reduced mitochondrial function via a defective SIRT1 pathway PARTS CLINICAL AND BASIC SCIENCE may contribute to developing distal axonopathy. Supported By: National Institute of Neurological Disorders and Stroke 56‑OR (1R01DK107007); U.S. Department of Veterans Affairs A Genetic Locus on Chromosome 2q24 Predicting Peripheral Neu‑ ropathy Risk in Type 2 Diabetes 58‑OR YALING TANG, JOSYF MYCHALECKYJ, BRUCE A. PERKINS, RODICA POP-BUSUI, Muscarinic Receptor Antagonist Improves Nerve Fiber Function in MICHAEL J. WAGNER, ALISON MOTSINGER-REIF, JOHN B. BUSE, HETAL SHAH, Subjects with Type 2 Diabetes and Peripheral Neuropathy ALESSANDRO DORIA, Boston, MA, Charlottesville, VA, Toronto, ON, Canada, Ann AARON I. VINIK, NIGEL A. CALCUTT, JOSHUA F. EDWARDS, JESSICA R. WEAVER, Arbor, MI, Chapel Hill, NC, Raleigh, NC MICHAEL D. BAILEY, PAUL FERNYHOUGH, LINDSEY B. CUNDRA, KATIE E. FRIZZI, Diabetic peripheral neuropathy (DPN) is a significant contributor to the HENRI PARSON, CAROLINA M. CASELLINI, Norfolk, VA, La Jolla, CA, Winnipeg, increased morbidity associated with type 2 diabetes (T2D). However, the MB, Canada risk of developing DPN varies among T2D patients, possibly due to genetic Degeneration of nerve fibers due to diabetic peripheral neuropathy (DPN) factors. We aimed to identify genetic predictors of DPN using an unbiased has been linked to mitochondrial dysfunction. Manipulation of mitochondrial genome-wide association (GWAS) approach in Action to Control Cardiovas- dysfunction through antagonism of muscarinic receptors (MR) promotes cular Risk in Diabetes (ACCORD)—a large trial of intensive glycemic control neurite outgrowth in adult sensory neurons in vitro and provides neuropro- in subjects with T2D and high CVD risk. Occurrence of DPN was defined tection in rodent models of DPN. The aim of the study was to assess the as a clinical Michigan Neuropathy Screening Instrument (MNSI) score >2.0 efficacy of MR antagonist topical 3% oxybutynin in structural and functional at baseline or during follow-up. Using logistic regression, we conducted measures of nerve fiber function in subjects with type 2 diabetes (T2DM) a GWAS, testing 6.8 million common single nucleotide polymorphisms and DPN. Pilot, randomized, placebo-controlled, double-blinded study in 40 (SNPs) among 4,384 DPN cases and 784 DPN-negative controls of white subjects assessed at baseline and after 20 weeks of treatment with oxy- origin for whom genetic data were available. A locus on chromosome 2q24 butynin or placebo with the following: intraepidermal nerve fiber density reached GWAS significance (p=1.9×10-9). The minor allele of the lead SNP (IENFD) on proximal and distal leg; neuropathy scores and quality of life (rs13417783, minor allele frequency=0.14) decreased DPN odds by 36% (OR (Norfolk QoL DN) questionnaire. Baseline demographic characteristics were 0.64, 95% CI 0.59-0.69)—an effect not influenced by the glycemic treat- similar between the treatment groups. IENFD improved significantly after 20 ment assignment (p for interaction=0.6). Adjacent to this SNP lies a cluster weeks for the treatment group. Neuropathy scores and Norfolk QoL DN also of genes coding for human voltage-gated sodium channels (SCN1A, SCN2A, improved significantly in the treatment group (Table 1). No improvements SCN3A, SCN7A, and SCN9A) that are expressed in neurons and are mutated were seen in the placebo group. In this study, oxybutynin proves to be effica- in monogenic forms of peripheral neuropathy and epilepsy. In an expres- cious in improving structural and functional measures of small fiber function, sion quantitative trait locus (eQTL) analysis, the minor (protective) allele of and quality of life in T2DM subjects. These results offer a promising novel this SNP was associated with higher expression of SCN2A (P=0.0009) in therapeutic approach for DPN that needs to be explored further. tibial nerve. Other loci reaching notable significance (p≤1x10-6) were 8p23 in the gene CSMD1, previously implicated in epilepsy, and 11q25 in OPCML, an opioid-binding protein. In summary, we have discovered a novel genetic locus predicting DPN risk. Pending further investigation, these findings may provide better mechanistic insights into the pathophysiology of DPN, hopefully leading to the development of novel pharmacological interventions to prevent or treat this diabetic complication. Supported By: National Institutes of Health

57‑OR Increased Deacetylation of Proteins in Sensory Neurons by Sirtuin 1 Protein Overexpression Reverses T2D Peripheral Neuropathy KRISH CHANDRASEKARAN, CHENG-YING HO, MOHAMMAD SALIMIAN, PRAN- ITH H. KUMAR, SAI SRUTHI REDDY KONDURU, JAMES W. RUSSELL, Baltimore, MD Sirtuin 1 (SIRT1) protein uses NAD+ as a substrate to deacetylate transcription factors, cofactors, and histones to enhance mitochondrial function. We tested the hypothesis whether increased expression of SIRT1 protein in dorsal root ganglion (DRG) neurons would rescue mice from peripheral Supported By: National Institutes of Health (DK102032) neuropathy induced by a high-fat diet (HFD). Neuron-specific, doxycycline ORALS (DOX)-inducible, SIRT1 protein over expressing C57BL6 transgenic mouse (nSIRT1OE Tg) were generated. Expression of SIRT1 protein was observed 59‑OR in small to medium sized DRG neurons. nSIRT1OE was shut off by feeding, Effect of Sacubitril/Valsartan vs. Valsartan on Vascular and Neural weaned Tg mice, with DOX in the diet for 12 weeks. The Tg mice were divided Complications in Type 2 Diabetic Rats into 3 groups. Group # 1: nSIRT1OE Tg mice fed with standard diet (STD) plus MARK A. YOREK, Iowa City, IA DOX for 4 months; Group # 2: nSIRT1OE Tg mice fed with HFD plus DOX for Previously we had shown that a vasopeptidase inhibitor, combination drug 4 months; Group # 3: nSIRT1OE Tg mice fed with HFD plus DOX for 2 months containing angiotensin converting enzyme and neprilysin inhibitors, was an until they develop neuropathy and then switched to HFD minus DOX for effective treatment for diabetes vascular and neural complications. How- additional 2 months. Neuropathy was determined by mechanical allodynia ever, once in clinical trials, side effects prevented further development of thresholds (MAT) and nerve conduction velocity (NCV) at 0, 2 and 4 months. this class of drugs. This led to development of sacubitril/valsartan, combina- Intraepidermal nerve fiber density (IENFD) was measured at 4 months. MAT, tion drug similar to vasopeptidase inhibitors containing angiotensin II recep- NCV and IENFD were decreased in HFD-fed (Group # 2) mice compared to tor blocker and neprilysin inhibitor that we hypothesized would be also an STD-fed mice (Group # 1) at 2 and 4 months. In Group # 3 mice, 2 months effective treatment for diabetic peripheral neuropathy. Using a rat model of after turning on nSIRT1OE, we observed a reversal of mechanical allodynia, type 2 diabetes, early and late intervention protocols, 4 and 12 weeks post NCV and attenuation of IENFD (Group # 3 compared to Group # 2). West- hyperglycemia, respectively, rats were treated daily by gavage with valsar- ern blot of protein extracts from DRG neurons showed that HFD increased tan or sacubitril/valsartan for 12 weeks followed by an extensive evaluation acetylation of proteins and SIRT1OE abolished the HFD-induced acetylation of vascular and neural endpoints. The results demonstrated that efficacy of of proteins. The mitochondrial bioenergetics profile of cultured adult DRG sacubitril/valsartan in improving vascular and neural function was superior neurons showed that hyperglycemia induced a decrease in the spare respi- to valsartan alone. In the late intervention protocol, treatment of the dia- ratory capacity in wild type (WT) DRG neurons. This was corrected in nSI- betic rats was initiated well after the appearance of vascular and neural RT1OE Tg DRG neurons. In type 2 diabetic neuropathy, altered acetylation of complications. Sacubitril/valsartan treatment was found to not only slow progression of vascular and neural deficits but also stimulate restoration of

ADA-Supported Research

A15 WHAT’S NEW IN DIABETIC NEUROPATHY? EQUAL PARTS CLINICAL AND BASIC SCIENCE

vascular reactivity, motor and sensory nerve conduction velocities and sen- 62‑OR sitivity/regeneration of sensory nerves of the skin and cornea. These studies Contemporary Prevalence of Diabetic Neuropathy in Type 1 Diabe‑ suggest that sacubitril/valsartan may be an effective treatment for diabetic tes (T1D)—Findings from the T1D Exchange peripheral neuropathy and since it already has FDA approval for treatment KARA R. MIZOKAMI-STOUT, CLAIRE T. BOYLE, VIRAL N. SHAH, GRAZIA ALEPPO, of heart failure advancing this drug to clinical trials could proceed quickly. JANET B. MCGILL, RICHARD PRATLEY, ELENA TOSCHI, LYNN ANG, RODICA POP- Supported By: Novartis (LCZ696BUS10T) BUSUI, Ann Arbor, MI, Tampa, FL, Aurora, CO, Chicago, IL, St. Louis, MO, Orlando, FL, Boston, MA 60‑OR Diabetic peripheral neuropathy (DPN) is a major cause of disability, mortality High-Fat Diet Induces Mitochondrial Dysfunction in Saphenous and poor quality of life in patients with T1D, with prior reported prevalence Nerve Axons—A Study by Confocal Microscopy In Vivo rates of up to 35%. The contemporary prevalence of DPN in T1D patients was MARIJA SAJIC, ANISH KANHAI, GIACOMO DENTONI, SHARLINI VARATHARA- evaluated in T1D Exchange Registry centers throughout the United States. JAH, LUCY M. HINDER, AMY RUMORA, EVA L. FELDMAN, KENNETH SMITH, The Michigan Neuropathy Screening Instrument (MNSI), a validated 15-item London, United Kingdom, Leiden, Netherlands, Stockholm, Sweden, Ann Arbor, MI self-administered questionnaire, was used to assess DPN in adults ≥18 Diabetic peripheral neuropathy (DPN) is a common, progressive and irre- years with ≥ 5 years of T1D duration. A score of ≥4 was used to define DPN. versible complication of diabetes (DM). Glucose regulation has no effect on Diabetes-related characteristics and laboratory data were obtained through the development and/or progression of the disease in type 2 DM and so, the most recent clinic update. Chi-square and t-tests were used to compare as the prevalence of DM increases, there is a growing unmet need for a demographic and diabetes-related characteristics between those with and mechanism-based therapy. without DPN. Linear regression was used to determine the effect of DPN on We have examined the effect of high fat diet (HFD, 60% kcal fat) on HbA1c, adjusted for possible confounders. In preliminary analyses of 5,058 axonal mitochondrial membrane potential and intra-axonal Ca2+ concen- participants across 62 sites (mean age 39±18 years, T1D duration 22±14 years, 2+ 56% female, 88% non-Hispanic white, mean HbA1c 8.1±1.6%), the prevalence tration [Ca ]i in C56Bl/6 mice, in vivo, using confocal microscopy. The HFD resulted in obesity, hyperglycaemia and glucose intolerance. Mitochondria of DPN was 10%. Those with DPN were older (52±17 vs. 37±18 years), more within saphenous nerve axons of mice fed a HFD for 16 or 36 weeks were likely to be female (61% vs. 55%), had longer T1D duration (32±16 vs. 21±13 significantly depolarised compared with those in mice fed a control diet. years), lower annual household income (37% vs. 59% earning ≥$75K), and Furthermore, HFD mitochondria did not depolarise in response to high fre- lower education level (55% vs. 69% with college degree) than those without quency electrical stimulation, in contrast to controls where depolarisation is DPN (all p<0.001). They also had higher systolic blood pressure (126±17 vs. associated with the increased energy demand of sustained impulse conduc- 123±14 mmHg), triglycerides (117±89 vs. 95±62 mg/dL), tobacco use (9% vs. tion. Confocal microscopy revealed that the number of mobile mitochondria 4%) and prevalence of established CVD (26% vs. 6%), despite higher use of was significantly greater in electrically active axons after 16 weeks of HFD, CVD-modifying agents such as statins (64% vs. 31%) and ACE-inhibitors/ARBs compared with controls, whereas after 36 weeks the number was signifi- (45% vs. 23%) (all p<0.001). Participants with DPN had higher HbA1c (8.4±1.7% cantly lower. Histologically, these changes were accompanied by signifi- vs. 8.1±1.6%), even after adjusting for multiple confounders (p <0.01). The prev- cantly increased labelling for the mitochondrial biogenesis marker PGC1α in alence of DPN in this national T1D cohort is lower than prior published reports, neuronal cell bodies at 16 weeks, but a reduction at 36 weeks, consistent reflecting current clinical care practices, and highlighting other non-glycemic with exhaustion of compensatory mechanisms activated early in the disease risk factors for DPN including CVD risk factors and socioeconomic status. 2+ Supported By: The Leona M. and Harry B. Helmsley Charitable Trust course. Interestingly, we found that [Ca ]i was decreased in mice fed a HFD. We conclude that a HFD impairs axonal mitochondrial function early in the course of type 2 MD. Impaired ATP production, especially during sustained 63‑OR impulse activity, may be a mechanism in the development of DPN. Differential Association of Inflammatory Markers and Growth Factors with Type 2 Diabetes and Polyneuropathy—A Multimarker Approach 61‑OR GIDON J. BÖNHOF, ALEXANDER STROM, WOLFGANG RATHMANN, MARGIT Alterations in Somatomotor Network Functional Connectivity in HEIER, CHRISTA MEISINGER, ANNETTE PETERS, MICHAEL RODEN, BARBARA THO- Painful Diabetic Neuropathy—A Resting State Functional Mag‑ RAND, CHRISTIAN HERDER, DAN ZIEGLER, Düsseldorf, Germany, Munich, Germany netic Resonance Imaging Study There is emerging evidence supporting a role of inflammation in the DINESH SELVARAJAH, MOHAMMED AWADH, RAJIV GANDHI, IAIN D. WILKIN- development and progression of diabetic sensorimotor polyneuropathy SON, SOLOMON TESFAYE, Sheffield, United Kingdom (DSPN) in patients with type 2 diabetes. We aimed to characterize inflam- Aims: Painful diabetic neuropathy (Painful-DN) is a common disabling con- matory signatures associated with DSPN and/or type 2 diabetes using a dition, with no objective biomarkers and less than optimal treatments. RS- multimarker approach. We measured 92 serum biomarkers including pro- and fMRI is a quick (5 minute) functional imaging method that evaluates regional anti-inflammatory cytokines, chemokines, and growth factors (GF) using the cortical interactions that occur when a subject is at rest. The aim of this study Proseek Multiplex INF I assay (OLINK Proteomics) in 304 individuals with was to explore resting functional connectivity of the somatomotor network in type 2 diabetes and polyneuropathy, defined by the Toronto Consensus painful DN as a possible objective biomarker for neuropathic pain. Criteria (2011), from the PROPANE study (DSPN) as well as 158 individuals with type 2 diabetes without DSPN (T2D) and 354 individuals with normal

ORALS Methods: 46 patients with diabetes (No DN, n=16; Painful DN, n=15; Pain- less DN, n=15) and 16 healthy volunteers underwent detailed clinical and glucose tolerance and without DSPN (CON) from the KORA F4 study (DSPN/ neurophysiological assessments. RS-fMRI data were acquired at 3T (Philips T2D/CON [mean±SD]: age: 68±9/71±6/69±5 years; male: 76/59/41%; BMI: Healthcare) and functional connectivity analysis was performed using FSL 30.8±5.3/30.8±4.4/26.9±3.7 kg/m²; diabetes duration: 13.5±9.6/7.6±5.8/- (www.fmrib.ox.ac.uk/fsl). years; HbA1c: 7.4±1.3/6.6+1.0/5.5+0.3%). After adjustment for multiple test- Results: There was reduced functional connectivity in the sensorimo- ing and sex, age, BMI, and smoking, a biphasic pattern of serum levels (nor- tor network (postcentral gyrus -42,-22,56; all TFCE, corrected p<0.05) malized protein expression values) with an increase in T2D and decrease in and default mode network (precuneus -6,-46,40; p<0.05), superior frontal DSPN was observed for four GFs (e.g., transforming GF (TGF)-α: 4.51±0.51 vs. gyrus (34,62,60; p<0.05), Heschl’s gyrus (-42,-22,12; p<0.05), insular cortex 4.63±0.56 vs. 3.91±0.51; vascular endothelial GF (VEGF): 10.9±0.5 vs. 11.0±0.5 (34,62,60; p<0.05) and superior parietal lobule (-22,-42,68; p<0.05). Somato- vs. 10.8±0.6), two chemokines (e.g., C-C motif ligand 4 (CCL4): 8.18±0.57 vs. motor network functional connectivity significantly correlated with quanti- 8.4±0.6 vs. 7.93±0.68) and one cytokine (oncostatin M: 4.83±0.62 vs. tative pain assessments (Short Form 36, r=-0.52; p=0.03 and Chronic Pain 5.06±0.63 vs. 4.35±0.75). Compared to T2D, the levels of another 12 biomark- Acceptance Questionnaire r=-0.55, p=0.045). ers were lower in DSPN (e.g., tumor necrosis factor (TNFSF)-14: 5.65±0.55 vs. Conclusion: These findings demonstrate that chronic pain has a wide- 4.90±0.77; matrix metalloproteinase (MMP)-1: 14.4±0.7 vs. 14.1±0.9), while spread impact on overall brain function in diabetes, and suggests that five were higher (e.g., CCL20: 5.2±1.1 vs. 5.79±1.23) (all P<0.00024). disruptions of the resting state networks may underlie the cognitive and In conclusion, deficits in growth factors promoting nerve regeneration/ behavioural impairments accompanying chronic pain. Specifically within the angiogenesis and a complex cross-talk between innate and adaptive immu- somatomotor network, we have demonstrated abnormal functional connec- nity may contribute to the development of DSPN in type 2 diabetes. tivity in painful DPN which correlates with clinical measures of pain and Supported By: European Union Seventh Framework Program (602273); German behaviour. RS-fMRI has the potential to serve as an objective biomarker for Diabetes Association; German Diabetes Foundation; Ministry of Science and the chronic pain condition in DPN. Research of the State of North Rhine-Westphalia; German Federal Ministry of Health; German Research Foundation (RA 459/3-1); German Federal Ministry of Education and Research

ADA-Supported Research

A16 EFFECTS OF EXERCISE ON METABOLIC HEALTH IN TYPE 1 AND TYPE 2 DIABETES

EFFECTS OF EXERCISE ON METABOLIC HEALTH IN Figure. TYPE 1 AND TYPE 2 DIABETES

64‑OR Maternal and Paternal Exercise Regulates Offspring Metabolic Health and Beta-Cell Phenotype JIA ZHENG, ANA B.A. WAGNER, NOAH B. PRINCE, KAWAI SO, JORAM MUL, ERCUMENT DIRICE, MICHAEL F. HIRSHMAN, KRISTIN I. STANFORD, ROHIT KULKARNI, LAURIE J. GOODYEAR, Beijing, China, Boston, MA, Worcester, MA, Amsterdam, Netherlands, Columbus, OH Maternal and paternal environment are critical factors in determining risk for obesity and diabetes in offspring. Maternal exercise in mice can prevent the detrimental effects of maternal high-fat feeding on offspring metabolic health, whereas the effects of paternal exercise on offspring are less clear. Little is also known about the effects of maternal or paternal exercise on offspring beta cells, a major factor in glucose homeostasis. We determined the effects of maternal, paternal and maternal+paternal exercise on off- Supported By: Insulet Corporation spring glucose tolerance and beta cell morphology. Male sires were housed ± running wheels for 3 weeks before breeding (4.4±0.8 km/day) and dams were housed ± running wheels for 2 weeks before breeding (8.4±0.5 km/day) 66‑OR and during gestation (4.7±0.5 km/day). All male and female breeders were Exercise Regulates the Concentration of Novel Circulating Factors fed a high-fat diet. Four groups of male offspring were studied: both parents in Humans sedentary (Sed); maternal exercise only (Mat Ex); paternal exercise only ROELAND MIDDELBEEK, JEFFREY J. RICHARD, NOAH B. PRINCE, LESLIE ROW- (Pat Ex); maternal+paternal exercise (Mat+Pat Ex) (n=5-8 litters/group). Off- LAND, JONATHAN DREYFUSS, HUI PAN, LAURIE J. GOODYEAR, Boston, MA spring were sedentary and separate cohorts were studied at weaning, 52, Exercise can improve metabolic health for all people and patients with and 80 weeks. Post-weaning, offspring were fed a chow diet. There was no diabetes; however, the molecular mechanisms that mediate the exercise effect of parental exercise on offspring glucose tolerance at weaning. At 52 response are not well understood. We and others have hypothesized that weeks, offspring of exercised parents had ~25% lower fasting blood glucose exercise causes factors to be released from skeletal muscles, adipose tis- concentrations vs. Sed (all p<0.01), and glucose tolerance was improved in sue, and possibly other tissues into the circulation, and that these factors Mat Ex (33%), Pat Ex (35%), and Mat+Pat Ex (50%) vs. Sed (p<0.001). Mat may contribute to the beneficial effects of exercise on health. To iden- Ex, Pat Ex and Mat+Pat Ex had decreased beta cell size (40%) and mass tify novel exercise-stimulated factors, healthy subjects (n=6 males and 6 (40%), whereas islet size and density were only decreased in offspring from females; age=29.2±0.6 years; BMI=22.5±0.7; VO2peak=40.6±1.3ml/kg/min) Mat+Pat Ex parents (28% and 40%, respectively). Older offspring (80 weeks) performed 45min of treadmill exercise at 75% VO2peak. Blood was collected from all three parental exercise treatments displayed improved glucose tol- at baseline, 15min, 45min during acute exercise, and 1-hour post-exercise erance (p<0.05). Maternal and paternal exercise in high-fat fed parents has and 1,310 circulating factors were measured using an aptamer-based pro- profound positive effects on offspring glucose tolerance during adulthood teomic method. Remarkably, this single bout of moderate intensity exer- and older age that may be linked to adaptations in the endocrine pancreas. cise significantly altered 247 or 19% of the measured circulating factors. In Supported By: American Diabetes Association (1-17-PMF-009 to A.B.A.W.); total, 171 unique proteins were increased and 76 proteins were decreased. National Institutes of Health (3R01DK101043) Importantly, based on our literature review, ~85% of these proteins have not been previously identified as exercise-stimulated circulating factors. Cardiorespiratory fitness (VO2peak) significantly correlated with the con- 65‑OR centration of 112 factors at rest, suggesting that some of these proteins Reducing Basal Insulin 90 Minutes before Exercise Protects may be biomarkers of cardiorespiratory fitness. Of these factors, baseline Against Hypoglycemia Better than Insulin Suspension at Exercise concentrations of 40S ribosomal protein S3, a multi-functional protein that Onset in T1D—The OmniTIME Results has been reported to be present in exosomes, correlated most strongly with DESSI ZAHARIEVA, SARAH M. MCGAUGH, RUBIN POONI, TODD VIENNEAU, VO2peak (p<0.0007). This data set will provide the basis for ongoing studies TRANG T. LY, MICHAEL RIDDELL, Toronto, ON, Canada, Oakville, ON, Canada, Bil- to unravel the mechanisms by which exercise mediates its beneficial effects lerica, MA on diabetes and overall health. Guidelines for T1D suggest reducing basal insulin delivery 60 to 90 min In summary, we have identified >200 novel exercise-stimulated proteins before aerobic exercise. In this study, participants with T1D on CSII (Omni- ® and >100 circulating proteins that correlate with cardiorespiratory fitness pod , Insulet) (n=13) were randomized in a crossover design to three basal level in healthy human subjects. rate reduction strategies pre-exercise. All participants completed three 60 Supported By: Daiichi Sankyo; National Institutes of Health (5P30DK36836, min aerobic exercise sessions (walking, ~50% VO2max). Basal insulin deliv- R01DK099511 to L.J.G.); Joslin Diabetes Center; National Institutes of Health ORALS ery was reduced by; a) -80%, 90 min pre-exercise; b) -50%, 90 min pre-exer- (T32DK07260-038 to R.M.); Boston Area Diabetes Endocrinology Research Center cise or; c) -100% at exercise onset. Basal rates were resumed post exercise (to R.M.) and participants then took 75% of usual bolus with a standardized meal. Pre- exercise blood glucose (BG) was similar in all conditions (9.5 ± 2.8 mmol/L). The mean BG drop during exercise was greater (p<0.05) in the -100% (-4.2 ± 67‑OR 3.3 mmol/L) compared to the -50% and -80% conditions (-3.3 ± 2.4 vs. -2.0 ± GDF15 Is a Contraction-Induced Myokine That Regulates Pancre‑ 2.1 mmol/L), respectively. In the -100% condition, seven (54%) participants atic β-Cell Function had hypoglycemia (hypo), whereas in the other two conditions only one hypo HUI ZHANG, ANNY MULYA, STEPHAN NIEUWOUDT, RUTH MCDOWELL, JOHN event occurred (8%, p<0.001). Meal related BG responses after exercise P. KIRWAN, Cleveland, OH were similar among all conditions. β-Cell dysfunction plays a major role in the onset of type 2 diabetes (T2D). In summary, reducing basal insulin delivery by 50-80% 90 min before aero- Exercise improves or restores β-cell function in both humans and rodents bic exercise better mitigates hypo risk vs. basal suspension or disconnection with T2D. The specific mechanism is unclear. We hypothesized that exer- at exercise onset. Basal rate reductions well in advance of exercise do not cise-induced myokines engage in cross-talk with the pancreas to help regu- cause pre-exercise hyperglycemia or meal related rebound hyperglycemia. late β-cell function and mass. To test our hypothesis, differentiated C2C12 myotubes were subjected to electrical pulse stimulation (EPS) to simulate in vivo exercise. EPS-conditioned media from the contracting myotubes significantly elevated glucose-stimulated insulin secretion (GSIS) inβ -cells vs. non-EPS control conditions (P<0.05). RNA-sequencing and bioinformatic analysis enabled us to identify Growth Differentiation Factor 15 (GDF15) as a novel contraction-induced myokine that potentially communicates with β-cells. At the cellular level, recombinant GDF15 protein treatment upregulated the GSIS response in β-cells by 55% (P<0.05), with no effect on cel-

ADA-Supported Research

A17 EFFECTS OF EXERCISE ON METABOLIC HEALTH IN TYPE 1 AND TYPE 2 DIABETES

lular apoptosis, cytotoxicity or proliferation. Furthermore, qRT-PCR analysis, p=0.03, respectively), which returned to baseline after detraining. T2D and ELISA, immunoblots, and intracellular assays demonstrated that GDF15 may IR CON increased suppression of endogenous glucose production (EGP) dur- regulate first-phase insulin secretion by increasing the intracellular ATP/ ing the high-insulin clamp (T2D: 91 ± 14% vs. baseline: 76 ± 11%, p=0.003 and ADP ratio, inhibiting K+ channels and increasing the intracellular Ca2+ with- IR CON: 107 ± 13% vs. 94 ± 14%, p=0.04). IR CON also increased suppression out affecting insulin synthesis. Using translational approaches, we found a of lipolysis during the low-insulin clamp (82 ± 9 vs. 84 ± 8%, p=0.04). In T2D positive correlation between fasting plasma GDF15 and insulin secretion in liver fat was decreased by 17% and remained decreased after detraining. prediabetic subjects (R=0.64, P<0.01, n=22). A 6 week supervised functional Liver fat content remained unchanged by HIIT training in both CON groups. high-intensity exercise intervention increased fasting plasma GDF15 by 35% Muscle maximal uncoupled respiration increased in T2D and CON by 44% in adults with T2D (P<0.01, n=12) and, interestingly, the increase in GDF15 and 48%, respectively, at 12 weeks and remained increased after detraining was positively correlated with an increase in the β-cell disposition index in all groups (p<0.01). We conclude that at the same level of insulin resis- (R=0.72, P<0.01, n=12), providing further support that GDF15 may be involved tance HIIT training improved hepatic and peripheral insulin sensitivity in in improving β-cell function. patients with T2D and insulin resistant controls whereas changes in mito- In summary, these findings expand our understanding of skeletal muscle chondrial function occurred irrespective of insulin resistance status. as an endocrine organ and identify GDF15 as a potential myokine for T2D therapy. 70‑OR Supported By: National Institutes of Health (R21AR067477) Aerobic Training Remodels Muscle Lipid Composition and Improves Intrinsic Mitochondrial Function in Men with Type 2 Diabetes 68‑OR MARIA F. PINO, NATALIE STEPHENS, FANCHAO YI, ANDREW P. HODGES, Exercise Prevents High-Fat Diet-Induced Impairment of Insulin- RICHARD E. PRATLEY, STEVEN R. SMITH, BRET H. GOODPASTER, LAUREN M. Induced Vasodilatation of Muscle Arterioles in Mice by Browning SPARKS, Orlando, FL, Cambridge, MA, La Jolla, CA Perivascular Adipose Tissue of Mice The effects of exercise training on the skeletal muscle (SKM) lipidome and BEATRIZ C.S. BOA, FERNANDA M. FERRÃO, ISABELA M. GONZAGA, LUIS FELIPE mitochondrial function have not been thoroughly explored in individuals with R. PINTO, VICTOR W.M. VAN HINSBERGH, ETTO C. ERINGA, Amsterdam, Nether- type 2 diabetes (T2D). We investigated the effects of exercise training on lands, Rio de Janeiro, Brazil molecular signatures within SKM of T2D, employing integrated multi-omics Perivascular adipose tissue (PVAT) surrounds most arteries and enhances data analyses including lipidomics [MS/MS-shotgun], global DNA methyla- insulin-induced vasodilatation through local release of adipokines. Obesity tion and transcriptomics [RNA-Seq]. Muscle tissue and cellular mitochondrial impairs the vasoregulatory function of PVAT on insulin-induced vasodilation, respiration was quantified by high resolution respirometry. Ten weeks of aer- thus contributing to cardiovascular risk. Exercise enhances insulin-induced obic training increased monolysocardiolipin (MLCL), a marker of mitochon- vasodilatation, and we evaluated whether exercise preconditions the muscle drial remodeling, reduced total and specific species of sphingomyelin (SM), microcirculation against HFD-induced impairment of insulin-induced vasodi- phosphatidylserine (PS) and ceramides lipid content and improved intrinsic latation. C57BL/6 mice were exercised for 4 weeks (EX), or fed a high-fat diet mitochondrial respiration in SKM of T2D without alterations in mitochon- (HFD) for 2 weeks. Exercise preconditioning was performed by exercising drial DNA copy number or cardiolipin content. Exercise training differentially mice for 4 weeks prior to HFD treatment (EX+HFD) or before and during HFD modified 50 DNA methylation sites. Among these genes, NOX4, acts as an (EX+HFD/EX). HFD increased femoral PVAT and exercise attenuated this effect oxygen sensor and catalyzes the reduction of reactive oxygen species (ROS), [(Control (C) 9±1mg vs. HFD 64±7mg - p<0.01) (EX+HFD 29±5mg; EX+HFD/ and PLCE1, catalyzes the hydrolysis of phosphoinositol 1,4,5-triphosphate EX 32±2mg; p<0.01 vs. HFD)]. Insulin-induced vasodilatation (0.5 IU/kg (IP3) and DAG, were hypo-methylated and hyper-methylated, respectively, i.p.) was evaluated in vivo in the cremaster muscle. HFD inhibited insulin- following training. Exercise training differentially regulated 70 transcripts in induced vasodilatation [C 22±5% dilatation; HFD -11±4.3% dilatation, p<0.01 SKM of T2D that resulted in enrichments of lipid and amino acid metabolism vs. C], but exercise precondition restored it [EX+HFD-24±5% and EX+HFD/ and β-oxidation pathways. EX-32±8.9% dilatation; p<0.05]. Browning of adipose tissue improves insu- In summary, aerobic training improves intrinsic and cell autonomous SKM lin sensitivity and adipokine secretion, and we observed browning by an mitochondrial function and modifies SKM lipid composition in men with T2D, increase in the mRNA of the browning marker Ucp-1 in aortic PVAT [72% with potentially advantageous augmentations of oxidative stress and insulin increase in EX+HFD/EX vs. C p<0.01] and in brown adipose tissue [C 3±0.3; signaling pathways. HFD 10±1; EX 8±0.9; *EX+HFD 19±2; *EX+HFD/EX 14±1.8; 2-Δct p<0.01 vs. Supported By: American Diabetes Association (7-13-JF-53 to L.M.S.) C]. mRNA of the vasoprotective adipokine adiponectin was increased in PVAT of EX and EX+HFD/EX mice (p<0.05 vs. C, HFD and EX+HFD), while the 71‑OR inflammatory TNF-α was substantially decreased (HFD vs. C, EX+HFD and MicroRNA Regulated Transcriptomic Changes during Aging in EX+HFD/EX vs. HFD; p<0.05). Human Skeletal Muscle Are Reversible with Exercise Training In conclusion, exercise reduces the HFD-induced increase in PVAT and JOE VALENTINE, SANGEETA GHOSH, BRANDON MILHOLLAND, JONATHAN A. preserves insulin-induced vasodilatation by increasing mitochondrial uncou- GELFOND, NICOLAS MUSI, San Antonio, TX, Boyds, MD, Bronx, NY pling and improving adipokine expression in PVAT. Muscle mass declines with age (sarcopenia) in both men and women Supported By: Carlos Chagas Filho Foundation; Netherlands Organization for

ORALS resulting in impaired physical function and altered glucose metabolism. Scientific Research Endurance exercise training can mitigate and even reverse some of the changes in skeletal muscle structure and function, which are observed at 69‑OR advanced age. To gain insight into the potential mechanisms driving sarcope- Effects on Insulin Sensitivity, but Not on Mitochondrial Function nia and the adaptive response to training, we conducted deep sequencing on Are Dependent on Insulin Resistance Status after High Intensity vastus lateralis muscle obtained from healthy, younger (n=14; age=27±0.89 Interval Training years; 8 women, 6 men) and older (n=14, age=71±1.39 years; 7 women, 7 men) MARIA APOSTOLOPOULOU, DOMINIK PESTA, YANISLAVA KARUSHEVA, SOFIYA subjects before and after a 4-month endurance training program. We ana- GANCHEVA, TOMAS JELENIK, ALESSANDRA BIERWAGEN, KARSTEN MÜSSIG, lyzed the sequencing data with bioinformatics tools to determine whether JULIA SZENDROEDI, MICHAEL RODEN, Düsseldorf, Germany miRNAs that change with age can be reverted back to youthful levels with High intensity interval training (HIIT) is a time-efficient training approach training. We found that skeletal muscle from women had 7 miRNAs and men to stimulate biogenesis in healthy populations. We hypothesized that HIIT had 8 miRNAs that changed with age and were reverted back to youthful would increase skeletal muscle insulin sensitivity due to improved muscle levels with training, respectively. In older trained females these 7 miRNAs mitochondrial function in type 2 diabetes (T2D) patients and age- and BMI- targeted 94 unique transcripts in the top 1000 differentially expressed matched controls (CON). We examined 18 sedentary male patients with genes between post-training compared to pre-training and these transcripts T2D and 23 healthy male CON (age: 58±5 vs. 57±4 years, BMI: 31.4±2.4 vs. were involved in lipid metabolism, glycolysis, and growth. In older men the 30.4±2.3 kg.m-2) that were enrolled in a 12-week HIIT cycling protocol. CON 8 miRNAs with youthful signatures after training had 146 unique targets were further grouped in insulin-sensitive (IS) and -resistant (IR) (baseline M involved in phosphoprotein and PI3K-AKT signaling, as well as cholinergic in mg.kg-1.min-1; 7.4 ± 1.3 vs. 4.2 ± 1.1, p<0.001). Two-step hyperinsulinemic- synapses. Notably, miR-29b-3p was primarily responsible for the regulation euglycemic clamps, skeletal muscle biopsies for high resolution respirom- of target transcripts within the PI3K-AKT pathway in older trained men; simi- etry and magnetic resonance spectroscopy for liver fat quantification were lar family members (miR-29a-3p and miR-29c-3p) have been directly linked to performed. After 12 weeks of HIIT, T2D and IR CON increased their insulin insulin sensitivity in preclinical models. All miRNAs with youthful signatures sensitivity (3.9±1.9 vs. baseline: 2.7±1.6, p=0.02 and 5.5±2.1 vs. 4.2 ± 1.1, in older trained subjects had opposing expression patterns to their target

ADA-Supported Research

A18 EMERGING TARGETS FOR DIABETES TREATMENT mRNAs. We conclude that some of the aging related changes to the skeletal cose AUC0-4h during an OGTT. LGD-6972 treatment also increased fasting muscle transcriptome are under the regulation of miRNAs and that exercise glucagon (1.9-3.1-fold) and total and active GLP-1 (1.3-1.4-fold and 1.1-1.4- training can reverse these changes. fold, respectively) from baseline. LGD‑6972 treatment was safe and well tolerated with a low incidence of hypoglycemia (8 mild, patient-reported events) and no severe hypoglycemia. Mild increases in aminotransferase EMERGING TARGETS FOR DIABETES TREATMENT levels (ALT and AST) were observed with LGD 6972 treatment (group means blood pressure or body weight Effects of the Novel Dual GLP-1R/GCGR Agonist SAR425899 on Post‑ observed in LGD-6972 dose groups compared to placebo. These findings prandial Glucose Metabolism in Overweight/Obese Subjects with indicate that glucagon receptor antagonism with LGD 6972 significantly low- Type 2 Diabetes ers HbA1c and fasting plasma glucose levels, and may be a promising new BRITTA GOEBEL, MICHELE SCHIAVON, ROBERTO VISENTIN, MICHELA RIZ, CHI- option to treat T2DM. The combination of safety and efficacy support further ARA DALLA MAN, CLAUDIO COBELLI, THOMAS KLABUNDE, Frankfurt, Germany, clinical development of LGD-6972. Padova, Italy SAR425899 (SAR) is a novel dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist. A randomized, double-blind, phase 74‑OR I multiple-ascending-dose study (NCT02411825) in 27 overweight/obese Fourteen-Day Study of Praliciguat, a Soluble Guanylate Cyclase subjects with type 2 diabetes receiving SAR demonstrated decreased Stimulator, in Patients with Diabetes and Hypertension body weight with a safety profile comparable with GLP-1R agonists. Here, JOHN P. HANRAHAN, JAMES D. WAKEFIELD, PHEBE J. WILSON, PAUL MILLER, we report additional analyses of the effect of SAR on key processes in glu- JENNIFER CHICKERING, LINDA MORROW, MICHAEL L. HALL, MARK CURRIE, G. cose metabolism. Subjects were randomized to receive daily subcutaneous TODD MILNE, ALBERT T. PROFY, Cambridge, MA, Raleigh, NC, Chula Vista, CA administrations of low-dose SAR (30, 60, 90 µg) or high-dose SAR (60, 120, Background: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, 180 µg) for 28 days; dose escalation occurred after Days 7 and 14. Mixed has been shown to augment nitric oxide (NO) signaling and reduce fasting meal tests were conducted before treatment (Day −1) and on Days 1 and 28. plasma glucose and proteinuria in an animal model of diabetic nephropathy. Oral glucose and C-peptide minimal models were used to quantify metabolic In healthy adults, repeated oral doses of 15-40 mg were tolerated and low- indices of glucose absorption, insulin sensitivity, and β-cell responsiveness. ered blood pressure (BP). Percent change in area under the curve for rate of meal glucose appearance Methods: This Phase 2a, double-blind, placebo (PBO)-controlled study between 0 and 120 min from Day −1 to Day 28 was −14% and −19% with low- assessed the effects of oral praliciguat in 26 patients with type 2 diabe- and high-dose SAR, respectively. Change in insulin sensitivity was 104% tes mellitus (T2DM) and hypertension on stable glycemic and BP-lowering and 262%, respectively. Change in β-cell function was 127% and 145%, therapy. Most (69%) were also on statins. Two praliciguat regimens were respectively (Table). After 28 days of treatment, SAR improved postprandial tested: (1) 40 mg QD for days 1-14 (N=10), and (2) 20 mg BID for days 1-7 then glucose control by significantly reducing glucose absorption rate, increasing 40 mg QD for days 8-14 (N=10). Assessments included serum chemistry, fast- insulin sensitivity, and enhancing β-cell function. ing plasma glucose, BP and heart rate, platelet function, reactive hyperemia index (RHI), plasma biomarkers, and adverse events (AEs). Results: Results were similar for both praliciguat regimens and are here combined. After 14 days of treatment, praliciguat-treated patients had notable differences in least squares mean change from baseline (95% CI) relative to PBO in 24-hour mean arterial pressure [-4.7 mmHg (-10, 0.8)], heart rate [2.9 bpm (-0.7, 6.5)], fasting glucose [-13 mg/dL (-32, 7)], HOMA-IR [-26 (-58, 7)], total serum cholesterol [-26 mg/dL (-44, -7)], and LDL cholesterol [-20 mg/ dL (-37, -3)]. Praliciguat had no effect on platelet function or RHI. Among the exploratory biomarkers tested, levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were lower in praliciguat vs. PBO recipients. Both praliciguat dose regimens were generally well tolerated. Except for one serious AE of upper gastrointestinal hemorrhage, AEs in praliciguat- treated patients were mild. Conclusion: In this initial trial, praliciguat had beneficial effects on BP and metabolic parameters in T2DM patients with hypertension on standard therapies. Further clinical investigation of praliciguat in T2DM patients is warranted.

Supported By: Sanofi

75‑OR ORALS Oral Sodium Nitrite Improves Insulin Sensitivity, Blood Pressure, 73‑OR and Arterial Thickening in Adults with Uncontrolled Hypertension Glucagon Receptor Antagonist LGD-6972 Significantly Lowers and Metabolic Syndrome HbA1c and Is Well Tolerated after 12-Week Treatment in Patients KARA S. HUGHAN, NICOLE L. HELBLING, STEVEN J. ANTHONY, JAMES DELANY, with Type 2 Diabetes Mellitus (T2DM) on Metformin BRET H. GOODPASTER, MARK GLADWIN, Pittsburgh, PA, Orlando, FL JEREMY PETTUS, ERIC G. VAJDA, JAMES PIPKIN, GRETCHEN WILLIAMSON, Introduction: Although nitrate and nitrite have traditionally been consid- MIRIAM A. ZANGMEISTER, YONG-XI LI, ROBERT R. HENRY, DAVID D’ALESSIO, ered to be inert and potentially toxic metabolites of nitric oxide (NO) oxida- JUAN P. FRIAS, LIN ZHI, KEITH MARSCHKE, San Diego, CA, Cincinnati, OH, Dur- tion, it is now appreciated that both can be recycled to bioactive NO. Recent ham, NC, Los Angeles, CA evidence suggests that the human nitrate-nitrite-NO pathway mediates Inappropriately elevated levels of glucagon are thought to contribute to signaling through NO to reduce blood pressure and endothelial function. The increased hepatic glucose production in people with T2DM and exacerbate effects of oral nitrite on insulin sensitivity, however, are not known. There- hyperglycemia. LGD-6972 is an orally bioavailable, small molecule glucagon fore, we developed the investigational drug, oral inorganic sodium nitrite, to receptor antagonist. In a Phase 2, double-blind, placebo-controlled study, assess its therapeutic effects. the safety and efficacy of LGD-6972 was evaluated in subjects with T2DM Methods: Our goal was to examine the effect of 12 weeks of open-label (HbA1c ≥7.0%-≤10.5%) on a stable dose of metformin. Patients were ran- oral sodium nitrite 40 mg three times daily in this pilot study. We employed domized to 5 mg (n = 43), 10 mg (n = 40), 15 mg (n = 42) LGD-6972, or placebo hyperinsulinemic-euglycemic clamps to assess insulin sensitivity, flow medi- (n = 41) daily for 12 weeks. Mean HbA1c at baseline was 8.2% and did not ated dilation (FMD) to measure endothelial function, and pulse wave velocity differ among groups. A statistically significant decrease from baseline in (PWV) and carotid intima media thickness (IMT) to measure arterial stiffen- HbA1c (p<0.0001) was observed at all LGD‑6972 doses during 12 weeks of ing and thickening, respectively, in obese adults with metabolic syndrome treatment [-0.90% (5 mg), -0.92% (10 mg), and -1.20% (15 mg) compared and uncontrolled hypertension. with placebo (‑0.15%)]. All doses of LGD-6972 significantly decreased fast- Results: Eighteen subjects were recruited with mean age 53 years, BMI ing plasma glucose (-30.1 to -39.3 mg/dL from baseline) and reduced glu- 41 kg/m2 and BP 147/88 mmHg and 72% were female. Following 12 weeks of

ADA-Supported Research

A19 EMERGING TARGETS FOR DIABETES TREATMENT

nitrite treatment, systolic (-10 mmHg), diastolic (-13 mmHg) and mean arterial Recently, we have developed a novel long-acting GLP-1/GIP/Glucagon triple pressures were reduced (-12 mmHg; all p<0.001). Insulin stimulated glucose agonist, HM15211, which showed potent weight loss in obese animal mod- disposal tended to improve (14%; 7.4±0.7 at baseline to 8.4±0.8 mg/kg/min els. When combined with basal insulin, HM15211 might maximize the insu- post, p=0.08) after oral nitrite treatment compared to baseline. Carotid IMT lin response and provide additional weight loss. Here, we investigated the decreased from 0.762±0.02 at baseline to 0.727±0.03 mm post (p<0.01), and PK/PD properties of HM15211 and HM12460A (once weekly basal insulin FMD and PWV trended towards improvement with oral sodium nitrite treat- developing in clinical stage) combination in animal models to evaluate the ment. There were no changes in body weight, BMI, fasting glucose, HbA1c, therapeutic benefits of this novel combination in diabetes. Firstly, drug- or lipids. to-drug interaction (DDI) was evaluated to assess the changes in intrinsic Conclusion: Oral sodium nitrite treatment is a promising therapeutic to activity and PK of the individual components. In vitro human insulin receptor improve insulin sensitivity, blood pressure, and arterial thickening in at-risk phosphorylation by HM12460A was not affected by concomitant HM15211 obese adults with metabolic syndrome and uncontrolled hypertension. treatment. Similarly, cAMP accumulation by HM15211 was unaffected. Anal- Supported By: National Institutes of Health ysis of PK demonstrated that the administration of HM15211 and HM12460A, either alone or in combination, did not change their extended half-lives in SD 76‑OR rats. Secondly, the glucose lowering and body weight loss by HM15211 in BZ043, a Novel Amylin Analog, in a Fixed Combination with Insulin combination with HM12460A was evaluated in db/db mice. After 4 weeks Glargine—Nonclinical Results treatment, a combination of HM15211 and HM12460A reduced the HbA1c CAIO V.F. NASCIMENTO, PAULO G. LACATIVA, FRANÇOIS BRIAND, LUIS M.T.R. more than the mono-treatment. Moreover, potent weight loss by HM15211 LIMA, Rio de Janeiro, Brazil, Labège, France was well maintained even when combined with HM12460A. Based on these Amylinomimetics are important adjunctive drugs for treating type 1 dia- observation, we propose that HM15211 might be an additional combination betes (T1D). Here we describe the amylin analog BZ043 and its potential to partner for long-acting basal insulin such as HM12460A providing improved be mixed with insulin glargine (GLAR) in a fixed combination (FC). The effect glycemic control and more favorable weight loss. of BZ043 over fed glycemia, either alone or co-administered with GLAR, was investigated in rats with streptozotocin-induced diabetes (STZ). Fasted 78‑OR STZ rats (3 h) received a single treatment of BZ043 (0.15, 0.6 or 1.2 mg/kg), MEDI0382, a GLP/Glucagon Receptor Dual Agonist, Significantly GLAR (1.5 IU/rat), or BZ043 plus GLAR (separate injections), and then had Reduces Hepatic Fat Content in Subjects with Type 2 Diabetes free access to food (5% glucose rich) and water. BZ043 dose-proportionally Mellitus prevented meal-induced increase of glycemia and the co-treatment (0.6 or MEENA JAIN, LAN-FENG TSAI, DARREN ROBERTSON, BOAZ HIRSHBERG, PAUL 1.2 mg/kg) with GLAR restored normoglycemia (100 mg/dL) without abrupt HOCKINGS, LARS JOHANSSON, PHILIP AMBERY, Cambridge, United Kingdom, variations of glycemia (Figure 1A), as further shown by comparison of the Gaithersburg, MD, Mölndal, Sweden area under the blood glucose curve (Figure 1B). Adding BZ043 to a commer- MEDI0382 is a balanced GLP-1/glucagon receptor dual agonist in develop- cial vial of GLAR at 0.1 or 0.5 mg/mL did not result in observable alteration ment for type 2 diabetes mellitus (T2DM). We investigated the effects of of the average molecular radius of the samples after 24 weeks of incubation MEDI0382 on hepatic fat content in overweight/obese subjects with T2DM at 4 °C, as shown by dynamic light scattering (Figure 1C). In STZ rats, BZ043 and a BMI of 27-40 kg/m2. In a phase 2a study (NCT02548585), 51 subjects showed a prolonged anti-hyperglycemic effect and, together with GLAR, were randomized to receive MEDI0382 200 µg or placebo daily during a promoted a long-lasting normoglycemia, with flat variation of glycemia. 41-day treatment period. Quantification of liver proton density fat fraction, While further investigation on the physicochemical stability of the combi- subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) was nation is ongoing, our data suggest that BZ043 and glargine in a FC might performed as an exploratory analysis in a subset of patients (MEDI0382 improve the control of T1D in humans. n = 17, placebo n = 21) who had MRI scans taken at baseline and day 41. Figure 1 A-C. Mean (SD) liver fat content at baseline was 15.7% (8.97) and 18.1% (8.38) in the MEDI0382 and placebo groups, respectively. Patients treated with MEDI0382 showed a highly significant relative reduction from baseline in hepatic fat content vs. placebo (Table). This was positively correlated with reductions in both body weight (r = 0.49, P = 0.002) and alanine aminotransferase (r = 0.42, P = 0.009). Treatment with MEDI0382 was also associated with reductions in both SAT and VAT vs. placebo. These data demonstrate the efficacy of MEDI0382 in reducing liver fat and its potential for the treatment of nonalcoholic fatty liver disease, including steatohepatitis. Table. Measurement Change from Baseline, P LS Mean (90% CI) MEDI0382 (n = 17) Placebo (n = 21) ORALS Hepatic fat content, -5.98 (-7.67, -4.29) -3.17 (-4.68, -1.65) 0.017 absolute change (%) Hepatic fat content, -39.12 (49.11, -29.14) -19.51 (-28.49, -10.53) 0.006 relative change (%) SAT (L) -0.39 (-0.48, -0.30) -0.21 (-0.30, -0.13) 0.008 VAT (L) -0.36 (-0.52, -0.19) -0.14 (-0.29, 0.01) 0.057

Supported By: National Council for Scientific and Technological Development of Brazil 79‑OR Effects of MEDI0382 on Pancreatic and Incretin Hormones 77‑OR VICTORIA E.R. PARKER, LAN-FENG TSAI, DARREN ROBERTSON, MARCELLA Novel Combination of a Long-Acting GLP-1/GIP/Glucagon Triple PETRONE, CRISTINA RONDINONE, BOAZ HIRSHBERG, LUTZ JERMUTUS, PHILIP Agonist (HM15211) and Once-Weekly Basal Insulin (HM12460A) AMBERY, Cambridge, United Kingdom, Gaithersburg, MD Offers Improved Glucose Lowering and Weight Loss in a Diabetic MEDI0382, a GLP-1/glucagon dual receptor agonist, is being developed Animal Model for the treatment of T2DM. GLP-1 receptor agonists promote glucose- JUNG KUK KIM, JONG SUK LEE, JAEHYUK CHOI, SUNG YOUB JUNG, SANG dependent insulin release, suppress glucagon, and increase or have minimal HYUN LEE, IN YOUNG CHOI, SUN JIN KIM, Seoul, Republic of Korea effect on glucose-dependent insulinotropic polypeptide (GIP); knowledge of Obesity is an established risk factor for prediabetes, diabetes and the the effect on GLP-1 is limited. We characterized fasting and postprandial aggravation of preexisting diabetes by negatively affecting lipid metabo- hormone profiles associated with MEDI0382 therapy. In a phase 2a study lism, insulin response, and β-cell function. Thus, effective body weight (NCT02548585), 51 subjects with T2DM and BMI 27-40 kg/m2 received loss not only prevents or delays the onset of T2DM, but also enhances MEDI0382 (uptitrated to 200 µg) or placebo (PBO) daily for 41 days. Glucose, the response to diabetes drugs including basal insulin in T2DM patients. insulin, glucagon, GIP, and GLP-1 were measured at baseline (day -1) and day

ADA-Supported Research

A20 TARGETING BETA-CELL APOPTOSIS AND DEATH

41 during a liquid mixed-meal tolerance test. MEDI0382 significantly reduced cell death. IME has been shown to prevent PTP opening in human endothelial fasting glucose (-49.9 vs. -19.2 mg/dL for PBO; P < 0.0001) and glucose AUC0-4h cells under oxidative stress. This study investigated whether IME could also (-32.8 vs. -10.2% for PBO; P < 0.0001) in association with increased fasting prevent PTP opening and cell death induced by high glucose or fructose con- insulin (2.2 mU/L vs. -3.9 mU/L for PBO; P = 0.016), but postprandial insulin centrations in INS-1 cells and human pancreatic islets. PTP status in intact AUC was unchanged. In contrast, fasting and postprandial endogenous glu- cells was assessed by confocal microscopy by measuring mitochondrial cagon, GIP (Figure), and active and inactive GLP-1 were suppressed, and a membrane potential (TMRM) and NAD(P)H (autofluorescence). Cell viabil- delay in their kinetics was observed after MEDI0382 therapy. The results ity was measured by flow cytometry. In INS-1 cells and human islets, 30 suggest that MEDI0382 is insulinotropic and might also cause delayed gas- mM Glucose or 2.5 mM Fructose for 24h led to PTP opening (increase in the tric emptying. The effects of MEDI0382 on GIP have not been observed with NAD(P)H/TMRM ratio), this phenomenon being prevented by 100µM IME. GLP-1 receptor mono-agonists and may represent a footprint of dual GLP-1/ Compared to normal glucose concentration (100%), cell viability significantly glucagon receptor agonism in this complex hormone signaling network. decreased (p<0.05) in cells exposed to 30 mM Glucose (76±5% and 47±18% Figure. in INS-1 cells and human islets, respectively) and to 2.5 mM Fructose for 72h (78±3% and 35±11% in INS-1 cells and human islets, respectively) but remained unchanged when these cells were preincubated with 100µM IME (in 30mM glucose: 94±3% and 98±3%/in 2.5mM fructose: 96±4% and 79±6% in INS-1 cells and human islets, respectively). We conclude that 1.) IME is able to prevent high glucose and fructose-triggered cell death through an inhibition of PTP opening and 2.) a PTP-targeted strategy can prevent beta cell death. In addition to its beneficial effect on beta cell function, IME may have the potential to preserve beta cell mass in a dysmetabolic environment via its inhibitory effect on PTP opening.

82‑OR Evidence for Necroptosis as a Mechanism of Islet Amyloid-Induced Beta-Cell Death ANDREW T. TEMPLIN, MEGHAN F. HOGAN, NATHALIE ESSER, SAKENEH ZRAIKA, REBECCA L. HULL, STEVEN E. KAHN, Seattle, WA Islet amyloid deposition occurs in the majority of individuals with type 2 diabetes (T2D) and contributes to beta-cell death by mechanisms which are not fully understood. Necroptosis is an immunogenic form of cell death dependent on tumor necrosis factor receptor 1 (TNFR1) signaling, receptor interacting protein kinase 3 (RIPK3) oligomerization, and mixed lineage kinase domain-like (MLKL) phosphorylation. We previously showed mRNA TARGETING BETA-CELL APOPTOSIS AND DEATH and protein levels of TNFa, a TNFR1 ligand, are increased in islets by amyloid deposition in vitro and in vivo, raising the possibility that amyloid formation 80‑OR induces necroptosis. Thus, we sought to determine whether the components miR-128 Targets Thioredoxin-Interacting Protein 3’UTR and Down‑ of necroptosis are present in beta cells, and if islet amyloid-induced TNFa regulates Its Expression in Pancreatic Beta Cells expression elicits beta-cell necroptosis. We observed protein expression of GU JING, JUNQIN CHEN, GUANLAN XU, ANATH SHALEV, Birmingham, AL TNFR1, RIPK3, and MLKL in the INS-1 beta-cell line, indicating necroptosis Thioredoxin-interacting protein (TXNIP) expression is induced by glucose, components are present in beta cells. In human islet amyloid polypeptide promotes beta cell apoptosis and is essential for glucotoxicity-induced beta (hIAPP) transgenic mouse islets cultured in 16.7 mM glucose, an in vitro cell death, whereas TXNIP deficiency protects against diabetes. Carbohy- model of islet amyloidosis, we found necroptosis signaling to be increased drate-response element-binding protein (ChREBP/MondoA) is the major compared to amyloid-free wild type (WT) islets. This manifested as transcription factor conferring glucose-induced TXNIP expression. How- increased oligomerization of RIPK3 protein (trimers: 2.7 ± 0.2 [hIAPP] vs. 1.0 ever, we recently discovered that glucose was still able to induce TXNIP [WT], n=3, p<0.01) and phosphorylation of the RIPK3 target MLKL (1.8 ± 0.3 in ChREBP knockout mouse islets suggesting the existence of other factors vs. 1.0, n=3, p=0.06), without a change in TNFR1 protein expression (1.1 ± 0.1 involved in glucose-induced TXNIP regulation. Analyses of the TXNIP 3’UTR vs. 1.0, n=2, p=0.74). As further support that amyloid induces immunogenic revealed several potential miRNA binding sites including miR-93, miR-106b cell death, immune cell mRNA abundance was increased in vivo in amyloid and miR-128. However, using INS-1 beta cells, primary mouse islets and T2D containing hIAPP transgenic islets vs. WT islets (macrophages: Emr1, 1.6 ± human islets, we found that only miR-128 was downregulated by glucose or 0.2 [hIAPP] vs. 1.1 ± 0.1 [WT], n=4, p=0.04, T cells: Cd3, 2.7 ± 0.5 vs. 1.0 ± 0.2, diabetes. This suggested that glucose might decrease miR-128 and thereby n=4, p=0.03, and B cells: Cd20, 2.3 ± 0.2 vs. 1.2 ± 0.5, n=2, p=0.17). These data suggest that necroptosis may be a previously unrecognized mechanism

promote TXNIP expression. In fact, we found overexpression of miR-128 ORALS significantly reduced TXNIP mRNA and protein expression whereas inhibi- of beta-cell death in response to islet amyloid formation. Further studies are tion of miR-128 increased beta cell TXNIP expression. Furthermore, lucif- needed to determine whether inhibition of necroptosis signaling may reduce erase assays using reporter constructs with the wild type TXNIP 3’UTR or beta-cell death in T2D. a mutated TXNIP 3’UTR confirmed that TXNIP is indeed a direct target of Supported By: U.S. Department of Veterans Affairs (BX001060 to S.E.K.); miR-128. Taken together, the results of these studies have identified a novel National Institutes of Health (F32DK107022 to A.T.T.) microRNA directly targeting TXNIP and revealed an alternative, post-transcriptional pathway by which glucose induces TXNIP expression. 83‑OR Supported By: National Institutes of Health; JDRF Pancreatic Beta Cell O-GlcNAc Transferase Overexpression Increases Susceptibility to Streptozotocin- and High-Fat Diet-Induced 81‑OR Diabetes in Female Mice Imeglimin Protects INS-1 Cells and Human Islets against High Glu‑ RAMKUMAR MOHAN, HAI-BIN RUAN, XIAOYONG YANG, EMILYN ALEJANDRO, cose- and High Fructose-Induced Cell Death by Inhibiting the Mito‑ Minneapolis, MN, New Haven, CT chondrial PTP Opening The nutrient-sensor O-GlcNAc transferase (OGT), the sole enzyme that SANDRINE LABLANCHE, EMILY TUBBS, CÉCILE COTTET-ROUSSELLE, FREDERIC adds an O-GlcNAc-modification onto proteins, was shown to regulateβ -cell LAMARCHE, ANAÏCK MOISAN, VIRGINIE PERSOONS, PIERRE Y. BENHAMOU, survival and function as β-cell deletion of OGT induces diabetes and islet fail- OE SOPHIE HALLAKOU-BOZEC, ERIC FONTAINE, Grenoble, France, Saint-Martin ure in mice. We hypothesized that β-cell OGT overexpression (Rip-cre;Ogt ) d’Hères, France, Saint-Ismier, France, Lyon, France would confer protection from streptozotocin (STZ)-induced β-cell failure and Imeglimin (IME) is a novel glucose-lowering agent improving insulin secre- high-fat diet (HFD)-induced diabetes. Female mice are typically resistant tion in response to glucose and insulin sensitivity by targeting mitochondrial from HFD- or STZ-induced diabetes. However, the molecular mechanisms bioenergetics. IME is currently in phase 3 in Japan. The toxicity of high glu- underlying this sexual dimorphism are unclear. Here, we uncovered that OE cose or fructose concentrations in INS-1 cells was linked to the opening of Rip-cre;Ogt mice show normal peripheral insulin sensitivity and glucose the permeability transition pore (PTP), a mitochondrial channel involved in tolerance in normal chow but when challenged female transgenic mice alone

ADA-Supported Research

A21 TARGETING BETA-CELL APOPTOSIS AND DEATH

develop glucose intolerance post 8-weeks of HFD and severe hyperglycemia folding, calcium storage, and regulating activity of the Perk, Ire1 and Atf6 1-week post STZ treatment. Surprisingly, male transgenic mice are neither unfolded protein response (UPR) pathways. Whole-body Grp78 deletion in protected nor impaired when compared to controls in both treatments. mice is lethal at E3.5 days. Despite its critical importance, the impact of Molecular interrogation of islets validated efficient overexpression of OGT Grp78 deletion in beta cells has not yet been reported. Mice lacking both in both sexes by confirming protein expression of the reporter transgene copies of Grp78 in beta cells (Grp78flox x Ins1CreThor) developed insulin- (GFP). However, significant increase in OGT protein was observed only in deficient diabetes by 2 weeks of age; heterozygotes remained normal. Islet islets of female Rip-cre;OgtOE mice, suggesting sex difference in OGT pro- architecture was disturbed in KO pups, with fewer beta cells than alpha tein regulation. Transcriptome analysis by RNA sequencing shows increased cells (14.8±1.7% beta cells-KO, 70.5±3.8%-WT), reduced beta cell mass expression levels of genes involved in acute inflammation (Ggt5, Ncf1, Ptgs1, (13.5±1.7mg-KO, 69.7±6.4mg-WT), and abnormal cellular arrangement, with Alox5ap) and immune response via agranulocyte adhesion and diapedesis alpha and beta cells interspersed throughout islets. Reduced beta cell num- (Glycam1) in islets of female Rip-cre;OgtOE mice, providing a mechanism of ber was likely due to increased cell death; TUNEL+ beta cells were increased susceptibility to STZ and HFD-induced islet failure. Indeed, pancreas analy- early, by 2 weeks of age (2.6±0.4%-KO, 0.1±0.0%-WT), whereas prolifera- sis shows increased islet infiltrating lymphocytes in female Rip-cre;OgtOE tion was not reduced until 4 weeks. Intriguingly, when BG was still normal, compared to control post 22 weeks of HFD treatment. Together, these data an increase in bi-hormonal glucagon/insulin cells was observed at 3-5 days highlight the novel role of OGT in inflammation induced by STZ and HFD in (7.1±0.6%-KO, 3.0±0.2%-WT) and 2 weeks (7.7±0.6% in KO, 2.2±0.2% in WT) islets and emphasize the importance of OGT dosage and O-GlcNAc cycling of age, suggesting defective fetal maturation, de-differentiation or trans- in β-cell survival. differentiation. Pdx1 staining was heterogeneously lost in insulin+ cells. Supported By: National Institutes of Health Acute deletion of Grp78 in ex vivo mouse islet cells, human islet cells and the human EndoC-BH1 beta cell line strongly reduced Grp78 RNA/protein, 84‑OR induced compensatory chaperone Grp94, and activated all three UPR pathways. Grp78 knockdown increased beta cell death (AnnexinV staining, Chop Caveolin-1 Deficiency Protects Pancreaticβ Cells against Palmi‑ tate-Induced Dysfunction and Apoptosis and Txnip) and caused loss of differentiation markers Pdx1, Nkx6.1, Mafa and WEN ZENG, KUNYING LIU, JIANSONG TANG, HAICHENG LI, HAIXIA XU, HON- Neurod1, and induction of Ngn3, a fetal beta cell marker. GYUN LU, HANGYA PENG, CHUWEN LIN, RILI GAO, SHUO LIN, KEYI LIN, YAN In summary, Grp78 is critically important for beta cell survival and plays an JIANG, LONGYI ZENG, Guangzhou, China unexpected role in maintaining beta cell differentiation markers. The regeneration of β-cell mass and preservation of its endocrine function Supported By: National Institutes of Health/National Institute of Diabetes and under lipotoxicity are long-sought goals in diabetes research. Membrane Digestive and Kidney Diseases protein caveolin-1 (Cav-1) is related to β-cell apoptosis and insulin secretion, however, the underlying mechanisms still remains unclear. 86‑OR Our objective is to explore whether Cav-1 depletion protects pancreatic Pancreatic Islets in Short Duration Type 2 Diabetes (T2D) Show β cells from lipotoxicity and investigate the related mechanisms. Here, we Macrophage Infiltration and Increased Inflammatory Signaling found that Cav-1 silencing significantly promotedβ -cell proliferation, inhib- JOHN T. WALKER, SHRISTI SHRESTHA, NRIPESH PRASAD, TIFFANY RICHARD- ited palmitate (PA)-induced apoptosis and enhanced insulin production and SON, RADHIKA ARAMANDLA, GREG POFFENBERGER, RITA BOTTINO, MARCELA secretion. These effects were associated with enhanced phosphorylation BRISSOVA, ALVIN C. POWERS, Nashville, TN, Huntsville, AL, Pittsburgh, PA of Akt and ERK1/2 protein, which then downregulated the expression of cell T2D is characterized by progressive pancreatic islet dysfunction; however, cycle inhibitors (FoxO1, GSK3β, P21, P27 and P53) and upregulated Cyclin dissecting causes of this dysfunction in humans is challenging due to disease D2 and Cyclin D3 expression. Subsequent inhibition of PI3K/Akt and ERK/ heterogeneity and the difficulty of obtaining relevant pancreatic samples. To MAPK pathways abolished Cav-1 depletion induced β-cell mass protection. address these challenges, we used an integrated approach to functionally Furthermore, under PA enhanced endoplasmic reticulum (ER) stress, Cav-1 and molecularly study the native pancreas and isolated islets from the same silencing significantly reduced eIF2α phosphorylation and expression of ER human T2D cadaveric donor. Using associated donor clinical information, we stress-responsive markers BiP and CHOP, which mediated the sensitization focused on donors with short disease duration (n=10, age 47-66 years, 2-7- to lipotoxicity. Our findings suggest the potential application of the Cav-1 year duration, oral medications). Compared to age-matched control islets, molecule as a target for effective T2DM treatment through preservation of T2D islets had dysfunctional insulin (decreased) and glucagon (increased) lipotoxicity-induced β-cell dysfunction and mass reduction. secretion despite similar insulin and glucagon content. Tissue-section analy- Figure 1. Caveolin-1 Expression in Mouse Pancreatic Islets and β Cells, as sis of T2D pancreata showed no difference in relative endocrine (α, β and well as Insulin Secretion after Islets Cav-1 Silencing. δ) cell composition. To assess for changes in the islet microenvironment, we analyzed macrophages by staining with Iba1 and endothelial cells with Cave- olin-1 and found a 2-fold increase in intraislet macrophages (T2D vs. control: 3.01% vs. 1.45% by area) but no change in intraislet endothelial cell area (T2D vs. control: 8.97% vs. 8.21% by area). Interestingly, costaining with Iba1 and thioflavin S demonstrated that T2D islets with amyloid did not have increased macrophage infiltration compared to T2D islets without amyloid ORALS although a close spatial association of macrophages and amyloid deposits was noted. RNA-sequencing analysis of FACS-purifiedα and β cells from T2D islets showed increased inflammatory signaling pathways in both cell types. Specifically, transcripts for cytokine receptors such as IL6R, IL1R1, and IFNGR1 as well as downstream regulators including NFKB1 and NFKB2 were elevated in T2D α and/or β cells. Together, these data suggest that early in the course of T2D, increased macrophages in the islet microenvironment may lead to elevated α and β cell inflammatory signaling and contribute to human α and β cell dysfunction. Supported By: National Institutes of Health; U.S. Department of Veterans Affairs; JDRF

87‑OR Supported By: National Natural Science Foundation of China Novel Small Molecule TXNIP Inhibitor Protects against Diabetes LANCE THIELEN, JUNQIN CHEN, GUANLAN XU, GU JING, TRUMAN GRAYSON, 85‑OR SEONGHO JO, ANATH SHALEV, Birmingham, AL Grp78 Is Critical for Beta-Cell Maturation and Survival Loss of functional beta cell mass represents a major factor in the patho- ROHIT B. SHARMA, XIAOYING ZHENG, BRIAN GABLASKI, JASON K. KIM, AMY genesis of diabetes. Currently, there are no therapies that halt this process; S. LEE, LAURA C. ALONSO, Worcester, MA, Los Angeles, CA however, thioredoxin-interacting protein (TXNIP) has recently emerged as Endoplasmic reticulum (ER) stress causes beta cell loss and dysfunction a promising therapeutic target. TXNIP was found to be the top glucose- in T1D and T2D. Glucose regulated protein 78 (Grp78) is an abundant, widely induced gene in a human pancreatic islet microarray, is increased in diabe- expressed ER chaperone with functions including peptide translocation and tes, and TXNIP overexpression results in beta cell apoptosis. TXNIP reduc-

ADA-Supported Research

A22 WHAT IS NEW IN DIABETIC KIDNEY DISEASE? tion has shown favorable effects in vivo, where whole body TXNIP-deficient 89‑OR and beta cell-specific TXNIP knockout mice have decreased beta cell apop- Evaluation of Glomerular Hemodynamic Function by Empagliflozin tosis, increased beta cell mass, elevated insulin levels, and are protected in Diabetic Mice Using In Vivo Imaging from diabetes. We have also shown that pharmacologic TXNIP inhibition, via KENGO KIDOKORO, YUJI SOGAWA, DAVID CHERNEY, VATHANY KULASINGAM, the anti-hypertensive medication verapamil, was able to mimic the protec- HAJIME NAGASU, MINORU SATOH, TAMAKI SASAKI, NAOKI KASHIHARA, Kura- tive effects of genetic TXNIP deletion and reversed overt diabetes in mice. shiki, Japan, Okayama, Japan, Toronto, ON, Canada In addition, it has been established that TXNIP downregulation is beneficial Improvement of glomerular hyperfiltration via tubuloglomerular feedback in multiple tissues, making it unnecessary and even undesirable to have a (TGF) is considered to be one of the possible pathways for renal protection beta cell-specific TXNIP inhibitor and suggesting that detrimental off target with SGLT2 inhibition (SGLT2i) in diabetic kidney disease (DKD). However, effects are unlikely. We have since undertaken a high-throughput screen the mechanisms that regulate renal function in response to SGLT2i have of 300,000 small molecules that has yielded a novel compound, TI-37330, not been fully elucidated. We explored the renal protective mechanisms of which is more effective than verapamil in inhibiting TXNIP expression. More- SGLT2i with empagliflozin (EMPA), with a focus on glomerular hemodynamic over, TI-37330 protected primary human islets and INS-1 beta cells against effects and TGF using in vivo multiphoton microscope (MPM) imaging tech- glucose- and cytokine-induced TXNIP and beta cell apoptosis. TI-37330 niques. C57BL/6 mice and spontaneously diabetic Ins2 Akita (AKITA) mice has also shown in vivo potential as it was found to have good pharmaco- were used. The mice were treated with EMPA (20mg/kg/day) and insulin for kinetic properties and was well tolerated in healthy mice. In the context of four weeks, and single-nephron GFR (SNGFR) was measured using MPM. An streptozotocin-induced diabetes, oral administration of TI-37330 promoted nNOS inhibitor (7-nitroindazole, 20mg/kg/day) or a COX2 inhibitor (SC58236, beta cell survival, prevented diabetes, and even significantly improved overt 6mg/L), or an adenosine A1 receptor (A1aR) antagonist (8-cyclopentyl-1,3-di- diabetes. Collectively, these data establish small molecule TXNIP inhibitors, propylxanthine, 1mg/kg/day) were administered to elucidate mechanisms especially TI-37330, as robust candidates for further development. of TGF signaling and SNGFR regulation. The urinary excretion of adenosine, Supported By: National Institutes of Health; JDRF nitric oxide metabolites (NOx) and the prostanoid PGE2 was also quantified. SNGFR in the AKITA group was higher compared to controls (C57BL/6; 4.9±1.3 nl/min vs. AKITA; 15.8±6.8 nl/min) and decreased in AKITA/EMPA to WHAT IS NEW IN DIABETIC KIDNEY DISEASE? 8.0±3.3 nl/min (p<0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (p<0.01) and increased glomerular permeability of albu- 88‑OR min in the AKITA group. EMPA ameliorated these changes (p<0.01). Urinary Proteomic Profile of Circulating Inflammatory Proteins Associated adenosine excretion in the AKITA/EMPA group was higher compared to with 10-Year Risk of ESRD in T1 and T2 Diabetes—Enrichment for AKITA (AKITA; 3.4±1.4 nmol/day, AKITA/EMPA; 11.2±3.0 nmol/day, p<0.05), TNF Receptor Superfamily Members while NOx and PGE2 did not differ. A1aR antagonism, but not nNOS or COX2 MONIKA NIEWCZAS, JAN K. SKUPIEN, VIJI NAIR, ADAM SMILES, JIHWAN inhibition, blocked the effect of EMPA on renal function. EMPA increased uri- PARK, EIICHIRO SATAKE, CHRISTOPHER A. SIMEONE, JOHN TSAY, WENJUN JU, nary adenosine excretion and reduced hyperfiltration via afferent arteriolar MATTHIAS KRETZLER, KATALIN SUSZTAK, ANDRZEJ KROLEWSKI, Boston, MA, constriction, effects that were abolished by A1aR blockade. The adenosine/ Kraków, Poland, Ann Arbor, MI, Philadelphia, PA, Princeton, NJ A1aR system plays a pivotal role in the regulation of SNGFR via TGF mecha- We conducted an untargeted proteomic profiling of circulating inflam- nisms in DKD. matory proteins using SOMASCAN platform to find those that predicted 10-year ESRD risk in two cohort study. Subjects with type 1 diabetes (T1D) 90‑OR served as a discovery cohort and a T2D cohort was used in validation. We Luseogliflozin Inhibits HIF-1α Expression in Renal Proximal Tubular identified a kidney risk inflammatory signature (KRIS) that robustly predicted Epithelial Cells progression to ESRD. The signature comprised 17 proteins out of 194 exam- RYOICHI BESSHO, YUMI TAKIYAMA, TSUGUHITO OTA, Asahikawa, Japan ined. KRIS was enriched for members of TNFR Superfamily (p=0.007) includ- Recent clinical trials have demonstrated the renoprotective effects of ing TNFR1, TNFR2, TNFRSF19, TNFRSF19L, TNFRSF21 and TNFRSF27. Other SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms KRIS proteins included IL15RA, IL17F, DAF, CLM6, TNFSF15, CCL14, CCL15, of SGLT2 inhibitors on prevention of DN have not been fully elucidated. CSF1, TIMD3, IL1R1 and IL18R1. Hazard ratio (95% CI) for the top KRIS pro- Hypoxia-induced tubulointerstitial fibrosis is considered to be a common tein, TNFR1 was 3.6 (2.8, 4.6), p < 10-25. KRIS was neither enriched in recep- pathway for various progressive kidney diseases including DN. Hypoxia tors for other cytokines nor in cytokine ligands. Pathway analyses pointed to inducible factor (HIF)-1α plays an important role in these pathological pro- candidate therapeutic targets and aligned them by ranks. Eight proteins are cesses. In this study, we assessed the effects of luseogliflozin, an SGLT2 currently targeted with compounds used for other clinical indications. Kid- inhibitor, on HIF-1α expression in both cultured human renal proximal tubular ney tissue expression analysis and urinary proteomics suggested a systemic epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for source of KRIS proteins. 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin In summary, our study identifies and validates a powerful protein sig- inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogli- nature enriched in TNFRSF members associated with 10-year ESRD risk in flozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and diabetes. These findings should inform future drug development strategies. GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex ORALS Figures A-B. I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Supported By: Japan Ministry of Education, Culture, Sports, Science and Tech- nology; Japan Society for the Promotion of Science (JP16H03035) Supported By: JDRF (5-CDA-2015-89-A-B to M.N.), (17-2013-311 to A.K.); National Institutes of Health (DK41526 to A.K.)

ADA-Supported Research

A23 NEW INSIGHTS INTO THE IMMUNOLOGY OF HUMAN TYPE 1 DIABETES

91‑OR 93‑OR Activation of PKM2 Protects against Diabetic Kidney Disease from Serum Galectin-3 and All-Cause Mortality in Type 2 Diabetes Mice to Men KATHRYN C.B. TAN, SAMMY SHIU, JOANNE K.Y. LAM, ALAN C. LEE, YING DANIEL GORDIN, TAKANORI SHINJO, RONALD ST-LOUIS, WEIER QI, LIANE J. WONG, Hong Kong, China TINSLEY, DAVID M. POBER, MEGAN J. BRISSETT, MONIKA NIEWCZAS, JONA- Background and Aims: Galectin-3, a member of the multifunctional galec- THAN DREYFUSS, HUI PAN, THORSTEN SADOWSKI, AIMO KANNT, HETAL tin family, is ubiquitously expressed and acts a broad-spectrum biological SHAH, HILLARY A. KEENAN, ANDRZEJ KROLEWSKI, GEORGE L. KING, Boston, response modifier. It is involved in tissue fibrosis, tumorigenesis, immunity MA, Frankfurt, Germany and inflammatory response. It can also act as a receptor for advanced glyca- We have reported that up-regulation of PKM2 in renal glomeruli isolated tion end products. Recent evidence suggests that galectin-3 is associated from patients with more than 50 years of type 1 diabetes (Joslin Medalist with heart failure and with progression of nephropathy in type 2 diabetes. Study) correlated with preservation of renal function even in the presence We have therefore evaluated whether serum galectin-3 level is associated of chronic hyperglycemia. Further, pharmacological activation of PKM2 in with all-cause mortality in patients with type 2 diabetes. T1D mice and podocyte-specific deletion of PKM2 in cultured cells and in Materials and Methods: Galectin-3 was measured in the baseline serum vivo strongly showed that PKM2 activation protects renal glomeruli from samples by ELISA in 1495 type 2 diabetic patients followed up in a teaching hyperglycemia/diabetes induced damage (Nat. Med 2017). To confirm these hospital specialist diabetes clinic. All-cause mortality was ascertained from findings in type 1 and type 2 diabetes, we performed the following: hospital electronic medical records. 1.) We showed the protein expressions of glycolytic (PKM1, PKM2, ENO1) Results: The mean age of the subjects was 55.3 ± 9.9 years and 54% and mitochondrial (MTCO2) enzymes to be significantly elevated in glomeruli were male. During a median follow-up of 11 years, 169 subjects died from of post mortem kidneys in people with T2D without DN compared to those all causes, and baseline serum galectin-3 levels were significantly higher with DN, or nondiabetic controls. in subjects with a fatal outcome (9.86 ± 3.11 ng/ml vs. 8.15 ± 2.73, p<0.01). 2.) Plasma PKM2 levels (SomaLogic aptamer assay) in the Medalists When galectin-3 was analyzed as quartiles, individuals in the third quartile (n=180) correlated with albumin-to-creatinine ratio (ACR;p=0.03) and eGFR [hazard ratio (HR) 2.0, 95% confidence interval (CI) 1.2-3.2, p<0.01] and the (p=0.0002) at baseline. In a replication cohort (n=219) of patients with T1D highest quartile (HR 3.9, 95% CI 2.6-6.0, p<0.01) had significantly elevated in the longitudinal Joslin Kidney Study (JKS) baseline plasma PKM2 also cor- risk of a fatal outcome compared to those in the lowest quartile. Multivari- related with ACR (p=0.0001). High baseline plasma PKM2 in JKS was inde- able Cox regression analysis showed a significant association between pendently associated with a slower decline in eGFR (p=0.004) and a slower serum galectin-3 level and all-cause mortality and the association remained progression to end-stage renal disease (p=0.0002). significant even after adjustment for potential confounders including age, 3.) TEPP-46, a PKM2 activator, prevented diabetes induced increases in sex, body mass index, duration of diabetes, smoking, baseline HbA1c, sys- mRNA expression of pro-fibrotic (Col3a1, Fn1, TGFb1) and inflammatory (Ccl2) tolic blood pressure, LDL and albuminuria status. genes, and enhanced key mitochondrial protein (Ppargc1a) in uninephrec- Conclusion: Elevated serum galectin-3 level was independently associ- tomized T2D db/db mice. No differences in ACR were observed between ated with all-cause mortality in patients with type 2 diabetes. Galectin-3 TEPP-46 treated and untreated diabetic mice. may potentially be useful as a predictor of adverse outcome. These findings support the hypothesis that PKM2 activation may protect or even reverse the initiation and progression of diabetic glomerular dysfunction and pathology in both T1D and T2D, suggesting that activating NEW INSIGHTS INTO THE IMMUNOLOGY OF PKM2 could be a clinically effective therapeutic target to stop the progres- HUMAN TYPE 1 DIABETES sion of DN. 94‑OR 92‑OR Metabolic Markers Associated with Increased Risk for Progression Short-Chain Fatty Acids Prevent Diabetic Nephropathy In Vivo and from Single to Multiple Autoantibodies in Type 1 Diabetes Relatives In Vitro EMANUELE BOSI, SUSAN GEYER, JAY SOSENKO, DOROTHY J. BECKER, MANU- WEI HUANG, YONG XU, YOUHUA XU, LUPING ZHOU, CHENLIN GAO, Luzhou, ELA BATTAGLIA, HEBA M. ISMAIL, MARK A. ATKINSON, LOUIS H. PHILIPSON, China, Macau, Macao JERRY P. PALMER, CARMELLA EVANS-MOLINA, Milan, Italy, Tampa, FL, Miami, FL, Diabetic nephropathy (DN) is the leading cause of end-stage renal disease Pittsburgh, PA, Gainesville, FL, Chicago, IL, Seattle, WA, Indianapolis, IN (ESRD). Over the past decade, the gut microbiota metabolites—short-chain Stage 1, defined as having >2 (i.e., multiple) autoantibodies (MA+) with fatty acids (SCFAs), have emerged as new therapeutic targets of systemic normoglycemia, is currently identified as the earliest pathogenetic phase inflammatory diseases and kidney injury such as acute kidney injury (AKI) of type 1 diabetes. There is limited information of a possible β-cell function via GPRs-β-arrestins signaling pathway and histone deacetylase-inhibiting decline during the preceding phase, defined as single autoantibody positivity (HDACi) properties. However, it is unclear whether SCFAs might prevent DN. (SA+). Therefore, we evaluated metabolic measures at time of SA+ determi- Therefore, we evaluated the role and mechanism of the three main SCFAs nation and assessed their predictive value for progression to MA+. Relatives (acetate, propionate, and butyrate) in streptozotocin (STZ) and high-fat diet participating in the TrialNet prospective Pathway to Prevention with SA+ (HFD)-induced type 2 diabetes (T2D) and DN mouse model in vivo and in high

ORALS (GADA, IAA, or IA-2A) were studied. Time from SA+ to MA+ was analyzed to glucose-induced mouse glomerular mesangial cells (GMCs) and human proxi- assess risk of conversion. Measures evaluated during OGTT included: mean mal renal tubular epithelia cells in vitro. Our results found that exogenous AUC for C-peptide, glucose, and insulin; fasting glucose and C-peptide; early SCFAs administration, especially butyrate, protected from experimental insulin and C-peptide responses (30-0 min); peak C-peptide; glucose and T2D, preventing the renal dysfunction and inhibiting oxidative stress and C-peptide sums (30 to 120 min) and Index60 (incorporating fasting C-peptide, NF-κB inflammatory signaling in vivo. The GPR43 expression decreased, 1-hour glucose, and 1-hour C-peptide). Clinical factors included age, sex, eth- however, β-arrestin-2 expression increased in both high glucose-stimulated nicity and autoantibody type. Of the 2,265 subjects identified as SA+, 57% renal intrinsic cells and in DN kidney tissue. We hypothesized that SCFAs- were female, 83% white, 14% Hispanic, with a median age of 14.8 years mediated GPR43-β-arrestin-2 signaling might modulate kidney injury. GPR43 (range: 1.0 to 48.7 years). Most (71%) had GADA, 25% IAA and 4% IA-2A; was over-expressed or suppressed by transfection with a GPR43 expressing 68% were tested also for ZnT8A. Overall, 407 (18%) SA+ progressed to MA+ vector or siRNA-GPR43 respectively. Exogenous SCFAs-mediated the anti- during a median follow-up of 1.8 years. Adjusting for age, sex, and ethnic- oxidative stress and anti-inflammatory effects were significantly facilitated ity we found that higher levels of mean AUC glucose (HR=2.4, p=0.004) or imitated by over-expression GPR43 or GPR43 agonist, but which were significantly increased risk of progression to MA+. Index60 was significant inhibited by siRNA-GPR43. Specifically, co-IP revealed that butyrate regu- both continuous (HR=1.17, p=0.004) and dichotomized (≥1 vs. <1; HR=2.28, lated the interaction between I-κBα and β-arrestin-2 via GPR43. Overall, p<0.0001). Reduced early C-peptide response was also significantly predic- this is the first observation of the potential therapeutic role of SCFAs in DN, tive (HR=0.94, p=0.007). Overall, we found that in early phase of SA+ there we demonstrated that SCFAs improved STZ and HFD-induced renal dysfunc- are already metabolic markers of beta cell dysfunction associated with tion and inhibited NF-κB activation through GPR43-β-arrestin-2 signaling, increased risk of progression to MA+. These findings provide insights into suggesting a likely mechanism for SCFAs-mediated therapeutic effect in DN. early phases of the disease and provide the rationale for interventions at Supported By: Collaborative Innovation Center for Prevention and Treatment of this stage. Cardiovascular Disease of Sichuan Province, China Supported By: National Institute of Diabetes and Digestive and Kidney Diseases

ADA-Supported Research

A24 NEW INSIGHTS INTO THE IMMUNOLOGY OF HUMAN TYPE 1 DIABETES

95‑OR 97‑OR Identification of Cross Reactive Insulin Immunogenic Epitopes from Decrease in Proportion of CD24hiCD38hi Beta Cells and Impairment Commensal Gut Microbes of Their Regulatory Capacity in Type 1 Diabetes Patients EMRAH ALTINDIS, ANTHONY N. VOMUND, I-TING CHOW, MARCOS DAMASIO, YUESHU WANG, XING WANG, YAO QIN, MEI ZHANG, Nanjing, China WILLIAM KWOK, EMIL R. UNANUE, C. RONALD KAHN, Boston, MA, St. Louis, Several studies have shown a subset of B cells play an important role in MO, Seattle, WA regulating the autoimmune response via the production of interleukin-10 Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by known as B10 cells. However, the role of B10 cells in type 1 diabetes (T1D) destruction of pancreatic β-cells. Because studies suggest an environmental and their relevance to the maintenance of peripheral tolerance in humans factor for the initiation of this autoimmunity, we hypothesized that intesti- remains elusive. The aim of this study was to investigate the role of B10 cells nal microbes might carry peptides with sequences similarity to insulin, a in the pathogenesis of T1D. Peripheral mononuclear blood cells of 62 patients major autoantigen. These peptides could modify the autoimmune response with T1D and 74 healthy volunteers were included. We showed that B10 cells through molecular mimicry. Using a bioinformatics approach, we identified in human peripheral blood belonged to a CD24hiCD38hi B cell subpopulation. seventeen B:9-23 epitope-like sequences in different microbial proteins Additionally, CD24hiCD38hi B cells from healthy individuals possessed regula- and investigated their capacity to stimulate insulin-specific reactive T cells tory capacity, inhibited IFN-γ, TNF-α and IL-17 production and promoted IL-4 obtained from T1D patients and NOD mice. In particular Parabacteroides dis- production and Foxp3 expression of CD4+ T cells via an IL-10-dependent path- tasonis contained a segment in the hypoxanthine phosphoribosyltransferase way. Compared with healthy controls, the proportion of B10 cells in patients (HPRT) protein with high sequence similarities to the B:9-23 segment of the with T1D was significantly lower (5.6±3.5% vs. 6.9±3.3%; P <0.05), produced insulin B chain. When synthesized, this peptide stimulated both human and less IL-10 (15.4±4.3% vs. 29.0±4.5%; P <0.001), and lacked the regulatory selected NOD mice T cells that recognized the B:9-23 peptide. Conversely, capacity. Pearson correlation analysis showed that the frequency of circulat- lymphocytes from mice immunized with this P. distasonis peptide showed ing B10 cells were negatively correlated with the frequency of CD4+IFN-γ+ increased secretion of cytokines when stimulated with the insulin B:9-23 and CD4+TNF-α+ T cells (r=-0.308 and r=-0.360, P=0.037 and P=0.021, respec- peptide. P. distasonis is a commensal microbe in the human gut microbiome. tively), and positively correlated with the frequency of CD4+ CD25+ Foxp3+ T Analysis of T1D metagenomic gut microbiome data from Finland, Estonia cells (r=0.018, P=0.001). There was also a negatively correlation between the and Russia showed that the abundance of P. distasonis significantly dif- frequency of circulating B10 cells and HbA1c (r=-0.339, P=0.010). These data fered between controls and seroconverting islet antibody-positive children provide direct evidence that there is a deficiency of circulating CD24hiCD38hi B at risk for T1D depending on the location of the study. In on-going analysis, cells in peripheral blood of patients with T1D, which might take part in the T1D compared to healthy control individuals, sera from T1D patients showed a immune imbalance, involved in the development of T1D. significant antibody response against multiple P. distasonis proteins. Supported By: National Natural Science Foundation of China (81670756, In summary, a peptide from HPRT of P. distasonis, a normal component of 81070622, 81370939); Jiangsu Higher Education Institutions (PAPD JX10231801) the gut microbiome, can elicit T-cell responses cross-reactive with human insulin-related peptides. We hypothesize that such cross-reactive peptides 98‑OR may become accessible to intestinal phagocytes for their presentation to T Characterization of T Cells Reactive to Hybrid Insulin Peptides in cells thus modulating T1D in genetically susceptible individuals. the Peripheral Blood of Type 1 Diabetes Patients Supported By: National Institutes of Health (R01DK031026, R01DK033201 to ROCKY L. BAKER, THOMAS DELONG, PETER GOTTLIEB, KATHRYN M. HASKINS, C.R.K.) Denver, CO, Aurora, CO We recently established, using the NOD mouse model, that hybrid insulin 96‑OR peptides (HIPs), formed spontaneously in islet beta-cells by fusion of insulin Costimulatory Molecules CD226 and TIGIT Impact CD8+ T-Cell C-peptide fragments to peptides of Chromogranin A (ChgA) or Islet Amyloid Activity and Function during Type 1 Diabetes Polypeptide (IAPP), are ligands for diabetogenic CD4 T cell clones. The goal WEN-I YEH, HOWARD SEAY, CLAYTON E. MATHEWS, TODD M. BRUSKO, of this study was to investigate whether HIP-reactive T cells were indicative Gainesville, FL of ongoing autoimmunity in human T1D patients. Investigations into the pathogenesis of type 1 diabetes (T1D) implicate To investigate their relevance to human disease, we used IFN-gamma antigen interaction and co-stimulation as key pathways in disease develop- ELISPOT to determine whether HIP-reactive T cells with an inflammatory ment. A non-synonymous polymorphism in CD226 (rs763361), that encodes phenotype were present in PBMCs of new onset T1D patients. We observed for a co-stimulatory molecule, has been linked to risk for T1D. The CD226 that PBMCs from new onset T1D patients responded to 8 of 16 HIPs tested activating pathway is constrained by TIGIT, the high-affinity co-inhibitory and that nearly half of the patients tested responded to one or more HIPs. receptor T cell immunoreceptor with Ig and ITIM domains, which competes We used a CFSE based assay to further characterize T cells reactive to with CD226 for ligands CD112/CD155. We have previously demonstrated HIP peptides. CD4+CFSEdimCD25+ T cells were sorted single into single that CD226 facilitates CD4+ T effector function, whereas TIGIT potenti- wells using FACS and cloned. T cell clonality was confirmed using TCR deep ates regulatory T (Treg) cell activity. Here, we report that both receptors sequencing and T cell reactivity to specific HIPs was confirmed by ELISA. A impact CD8+ T cell pathogenesis. Both CD226 and TIGIT are upregulated total of 6 T cell clones were isolated and show a variety of TCR usage. One T upon human CD8+ T cell activation and are divergently expressed by memory cell clone was isolated from the same patient on two different blood draws, CD8+ T cell subsets. These results support our finding in the positive correla- indicating a high frequency of this T cell clone in the peripheral blood. While ORALS tion between age and CD226 or TIGIT expression (P=0.0002 and P<0.0001, all the T cell clones isolated secreted IFN-gamma in response to HIPs, they respectively). Interestingly, TIGIT is co-expressed with the transcription fac- also produced other inflammatory cytokines such as TNF-alpha and GM-CSF tors T-bet and EOMES, whereas ~80% of cytokine producing cells express and reacted to HIPs in the low nanomolar range. CD226, demonstrating the coordinated expression of these factors with dif- Our findings provide new evidence that HIP-reactive T cells are critical ferentiated T cell lineages. We next sorted CD8+ T cells based on CD226 and players in the pathogenesis of T1D and indicate that HIP-reactive T cells TIGIT expression and assessed effector activity as well as gene expression present in the peripheral blood can potentially serve as a biomarker of dis- profile following in vitro activation. Using a multiplex assay, we showed that ease in a subset of patients. CD226+ cells produced cytokines, chemokines, and cytolytic proteins (e.g., Supported By: American Diabetes Association (1-15-JF-04 to R.L.B.); JDRF; IFNγ, MIP-1α, granzyme B, and perforin) in a manner independent of TIGIT. National Institutes of Health Importantly, CD8+ T cell cytotoxicity to human β-cell line βLox5 that express high levels of ligands CD112 and CD155 is attenuated by blocking CD226 99‑OR and enhanced by hindering TIGIT through a granzyme B dependent mecha- Supramolecular Biomaterials as a Biomimetic Platform for Inves‑ nism. These data support this co-stimulatory axis as a potential therapeutic tigating Immunopathological Processes of Human Type 1 Diabetes pathway in T1D that could either attenuate CD226 co-stimulation of effector MATTHEW BECKER, DILLON SEROSKI, SCOTT STIMPSON, CLAYTON E. T cells, or inversely support tolerance induction by TIGIT signaling on Tregs. MATHEWS, GREG HUDALLA, EDWARD A. PHELPS, Gainesville, FL Supported By: American Diabetes Association (1-16-PDF-131 to W-I.Y.) A major goal of type 1 diabetes (T1D) research is development of state-of- the-art devices that recreate the human islet microenvironment to help elucidate the molecular and cellular mechanisms of beta cell loss due to autoimmunity. We recently reported a novel synthetic biomaterial technology to form hydrogels from a pair of oppositely-charged peptides that co-assemble into a network of interpenetrating β-sheet nanofibers. Here, we explored the poten-

ADA-Supported Research

A25 HYPOGLYCEMIA—REDUCING THE RISK

tial of this biomaterial, referred to as “CATCH” for Co-Assembly Tags based on blood was stimulated with increasing amounts of IL-2 [0, 0.5, 1, 5, 10 U/ml] Charge complementarity, to serve as a 3D microenvironment for studies of the for 30 minutes. The expression of phosphorylated STAT5 (pSTAT5) in Tregs islet—immune cell interface within the context of human T1D. Supramolecular was quantified by flow cytometry. CATCH hydrogels offer a number of key advantages for engineering islet biomi- Results: Tregs from T1D patients showed significantly decreased levels metic microenvironments: 1.) they are fabricated via self-assembly at neutral of pSTAT5 when compared to healthy controls after stimulation with 0.5 U pH and 37°C; 2.) require no chemical reaction to form; and 3.) are intrinsically IL-2 (mean fluorescence intensity, MFI: 896 ± 502 vs. 3435 ± 1035, p<0.001) capable of installing folded proteins or peptides via covalent fusion to one of and after stimulation with 1 U IL-2/ml (MFI: 1095 ± 942 vs. 3835 ± 1415, the synthetic peptides sequences. In initial experiments, we established that p=0.004). Tregs from RA patients showed decreased levels after stimulation CATCH hydrogels are cyto-compatible with human beta cells via encapsulation with 0.5 U IL-2/ml (p=0.043) but not after stimulation with 1U IL-2/ml when of BetaLox5 cells (βL5), an immortalized human beta cell line, by demonstrat- compared to healthy controls. No significant differences between cells from ing strong viability (Calcein-AM/Ethidium homodimer-1 stain) of encapsulated CRC patients and controls were found. Stimulation with 5 or 10 U IL-2/ml led βL5 at 3 and 24 hours after gelation similar to βL5 seeded on 2D surfaces. to comparable levels of pSTAT5 in all investigated samples. CATCH gels also showed promise as a viable platform for interrogating the Conclusion: The response towards low doses of IL-2 is significantly engagement of autoreactive immune cells with human beta cells. Engineered decreased in Tregs from T1D and RA patients. This might contribute to the CD8+ human T cell avatars expressing human T cell receptors for the islet anti- loss of tolerance and increased autoimmunity in those diseases. However, gen pre-proinsulin co-embedded into CATCH hydrogels with βL5 cells show low dose IL2 signaling does not seem to be involved in the increased sup- strong beta cell killing in 3D by T cells over an 18-hour period (assessed via loss pressive capacity of Tregs in CRC patients. of TMRM staining of mitochondrial membrane potential). These initial suc- Supported By: Austrian Federal Government cesses are guiding our development of CATCH hydrogels for further detailed 3D studies of islet—immune cell interactions relevant to human T1D. Supported By: JDRF (1-FAC-2017-367-A-N); National Institutes of Health HYPOGLYCEMIA—REDUCING THE RISK (R01DK074656, UC4DK104194, P01AI042288) 102‑OR 100‑OR Low Doses of Dasiglucagon Consistently Increase Plasma Glucose Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells among (PG) Levels in Hypoglycemia and Euglycemia in People with Type 1 the Three Subtypes of Type 1 Diabetes Diabetes Mellitus (T1DM) DAISUKE CHUJO, AKITSU KAWABE, NOBUYUKI TAKAHASHI, MAYA MATSUSH- ULRIKE HOVELMANN, MINNA BRAENDHOLT OLSEN, ULRIK MOURITZEN, ITA, CHIHARU TSUTSUMI, FUMITAKA HASEDA, AKIHISA IMAGAWA, TOSHIAKI DANIELA LAMERS, BIRGIT KRONSHAGE, TIM HEISE, Neuss, Germany, Glostrup, HANAFUSA, KUNIMASA YAGI, MASAYUKI SHIMODA, KOHJIRO UEKI, HIROSHI Denmark KAJIO, Tokyo, Japan, Takatsuki, Japan, Sakai, Japan, Toyama, Japan To develop a dual hormone artificial pancreas (DHAP) a liquid stable glu- Type 1 diabetes (T1D) is classified into three subtypes in Japan based on cagon component is needed. Currently available glucagon products have the manner of disease progression: acute onset (AT1D), slowly progressive limited stability after reconstitution and are therefore not suited for DHAP (SP1D), and fulminant (FT1D). While cellular immunity is considered to play usage. Dasiglucagon is a novel liquid stable glucagon analog, which does not a major role in pancreatic β cell destruction in T1D, the phenotypes of auto- require mixing prior to use. In this randomized double-blind trial 17 patients reactive T cells in each subtype remain largely unknown. In this study, we with T1DM received four single subcutaneous doses (0.03, 0.08, 0.2 and performed an integrated assay that permits the determination of ex-vivo 0.6 mg) of dasiglucagon (4 mg/ml) under hypoglycemic (plasma glucose (PG) cellular immune responses and T cell repertoires specific for islet antigens. 56-66 mg/dL) or euglycemic (PG 100 mg/dL) conditions. For comparison, Briefly, peripheral blood mononuclear cells obtained from 20 AT1D, 17 SP1D, identical doses of Lilly Glucagon™ (GLUCA) were investigated at euglycemia. 18 FT1D patients, and 17 nondiabetics (ND) were stimulated for 48 hours In the euglycemic setting dasiglucagon showed rapid and dose-dependent with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters, after which PG-increases of mean 39, 56, 72 and 80 mg/dL at 30 minutes post-dosing cytokine/chemokine secretion was measured (ex-vivo assay). Subsequently, (30, 49 and 71 mg/dl for GLUCA) (Figure). During hypoglycemia, dasiglucagon peptide-reactive T cells were further expanded with IL-2 to identify CD4+ provided similar PG-increases of 24, 64, 87 and 94 mg/dL 30 minutes post T cell subsets based on intracellular cytokine staining (ICS assay). In the dose and rapidly corrected PG levels to normoglycemia (PG ≥70 mg/dL) with ex-vivo assay, GAD65-specific IL-6 and IP-10 responses, and PPI-specific a median time of 14, 10, 6 and 6 minutes. IP-10 responses were significantly upregulated in AT1D as compared to In conclusion, low doses of dasiglucagon rapidly and effectively increase ND. Furthermore, GAD65-specific IL-1β and G-CSF, and PPI-specific G-CSF PG from euglycemia and hypoglycemia in a titratable manner. Therefore, responses were significantly upregulated in FT1D when compared to ND. dasiglucagon should be a good candidate for use in dual-hormone closed- The ICS assay further revealed that GAD65- and PPI-specific CD4+ T cells loop AP systems. were skewed towards Th1 cells in AT1D (P = 0.007 and P = 0.004, respec- Figure. Different Doses of Dasiglucagon and GLUCA, PG Levels (Mean ±SEM). tively), whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D (P = 0.01 and P = 0.03, respectively) than in ND. Interestingly, GAD65- specific Th1 cells were more abundant in SP1D with HLA-DR9 than in those ORALS without DR9 (P = 0.03). Of note, FT1D displayed significantly less Tr1 cells specific for all four antigens and GAD65-specific Th2 cells than ND. In conclusion, the phenotypes of islet antigen-specific CD4+ T cells differ among the three T1D subtypes. These distinct phenotypes of pathogenic T cells may be associated with the manner of progression of β cell destruction. Supported By: Japan Society for the Promotion of Science

101‑OR Low-Dose IL-2 Signaling Is Specifically Impaired in Regulatory T Cells from T1D Patients BARBARA PRIETL, SELINA KOFLER, STEFANIE STANZER, BARBARA M. OBER- MAYER-PIETSCH, THOMAS R. PIEBER, HARALD SOURIJ, Graz, Austria Background: The response to interleukin-2 (IL-2) is essential for the stability and function of regulatory T cells (Tregs) in vivo. Impaired IL-2 receptor signaling might play a crucial role in the decreased function of Tregs in autoimmunity but might also contribute to the increased suppressive capacity of Tregs in cancer. In this study we aimed to examine differences in the phosphorylation of STAT5 as a downstream measure of IL-2 signaling in type 1 diabetes (T1D), rheumatoid arthritis (RA), colo-rectal cancer (CRC) and healthy controls. Material and Methods: Phosphorylation of STAT5 was quantified in 10 T1D, 10 RA, 10 CRC and 10 healthy control samples. Freshly drawn whole

ADA-Supported Research

A26 HYPOGLYCEMIA—REDUCING THE RISK

103‑OR 105‑OR Glycemic Management as the Primary Determinant of One-Year Prediction of Nonsevere Hypoglycemia—The Influence of Glycemic Mortality after CABG Variability in the HypoAna Study LUCAS HELLER, MARGARET ZUPA, PETER L. PERREIAH, R. HARSHA RAO, RIKKE M. AGESEN, PETER L. KRISTENSEN, SR., HENNING BECK-NIELSEN, SR., Pittsburgh, PA KIRSTEN NØRGAARD, TONNY JENSEN, SR., BIRGER THORSTEINSSON, SR., LISE Factors linked to mortality after coronary artery bypass graft (CABG) TARNOW, ULRIK PEDERSEN-BJERGAARD, Hillerød, Denmark, Odense, Denmark, include post-operative hypoglycemia and graft source. However, it is Copenhagen, Denmark unknown if there is an interaction between these two factors, namely Introduction: In the struggle for tight glycemic control many type 1 diabe- whether arterial grafts withstand hypoglycemia better than veins after tes patients daily experience large blood glucose fluctuations. It has been CABG. In a cohort of 1323 patients undergoing CABG with intensive insulin demonstrated that increased glucose variability is a predictor of hypoglyce- therapy (IIT) in the ICU, we identified graft sources, ICU glucometrics, diabetes mia, which is the main limiting factor for achieving the recommended HbA1c (DM) status and one-year mortality. Glucometrics included mean BG in ICU, target. The relationship between glucose variability and hypoglycemia might and hypoglycemic excursions (Incidence, Severity [nadir <40, 40-69mg/dl], seem intuitive, but the exact nature of the relationship is difficult to elu- Repetitive Frequency [number of excursions/patient]). The impact of differ- cidate. In this study we wanted to clarify the relationship between occur- ent factors, individually and in combination, was analyzed by Chi-squared rence of non-severe hypoglycemia and glycemic variability in type 1 diabetic Automatic Interaction Detection (CHAID). One year mortality was highest in patients at high risk of severe hypoglycemia. those with BG<40mg/dl in the ICU (12/26, 46%) vs. those without (67/1283, Material and Methods: 72 patients participated for 4 x 3 days of blinded 5%, p<0.001). No other factor was significant in those with BG<40. Of those CGM recordings during a 2-year randomized balanced cross-over study with without BG<40, repetitive (≥2) excursions with BG<70mg/dL had the highest human insulins (regular/NPH) and insulin analogs (aspart/detemir). CGM mortality (17/96, 17%, p<0.001), vs. those with one (13/190, 7%) or no excur- recordings were used to calculate the coefficient of variation (CV) in glucose. sions (37/997, 4%). Graft source was dominated by other factors in one year Results: Treatment with insulin analogs resulted in a significant lower mortality, except for the left internal thoracic artery which was protective nocturnal CV (mean 29.2%, SD 9.1) than treatment with human insulin only in patients who had no hypoglycemia. Hyperglycemia was a contributor (mean 36.9%, SD 9.1) (p<0.0001). CV was a significant risk marker of noc- to mortality in patients who had a single hypoglycemic excursion <70 (ICU turnal asymptomatic hypoglycemia ≤3.9 mmol/l in a multivariate analysis. Mean BG>152mg/dl 4/17, 24% vs. ≤152 9/173, 5%, p=0.039). Preexisting DM A 1% increase in CV corresponds to a 5% (p<0.0001) increase in asymptom- was protective in patients with repetitive hypoglycemia (DM 2/43, 5% vs. atic hypoglycemia. CV did not predict hypoglycemia during daytime at this 15/53 28%, p=0.003). Hypoglycemia in the ICU has a durable impact on one- threshold. year mortality post-CABG, superseding graft source, which is only signifi- Conclusion: Treatment with insulin analogs provides less glycemic vari- cant without hypoglycemia. Severe hypoglycemia poses the greatest risk, ability during night-time. The coefficient of variation in patients suffering but even mild repetitive BG<70 is a threat to one year survival. from recurrent severe hypoglycemia predicts the occurrence of especially In conclusion, hypoglycemia erases the survival benefit from arterial nocturnal hypoglycemia. grafts, suggesting that it may be equally damaging, regardless of graft Supported By: Novo Nordisk A/S source, and far more durable than hitherto envisioned. 106‑OR 104‑OR Prevalence of Impaired Awareness of Hypoglycemia and Severe Metoclopramide Restores the Sympathoadrenal Response to Hypo‑ Hypoglycemia among People with Type 2 Diabetes Treated with glycemia in a Novel Model of HAAF Insulin—The Dutch Diabetes Pearl Cohort ADRIANA VIEIRA DE ABREU, RAHUL AGRAWAL, PARKER HOWE, SIMON J. LIAN VAN MEIJEL, FEMMIE DE VEGT, CORNELIS TACK, EVERTINE J. ABBINK, FISHER, Salt Lake City, UT FEMKE RUTTERS, JACQUELINE M. DEKKER, BRUCE H. WOLFFENBUTTEL, FRITS We sought to develop a novel animal model of hypoglycemia-associated HOLLEMAN, J. HANS DEVRIES, NATHALIE MASUREL, HANNO PIJL, BEHIYE autonomic failure (HAAF) and investigate the pathological process involved. OZCAN, BIANCA SILVIUS, BASTIAAN E. DE GALAN, Nijmegen, Netherlands, We previously noted that recurrent 2-deoxyglucose (R2DG) administration Amsterdam, Netherlands, Groningen, Netherlands, Leiden, Netherlands, Rotterdam, induced hypoglycemia unawareness (indicated by a blunted food intake Netherlands, Utrecht, Netherlands response to hypoglycemia). We now evaluate whether R2DG also induces Aim: Most people with type 2 diabetes eventually require insulin treat- an impaired counterregulatory response and whether this pathological ment because of progressive loss of beta-cell function. The most common process is mediated by dopaminergic receptor activation. Sprague-Dawley adverse effect of insulin therapy is hypoglycemia. Recurrent hypoglycemia rats were randomized to one of three, 3-day treatments: 1.) recurrent saline may lead to impaired awareness of hypoglycemia (IAH) and a consequently (Control), 2.) R2DG (200 mg/kg/d, SQ), or 3.) R2DG and antecedent pretreat- high risk for severe hypoglycemia. The aim of this study was to determine ment with the dopaminergic antagonist metoclopramide (R2DG+Meto, the prevalence of IAH and severe hypoglycemia in a large cohort of people 3 mg/kg/d, IP). On the fourth day, all rats underwent hyperinsulinemic (50 with insulin-treated type 2 diabetes. mU/kg/min) hypoglycemic (~50 mg/dl) clamps. Notably, recurrent 2DG abro- Methods: The Dutch Diabetes Pearl is a contemporary cohort of people gated the epinephrine response to hypoglycemia as compared to controls. In with type 2 diabetes from primary, secondary and tertiary medical centers addition to restoring hypoglycemia awareness (i.e., the food intake response in the Netherlands. We collected data from people on insulin therapy who ORALS to hypoglycemia), metoclopramide normalized the epinephrine response to had completed the validated Dutch version of the Clarke questionnaire on hypoglycemia in 2DG treated rats. Glucagon showed a similar pattern of IAH, where 3 out of 5 points indicate IAH. Descriptive statistics, T-tests and suppression with R2DG and restoration with R2DG+Meto. In this model of Chi-square tests were performed. HAAF, our data identifies a novel role of dopaminergic receptor activation Results: Our study included 1923 patients of whom 59% were men. in the development of HAAF and a potential role for metoclopramide as a Median age was 62.1 years (interquartile range 55.1-68.3), median diabetes potential therapeutic agent to prevent HAAF. duration was 13.9 years (8.8-20.1) and median HbA1c was 7.6% (7.0-8.5). 208 Figure. individuals (10.8%) were classified as having IAH; these people were more likely to be non-Caucasian and have a lower educational level, and less likely to have a partner. Severe hypoglycemia was reported in 617 patients (32.1%) and severe hypoglycemia requiring medical intervention in the past year in 166 patients (8.6%). People with severe hypoglycemia were more likely to be non-Caucasian, to have a lower educational level, to have a history of cardiovascular events and neuropathic pain, and to use over 10 different types of drugs. No significant associations between HbA1c-levels and IAH or severe hypoglycemia were observed. Conclusion: IAH and severe hypoglycemia are common in people with type 2 diabetes treated with insulin. Non-Caucasian ethnicity, lower educational level, and being single may be risk factors for IAH in patients with type 2 diabetes.

ADA-Supported Research

A27 HYPOGLYCEMIA—REDUCING THE RISK

107‑OR ment and age group. Degludec was associated with a lower risk of severe Insulin Degludec Has Lower Hypoglycemia Risk than Insulin hypoglycemia vs. glargine U100, regardless of age. This analysis supported Glargine U100 in Older People with Type 2 Diabetes (T2D) the CV safety of degludec and demonstrated a lower risk of severe hypogly- SIMON R. HELLER, J. HANS DEVRIES, CAROL H. WYSHAM, CHARLOTTE T. HAN- cemia vs. glargine U100 in older patients with T2D. SEN, MELISSA V. HANSEN, BRIAN M. FRIER, Sheffield, United Kingdom, Amster- dam, Netherlands, Spokane, WA, Søborg, Denmark, Edinburgh, United Kingdom Vulnerability to hypoglycemia increases with age. To further assess the safety of insulin in older patients, the risk of hypoglycemia was investigated post-hoc in the SWITCH 2 treat-to-target, 64-week, crossover trial. Patients with T2D and high risk of hypoglycemia were randomized, double-blind, to either degludec or insulin glargine U100 ± OADs. The primary endpoint was the number of positively adjudicated severe (external assistance) or symptomatic hypoglycemic events (plasma glucose <56 mg/dL) during the two 16-week maintenance periods. For patients ≤65 (n=450) and >65 (n=270) years, baseline median [range] diabetes duration was 12.0 [1-40] vs. 15 [1-54] years, mean A1C was 7.7 vs. 7.4%, and mean eGFR was 87.0 vs. 63.7 mL/min/1.73m2. No significant differences in A1C reduction (degludec vs. glargine U100) were seen for patients ≤65 and >65 years. During the maintenance period, degludec had a lower risk of hypoglycemia (overall/nocturnal symptomatic) vs. glargine U100 in patients ≤65 (31/43%) and >65 years (30/41%) (Figure). The number of severe hypoglycemia episodes was not significantly lower. The adverse event rate was 3.2 and 3.3 events/patient-year for ≤65 years and 3.5 and 4.1 events/patient-year for >65 years, for degludec and glargine U100, respectively. Degludec was safe and effective, and the frequency of hypoglycemia was lower than glargine U100 in patients ≤65 and >65 years with T2D. Figure.

Supported By: Novo Nordisk Supported By: Novo Nordisk A/S

108‑OR 109‑OR Effect of Combination Therapy with Liraglutide plus Canagliflozin

ORALS Cardiovascular (CV) Safety and Severe Hypoglycemia Benefit of on HGP and HbA1c vs. Each Therapy Alone in T2DM Insulin Degludec vs. Insulin Glargine U100 in Older Patients ( 65 ≥ MUHAMMAD ABDUL-GHANI, ALI M. ALI, ROBERT A. MARTINEZ, JOHN M. Years) with Type 2 Diabetes (T2D)—Observations from DEVOTE ADAMS II, EUGENIO CERSOSIMO, CURTIS L. TRIPLITT, RALPH A. DEFRONZO, San RICHARD E. PRATLEY, SCOTT S. EMERSON, EDWARD FRANEK, MATTHEW P. Antonio, TX GILBERT, STEVEN P. MARSO, DARREN K. MCGUIRE, THOMAS R. PIEBER, NEIL Background: We previously have shown that SGLT2 inhibitors cause an R. POULTER, CHARLOTTE T. HANSEN, MELISSA V. HANSEN, THOMAS MARK, increase in HGP accompanied with an increase in plasma glucagon concen- ALAN C. MOSES, BERNARD ZINMAN, ON BEHALF OF THE DEVOTE STUDY tration. We hypothesized that the increase in plasma glucagon concentra- GROUP, Orlando, FL, Seattle, WA, Warsaw, Poland, South Burlington, VT, Kansas City, tion is, at least in part, responsible for the increase in HGP. The aim of the MO, Dallas, TX, Graz, Austria, London, United Kingdom, Søborg, Denmark, Plainsboro, present study was to examine whether inhibition of glucagon secretion by NJ, Toronto, ON, Canada liraglutide can prevent the increase in HGP. There is limited comparative evidence for the CV safety and hypoglyce- Research Design and Methods: 51 T2DM patients (age=51±1 year; mia risk of basal insulins in older patients with T2D. This secondary analysis 40% female; BMI=34.6±0.7; diabetes duration=6.8±0.8 year; FPG=175±7; from DEVOTE investigated the CV safety and severe hypoglycemia risk of HbA1c=8.3±0.1%) were randomized to receive for 16 weeks: (i) canagliflozin insulin degludec (degludec) vs. insulin glargine 100 units/mL (glargine U100) 300 mg; (ii) liraglutide 1.8 mg; or (iii) canagliflozin 300 mg plus liraglutide 1.8 in older patients (65-74 and 75 years age groups). Data from DEVOTE (a ≥ mg. HGP (measured with 3-3H-glucose infusion) and plasma glucagon con- treat-to-target, randomized, double-blind trial evaluating 7637 patients with centration were measured before and after 16 weeks of treatment. T2D over a median period of 2 years at high risk of CV events) were stratified Results: Canagliflozin monotherapy caused a significant reduction in by three age groups. At baseline, A1C, fasting plasma glucose and estimated HbA1c (-1.1±0.2%, p<0.01) accompanied with an increase in plasma glucagon glomerular filtration rates decreased with increasing age. Older patients had concentration (by 28%, p<0.05) and HGP (by 15%, p<0.05) which lasted for a higher risk of major adverse cardiovascular events (MACE), all-cause mor- 16 weeks. Conversely, liraglutide monotherapy caused a 1.6±0.5% (p<0.01) tality and severe hypoglycemia, regardless of treatment. Treatment differ- reduction in HbA1c accompanied by a small (6%) reduction in HGP (P=NS) ences for MACE, all-cause mortality and severe hypoglycemia were similar without significant change in fasting plasma glucagon concentration. The across age groups (Figure), with no significant interactions between treat-

ADA-Supported Research

A28 RESEARCH ADVANCES IN THE DIABETIC FOOT combination of canagliflozin plus liraglutide caused a greater reduction in (i.e., congestive heart failure and venous stasis) can significantly impact the HbA1c (1.9±0.5%, p<0.05 vs. canagliflozin and p=NS vs. liraglutide) and healing of skin grafts and should be taken into consideration when contem- attenuated the increase in fasting plasma glucagon and basal HGP at 16 plating split thickness skin grafting in a diabetic patient. weeks. Conclusion: These results: 1.) support a possible role for increased plasma 112‑OR glucagon levels in the long-term maintenance of increase in HGP caused Bedside Blind Bone Biopsy (B4) for Suspected Diabetic Foot Oste‑ by SGLT2i, and 2.) suggest that factors other than/in addition to glucagon itis—A Reliable Tool to Manage Medical Treatment? contribute to the initiation of the increase in HGP. FLORINE FÉRON, DIANE-CÉCILE GAUTHIER, JEAN-FRANÇOIS GAUTIER, JEAN- Supported By: Janssen Pharmaceuticals PHILIPPE KEVORKIAN, Paris, France Performed by a diabetologist, B4 has been recently shown as an easy and safe procedure. To assess Exclusive Medical Treatment (EMT: offload- RESEARCH ADVANCES IN THE DIABETIC FOOT ing, wound care ± antibiotherapy) determined by B4 in suspected Diabetic Foot Ulcer (DFU) osteitis, we compared DFU outcome between proved (B4+) 110‑OR and ruled out osteitis (B4-) with microbial culture. A prospective obser- Are We Misdiagnosing Diabetic Foot Osteomyelitis? vational study was conducted in our Diabetology Unit during a 16-month PETER A. CRISOLOGO, JAVIER LA FONTAINE, KATHRYN DAVIS, LAWRENCE A. period (12.17.2015-09.04.2017). Among 244 patients admitted with DFU, we LAVERY, Dallas, TX included 31 (13%) consecutive patients with type 2 diabetes mellitus (T2DM) Rationale and Aim: To compare the incidence of osteomyelitis based on and clinical and/or radiological suspicion of osteitis. For each, B4 was different operational definitions using the gold standard of bone biopsy. decided by our DFU multidisciplinary board. B4 was performed ≥ 2 weeks Methods: We prospectively enrolled 35 consecutive patients who met the after antibiotics discontinuation in case of prior exposure. A 6-weeks tar- criteria of ≥21 years of age, a moderately or severely infection based on the geted antibiotherapy was indicated for proven osteitis by prolonged culture Infectious Diseases Society of America. Bone samples were obtained from (B4+). In case of negative cultures (B4-), no antibiotherapy was prescribed. all participating patients either by percutaneous bone biopsy or intraop- The primary outcome was the complete DFU healing with EMT at 6 months. erative if the patient required surgery in the operating room. Bone samples Patients characteristics and results are summarized (Table 1). Around 50% were analyzed for conventional culture, histology examination, and16S rRNA of suspected osteitis was B4-, which prevented from unneeded antibiotics qPCR. We evaluated five definitions for osteomyelitis: 1.) traditional culture without poorer healing outcomes. B4 might be a reliable tool for managing 2.) traditional histology 3.) genetic sequencing 4.) traditional culture and his- EMT in suspected DFU osteitis. tology 5.) genetic sequencing and histology. Results: There was significant variability in the incidence of osteomyelitis based on the criteria for diagnosis. Traditional cultures identified significantly more cases of osteomyelitis than histology alone (68.6% vs. 45.7%, p=0.02). In every case that histology reported osteomyelitis; the bone culture was positive using traditional culture methods and genetic sequencing. 16S rRNA testing identified more cases of osteomyelitis compared to histology (82.9% vs. 45.7%, p=0.001) and compared to traditional cultures (82.9% vs. 68.6%, p=0.31). When both histology and traditional culture (68.6%) or histology and genetic sequencing based cultures (82.9%) were used to define osteomyelitis the incidence of osteomyelitis did not change. Conclusions: There is considerable variability in the incidence of osteomyelitis based on how the gold standard of bone biopsy is defined in diabetic foot infections. Supported By: National Institutes of Health (3U24DK076169-08S4)

111‑OR Outcomes of Split Thickness Skin Grafting on Lower Extremity Wounds in Diabetics ELLIOT WALTERS, GREG STIMAC, NEHA RAJPAL, IRAM NAZ, TAMMER ELMAR- 113‑OR SAFI, JOHN STEINBERG, KAREN EVANS, CHRISTOPHER ATTINGER, PAUL KIM, Risk of Foot Ulcer and Lower Extremity Amputation among Partici‑ Washington, DC pants in the Diabetes Control and Complications Trial/Epidemiology Background: Split thickness skin grafting is a widely used surgical tech- of Diabetes Interventions and Complications Study (DCCT/EDIC) nique for wound coverage. Despite the popularity of this procedure, out- EDWARD J. BOYKO, LEILA ZELNICK, BARBARA BRAFFETT, RODICA POP-BUSUI,

comes data has been poorly reported. CATHERINE C. COWIE, GAYLE M. LORENZI, ROSE GUBITOSI-KLUG, BERNARD ORALS Methods: Under IRB approval we reviewed the records of all patients ZINMAN, IAN DE BOER, Seattle, WA, Rockville, MD, Ann Arbor, MI, Bethesda, MD, receiving split thickness skin grafting for lower extremity wounds from 2014 La Jolla, CA, Cleveland, OH, Toronto, ON, Canada to 2016. We have defined success as wound healing of≥ 99.5%. We examined whether prior intensive glycemic control during the DCCT Results: 180 diabetic patients were included in this study. 103 (95%) had reduced the risk of diabetic foot ulcer (DFU) and lower extremity amputa- hypertension, 75 (69%) had hyperlipidemia, 29 (27%) had congestive heart tion (LEA) among participants with type 1 diabetes in the EDIC study. DFU failure, and 22 (20%) had venous stasis. Complete epithelialization was and LEA occurrence were determined by physical examination during annual obtained for 29 (22%) patients at 30d, 48 (36%) patients at 60d, 63 (48%) EDIC follow-up visits that began about one year following DCCT completion. patients at 90d, and 77 (58%) patients at 365d of follow-up. At last available We also assessed DFU and LEA risk by neurovascular measurements, diabe- follow-up 79 (60%) wounds obtained complete healing (≥99.5% coverage). tes characteristics, and clinical factors. Data were available from 23 years Venous stasis led to inadequate healing at 30d (p<0.0371), 90d (p<0.0006), of EDIC follow-up. Recurrent DFU was defined if reported on a different limb and 365d (p<0.0005) and trended toward significance at 60d (p<0.0809). in subsequent years or the same limb in non-consecutive years. Multivari- Congestive heart failure led to inadequate healing at 60d (p<0.0334), and able Cox models were used to estimate the association of intensive glycemic 90d (p<0.0015). The presence of bacteria on wound bed cultures did not control and other time-updated risk factors with incident or total (incident + show a significant effect on wound healing. The most common bacteria were recurrent) DFU or incident LEA. Incident DFU was observed in 86 of the 699 coagulase negative Staphylococcus, MRSA, and Enterococcus faecalis. Only former intensive and 109 of the 708 former conventional treatment partici- enterococcus faecalis showed significance at 30d if present following the pants (HR 0.78, 95% CI 0.59 to 1.03). Total DFU numbered 117 in intensive initial debridement. (7.3/1000 person-years (p-y)) and 153 in conventional treatment participants Conclusion: The data suggests that split thickness skin grafting can be (9.6/1000 p-y) for a significant risk reduction (HR 0.76, 95% CI 0.60 to 0.97, a successful coverage technique for diabetic patients with chronic lower p=0.03). Incident LEA occurred in 15 intensive (1.0/1000 p-y) compared to 21 extremity wounds. The bacterial milieu of chronic wounds in diabetics is conventional treatment participants (1.4/1000 p-y, HR 0.70, 95% CI 0.36 to diverse but with excisional debridement these bacteria have little impact 1.36, p=0.30). Greater HbA1c, lower eGFR, albuminuria, confirmed clinical on wound healing after split-thickness skin grafting. However, comorbidities neuropathy, lower nerve conduction velocity, abnormal cardiovascular auto-

ADA-Supported Research

A29 IT’S NOT TOO LATE, BABY—OUTCOMES IN GESTATIONAL DIABETES MELLITUS

nomic function, proliferative retinopathy, and macular edema were indepen- Figures A-D. dently associated (p<0.05) with higher risk of incident DFU and LEA. Current smoking was associated with higher risk of LEA only. Ankle-brachial index measured continuously was not associated with risk of either outcome. We conclude that intensive glycemic control decreases the risk of DFU, the most important antecedent in the causal pathway to LEA. Supported By: National Institutes of Health

114‑OR Accuracy of Daily Foot Temperature Monitoring for Patients with Recently Healed Diabetic Foot Ulcers or History of Amputation IAN L. GORDON, AKSONE NOUVONG, BIJAN NAJAFI, GARY M. ROTHENBERG, BRIAN PETERSEN, JONATHAN D. BLOOM, ROBERT FRYKBERG, Long Beach, CA, Sylmar, CA, Houston, TX, Ann Arbor, MI, Somerville, MA, Phoenix, AZ Diabetic foot ulcers (DFU) result in significant morbidity, mortality, and healthcare costs. Fortunately, recent data suggest as many as 75% of DFU and 60% of lower extremity amputations (LEA) may be preventable. Clinical practice guidelines recommend evidence-based foot temperature monitoring (thermometry) for preventing DFU and its complications in high- risk patients. Despite this, several authors have noted that thermometry is not commonly practiced. This is potentially due to lack of evidence on the accuracy of thermometry in some of the highest-risk cohorts, including IT’S NOT TOO LATE, BABY—OUTCOMES IN those with a recently-healed DFU or with history of LEA. The former may GESTATIONAL DIABETES MELLITUS present with ipsilateral inflammation as part of normal healing, while the latter may lack the anatomy required for direct contralateral comparison of 116‑OR temperatures. Whether these concerns affect the accuracy of thermometry Gestational Diabetes (GDM) and Childhood Disorders of Glucose needs to be assessed. A recent study suggests that a novel thermometric Metabolism—Hyperglycemia and Adverse Pregnancy Outcome mat may address some barriers to adoption. The mat was found to predict Follow-Up Study (HAPO FUS) as many as 97% of non-traumatic plantar DFU with an average lead time WENDY BRICKMAN, PATRICK CATALANO, PETER E. CLAYTON, CHAICHARN of five weeks in a cohort with history of DFU. In the current investigation, DEEROCHANAWONG, JILL HAMILTON, PAULA M. LASHLEY, JEAN M. LAW- we hypothesized no difference in the predictive accuracy of thermometry RENCE, YAEL LEBENTHAL, DAVID R. MCCANCE, WING H. TAM, ALAN KUANG, in two subgroups: those who healed from a DFU fewer than three months LYNN P. LOWE, BOYD E. METZGER, DENISE SCHOLTENS, WILLIAM LOWE, JR., before enrolling, and those with a history of partial LEA (Syme amputation or HAPO FOLLOW-UP STUDY COOPERATIVE RESEARCH GROUP, Chicago, IL, Cleve- more distal). Of the 129 subjects studied, 31% had recently-healed DFU and land, OH, Manchester, United Kingdom, Bangkok, Thailand, Toronto, ON, Canada, 56% had history of LEA. Although no difference in DFU incidence was found Saint George, Barbados, Pasadena, CA, Tel Aviv, Israel, Belfast, United Kingdom, in the LEA subgroup, we found significantly higher incidence in those with Hong Kong, China recently-healed DFU (0.88 vs. 0.63 DFU/subject/year). Importantly, we found While overt diabetes during pregnancy is associated with a higher child- no differences in accuracy or lead time in either cohort. These data suggest hood risk of altered glucose metabolism, the contribution of GDM to this thermometry is appropriate for monitoring both recently-healed DFU or par- disorder is less clear. Using data from HAPO FUS, we examined associa- tial LEA patients. Utilization of thermometry in these high-risk patients may tions of maternal GDM not confounded by treatment with childhood glucose significantly reduce morbidity, mortality, and resource utilization. metabolism. Maternal glycemic status was based on a 75 g oral glucose Supported By: Podimetrics, Inc. tolerance test (OGTT) at ~28 weeks gestation. Offspring disorders of glucose metabolism (impaired glucose tolerance or type 2 diabetes using American 115‑OR Diabetes Association criteria) were assessed at the HAPO FUS visit using Increased Rates of Readmission, Reoperation, and Mortality fol‑ an OGTT in 4106 children at mean age 11.4 (range 7.9-15.5) years. Insulin lowing Open Reduction and Internal Fixation of Ankle Fractures Are sensitivity (IS) was calculated using the Matsuda index and insulin secretion Associated with Diabetes Mellitus using the insulinogenic index. GDM was defined by International Association JENNIFER LIU, JUNHO AHN, KATHERINE M. RASPOVIC, DANE WUKICH III, of Diabetes in Pregnancy Study Groups criteria. Among offspring of moth- Dallas, TX ers with and without GDM, 10.6% and 5.2%, respectively, had a disorder The purpose of this large-scale retrospective study was to calculate the of glucose metabolism. After adjusting for field center, maternal pregnancy increased risk for postoperative unplanned readmission, unplanned reopera- variables and child age, Tanner stage and family history of diabetes at HAPO FUS, the odds ratio and 95% confidence interval (CI) for a disorder of glucose

ORALS tion, and mortality after ankle fracture ORIF in patients with diabetes melli- -5 tus (DM). Patients who underwent ORIF for ankle fractures from 2006 to 2015 metabolism was 1.98 (1.44-2.74), p=3.1 x 10 . Adjusting for maternal BMI were identified in ACS-NSQIP database. Patient demographics and 30-day at OGTT, child’s BMI z score, or both did not attenuate risk. Offspring of postoperative readmission, reoperation, and mortality rates were compared GDM mothers had lower IS (29.7 + 12.5, mean + SD) compared to offspring in 2,044 patients with DM and 15,420 patients without DM. Adjusted odds of non-GDM mothers (33.7 + 13.8) with a mean difference of -2.1 (95% CI -4 ratios controlling for age differences were calculated for each parameter -3.2 - -1.0), p= 1.9 x 10 after adjusting for field center, maternal pregnancy with a 95% confidence interval. Patient factors and pre-operative laboratory variables and child age, Tanner stage and family history of diabetes at HAPO statistics are summarized in Figure D, and the distribution of ankle fractures FUS. The association was attenuated but still significant after adjusting for for each group are shown in Figure A and B. Comparing patients with DM vs. maternal BMI and/or child BMI z score. Maternal GDM was not associated without DM, the rate of readmission was 4.35% vs. 1.50%, rate of reopera- with insulinogenic index. tion was 2.30% vs. 0.75%, and rate of mortality was 0.73% vs. 0.21%. As In summary, exposure to untreated GDM in utero is significantly and inde- shown in Figure C, patients with DM had a 2.66 times increased risk of read- pendently associated with childhood disorders of glucose metabolism, in mission, a 2.76 increased risk of reoperation, and a 2.34 increased risk of part through insulin resistance. mortality. Interestingly, we also found a 22.06 increased risk of amputation, Supported By: National Institutes of Health (1U01DK094830, R01HD34242, though the rate of amputation in both groups was very small. Ankle fractures R01HD34243) requiring ORIF in patients with DM are serious events and associated with substantial morbidity. 117‑OR Maternal Type 1 Diabetes and Risk of Autism in Offspring ANNY XIANG, XINHUI WANG, MAYRA P. MARTINEZ, KATHLEEN A. PAGE, THOMAS A. BUCHANAN, Pasadena, CA, Los Angeles, CA Children exposed in utero to maternal type 2 diabetes (T2D) or gestational diabetes (GDM) diagnosed by 26 weeks gestation are at increased risk of autism spectrum disorders (ASD). Here we assessed the association

ADA-Supported Research

A30 IT’S NOT TOO LATE, BABY—OUTCOMES IN GESTATIONAL DIABETES MELLITUS between ASD and in utero exposure to maternal type 1 diabetes (T1D). Data Methods: LIFE-Moms was a consortium of randomized clinical trials of were from 419,425 singleton children born at 28-44 weeks’ gestation from lifestyle interventions aimed at limiting gestational weight gain compared 1995-2012 at Kaiser Permanente Southern California (KPSC) hospitals. Chil- with standard care in 1,150 pregnant women with BMI ≥25 kg/m2 recruited dren were tracked from birth until the first of the following: date of clini- from different racial/ethnic groups in the U.S. women with known or prob- cal diagnosis of ASD, last date of continuous KPSC membership, death due able diabetes preceding this pregnancy were excluded. As previously to any cause, or December 31, 2016. Relative risks of ASD were estimated reported, the lifestyle interventions modestly reduced excessive gestational by hazard ratio (HR) using Cox regression models adjusted for birth year, weight gain (the primary outcome) but had no significant effect on the preva- maternal age, parity, education, household income, race/ethnicity, history lence of GDM at 24-31 weeks. We report here results of 75 g 2-hour OGTTs of comorbidity, and sex of the child. A total of 621 children were exposed performed sequentially during pregnancy in a subset of 296 women tested to T1D, 9,453 to T2D, 11,922 to GDM diagnosed by 26 weeks gestation, and at <16 weeks (mean 12.6 weeks) gestation and again at 24-31 weeks. Tests 24,505 to GDM diagnosed after 26 weeks gestation. During a median of 6.3 were interpreted by the International Association of Diabetes in Pregnancy (interquartile range of 3.4-11.2) years after birth, 5,624 children were diag- Study Groups (IADPSG) criteria. nosed with ASD (19 exposed to T1D, 221 to T2D, 250 to GDM by 26 weeks, Results: Plasma or serum glucose declined from the early to later tests 360 to GDM after 26 weeks, and 4,774 not exposed to maternal diabetes). by a mean of 2.5 mg/dl (fasting), 10.9 mg/dl (1-hr), and 9.3 mg/dl (2-hr), each Unadjusted annual ASD incidence rates per 1,000 children were 4.7 (T1D), p<0.0001. The prevalence of GDM by IADPSG criteria declined from 20% at 3.7 (T2D), 3.1 GDM by 26 weeks, 2.2 (GDM after 26 weeks) and 1.9 (unex- <16 weeks gestation to 15% at 24-31 weeks (p=0.04). Only 9% of women posed). Relative to no diabetes exposure, the adjusted HRs for exposure to without GDM at <16 weeks gestation developed it by 24-31 weeks, whereas maternal diabetes were 2.5 (95% CI, 1.4-4.4) for T1D, 1.5 (95% CI, 1.3-1.7) 65% of those meeting GDM criteria at <16 weeks gestation did not meet for T2D, 1.4 (95% CI, 1.2-1.6) for GDM by 26 weeks, and 1.0 (95% CI, 0.9- GDM criteria at follow-up. None of the changes in glucose or GDM preva- 1.1) for GDM after 26 weeks gestation respectively. Thus, exposures to T1D, lence differed significantly by treatment group. T2D and GDM diagnosed by 26 weeks gestation were all associated with an Conclusions: In pregnant women with BMI ≥25 kg/m2 who are otherwise increased risk of autism spectrum disorders in offspring. Exposure to T1D healthy, glucose tolerance improves on average rather than deteriorates imparted the highest risk, followed by exposure to T2D and GDM diagnosed during gestation. Testing for GDM early in pregnancy is needed to under- by 26 weeks. Rising rates of maternal diabetes may be one factor contribut- stand pregnancy effects on glycemia and the clinical implications of earlier ing to rising rates of ASD. detection of GDM. Supported By: National Institutes of Health 118‑OR Pregnancy Glycemia Reflects Life Course Glycemia of the Mother 120‑OR NILAM S. MEMANE, DATTATRAY BHAT, DEEPA A. RAUT, SOMNATH J. BOND- Placental DNA Methylation of miR-548 and WWTR1 Genes Influ‑ ARDE, RASIKA LADKAT, PALLAVI C. YAJNIK, CAROLINE FALL, CHITTARANJAN ence Insulin Sensitivity during Pregnancy S. YAJNIK, Pune, India, Southampton, United Kingdom MARIE-FRANCE HIVERT, CATHERINE M. BRIGGS, ANDRES CARDENAS, PATRICE Background: Current practice of diagnosis and treatment of GDM is largely PERRON, LUIGI BOUCHARD, Boston, MA, Brookline, MA, Sherbrooke, QC, Canada restricted to late pregnancy, ignoring peri-conceptional window which Insulin sensitivity decrease in pregnancy is a physiologic adaptation to is crucial for fetal programming of diabetes. A few longitudinal studies derive nutrients for the growing fetus. The placenta has a major role in this reported that metabolic risk factors of GDM are present before pregnancy phenomenon, but exact mechanisms remain unknown. A better understand- but there are no reports on a ‘lifecourse’ trajectory of pregnancy glucose ing of the placental role in maternal insulin sensitivity regulation could help insulin metabolism. Pune Maternal Nutrition Study (PMNS) offers a unique us reveal pathophysiologic pathways of gestational diabetes. We tested opportunity to investigate the association between gestational glycaemia associations between insulin sensitivity and DNA methylation (DNAm) and pre-pregnancy lifecourse glycaemia because of serial measurements across the genome in 439 placenta samples from our prospective cohort from childhood to pregnancy. Gen3G using Illumina EPIC arrays. Women were recruited at 1st trimester of Methods: We studied 328 female offspring of PMNS born in 1993-96 for pregnancy and performed a 2-hour 75g oral glucose tolerance test (OGTT) at glucose insulin measurements at 6, 12, and 18 year of age. Girls who became 24-28 weeks: we measured glucose and insulin levels at each OGTT point pregnant underwent a 75 gm OGTT at 28 weeks gestation. We studied the to calculate Matsuda insulin sensitivity index. We applied standard qual- association between gestational fasting plasma glucose (FPG) with their ity control procedures for DNAm arrays analyses and adjusted for techni- earlier measurements using multiple linear regression analysis. cal batch effects. We tested 720,077 individual CpGs using robust linear Results: Up to October 2017, 110 women had an OGTT at 28 weeks ges- regression on M-values (adjusted for maternal age, BMI, smoking, parity, tation and have delivered. Thirteen (11.8%) were diagnosed GDM (IADPSG gestational age at birth, and child sex) and used Bonferroni to account for criteria 2011) In lifecourse analysis, FPG tracked from 6 years of age to preg- multiple testing. We found that maternal insulin sensitivity was associ- nancy through 12 and 18 years. FPG at 28 weeks was directly associated ated with DNAm in placenta at 318 CpGs across the genome (P<6.94 x10-8). with her own FPG at 18, 12 and 6 years of age (β= ~0.3, p<0.001), and it Among these, we found differentially methylated CpGs at loci well known was inversely associated with disposition index at 18 and 12 years (p<0.05). for their role in diabetes (TCF7L2, SPRY2), brown adipose tissue regulation Conclusions: The world’s first description of lifecourse evolution of preg- (PRDM16), and near the Prolactin Receptor. Mendelian Randomization (MR) nancy glycaemia suggests that it tracks from early childhood and is associ- analyses supported that placental DNAm causally influence maternal insulin ORALS ated with impaired beta cell function. The analysis provides an important sensitivity at 28 CpGs (P<0.05). Among these, our two strongest MR asso- clue that GDM women have higher pre- and peri-conceptional glycaemia ciations (FDR adjusted P<0.05) pointed at CpGs near miR-548 (P=5.6x10-5) which is a risk factor for fetal programming of diabetes. Current clinical and WWTR1 (P=9.8x10-5), a gene implicated in PPARalpha regulation and approach to GDM ignores this important window. This could be a potential previously associated with preeclampsia. Our findings highlight the role of explanation of the failure of current practice of GDM treatment in late preg- placenta epigenetic regulation in insulin sensitivity during pregnancy and nancy to reduce long term risks in the offspring. reveal novel potential biologic targets implicated in gestational metabolic Supported By: Government of India Department of Biotechnology disorders. Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-15- 119‑OR ACE-26 to M-F.H.); Fonds de recherche du Québec en santé (20697 to M-F.H.); Changing Glucose Tolerance during Pregnancy—Implications for Canadian Institutes of Health Research (MOP115071 to M-F.H.), (PJT152989 to L.B.); Diagnosis of Gestational Diabetes Mellitus (GDM) Diabète Québec (to P.P., L.B.) WILLIAM C. KNOWLER, KIMBERLY DREWS, LEANNE REDMAN, KAUMUDI J. JOSHIPURA, S. SONIA ARTEAGA, XAVIER PI-SUNYER, LINDA VAN HORN, RENA 121‑OR R. WING, SAMUEL KLEIN, LIFE-MOMS RESEARCH GROUP, Phoenix, AZ, Rockville, Prevalence and Trends in Gestational Diabetes Mellitus among MD, Baton Rouge, LA, San Juan, PR, Bethesda, MD, New York, NY, Chicago, IL, Provi- Women in the United States, 2006-2016 dence, RI, St. Louis, MO TAO ZHOU, DIANJIANYI SUN, XIANG LI, YORIKO HEIANZA, HOIRUN NISA, GANG Background: ADA recommends testing for GDM at 24-28 weeks gestation. HU, XIAOFANG PEI, XIAOYUN SHANG, LU QI, New Orleans, LA, Baton Rouge, LA, This recommendation is based on the assumption that glucose tolerance Chengdu, China, Marrero, LA worsens during pregnancy and, therefore, testing earlier in pregnancy will Gestational diabetes mellitus (GDM) is a common medical complication in miss many cases that develop only later. We tested this assumption in the pregnancy, carries adverse health outcomes for both mothers and offspring. LIFE-Moms consortium. However, national data on the prevalence and secular trends of GDM during

ADA-Supported Research

A31 UPDATES FROM TYPE 2 DIABETES TRIALS

the past 10 years in the U.S. is lacking. This study included 26,340 ever- pregnant women aged ≥ 18 years from the National Health Interview Survey in 2006 and 2016. We examined GDM prevalence in 2006 and 2016. The prevalence of GDM increased from 4.6% in 2006 to 8.2% in 2016 (P<0.001). non-Hispanic white women showed less increase in the prevalence (2.8%) than non-Hispanic black women (3.8%), Hispanic women (4.1%), and women with other race/ethnicity (8.4%). The prevalence of GDM in non-Hispanic white women was higher than in non-Hispanic black (P=0.01) and women with other race/ethnicity (P=0.01) in 2006; but similar with in non-Hispanic black and lower than in women with other race/ethnicity (p=0.02) in 2016. The prevalence of GDM in non-Hispanic white and Hispanic women was similar in 2006 and 2016. In addition, the increase of GDM was more evident among women who were overweight, had low income, aged between 45-64 years, and had insufficient physical activity. In conclusion, the prevalence of GDM increased by 3.6% from 2006 to 2016; and the rise was more marked among non-white, overweight, low income, age 45-64 years, and insufficient activity groups. Figure.

Supported By: National Heart, Lung, and Blood Institute (HL071981, HL034594, Supported By: Novo Nordisk HL126024), (R01DK100790 to G.H.); National Institute of Diabetes and Digestive and Kidney Diseases (DK091718, DK100383, DK078616); Boston Obesity Nutrition Research Center (DK46200); United States-Israel Binational Science Foundation 123‑OR (2011036); American Heart Association (0730094N to L.Q.); National Institute of Durable HbA1c Reduction with Initial Combination Therapy with General Medical Sciences (U54GM104940 to G.H.) Metformin/Pioglitazone/Exenatide in Subjects with New-Onset Diabetes—Six-Year Follow-Up of the EDICT Study MUHAMMAD ABDUL-GHANI, CURTISS L. PUCKETT, JR., CURTIS L. TRIP- UPDATES FROM TYPE 2 DIABETES TRIALS LITT, DAVID MAGGS, JOHN M. ADAMS II, EUGENIO CERSOSIMO, RALPH A. DEFRONZO, San Antonio, TX, Lexington, MA Background: We previously have shown that initial combination therapy 122‑OR with metformin/pioglitazone/exenatide (Triple Therapy) produced greater Efficacy of Semaglutide vs. Dulaglutide across Baseline HbA1c in reduction in A1c than sequential add-on therapy in subjects with new onset SUSTAIN 7 of type 2 diabetes (T2D). RICHARD E. PRATLEY, JUAN P. FRIAS, HARISH KUMAR, JOHN PETRIE, ANDREA Research Design and Methods: The EDICT study is a randomized open- NAVARRIA, MORTEN ABILDLUND NIELSEN, WOLFGANG E. SCHMIDT, Orlando, label study in which newly diagnosed drug-naïve T2D subjects with new FL, Los Angeles, CA, Kochi, India, Glasgow, United Kingdom, Søborg, Denmark, Aal- onset diabetes were randomized to receive Triple Therapy (n=132) or escalat-

ORALS borg, Denmark, Bochum, Germany ing dose of metformin followed by sequential addition of glipizide and then Semaglutide, a new GLP-1 analog for T2D, showed significant and clini- glargine insulin (Conventional Therapy) (n=146) to maintain A1c< 6.5. Here, cally meaningful HbA1c and body weight (BW) reductions across the SUS- we report the 6-year follow-up results. TAIN clinical trial program. This post-hoc analysis of the phase 3b SUSTAIN Results: Subjects receiving Triple Therapy experienced a significantly 7 trial evaluated once-weekly subcutaneous semaglutide 0.5 mg vs. dulaglu- greater reduction in A1c after a mean follow-up of 6 years vs. Conventional tide 0.75 mg and semaglutide 1.0 mg vs. dulaglutide 1.5 mg by baseline (BL) Therapy (5.8% vs. 6.7%, p<0.001) (Figure 1). Further, more subjects main- HbA1c subgroups in subjects with T2D. tained HbA1c <6.5% in the Triple Therapy group (25%) than in the Conven- At week 40, improvements in HbA1c (Figure 1) and BW were similar or tional Therapy (52%). Progression of carotid IMT (read blindly) was reduced favored semaglutide vs. dulaglutide across subgroups (p-value for interac- by >50% (P<0.001) in Triple Therapy vs. Insulin Therapy. tion: HbA1c, p=0.02; BW, p>0.05); estimated treatment effects were similar Conclusion: The results of this study demonstrate that combination or favored semaglutide. More subjects with BL HbA1c >9% achieved HbA1c therapy with metformin/pioglitazone/exenatide in newly diagnosed T2DM targets with semaglutide vs. dulaglutide (Figure 2). patients produces greater and more reduction in A1c than sequential add-on Semaglutide was associated with similar or greater HbA1c and BW reduc- therapy with metformin, sulfonylurea, and basal insulin. tions vs. dulaglutide in all subjects regardless of BL HbA1c.

ADA-Supported Research

A32 UPDATES FROM TYPE 2 DIABETES TRIALS

Figure 1. 125‑OR Remission of Type 2 Diabetes in 12% (16 of 133) Hypogonadal Men Receiving Long-Term Testosterone Therapy in a Real-Life Registry Study AHMAD HAIDER, KARIM S. HAIDER, FARID SAAD, Bremerhaven, Germany, Berlin, Germany Background: According to 2016 AACE/ACE obesity guidelines, hypogonad- ism should be assessed and treated in indicated in men with type 2 diabetes mellitus (T2DM). Methods: In a registry of hypogonadal men (testosterone ≤12.1 nmol/L) conducted since 2004 in a urological setting, 133/400 patients (33.3%) had T2DM diagnosed and treated in a diabetes center. All men received testosterone undecanoate (TU) injections 1000 mg/12 weeks (T-group) following an initial 6-week interval. Anthropometric and metabolic parameters were measured at least twice a year and fasting insulin obtained from the diabetes center to calculate HOMA-IR. Results: Mean follow-up was 6.9, maximum 11 years. 16 patients (12%) Supported By: American Diabetes Association (1-15-CE-01 to M.A-G.); National were in remission at the last measurement. Their average baseline age was Institutes of Health 60.1 years. HbA1c dropped from 8.3% at baseline to 5.7% at the last measurement, fasting glucose from 7.8 mmol/L to 5.4 mmol/L. Fasting insulin 124‑OR declined from 24.7 to 7.6 µU/mL. HOMA-IR dropped from 8.7 to 1.8. Triglyc- Comparing Treatment Strategies for Patients with Very Elevated erides decreased from 3.2 to 2.2 mmol/L. Anthropometric parameters: Body HbA1c—A Randomized Trial weight declined progressively from 107 to 89 kg, waist circumference from MARCONI ABREU, ANNA TUMYAN, AHMED ELHASSAN, OLIVIA PAPACOSTEA, 108 to 97 cm. All patients had been on oral antidiabetic medication (metfor- KATHERINE PEICHER, PERIHAN DIMACHKIE, MUHAMMAD S. SIDDIQUI, BEV- min) at baseline. Five patients were on insulin at an average dose of 21.6 ERLEY ADAMS-HUET, XILONG LI, LAURENTIU POP, ILDIKO LINGVAY, Dallas, TX, units per day. The average observation time was 106.3 months (8.9 years). Redlands, CA, Oklahoma City, OK, McKinney, TX The average time to discontinuation of diabetes medication was 74.8 months Basal-bolus insulin (BBI) is the most established treatment strategy for (6.2 years). One patient was diagnosed with low-grade prostate cancer and patients with very elevated HbA1c (>10%), especially if symptomatic. The developed new onset T2DM while off TU. He went into remission 48 months combination of metformin (Met), GLP-1 receptor agonist (GLP-1RA) and basal after resuming TU therapy. Medication adherence to TU was 100 per cent insulin (BI) is a very effective glucose lowering strategy, but it has not been as all injections were administered in the urology office and documented. evaluated in patients with very elevated HbA1c. This is the first randomized Discussion: The registry was not designed to study the effects of testos- trial comparing a GLP-1RA+BI treatment regimen to a BBI treatment regimen terone on T2DM. Remission of T2DM has not been described in the literature in patients with very uncontrolled (HbA1c>10%) T2D. Basal insulin detemir and was unexpected. was either continued at the prior dose or initiated at 0.3 units/kg and self- Conclusions: Long-term testosterone treatment in hypogonadal men may titrated. GLP-1RA liraglutide was titrated according to label to 1.8 mg/day. result in remission of T2DM. Meal-time insulin aspart was initiated at 0.1 units/kg/meal and self-titrated. Supported By: Bayer AG metformin was continued or (re)initiated if tolerated, all other hypoglycemic agents were discontinued. The primary outcome was change in HbA1c at 126‑OR 6 months, analyzed using a mixed model repeated measures analysis. We Efficacy and Safety of an Expanded Dulaglutide Dose Range—A randomized 120 patients with a mean age (SD) 47.4 (9.5) years, Hispanic Phase 2, Placebo-Controlled Trial in T2D Patients on Metformin 48%, diabetes duration of 10.54 (7.19) years, 76% were already treated JUAN P. FRIAS, ALAN G. WYNNE, BEATA MATYJASZEK-MATUSZEK, DAGMAR with insulin, HbA1c 12.1 (1.4) %, BMI 37.2 (10.3) kg/m2, 86% had one or more BARTASKOVA, DAVID COX, BRAD WOODWARD, GRACE LI, ZVONKO MILICEVIC, symptoms of hyperglycemia. Los Angeles, CA, Topeka, KS, Lublin, Poland, Prague, Czech Republic, Indianapolis, Both treatment strategies lead to dramatic improvements in HbA1c, treat- IN, Vienna, Austria ment with GLP-1RA+BI, compared with BBI, resulted in significantly better Dulaglutide is approved at two doses (0.75 and 1.5 mg) for treatment of glycemic control, weight, insulin dosage, and hypoglycemia (Table). T2D. There has been limited assessment of higher doses. We hypothesized Table. Effects of Treatment with GLP-1RA + Basal Insulin (GLP-1RA+BI) that higher doses of dulaglutide may provide further improvement in glucose Compared with Basal-Bolus Insulin (BBI) on Primary and Secondary and body weight control. In this study, 3 and 4.5 mg doses were evaluated Outcomes. for safety/efficacy after 18 weeks (weeks) of treatment, including a 6 week Variable GLP-1RA+BI Group BBI Group Estimated P value dose escalation. Patients (N=318) on ≥1500 mg metformin, were randomized Treatment between (1:1:1:1) to placebo (n=82), dulaglutide 1.5 mg (n=81), dulaglutide 3 mg (n=79), Difference groups dulaglutide 4.5 mg (n=76). The primary objective was superiority of dulaglu- ORALS Baseline 6 months Baseline 6 months tide doses over placebo in HbA1c reduction at 18 weeks. Table 1 presents the HbA1c (%) 12.2 8.1 11.8 8.8 1.1 0.03 primary and selected secondary efficacy data. Reductions in HbA1c and body (11.8 to 12.6) (7.4 to 8.7) (11.5 to 12.2) (6.0 to 9.1) (0.1 to 2.0) weight were significant for each dose vs. placebo. Incidence of gastrointes- Weight (kg) 98.2 97.5 104.5 107.6 3.7 <0.01 tinal events (mostly mild to moderate) were dose-dependent for nausea (pla- (89.7 to 106.6) (89.1 to 106.0) (96.2 to 112.8) (99.3 to 115.9) (1.53 to 5.9) cebo, 4.9%; dulaglutide 1.5 mg, 22.2%; dulaglutide 3 mg, 24.1%; dulaglutide TDD Insulin (U/day) 25.0 64.0 60.0 100.0 0.3* 0.01 4.5 mg, 30.3%) but not for vomiting (placebo 4.9%; dulaglutide 1.5 mg, 11.1%; (median, 25th to 75th (22.0 to 36.0) (39.5 to 80.0) (43.0 to 72.0) (66.0 to 155.0) (0.1 to 0.5) dulaglutide 3 mg, 10.1%; dulaglutide 4.5 mg, 13.2%). No patients experi- percentile) enced severe hypoglycemia. The results of this trial show that 3 mg and HbA1c ≤7% NA 20 (44.4) NA 9 (20.5) Odds Ratio 0.32 0.02 4.5 mg doses, compared to the 1.5 mg dose, may provide additional glycemic N (%) (0.13 to 0.82) benefit and weight reduction with an acceptable safety profile in treatment of T2D patients, providing support for further phase 3 development. Patients with any NA 19 (35.2) NA 37 (66.1) NA <0.01 hypoglycemia (glucose <70 mg/dL) N (%) Data represents Least Squares Means (95% confidence intervals) unless otherwise noted. *ETD for the log transformed value.

ADA-Supported Research

A33 UPDATES FROM TYPE 2 DIABETES TRIALS

128‑OR Efficacy and Safety of Evolocumab in Patients with Type 2 Diabetes Mellitus and Hypercholesterolemia or Mixed Dyslipidemia ROBERT S. ROSENSON, MARTHA L. DAVIGLUS, PETER REAVEN, PAOLO POZZILLI, HAROLD BAYS, MARIA LAURA MONSALVO, MARY ELLIOTT, RANSI SOMA- RATNE, YEHUDA HANDELSMAN, New York, NY, Chicago, IL, Phoenix, AZ, Rome, Italy, Louisville, KY, Thousand Oaks, CA, Cambridge, United Kingdom, Tarzana, CA Aim: To examine the efficacy of 12 weeks of monthly evolocumab (EvoMab) 420 mg vs. placebo (Pbo) in lowering LDL-C in those with type 2 diabetes mellitus (T2DM) and hypercholesterolemia or mixed dyslipidemia and on statin therapy. Methods: Patients ≥18 years with T2DM, HbA1c <10%, on stable pharmacotherapy for diabetes for ≥6 months, and taking a statin were eligible; 421 Supported By: Eli Lilly and Company were randomized (2:1 EvoMab:Pbo) and dosed. Co-primary endpoints: mean % change in LDL-C from baseline to week 12 and mean % change in LDL-C 127‑OR from baseline to the mean of weeks 10 and 12. Additional measures included Superior Efficacy of Insulin Degludec/Liraglutide (IDegLira) vs. change in non-HDL-C, LDL-C goals, and measures of glycemic control. Insulin Glargine (IGlar U100) as Add-On to Sodium-Glucose Cotrans‑ Results: Mean participant age (SD) was 62 (8) years; 44% women; 77% porter-2 Inhibitor (SGLT2i) ± Oral Antidiabetic Drug (OAD) Therapy caucasian. In modified, intent to treat analyses (all randomized and dosed in Patients with Type 2 Diabetes (T2D)—DUAL IX Trial patients) EvoMab significantly reduced LDL-C and non-HDL-C vs. Pbo, and did ATHENA PHILIS-TSIMIKAS, LIANA BILLINGS, ROBERT S. BUSCH, CRISTOBAL not impact glycemic control. The incidence of AEs was comparable between MORALES PORTILLO, RAKESH SAHAY, NATALIE HALLADIN, RUTA GRONSKYTE EvoMab and Pbo, with no clinically meaningful differences in serious AEs. JUHL, STEWART HARRIS, San Diego, CA, Evanston, IL, Albany, NY, Gelves, Spain, (Table). Hyderabad, India, Søborg, Denmark, London, ON, Canada Conclusion: In statin-treated patients with T2DM and hypercholester- This study investigated the safety and efficacy of IDegLira as add-on to olemia or mixed dyslipidemia, evolocumab safely and effectively lowered SGLT2i. In this 26-week, phase 3b, open-label trial, 420 patients with T2D LDL-C. uncontrolled on SGLT2i ± OADs were randomized 1:1 to receive add-on ther- Table. apy of IDegLira or IGlar U100 (100 units [U]/mL). Starting doses were 10 U in both treatment arms. Doses were titrated twice-weekly to a fasting glucose Placebo Evolocumab target of 72 to 90 mg/dL; only IDegLira had a maximum dose (50 U). (N=141) (N=280) Mean A1C decreased from 8.2% at baseline to 6.3% at week 26 for IDe- Baseline LDL-C (mg/dL), mean (SD) 110.4 (33.0) 108.6 (31.0) gLira and from 8.4 to 6.7% for IGlar U100; IDegLira superiority confirmed Baseline non-HDL-C (mg/dL), mean (SD) 145.5 (33.9) 144.6 (34.9) (p<0.0001). IDegLira treatment resulted in unchanged mean body weight Any adverse event (AE), n (%) 52 (36.9) 110 (39.3) vs. 2.0 kg weight gain with IGlar U100 (p<0.0001). The rate of treatment Serious AE, n (%) 2 (1.4) 14 (5.0) 14 emergent severe or blood glucose confirmed symptomatic hypoglycemic a episodes was 58% lower (p=0.0035) with IDegLira (0.37 events/patient-year (5.0)14 (5.0) of exposure [PYE]) vs. IGlar U100 (0.90 events/PYE). Total daily insulin dose Mean of Weeks 10 and 12 after 26 weeks was 36 U for IDegLira vs. 54 U for IGlar U100 (p<0.0001). Lipids Percent change from -0.8 (1.8) -65.0 (1.3)b Adverse event rates were low in both treatment arms with no unexpected baseline in LDL-C, safety issues. mean (SE) In conclusion, superiority of IDegLira vs. IGlar U100 as add-on to SGLT2i Percent change from -0.05 (1.6) -56.6 (1.2)b was confirmed for glycemic control, body weight, hypoglycemia rate and baseline in non-HDL-C, total daily insulin dose. mean (SE) Table. Achievement of LDL-C 20 (14.8) 253 (92.7)b < 70 mg/dL, n, % Achievement of ≥50% 1 (0.7) 230 (84.2)b reduction in LDL-C, n, % Week 12 Glycemic Control Change from baseline in 4.0 5.0 FSG in mg/dL, median (-15.0, 29.0) (-13.5, 29.5) (Q1, Q3)

ORALS Change from baseline 0.1 0.1 in HbA1c in percent, (-0.2, 0.5) (-0.2, 0.5) median (Q1, Q3) aNo pattern in differences in serious AEs was observed. Chronic obstructive pulmonary disease was the only serious AE reported by ≥ 1% of patients in the evolocumab treatment group. bP < 0.0001 for evolocumab vs. placebo comparison. FSG, fasting serum glucose; HbA1c, glycated hemoglobin; LDL-C, low-density lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; SE, standard error. Supported By: Amgen Inc.

129‑OR More Subjects Achieved Composite Reductions of ≥1% HbA1c, ≥5% Body Weight, and ≥5 mmHg SBP with Semaglutide vs. Comparators (SUSTAIN 1-5, 7) KATHLEEN M. DUNGAN, VANITA ARODA, FILIP K. KNOP, LAWRENCE A. LEITER, NANNA L. LAUSVIG, SØREN LINDBERG, JURIS J. MEIER, Columbus, OH, Hyatts- ville, MD, Hellerup, Denmark, Toronto, ON, Canada, Søborg, Denmark, Bochum, Germany Semaglutide is a new GLP-1 analog for the once-weekly treatment of Supported By: Novo Nordisk A/S type 2 diabetes (T2D). Across the SUSTAIN clinical trial program, subjects with T2D achieved significantly greater reductions in three cardiovascular (CV) risk factors with semaglutide vs. placebo or comparators (dulaglutide,

ADA-Supported Research

A34 THE IMPACT OF DIABETES INTERVENTIONS AND BEHAVIORS ON OUTCOMES exenatide extended release, insulin glargine, or sitagliptin): HbA1c, body 131‑OR weight and systolic blood pressure (SBP). Six SUSTAIN trials (SUSTAIN The Pros and Cons of Gastric Bypass Surgery in Obese Individuals 1-5 and 7) were assessed post-hoc to determine to what extent subjects with Type 2 Diabetes—Nationwide, Matched, Observational Cohort achieved clinically meaningful reductions in all of these risk factors (compos- Study ite endpoint: ≥1% decrease in HbA1c, ≥5% body weight loss, and ≥5 mmHg VASILEIOS LIAKOPOULOS, ANN-MARIE SVENSSON, INGMAR NASLUND, BJORN SBP reduction). Across trials, mean baseline HbA1c, body weight and SBP ELIASSON, Gothenburg, Sweden, Orebro, Sweden ranges were 8.1-8.4%, 89.5-95.8 kg and 128.8-134.8 mmHg, respectively. Introduction: Long-term effects of gastric bypass (GBP) surgery have been Significantly more subjects achieved the composite endpoint with sema- presented in observational and randomized studies, but there is still only glutide (0.5 mg: 14-20%; 1.0 mg: 15-37%) vs. comparators (1-12%; p<0.001 limited data for obese persons with type 2 diabetes (T2DM), in particular for all comparisons; Figure). With semaglutide, significantly more subjects regarding postoperative complications. We investigated postoperative out- achieved clinically meaningful improvements in the composite of HbA1c, comes after GBP in a nation-wide cohort. body weight and SBP reductions vs. comparators, which may promote a bet- Research Design and Methods: In this observational study, we merged ter overall CV risk profile with semaglutide vs. comparators. data from the Scandinavian Obesity Surgery Registry, the National Diabetes Register and national databases. We matched persons with type 2 diabetes who had undergone GBP with persons not surgically treated for obesity, based on sex, age, BMI and propensity score. The risks of postoperative outcomes were assessed using Cox regression model adjusted for sex, age, BMI and socioeconomic status. Results: 5321 T2DM patients who had undergone GBP and 5321 control persons were followed for up to 9 years. We confirm lower risks of all-cause mortality (49%) and cardiovascular disease (34%), found positive effects on severe kidney disease, but also demonstrate significantly increased risks (2 to 9-fold) of several short-term complications after GBP. There were higher rates of abdominal pain and gastrointestinal conditions frequently requiring additional surgical procedures, apart from reconstructive plastic surgery. Long-term, the risk of anemia was 92% higher, malnutrition appeared approximately 3-fold more often, while psychiatric diagnoses were 33% increased, and alcohol abuse was 3-fold higher than in the control group. Supported By: Novo Nordisk Conclusions: This nation-wide study confirms the benefits but also describes the panorama of adverse events after bariatric surgery in obese persons with T2DM. In order to maximize the benefit and minimize the risk THE IMPACT OF DIABETES INTERVENTIONS AND of unfavorable results after bariatric surgery, a thorough and long-term fol- BEHAVIORS ON OUTCOMES low-up and support of these patients seems paramount. Better selection of patients for such treatment could probably also improve results. 130‑OR Lifestyle Interventions Lower Cardiovascular Disease in Chinese 132‑OR Adults with IGT—Thirty-Year Follow-Up of Da Qing Diabetes Pre‑ Cost and Cost-Effectiveness of a Faith-Based Lifestyle Intervention vention Study for Diabetes Prevention among African-Americans—A Within-Trial QIUHONG GONG, PING ZHANG, JIXIANG MA, JINPING WANG, YALI AN, YIL- Analysis ING J. CHENG, BO ZHANG, HUI LI, YANYAN CHEN, YINGHUA HU, EDWARD W. ELIZABETH C. RHODES, EESHWAR CHANDRASEKAR, SHIVANI PATEL, K.M. VEN- GREGG, PETER H. BENNETT, GUANGWEI LI, Beijing, China, Atlanta, GA, Daqing, KAT NARAYAN, THOMAS V. JOSHUA, LUCY MARION, LOVORIA B. WILLIAMS, China, Phoenix, AZ MOHAMMED K. ALI, Atlanta, GA, Augusta, GA Lifestyle interventions can prevent or delay type 2 diabetes (T2DM) but Background: Estimates of the economic value of investing in real-life dia- their long-term effect on cardiovascular disease (CVD) remains unclear. betes prevention lifestyle interventions are scarce. We assessed the cost We assessed the effect of lifestyle interventions on the incidence of CVD of implementation and cost-effectiveness of a church-based lifestyle inter- events among Chinese adults with impaired glucose tolerance (IGT) who vention (Fit Body and Soul [FBAS]), in which African American participants participated in the Da Qing Diabetes Prevention Study (DQDPS) and who received 18 lifestyle education sessions. were followed for up to 30 years after randomization. DQDPS was a 6-year Methods: An incremental cost-effectiveness ratio (ICER) was calculated clinical trial conducted in Da Qing, China from 1986 to 1992 in which 576 using data from a cluster randomized controlled trial comparing FBAS with adults with IGT were randomized by clinic to a control group (138) or one a health education (HE) control group among 604 overweight participants of three lifestyle intervention groups (438) (diet or exercise or both). After in 20 churches. We calculated the ICER as the difference in costs to deliver the 6-year active intervention, participants were followed to assess their FBAS vs. HE over the difference in weight change at one-year follow-up. ORALS first CVD events until December 31st 2016. CVD was defined as fatal or non- Costs included those incurred for participant identification (screening at fatal CVD, which included myocardial infarction, congestive heart failure, baseline) and program implementation (payment for health advisors to lead stroke, and amputation. The intervention effect on CVD was assessed using sessions, materials, equipment, and incentives to churches and partici- Cox proportional hazards analysis. 542 (94.1%) of the original participants pants). To estimate adjusted difference in weight (kg) change between FBAS had data for CVD events. 192 in the combined intervention group and 79 in and HE, we fit mixed linear models, accounting for clustering of participants the control group had a first CVD event during the follow-up. The 30-year within churches and for age, sex, and educational attainment. We repeated cumulative incidence of CVD was 51.8% (95% CI 47.1-56.5) in the interven- this analysis for waist circumference (cm). tion group and 65.7% (95% CI 57.8-73.6) in the control group. CVD incidence Results: The per-person intervention cost of FBAS was $50.39 more per 1000 person-years was 21.8 (95% CI 18.8-25.1) in the intervention group than HE (per-person FBAS cost: $442.22; per-person HE cost: $391.83). The compared to 29.0 (95% CI 23.0-36.2) in the control. After accounting for adjusted difference in weight change between the FBAS group and the HE clinic cluster, the intervention reduced the risk of the first CVD event by 27% group was 1.9 kg (95% CI 1.0, 2.8), and the adjusted difference in waist cir- (HR 0.73, 95% CI 0.55-0.96; p=0.024). Our study is the first to show that a cumference change was 2.4 cm (95% CI, 1.3 to 3.4). Compared to HE, imple- lifestyle intervention program in persons with IGT significantly reduces the mentation of FBAS cost an additional $26.52 per kg weight lost and $21.00 incidence of CVD. Our results provide further justification to adopt lifestyle per cm reduction in waist circumference. interventions as public health measures to prevent T2DM. Conclusion: For a modest increase in cost, FBAS led to greater weight loss and reduction in waist circumference among African Americans compared to standard HE at a church setting. Supported By: National Heart, Lung, and Blood Institute; National Institutes of Health

ADA-Supported Research

A35 THE IMPACT OF DIABETES INTERVENTIONS AND BEHAVIORS ON OUTCOMES

133‑OR Table. Long-Acting Insulin Analogues and Risk of Breast Cancer— 12 years post- 14 years post- Odds of frailty vs. Meta-analysis randomization randomization non-frailty pooled over MARIA BOTA, PHILIPPE AUTIER, PETER BOYLE, Écully, France, Lyon, France both exams Observational studies on the association between prescription of long Treatment Prevalence Prevalence OR (95% CI) P-value acting insulin analogues (glargine and detemir) and the risk of breast cancer Group compared (BC) report conflicting results. We performed a meta-analysis of prospec- with placebo tive studies on the risk of breast cancer among diabetic patients exposed to Pre-Frailty Frailty Pre-Frailty Frailty long acting insulin analogues following PRISMA guidelines. Summary rela- Placebo 42.8% 5.7% 43.7% 5.4% 1 (referent) n/a tive risks (SRR) were computed using a random-effect model. Thirteen studies were identified which reported data on insulin glargine which included Metformin 45.8% 5.4% 42.5% 5.3% 0.994 0.976 438,214 subjects and 4,041 BC cases over a median follow-up of 2.8 years. In (0.694-1.424) 11 studies, including 94.8% of BC, the comparator groups included diabetic Lifestyle 46.8% 3.0% 46.3% 3.6% 0.625 0.022 patients treated with non-glargine insulin. The SRR was 1.11 (95% CI: 0.98, (0.419-0.934) 1.24), with little heterogeneity (I²=27%). Among prevalent users of insulin, based on 7 studies, the SRR of BC (1,425 cases) associated with prescrip- Supported By: National Institute of Diabetes and Digestive and Kidney Diseases tion of glargine was 1.05 (95% CI: 0.90, 1.23), with moderate heterogeneity (U01DK048489) among studies (I²=40%). One influential study (Wu et al. 2017) accounted for most of the heterogeneity. When it was removed from the meta-analysis, 135‑OR the I2 dropped to 0% with a SRR of 0.99 (95% CI: 0.88, 1.12). Among new Evaluation of Treatment Persistence in Individuals with Type 2 Dia‑ users of insulin, based on 10 studies, the SRR of BC (3,847 cases) associated betes in a Real-World Setting with prescription of glargine was 1.12 (95% CI: 0.96, 1.29), with moderate WILLIAM T. CEFALU, TAMARA DARSOW, MATT PETERSEN, LIISA PALMER, heterogeneity among studies (I²=41%). One influential study (Ruiter et al. ELLEN THIEL, LISA LATTS, Arlington, VA, Baltimore, MD, Ann Arbor, MI, Denver, CO 2012) accounted for most of the heterogeneity. When it was removed from Medication persistence is imperative for successful treatment of type 2 the meta-analysis, the I2 dropped to 0% with a SRR of 1.07 (95% CI: 0.96, diabetes (T2D). Prior research has shown that discontinuation of prescribed 1.21). Only three studies reported on detemir, with a SRR for BC (1,947 cases) medication within the first year is common for number of chronic disease of 1.09 (95% CI: 0.77, 1.57). There seems to be consistent evidence showing treatments and that this is associated with poor clinical outcomes. no association between the prescription of long acting insulins and risk of We performed a retrospective, observational study using claims data BC. However, most of these studies had a short duration of follow-up. In from the Truven Health MarketScan® Commercial and Medicare Supplemen- addition, only three studies were identified reporting the risk of BC associ- tal Databases (2013-2016) to identify T2D patients initiating T2D therapy. ated with use of detemir. Patients with at least one diagnosis for T2D and one outpatient pharmacy claim for a T2D medication preceded by 12 months without such medications 134‑OR were included. Patients were required to have at least 12 months of continu- Association of Intensive Lifestyle Intervention and Metformin with ous enrollment in the database before and after the therapy initiation date. Frailty in the Diabetes Prevention Program (DPP) Outcomes Study The primary outcome was initial treatment regimen discontinuation, defined (DPPOS) as a gap of at least 45 days with no T2D medications on hand. Re-starting HELEN P. HAZUDA, QING PAN, JILL P. CRANDALL, HERMES FLOREZ, JOSE A. therapy with any T2D medication from the discontinuation date through the LUCHSINGER, ELIZABETH M. VENDITTI, ANDREA KRISKA, SHERITA GOLDEN, end of the 12-month follow-up was also assessed. Those with continuous GEORGE BRAY, DPP RESEARCH GROUP, San Antonio, TX, Washington, DC, Bronx, treatment were censored at the end of the 12-month follow-up period. NY, Miami, FL, New York, NY, Pittsburgh, PA, Baltimore, MD, Baton Rouge, LA, A total of 324,136 patients initiating therapy were identified. The sample Rockville, MD was 46% female and had an average age of 55 years. Following initia- Objective: To examine whether odds of frailty differ by DPP intervention tion, 31% of patients discontinued within the first 3 months, 44% within 6 arms (intensive lifestyle intervention [ILS], metformin [MET], placebo [PLB] months and 58% within 12 months. Among those who discontinued within after long-term follow-up in the DPPOS. the 12-month follow-up, 27% restarted therapy within 60 days, and 39% Methods: DPP, a randomized trial of individuals at high risk of diabetes, re-started therapy anytime during the 12-month follow-up. Those who dis- lasted 2.8 years, after which there was a 13-month bridge period in which continued and re-started therapy spent a mean of 107 days without treat- 16 sessions of group ILS was offered to all participants. During DPPOS, ment on hand. ILS was offered lifestyle reinforcement semi-annually and MET received This study provides real-world evidence that a majority of T2D medica- unmasked metformin. Frailty was measured initially in DPPOS Year 8 in 2385 tion patients discontinue within one year of initiation. Interventions aimed participants and repeated in 2264 of these in Year 10 (12 and 14 years after at improving treatment persistence should be targeted early after initiation randomization). Using Fried criteria, presence of 3 of 5 characteristics (slow to avoid gaps in therapy and associated clinical consequences. walking speed, weak grip strength, unintentional weight loss, low physical ORALS activity, and exhaustion) was classified as frail, 1 or 2 pre-frail, and 0 non- 136‑OR frail.: GEE models adjusting for visit year were employed with repeated mea- Gluten Intake and Risk of Islet Autoimmunity and Progression to sures pooled from Years 8 and 10 to estimate pairwise odds ratios between Type 1 Diabetes in Children at Increased Risk of Disease ILS, MET and PLB for the outcomes of frail and pre-frail vs. non-frail. NICOLAI A. LUND-BLIX, FRAN DONG, KARL MARILD, ANNA E. BARON, JEN- Results: Odds of frailty vs. non-frailty were 37% lower in ILS compared to NIFER A. SEIFERT, KATHLEEN WAUGH, GEIR JONER, KETIL STORDAL, GERMAN PLB (Table) and MET (OR=0.629, CI= 0.420-0.942, p=0.024). There were no TAPIA, LARS C. STENE, RANDI K. JOHNSON, MARIAN REWERS, JILL NORRIS, significant treatment group differences in odds of pre-frailty. Aurora, CO, Denver, CO, Oslo, Norway Conclusion: ILS intervention in persons at high risk of diabetes may reduce Background: Few studies have examined gluten intake as a risk factor for frailty in later life. islet autoimmunity (IA) and type 1 diabetes (T1D). Our aim was to study the association of gluten intake with development of IA and progression to T1D, accounting for HLA genotype. Methods: The Diabetes Autoimmunity Study in the Young (DAISY) follows children with an increased risk of T1D based on presence of a first-degree relative with T1D, or HLA genotype. HLA genotypes were categorized in three groups as DR3/4 (DR3/4-DQ2/8), DR3 (DR3/3 or 3/X), and DR4 (DR4/4, 4/X or X/X, where X was neither 3 nor 4). Blood samples were collected at age 9, 15 and 24 months, and annually thereafter. IA was defined by the appearance of at least one autoantibody against insulin, IA-2, GAD or ZnT8 in at least two consecutive blood samples; T1D was defined using ADA criteria. Using FFQs we estimated the gluten intake (g/d) annually from 1 year of age. Cox regression was used to estimate hazard ratios adjusted for potential confounders.

ADA-Supported Research

A36 CLINICAL PROGRESS IN ISLET TRANSPLANTATION—CLINICAL SCIENCE

Results: This report includes 1,916 subjects followed from birth to the 19 subjects have been implanted and evaluated throughout implantation median age 13.5 years; 178 developed IA and 56 of these progressed to duration until final product explant. Product has been safe and well toler- T1D. At the first assessment (median age 1.4 years) the average daily glu- ated; adverse events have been relatively minor and generally related to the ten intake was 11.4 g (SD: 5.6). The amount of gluten intake did not predict surgical procedure. The delivery device has provided protection against the risk of IA (HR per 4 g 0.93; 95% CI 0.78-1.11) or progression from IA to T1D host adaptive immune system with no evidence of allogeneic or autoimmune (HR 1.30; 95% CI 0.94-1.78). Four grams of gluten equal to a slice of bread. rejection or sensitization based upon a sensitive panel of immune function However, we found a significant interaction with HLA (p=0.02), where the tests. No off-target growth of donor cells has been detected. The potential association between gluten and IA were significantly different between all for prolonged cell survival has been demonstrated, as long as 24 months, groups. Moreover, we found a significant interaction for progression to T1D but has been inconsistent among subjects and primarily limited by a for- (p=0.04) where in children with IA, gluten intake was associated with T1D eign body response to the device component. Positive immunohistochemical among children with DR3/4 (HR 1.66 95% CI 1.10-2.51), but not in children staining for pancreatic islet cell markers NKX6-1, insulin, and glucagon has with the other HLA groups. been observed. These preliminary results demonstrate the immune-protec- Conclusion: Gluten intake may have different effects on development of tive nature of the encapsulation device and safety of this human stem cell- IA and progression from IA to T1D depending on HLA genotype. derived product, but also indicate the ongoing requirement for optimization of the host response to device materials. 137‑OR Dietary Behaviors throughout Childhood Are Associated with Body 139‑OR Mass Index and Estimated Insulin Resistance in Adolescence—A Clinical Islet Transplantation Outcomes Are Comparable in Obese Longitudinal Study Type 1 Diabetes Recipients VERONIQUE GINGRAS, SHERYL RIFAS-SHIMAN, ELSIE M. TAVERAS, EMILY ANNA LAM, KATHRYN J. POTTER, SHAREEN FORBES, SHARLEEN IMES, OKEN, MARIE-FRANCE HIVERT, Boston, MA ANDREW J. MALCOLM, ANDREA HAQQ, A.M. JAMES SHAPIRO, PETER A. Background: Prediabetes is increasingly common in adolescent popula- SENIOR, Edmonton, AB, Canada, Edinburgh, United Kingdom tions, yet, little is known about the contributing role of dietary behaviors Many clinical islet transplant programs have body mass index (BMI) and/ throughout childhood to risk of prediabetes in adolescence. or weight based thresholds for eligibility. Large subjects may require higher Objective: We examined longitudinal associations of dietary behaviors islet mass to reliably achieve insulin independence. However, for long term throughout childhood with body mass index z-scores (BMI-z) and HOMA-IR graft function and protection from hypoglycemia, data supporting a BMI index in adolescence. based exclusion is lacking. Design: Among 596 children from Project Viva, a pre-birth cohort from Methods: Subjects undergoing islet transplantation at a single center, eastern Massachusetts, we examined associations of maternal- or child- between 1999 and 2017 were included. Graft survival as measured by reported dietary behaviors annually from ages 4 to 12 years with BMI-z and C-peptide positivity from last CIT was compared between recipients with HOMA-IR in adolescence (median 12.8 years). We used mixed effects mod- BMI<30 kg/m2 (n=237, 24.8+2.8 kg/m2) and BMI >30 kg/m2 (n=24, mean+SD els adjusted for: maternal education, pre-pregnancy BMI, marital status and 31.9+2.1 kg/m2) pre-transplant. parity, and child’s age, sex, and race/ethnicity. Results: Apart from BMI and weight (BMI<30 vs. BMI>30, 70.9+11.3 kg vs. Results: The frequency of healthful dietary behaviors decreased with 82.8+9.5 kg; P<0.05) there were no difference between groups in terms of advancing age; at age 12, 64% of children ate breakfast daily, 32% ate din- baseline recipient characteristics, including in daily insulin dose (0.58+0.16 ner together with family daily, 59% ate fast food meals <1x/week, and 50% U/kg vs. 0.59+0.18 U/kg; P=0.34). Both groups received similar total number watched television during meals <1x/week. BMI-z (mean 0.44 units, SD 1.06) of islet infusions (2.4+0.9 and 2.3+0.6, p=0.6), total islet equivalents (1 053 in adolescence was lower in children who reported daily breakfast eating (β 022+471 532 vs. 1 182 498+366 368, P=0.19) and total islet equivalents per -0.17; 95% CI -0.25, -0.08), daily family dinner (-0.11; -0.17, -0.05), fast food recipient body weight (15 208+6 373 IE/kg vs. 14 120+5 041 IE/kg, P=0.42). <1x/week (-0.11; -0.18, -0.05) and television during meals <1x/week (-0.07; Long term C-peptide survival was not different between groups (P=0.18). -0.13, -0.01) throughout childhood. Log-transformed HOMA-IR (mean 0.99, Conclusion: T1D patients with BMI>30 appear to have similar islet mass SD 0.62) in adolescence was also lower with daily breakfast (β -0.12; 95% requirements and graft survival compared to those with BMI<30. This likely CI -0.17, -0.07), daily family dinner (-0.05; -0.09, -0.01), eating fast food <1x/ reflects careful selection of smaller (weight <90 kg), insulin sensitive (<0.6 week (-0.05; -0.09, -0.01) and television during meals <1x/week (-0.05; -0.08, U/kg) subjects with only class I obesity. Given the prevalence of obesity in -0.01) throughout childhood. Association of daily breakfast eating with lower T1D, future studies should evaluate the roles of obesity, weight and insulin HOMA-IR remained significant after further adjustment for child’s BMI-z in sensitivity to ensure rational and equitable access to transplantation. adolescence (β -0.08; 95% CI -0.13, -0.04). Conclusions: Children with healthful dietary behaviors throughout child- 140‑OR hood have lower BMI-z and less insulin resistance in adolescence. Long-Term Function of Islet Allografts Transplanted on the Omen‑ Supported By: National Institutes of Health tum Using a Biological Scaffold DAVID BAIDAL, CAMILLO RICORDI, DORA M. BERMAN, ANTONELLO PILEGGI,

ANA M. ALVAREZ GIL, NATHALIA PADILLA, GAETANO CIANCIO, ELINA LINETSKY, ORALS CLINICAL PROGRESS IN ISLET TRANSPLANTATION— RODOLFO ALEJANDRO, Miami, FL CLINICAL SCIENCE The liver is the preferred site for islet transplantation (ITx) but not ideal due to limitations affecting engraftment. 138‑OR We evaluated the safety and efficacy of ITx in the omentum using a Initial Clinical Evaluation of VC-01™ Combination Product—A Stem resorbable biological scaffold in 3 subjects with type 1 diabetes and nega- Cell-Derived Islet Replacement for Type 1 Diabetes (T1D) tive C-peptide. Islets were combined with autologous plasma and thrombin ROBERT R. HENRY, JEREMY PETTUS, JON WILENSKY, A.M. JAMES SHAPIRO, to generate a biologic scaffold and layered laparoscopically on the omen- PETER A. SENIOR, BART ROEP, RICHARD WANG, EVERT J. KROON, MICHAEL tum. Induction was with anti-thymocyte globulin and etanercept. Mainte- SCOTT, KEVIN D’AMOUR, HOWARD L. FOYT, San Diego, CA, Edmonton, AB, Can- nance was with mycophenolate sodium and tacrolimus. Demographics, islet ada, Duarte, CA, North York, ON, Canada dose and metabolic data are shown in the Table. VC-01 is a novel cell replacement therapy being developed as a poten- Subject 1 was insulin independent for 15 months and maintains stable tial long-term diabetes therapy to more effectively control glycemia, reduce glycemic control. Subject 2 had marginal graft function with persistence of hypoglycemia risk and mitigate diabetes-related complications. VC-01 is severe hypoglycemia (SH) and then underwent intrahepatic ITx resulting in derived from directed differentiation of human embryonic stem cells to pan- insulin independence. Subject 3 had a 55% reduction in insulin dose and creatic progenitor cells, which are loaded into a durable, retrievable delivery maintains excellent glycemic control. device and then implanted subcutaneously. Once implanted and matured, Our initial experience demonstrates feasibility and safety of ITx on the the cells secrete insulin and other islet hormones in response to blood glu- omentum. Graft function persisted throughout follow-up (6-24 months) result- cose levels. The delivery device is immunoprotective to implanted cells. ing in improved glycemic control and absence of SH (subjects 1 and 3). VC-01 is currently being evaluated in a 24-month open-label, dose-escalat- Results suggest a significant loss of islets early post-ITx followed by ing Phase 1/2 study in T1D patients with minimal insulin-producing β-cell gradual functional decline similar to intrahepatic ITx. Strategies to improve function. Cohort 1 has evaluated product at a subtherapeutic dose to assess oxygen delivery and neo-vascularization and minimize immunosuppression initial safety and tolerability and establish implantation techniques. To date, are needed to improve long-term outcomes at this site.

ADA-Supported Research

A37 CLINICAL PROGRESS IN ISLET TRANSPLANTATION—CLINICAL SCIENCE

Table. of PRA+ remains a concern due to potentially prolonged wait list time for Subject Age Duration BMI IEQ†/Kg MMTT‡ Insulin dose HbA1c% subsequent organ transplantation if needed. # (years) T1DM (Kg/m2) Stimulated Glucose U/Kg/day (years) (mg/dl)/C-peptide (ng/ml) (Units/day) 6 months 12 months 24 months Pre-ITx Latest Pre-ITx Latest 1 43 26 21.5 11,386 181/3.32 277/1.79 317/0.49 0.62 (33) 0.14 (7) 6.8 5.3 2 32 16 25.3 9,635 372/0.88 374/0.65 332/0.52* 0.45 (31) 0.45 (30) 5.7 5.6 3 46 42 24 12,648 277/2.47 - - 0.45 (20) 0.2 (11) 6.3 6.3 * 18 months Post-ITx. † IEQ= islet equivalents. ‡ MMTT = mixed meal tolerance test. Supported By: JDRF; The Leona M. and Harry B. Helmsley Charitable Trust; Florida Diabetes Research Institute Foundation; University of Miami Clinical and Translational Science Institute/National Institutes of Health

141‑OR Enhanced Recipient Cardiovascular Health Pretransplant Predicts Better Clinical Outcomes following Islet Cell Transplant for Type 1 Diabetes KIRSTIE K. DANIELSON, REBECCA S. MONSON, YI LI, BRETT RYDZON, Chicago, IL Increased cardiovascular disease (CVD) risk is a clinically significant feature of type 1 diabetes (T1D). Several CVD markers were shown to regress or stabilize after functionally curing T1D via islet cell transplant (ICT), yet the inverse is unknown: does recipients’ CVD health at first ICT predict clinical outcomes following ICT. In 30 patients receiving 1-3 ICTs, CVD markers and basic labs were measured at first ICT (e.g., lipids, blood pressure, blood counts/chemistries, and carotid intima-media thickness [CIMT]), along with Supported By: Florida Diabetes Research Institute Foundation clinical outcomes (e.g., C-peptide/insulin from four distinct metabolic tests) measured serially after first ICT (mean [range] 2.1 [0.3-8.7] years). Multivari- 143‑OR able regression of repeated measures determined longitudinal associations, CGM Shows Islet Transplantation Prevents Hypoglycemia, Correct‑ adjusting for recipient demographics, BMI, ICT number, and islet equivalents ing Time in Range and Reducing Glycemic Variability, Despite Sub‑ transplanted. Mean (SD) age, T1D duration, and BMI at first ICT were 47.3 normal Beta-Cell Function (11.5) and 29.4 (12.0) years, and 22.9 (1.9), respectively; 80% female. Lower SHAREEN FORBES, TOLU OLUTOYIN OLATEJU, ANNA LAM, JOHN CASEY, JOHN LDL/HDL pre-ICT predicted greater fasting C-peptide (n=759 observations, CAMPBELL, LAURA REID, RICHARD A. ORAM, ANDREW J. MALCOLM, A.M. p=0.01) and multiple measures of stimulated C-peptide and insulin (n=68- JAMES SHAPIRO, PETER A. SENIOR, Edinburgh, United Kingdom, Edmonton, AB, 76, all p≤0.04); lower triglycerides pre-ICT (n=811, p=0.003) and systolic Canada, Exeter, United Kingdom blood pressure day of first ICT (n=1142, p=0.007) predicted greater fasting The impact of clinical islet transplant (CIT) on glycemic variability com- C-peptide. Low (normal) hemoglobin and percent eosinophils the day of first pared with type 1 diabetes (T1D) subjects and nondiabetic controls has not ICT also predicted greater fasting C-peptide (n=1142, all p=0.002) and stimu- been explored. lated C-peptide and insulin (n=73-107, all p≤0.048). Lower recipient CIMT Aim: To evaluate the relationship between glycemic variability (by CGM) pre-ICT predicted greater acute insulin response post-ICT (n=42, p=0.04). and islet graft function in CIT vs. T1D subjects on Continuous Subcutaneous Multiple traditional CVD protective factors, along with lower hemoglobin Insulin Infusions (CSII), using healthy, nondiabetic subjects as reference. and eosinophils (both recently shown to be CVD protective factors), at first Methods: CIT recipients n=39, (insulin independent vs. dependent: n=15 ICT predicted better outcomes. These results may be clinically significant as vs. n=24) and subjects on CSII therapy (n=10) all with T1D were assessed at 1 improving CVD health pre-ICT (e.g., with diet, exercise, and/or medications) year post-intervention with CGM. Published data from nondiabetic controls may further support its success. were used for comparison. Fasting glucose, C-peptide and HbA1c concentra- Supported By: American Diabetes Association (1-16-ICTS-022 to K.K.D.) tions and insulin dose were recorded (including in a control cohort, n=28) where applicable; BETA-2 scores were calculated in those with endogenous 142‑OR C-peptide. Persistence of Allosensitization after Islet Allograft Failure Results: CIT recipients had decreased coefficient variation of glucose and PAOLA RIOS, DAVID BAIDAL, NATHALIA PADILLA, ANA M. ALVAREZ GIL, SRI- lower time in hypoglycemia vs. those on CSII therapy; BETA-2 scores were ORALS GITA MADIRAJU, JONATHAN AMBUT, ALEJANDRO M. MANTERO, SHARI ~20% lower in CIT recipients vs. controls (p<0.05. MESSINGER CAYETANO, CAMILLO RICORDI, RODOLFO ALEJANDRO, Miami, FL, Conclusion: Islet transplant recipients have diminished glycemic variability Homestead, FL with abrogation of hypoglycemia vs. CSII at 1 year. Normalisation of time Allosensitization (PRA+) has been reported after discontinuation of immu- in range and glycemic variability was observed in insulin independent CIT nosuppression (IS) following graft failure (GF) in islet transplant (ITx) recipi- recipients despite beta cell function ~20% lower than in nondiabetic subjects. ents. Persistence of PRA+ after GF is unknown. Supported By: Diabetes UK; UK Islet Transplant Consortium; UK NHS Blood and Thirteen ITx alone (IA) and 5 ITx subjects who also received donor CD34+ Transplant; Alberta Health Services; Government of Alberta; Alberta Innovates hematopoietic stem cells (IBM) and developed GF enrolled in the study “Allo- Health Solutions; JDRF International; National Institutes of Health; Diabetes sensitization After Islet Transplantation.” Panel reactive antibodies (PRA) Research Institute Foundation of Canada were measured yearly while having graft function, within 1 year post-GF and yearly after study enrollment. Two subjects who developed GF but remained on mycophenolate mofetil (MMF) for 2 years and 18 subjects with persistent graft function (15 IA and 3 islet after kidney [IAK]) were also evaluated. 80% IBM subjects developed high PRA+ (>89%) (Figure 1a) while 70% IA subjects were PRA+ at enrollment (0.8-6 years post-GF) and 42%-55% were PRA+ throughout follow-up. PRA results were variable (Figure 1b). The 2 subjects taking MMF post-GF and 1 subject who required IVIG pre-GF (for parvo- virus infection) were not PRA+. Four subjects with persistent graft function by 10 years became PRA+ (2 IA and 2 IAK; Figure 1c). After stopping IS, 70% IA subjects became PRA+ with persistence of PRA (although variable) for at least 10 years post-GF. PRA+ was higher in IBM compared to IA subjects. While receiving IS, PRA+ was minimal. Persistence

ADA-Supported Research

A38 G IS FOR GLP, GIP, AND GLUCAGON—A NOD TO THE GUT-LIVER AXIS

G IS FOR GLP, GIP, AND GLUCAGON—A NOD TO THE Figure. GUT-LIVER AXIS

144‑OR Extracellular Volume Expansion Uncovers a Natriuretic Action of Glucagon-Like Peptide-1 ALI ASMAR, PER K. CRAMON, LENE SIMONSEN, MEENA ASMAR, CHARLOTTE M. SORENSEN, STEN MADSBAD, SR., CEDRIC MORO, BOLETTE HARTMANN, JENS J. HOLST, BOYE L. JENSEN, JENS BÜLOW, Copenhagen, Denmark, Toulouse, France, Odense, Denmark We have previously demonstrated that the gut hormone glucagon-like peptide-1 (GLP-1) does not affect renal hemodynamics or function at baseline conditions in healthy individuals and in patients with type 2 diabetes mellitus. However, it is possible that GLP-1 promotes renal NaCl excretion under conditions with addition of salt and water to the extracellular fluid (mimicking a mixed meal/daily NaCl load). The present study was designed to investigate whether GLP-1 induces natriuresis in healthy, volume overloaded individuals. Under fixed sodium intake, 8 healthy men were examined twice in random order during a 3-hour infusion of either GLP-1 (1.5 pmol kg-1 min-1) or vehicle together with an intravenous infusion of 0.9% NaCl (750 ml/hour) given throughout both experiments. Timed urine collections were conducted Supported By: European Foundation for the Study of Diabetes; Novo Nordisk throughout the experiments at voluntary voiding, and subjects remained Foundation supine during the experiments. Renal plasma flow (RPF), glomerular filtration rate (GFR), and uptake/release of hormones and ions were measured using Fick’s Principle after catheterization of a renal vein and an artery. During 146‑OR GLP-1 infusion, urinary sodium excretion increased compared to vehicle infu- Mechanisms by Which Glucagon Acutely Stimulates Hepatic Mito‑ sion (193 ± 40 mmol/180 min vs. 89 ±10 mmol/180 min, p<0.05), whereas chondrial Oxidation and Gluconeogenesis GLP-1 had no significant effect on urine flow rate (1666 ± 284 ml/180 min vs. RACHEL J. PERRY, YONGLIANG WANG, ALLISON L. BRILL, LIANG PENG, DONG- 1189 ± 132 ml/180 min, p=0.184). Plasma renin levels decreased similarly on YAN ZHANG, SYLVIE DUFOUR, YE ZHANG, XIAN-MAN ZHANG, YUICHI NOZAKI, both days, whereas angiotensin II levels decreased significantly only during GARY CLINE, BARBARA E. EHRLICH, KITT PETERSEN, GERALD I. SHULMAN, New GLP-1 infusion. During GLP-1 infusion, GLP-1 was extracted by the kidneys by Haven, CT, Beijing, China about 50%. RPF and GFR remained unchanged on both days. Plasma proANP, Glucagon has been suggested to be both pathogenic and protective ANP, and BNP levels remained unchanged on both days. This study shows against type 2 diabetes (T2D) reflecting our poor understanding of the that physiologically relevant changes in GLP-1 induce significant natriuresis molecular mechanisms by which glucagon alters hepatic metabolism. Using independent of measurable changes in RPF and GFR in volume overloaded Positional Isotopomer NMR Tracer Analysis (PINTA) we found that acute subjects. The natriuresis is likely brought about via a tubular mechanism glucagon infusion stimulates rates of hepatic gluconeogenesisin glycogen- secondary to suppression angiotensin II. depleted mice by promoting increases in intrahepatic lipolysis and hepatic acetyl-CoA content (75±6 vs. 101±6 nmol/g, p=0.01), pyruvate carboxylase flux (VPC, 70.9±3.9 vs. 99.9±7.1 µmol/[kg-min], p=0.008) and mitochondrial 145‑OR oxidation (90.0±12.9 vs. 452.1±78.3 µmol/[kg-min], p=0.001) in vivo. Each Postprandial Effects of Individual and Combined GIP and GLP-1 of these effects was abrogated in liver-specific Inositol Trisphosphate Receptor Antagonization in Healthy Subjects Receptor-I (InsP3R-I) knockout mice demonstrating that intracellular calcium LÆRKE S. GASBJERG, MADS M. HELSTED, ALEXANDER H. SPARRE-ULRICH, signaling is necessary for these processes. Liver specific knockdown of AMALIE R. LANNG, SIGNE STENSEN, METTE HØY JAKOBSEN, BOLETTE HART- adipocyte triglyceride lipase abolished acute glucagon stimulation of glu- MANN, MIKKEL B. CHRISTENSEN, JENS J. HOLST, TINA VILSBØLL, METTE M. coneogenesis (control 70.5±2.9 vs. 103.3±3.3 µmol/[kg-min], p<0.0001 and ROSENKILDE, FILIP K. KNOP, Copenhagen, Denmark, Hellerup, Denmark, Gentofte, ATGL knockdown 69.7±1.7 vs. 69.6±2.0 µmol/[kg-min], p=0.9) and VPC (control Denmark 63.3±5.0 vs. 100.0±4.8 µmol/[kg-min], p=0.0003, ATGL knockdown 58.9±4.9 The gut-derived incretin hormones glucose-dependent insulinotropic poly- vs. 59.2±3.2 µmol/[kg-min], p=0.9) in WT mice demonstrating the necessity peptide (GIP) and glucagon-like peptide-1 (GLP-1) are known for insulinotropic of hepatocellular lipolysis in mediating this process. Chronic increases in and glucose-lowering effects. However, their individual roles in postprandial plasma glucagon concentrations reversed high fat diet-induced hepatic ste- glucose homeostasis are unknown. We infused the GIP receptor antagonist atosis (liver triglyceride 50.1±8.5 vs. 28.3±5.1 mg/g, p=0.04; liver membrane GIP(3-30)NH and the GLP-1 receptor antagonist exendin (9-39) during a meal 2 diacylglycerol 801±142 vs. 448±47 nmol/g, p=0.03, PKCε membrane/cytosol to determine the roles of endogenous GIP and GLP-1 on postprandial glu- 1.76±0.40 vs. 0.23±0.04, p=0.0009) and glucose intolerance (GTT glucose ORALS cose metabolism. On four separate days, 12 healthy men (age 19-65 years, 2 AUC 1.44±0.06 vs. 1.13±0.05 mM-min, p=0.003, insulin AUC 226.8±18.7 vs. BMI 20-25 kg/m ) received a liquid mixed meal test with randomized and 90.2±11.2 nM-min, p<0.0001). These results provide new insights into glu- double-blinded infusions of GIP(3-30)NH2 (800 pmol/kg/min) + Ex(9-39) (450 cagon biology and suggest that glucagon and InsP3R-I may be therapeutic pmol/kg/min) (A), GIP(3-30)NH2 (B), Ex(9-39) (C), and saline (D). On Day A, B targets to reverse nonalcoholic fatty liver disease and T2D. and C, glucose excursions were significantly increased by 85%, 55% and Supported By: National Institutes of Health (K99CA215315, R01DK40936, 15%, respectively, compared to D. Day A and B excursions were significantly R01DK113984, P30DK059635); MicroMouse (16AU3737) higher than C (Figure). Glucagon levels at 60 min differed between Day A and B (11.5±1.2 vs. 7.5±0.6 pmol/l, P=0.01), and Day B and C (11.5±1.2 vs. 12.9±1.5 pmol/l, P=0.008). GLP-1 was higher on Day A (P=0.01) and C (P=0.02) than D. 147‑OR No significant effects on insulin, C-peptide, gastric emptying, or GIP were Glucagon Resistance at the Level of Amino Acid Turnover and Ure‑ observed. We conclude that endogenous GIP affects postprandial plasma agenesis in Obese Subjects with Hepatic Steatosis glucose excursions more pronouncedly than GLP-1, and that both hormones MALTE P. SUPPLI, JONATAN I. BAGGER, ASGER B. LUND, NICOLAI J. WEWER additively contribute to control postprandial glycemia in healthy subjects. ALBRECHTSEN, JENS J. HOLST, TINA VILSBØLL, FILIP K. KNOP, Hellerup, Den- mark, Copenhagen, Denmark, Gentofte, Denmark Hyperglucagonemia is considered an important pathophysiological trait of type 2 diabetes and occurs also in individuals with obesity. The underlying mechanisms remain unclear. We hypothesized that hyperglucagonemia is a consequence of steatosis-induced glucagon resistance. We examined 15 healthy (BMI: 23.2±1.6 (mean±SD) kg/m2, age 40.9±12.8 years, hepatic steatosis 2:15) and 15 obese individuals (BMI: 33.6±2.1 kg/m2, age 35.8±9.4 years, hepatic steatosis 14:15) in a pancreatic somatostatin clamp with basal insulin (1 mU/kg/h) and low and high glucagon infusion rates (0.6 and

ADA-Supported Research

A39 G IS FOR GLP, GIP, AND GLUCAGON—A NOD TO THE GUT-LIVER AXIS

3.0 ng/kg/min). Glucagon sensitivity was assessed as changes in plasma would prevent NAFLD and insulin resistance in normal rats fed a low fat (12% amino acids (AA) and endogenous glucose production (EGP) and urea syn- calories from fat), sucrose containing diet (17% calories from fructose). KHK thesis (U.S.) measured by tracer techniques. Basal glucagon and AA were ASO decreased hepatic KHK mRNA and protein expression by 96 and 90%, significantly higher in obese compared to lean individuals, but no differ- respectively. KHK ASO decreased WAT mRNA expression by 65%. KHK ASO ences in basal EGP or U.S. were observed. In the lean group AA significantly did not alter body mass. KHK ASO decreased fasting plasma glucose (102±2 dropped in response to glucagon infusion (p<0,001) whereas no response vs. 94±2 mg/dL, P=0.01), fasting plasma triglyceride 60% (52±7 vs. 21±2 mg/ was observed in the obese group. Accordingly, both glucagon infusion rates dL P<0.01) and liver triglyceride by 30% (8.2±0.4 vs. 5.6±0.3 mg TG/g liver, resulted in significantly greater EGP and U.S., respectively, in lean vs. obese P<0.001) but not plasma insulin or non-esterified fatty acids. Insulin sensitiv- individuals. ity was assessed by a 4 mU·kg-1·min-1 hyperinsulinemic-euglycemic clamp. In conclusion, our results point to steatosis-induced hepatic glucagon Surprisingly, there were no differences in rates of EGP or suppression of resistance and compensatory glucagon secretion mediated by glucagono- EGP. Instead there was a 25% improvement in insulin stimulated whole body tropic amino acids as a likely explanation of obesity-associated hyperglu- glucose metabolism (36.6±0.8 vs. 45.6±2.2, P<0.01). We attribute this to a cagonemia. 40% decrease in hepatic DNL (53±4 vs. 32±4%, P<0.01) and VLDL secretion Figure. rate (11.0± 0.8 vs. 5.5± 0.5 mg/dL-min P<0.01) independent of changes in SREBP1 or ChREBP mRNA expression. Thus, decreased DNL and VLDL export prevents fructose induced peripheral insulin resistance. Conclusion: KHK ASO prevents NAFLD, decreases plasma triglyceride and improves insulin sensitivity. Supported By: U.S. Department of Veterans Affairs

150‑OR Early Effects of Sleeve Gastrectomy (SG) on Systemic and Organ- Specific Dietary Fatty Acid Uptake, Postprandial Free Fatty Acids (FFA), and Glucose Metabolism in Type 2 Diabetes (T2D) ANNE-MARIE CARREAU, CHRISTOPHE NOLL, BRIGITTE GUERIN, LAURENT BIER- THO, ERIC E. TURCOTTE, ANDRE TCHERNOF, ANDRÉ CARPENTIER, Sherbrooke, QC, Canada, Quebec City, QC, Canada 148‑OR Postprandial dietary fatty acid (FA) biodistribution is impaired in subjects NOD1 Mediates Insulin Resistance in Response to Circulating Free with glucose intolerance, with decreased FA storage in adipose tissues Fatty Acids (AT) and increased FA uptake by the myocardium. These abnormalities are SYDNEY L. RIVERS, LUCY SHU NGA YEUNG, KENNY L. CHAN, YUSAKU MORI, improved after 1 year of lifestyle-induced weight loss. Bariatric surgery DANA PHILPOTT, AMIRA KLIP, ADRIA GIACCA, Toronto, ON, Canada leads to early metabolic improvements before weight loss, but the early Insulin resistance (IR) in type 2 diabetes (T2D) is associated with chronic effects on dietary FA metabolism and biodistribution have never been stud- low-grade inflammation. We have shown that nucleotide binding oligomer- ied. Nine subjects with T2D underwent a 6-hour postprandial metabolic pro- 13 ization domain (NOD)1, an intracellular pattern recognition receptor for bac- tocol before and 12 days after SG. A liquid meal containing [U- C]-palmitate 2 3 terial wall peptidoglycans, is a factor linking the innate immune system to and [ H]-glucose was ingested at time 0, with an IV perfusion of [ H]-glucose 2 the development of high fat diet-induced IR. Although high fat diet may result and [7,7,8,8- H]-palmitate, allowing quantification of glucose absorption, in NOD1 activation via increased intestinal absorption of bacterial products, endogenous glucose production (EGP), lipolysis and dietary FA spillover. A 18 NOD1 has also been shown to be activated by saturated fatty acids in capsule containing a positron-emitting long-chain FA analog ( FTHA) was intestinal cells and adipocytes. We hypothesize that NOD1 plays a role in taken with the meal for the quantification of organ-specific dietary FA uptake IR caused by chronic high levels of circulating free fatty acids (FFA) as found using PET/CT (n=6). Twelve days after SG, whole body and hepatic insulin in obesity and T2D. Wild type (WT) and NOD1-/- mice were infused with sensitivity increased significantly whereas fasting and postprandial EGP ethylpalmitate (resulting in raised circulating palmitate) or ethanol vehicle decreased. Postprandial GLP-1, GIP, PYY and glucagon were higher after vs. as a control, for 48 hours to model prolonged elevation of circulating FFA in before SG. Postprandial AUC of FFA (175 vs. 228 mmol/Lx min; p< 0.05) and vivo. Hyperinsulinemic-euglycemic clamp with tracer methodology was then total FA oxidation were higher (p< 0.02 from 180 min to 360 min), but dietary used to assess peripheral and hepatic insulin sensitivity. WT mice treated FA oxidation was reduced after SG (p< 0.002 from 180 min to 360 min). After with ethylpalmitate had decreased peripheral and hepatic insulin sensitivity SG, uptake of dietary FA 6 hours after the meal was reduced in the myocar- expressed as stimulation of glucose utilization (Control= 123%±30 and Ethyl- dium (mean SUV 2.07± 0.66 vs. 1.36± 0.44; p=0.06) and was increased in palmitate= 25%±25, p<.05) and suppression of glucose production (Control= visceral AT (mean SUV 0.24± 0.2 vs. 0.42± 0.2; p=0.03), without changes in 51%±10 and Ethylpamitate= 22%±2, p<.05). NOD1-/- mice were protected other organs. from palmitate induced IR, showing no difference in stimulation of glucose In conclusion, there is reduced myocardial and increased visceral AT uptake of dietary FA associated with reduced dietary FA oxidation and

ORALS utilization (Control= 289%±27 and Ethylpalmitate= 352%±64) and suppression of glucose production (Control= 63%±16 and Ethylpalmitate= 95%±25) increased FFA mobilization and oxidation, concomitant with reduced EGP with or without palmitate. Thus, our data provide compelling evidence that and insulin sensitization in patients with T2D early after SG. NOD1 activation via elevated plasma FFA contributes to the development of Supported By: Diabetes Canada; Canadian Institutes of Health Research (TB2- peripheral and hepatic IR. 138776-2014-2019) Supported By: Canadian Institutes of Heath Research 151‑OR 149‑OR Ileum-Specific Transgenic Expression of Human Chimeric Intes‑ Targeting Ketohexokinase (KHK) with a Novel Antisense Oligo‑ tinal Alkaline Phosphatase (IAP) Attenuates Western Diet (WD)- nucleotide (ASO) Decreases De Novo Lipogenesis and Improves Induced Barrier Dysfunction and Glucose Intolerance Insulin-Mediated Whole Body Glucose Metabolism HONGLIANG HE, SIDDHARTHA S. GHOSH, PAUL J. YANNIE II, JING WANG, DONGQING LIU, JOHN A. STERPKA, DANIEL F. VATNER, MELANIE BELL, SUE SHOBHA GHOSH, Richmond, VA MURRAY, SANJAY BHANOT, GARY CLINE, VARMAN SAMUEL, New Haven, CT, Intestinal epithelial cell derived IAP dephosphorylates/detoxifies bac- Carlsbad, CA, West Haven, CT terial endotoxin LPS in the gut lumen. We have earlier demonstrated that Increased sucrose consumption is associated with rising rates of obesity improvement of intestinal barrier function was accompanied by an increase and related conditions [e.g., insulin resistance, type 2 diabetes and nonalco- in IAP activity. Herein, we developed IAP transgenic mice where expres- holic fatty liver disease (NAFLD)]. Ketohexokinase (KHK) catalyzes the first sion of human chimeric IAP is under the control of intestine specific villin step of fructose metabolism and is highly expressed in liver, kidney and brain, promoter. As shown in Panel A, compared to non-transgenic wild type (WT) though found in many tissues. Essential fructosuria (hepatic KHK deficiency) littermates, IAP expression (identified by c-myc tag) was noted in colon, is a benign condition, suggesting that KHK is a viable drug target. Inhibition ileum jejunum and duodenum of IAP-transgenic (IAPTg) mice. IAP was almost of hepatic KHK would block metabolism of fructose (50% of dietary sucrose) uniformly expressed along the length of the ileum (Panel B) and while IAP and improve numerous facets of metabolism. ASO’s potently decrease tar- activity reduced from proximal P1 segment to distal P9 segment in WT mice, get expression in liver and WAT and we hypothesized that a novel KHK ASO this activity was maintained in the IAPTg mice (Panel C). Dietary challenge

ADA-Supported Research

A40 MUSCLING IN ON THE HEART OF METABOLISM AND INSULIN SENSITIVITY with WD impaired glucose tolerance in WT mice and this intolerance was controls, suggesting that cardiac mitochondrial Akt signaling may remotely attenuated in IAPTg mice (Panels D and E). Significant decrease in fecal zonu- modulate fat metabolism in the liver. lin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice (Panel F) In summary, activating cardiac mitochondrial Akt protected against dia- and a corresponding decrease in translocation of oral 4kDa FITC dextran to betic cardiomyopathy, improved glucose tolerance, and attenuated fatty plasma (Panel G) suggests that IAP overexpression improves intestinal bar- liver in HFF-induced diabetes. rier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD induced intestinal dysfunction and glucose intolerance. 153‑OR Panels A-G. Caloric Restriction Increases the Resistance of Aged Heart to Myo‑ cardial Ischemia/Reperfusion Injury ZHIJIA GUO, THOMAS ROUSSELLE, JI LI, Taiyuan, China, Jackson, MS Ischemic tolerance of heart decreases with age. Caloric restriction (CR) is the most reliable intervention to extend lifespan and prevent age-related disorders. We have revealed that both AMPK and SIRT1 signaling pathways are involved in the impairments occurred in aged hearts. We hypothesize that cardiac AMPK-SIRT1 signaling cascade mediates the increased tolerance of aged heart to ischemic insults by caloric restriction. Aged (18-20 months) mice were divided into 2 groups: al libitum (AL) food intake group and caloric restriction (diet 70% of standard food intake for 6 weeks) (CR) group. Each group was divided into sham operations and ischemia/reperfusion (I/R) semi-groups. I/R groups were subjected to ligation of left anterior descending coronary artery for 30 min of ischemia and 24 hours of reperfusion. The body fat composition was significantly decreased in CR group vs. AL group, moreover, the maximum of oxygen consumption was significantly improved in CR group vs. AL group. Importantly, there is a significantly smaller myocardial infarction size induced by ischemia (30 min) and reperfusion (24 hours) in CR group than that in AL group. The immunoblotting results demonstrated that the impaired ischemic AMPK activation in AL group was augmented in CR group, and the AMPK downstream acetyl CoA carboxylase (ACC) phosphorylation and PGC-1alpha phosphorylation were also augmented in CR vs. AL group in response to ischemic insults. Intriguingly, the expression levels of a longevity protein, SIRT1, were upregulated in the heart of CR group vs. the corresponding AL group. Furthermore, the inducible cardiomyocyte SIRT1 deficiency mice (icSIRT1 KO) did not show any recused impaired ischemic AMPK activation in the CR group vs. icSIRT1 KO AL group. Thus, caloric restriction is a non-pharmacological approach to increase the tolerance of aged heart to ischemic insults. The AMPK-SIRT1 signaling cascade plays a critical role in the cardiac CR benefits in the elderly. Supported By: American Diabetes Association (1-17-IBS-296 to J.L.); National Institutes of Health (R01AG049835)

154‑OR Supported By: American Diabetes Association (1-16-IBS-105 to S.G.) Effects of Hormone-Sensitive Lipase Overexpression on Lipotoxic Cardiomyopathy in Mice Lacking Adipose Triglyceride Lipase MIKA YAMADA, JINYA SUZUKI, MASAMICHI HIROSE, TAKAHIRO NAKAYA, MAI MUSCLING IN ON THE HEART OF METABOLISM AND ICHIKAWA, SATSUKI SATO, MICHIKO IMAGAWA, YASUO ZENIMARU, FREDRIC INSULIN SENSITIVITY B. KRAEMER, TADASHI KONOSHITA, TAMOTSU ISHIZUKA, Fukui, Japan, Iwate, Japan, Stanford, CA 152‑OR Lipotoxic cardiomyopathy, which is associated with obesity and diabetes, Activation of Cardiac Mitochondrial Akt Signaling Improved Dia‑ is characterized by intracellular triacylglycerol (TAG) droplet accumulation betic Cardiomyopathy and Induced Liver Metabolic Remodeling (steatosis). The first step of TAG hydrolysis is catalyzed by adipose triglyc- ALBERT TA, YU-HAN CHEN, YUMAY CHEN, HYE-JIN LEE, HUGO YOU-HSIEN LIN, eride lipase (ATGL), and its deficiency results in extreme cardiac steatosis in

PING H. WANG, Irvine, CA, Seoul, Republic of Korea mice and humans. Although hormone-sensitive lipase (HSL) functions as a ORALS Insulin stimulates Akt translocation to cardiac mitochondria, and we have diacylglycerol (DAG) lipase in the heart, we hypothesized that activated HSL recently shown that inhibition of cardiac mitochondrial Akt led to mito- might catalyze cardiac TAG hydrolysis. To test this hypothesis we gener- chondria dysfunction and cardiomyopathy. However, it is unknown whether ated transgenic mice with cardiac-specific HSL-overexpression (cHSL) and activation of mitochondrial Akt in cardiac muscle can protect against the crossed them with ATGL-knockout (KO) mice. By crossing ATGL-KO and cHSL development of diabetic cardiomyopathy. To this end, we have generated an lines, we generated homozygous ATGL-KO (AKO) mice, AKO mice with car- inducible, heart-specific, transgenic mice harboring a mitochondria-targeting diac HSL-overexpression (AKO/cHSL), and wild type (Wt) mice. Cardiac lipid constitutively active Akt (CAMAAKT). After 5 months of high fat-high fruc- content was measured and its function was assessed by ultrasonography. tose diet (HFF), the control mice developed myocardial fibrosis and cardio- Histological investigation was performed by transmission electron micros- megaly. In contrast, myocardial fibrosis and cardiomegaly were attenuated copy. Cardiac TAG content of AKO mice was 160-fold greater than that of in the CAMAAKT mice on HFF. Compared to the control mice, the expression Wt mice, whereas that of AKO/cHSL mice was as low as that of Wt hearts. of myocardial BNPa, BNPb, and Myh7 (heart failure genes) were significantly Cardiac systolic function (fractional shortening; FS) was markedly reduced in suppressed in the CAMAAKT mice by 80%, 90%, and 80%, respectively (all AKO mice compared to that of Wt mice (28% vs. 49%), while AKO/cHSL mice p<0.01). Col1a1 and Col3a1 expression were reduced in the HFF-CAMAAKT showed comparable FS to that of Wt mice (48% vs. 49%). Electron micros- mice, corroborating the reduction of myocardial fibrosis. These data indi- copy showed accumulation of huge lipid droplets, damaged mitochondria cated that activation of mitochondrial Akt protected against cardiomyopathy with destroyed cristae and vacuole degeneration, and disrupted intercalated in diet-induced diabetes. In OGTT, fasting glucose level was unchanged but discs (where gap junctions and desmosomes are localized) in the cardio- postprandial glucose levels were lower in the CAMAAKT mice. In another myocytes of AKO mice. In contrast, the cardiomyocytes of AKO/cHSL mice heart-specific transgenic mice harboring a mitochondria-targeting dominant showed no such morphological characteristics. These results indicate that negative Akt (CAMDAKT), the glucose curve in OGTT was significantly higher cardiac overexpression of HSL normalizes lipotoxic cardiomyopathy in AKO than the control mice. Therefore, mitochondrial Akt signaling in the heart mice and suggest that activation of cardiac HSL could be a potent strategy could modulate whole body glucose homeostasis. Interestingly, the diet- to regulate the lipotoxic cardiomyopathies that occur in metabolic disorders. induced fatty liver in the CAMAAKT mice was significantly less than the

ADA-Supported Research

A41 MUSCLING IN ON THE HEART OF METABOLISM AND INSULIN SENSITIVITY

155‑OR kg/m2) before and for 52 w after bariatric surgery. Genome-wide gene expres- Mitochondrial Glycerol 3-Phosphate Dehydrogenase Improves sion and DNA methylation were analyzed in a subgroup of 16 OBE. Initial Skeletal Muscle Regeneration in Obesity and Diabetes weight loss increases muscle oxidative capacity by 11% at 2 w, but transient HUA QU, YI ZHENG, Chongqing, China elevation of certain muscle diacylglycerols resulting from unrestrained adipose While adult mammalian skeletal muscle is stable due to its post-mitotic lipolysis prevents from rapid improvement in IS. At 52 w, both mitochondrial nature, muscle regeneration is still essential throughout life for maintaining function and intracellular lipids are comparable to lean humans. Acute altera- functional fitness. During certain diseases, such as the modern pandemics of tions in expression of 1287 genes involved primarily in mitochondrial function, obesity and diabetes, the regeneration process becomes impaired, leading transcriptional regulation, protein transport, fatty acid metabolism and inflam- to the loss of muscle function, and contributing to the global burden of these matory processes, but not changes in DNA methylation, underlie the transient diseases. However, the underlying mechanisms behind the impairment are upregulation of mitochondrial function and lipolysis. At 52 w, 109105 CpGs are not well defined. Here we identify mitochondrial glycerol 3-phosphate dehy- differentially methylated, which relates to improved IS. Specifically, epigen- drogenase (mGPDH) as a critical regulator of skeletal muscle regeneration. etic alterations at 52 w in FTO gene, encoding an α-ketoglutarate dependent Specifically, it regulates myogenic markers and myoblast differentiation by dioxygenase, and TOMM7 gene, encoding a translocase of the outer mito- controlling mitochondrial biogenesis via CaMKKβ/AMPK. mGPDH deletion chondrial membrane, contribute to reprogramming transient changes in mRNA (mGPDH-/-) attenuated skeletal muscle regeneration in vitro and in vivo while expression at 2 w. mGPDH overexpression improved the insufficient regeneration in mdx mice. In conclusion, initial metabolic changes after weight loss induce epigen- Moreover, in patients and animal models of obesity and diabetes, mGPDH etic modification of genes involved in muscle energy metabolism, which in expression in skeletal muscle was reduced, further suggesting a direct cor- turn leads to long-term beneficial changes in gene expression. relation between its abundance and muscular regeneration capability. Res- cuing mGPDH expression in obese (HFD, ob/ob) and diabetic (STZ) mice by 157‑OR AAV led to a significant improvement in their muscle regeneration (Figure 1). Effect of Metformin on Mitochondrial Pathways in Human Skeletal Our study provides a new mechanism and potential therapeutic target for Muscle Cells skeletal muscle regeneration impairment. JESSICA SACKS, ANNY MULYA, CIARAN E. FEALY, KELLY M. FITZGERALD, EMILY HUANG, JOHN P. KIRWAN, Cleveland, OH Metformin (Met) is the most commonly prescribed antidiabetic agent worldwide. Although it has been marketed in the U.S. for over two decades, the exact molecular mechanisms of Met remain controversial. In addition to suppression of hepatic gluconeogenesis, increasing evidence suggests Met induces extrahepatic tissue-specific effects, such as in skeletal muscle (SKM), where the mitochondrion is believed to be the primary cellular target. We investigated the effects of Met on mitochondrial pathways in human SKM cells isolated by percutaneous needle biopsy from lean control, obese nondiabetic, and type 2 diabetic (T2D) volunteers. Mitochondrial function was assessed by Seahorse XF24 analyzer in SKM cells treated with or without Met for 24 hours. Glycolytic rate increased in Met-treated SKM cells from lean (P<0.01), obese (P<0.0001), and T2D subjects (P<0.01) compared to untreated cells. In contrast, basal respiration and ATP production were dramatically reduced in SKM cells treated with Met (P<0.0001 for all phenotypes). Evaluation of the insulin signaling pathway revealed increased Akt phosphorylation at Ser473 (P<0.05) and Thr308 (P<0.01) in cells pre-treated with Met compared to insulin stimulation alone, indicating an additive effect of Met on insulin action. As expected, muscle cells incubated with Met showed enhanced AMPK phosphorylation at Thr172 (P<0.001). Recent studies have identified a direct link between AMPK activation and mitochondrial dynamics pathways. We therefore assessed changes in markers of mitochondrial fission and fusion and found that Met treatment significantly induced phosphorylation of mitochondrial fission factor (MFF; P<0.01) in human SKM cells. Our data suggest Met may mediate improvements in glycemic control by activating AMPK to directly regulate the mitochondrial fission machinery and alter cellular bioenergetics. Functional morphometric and biochemical studies are ongoing to validate mechanisms by which Met exerts its antihyperglycemic effects in SKM. ORALS Supported By: National Institutes of Health (R01DK108089)

158‑OR Sex Differences in Insulin Sensitivity Are Related to Muscle Tissue Supported By: National Key Technology R&D Program of China; National Natu- Acylcarnitines and Serum Lysophosphatidylcholines but Not Sub‑ ral Science Foundation of China cellular Lipid Distribution in Humans JOSIANE L. BROUSSARD, LEIGH PERREAULT, SEAN A. NEWSOM, DARCY E. 156‑OR KAHN, ANNA KEREGE, KATHLEEN A. HARRISON, BRYAN BERGMAN, Boulder, Epigenetic and Metabolic Changes in Skeletal Muscle Underlying CO, Aurora, CO, Corvallis, OR the Improvement of Insulin Sensitivity after Bariatric Surgery in Sex differences in insulin sensitivity are present throughout the lifespan, Humans though mechanisms that account for these differences are unclear. Accumula- SOFIYA GANCHEVA, MERIEM OUNI, CHRYSI KOLIAKI, TOMAS JELENIK, DAN- tion of diacylglycerol (DAG) and sphingolipids in skeletal muscle are thought to IEL F. MARKGRAF, JULIA SZENDROEDI, MATTHIAS SCHLENSAK, ANNETTE promote insulin resistance, and localization plays an important role. Other lip- SCHUERMANN, MICHAEL RODEN, Düsseldorf, Germany, Potsdam, Germany, Ath- ids and lipid intermediates found in muscle and plasma such as acylcarnitines ens, Greece, Nuthetal, Germany also relate to insulin sensitivity. However, it is not known whether there are Weight loss-induced improvement in insulin sensitivity (IS) has been related sex differences in accumulation of these lipids that may affect insulin sensitiv- to enhanced muscle energy metabolism. Thus, we hypothesized that weight ity. We evaluated insulin sensitivity and sub-cellular localization of skeletal loss due to bariatric surgery may induce epigenomic changes, which in turn muscle DAG and sphingolipids, as well as muscle acylcarnitines and serum modify mitochondrial function and intracellular lipids. We previously reported lipidomics, in 25 obese, nondiabetic men and women (13F). Insulin sensitiv- that IS fails to improve at 2 weeks (2 w), but then continuously increases until ity was assessed with a hyperinsulinemic-euglycemic clamp. Muscle biopsies 52 weeks (52 w) after surgery. Now, we monitored muscle mitochondrial func- were taken during basal and insulin stimulated conditions and fractionated tion and lipid intermediates in 49 obese humans (OBE; 40±10 years, BMI 51±7 into sub-cellular compartments. Lipids were measured using LC/MS/MS.

ADA-Supported Research

A42 PREDICTION AND PREVENTION OF CARDIOVASCULAR DISEASE

Insulin sensitivity was significantly lower in men (p=0.05), however we found including T2DM and baseline CAD (HR 0.55 [95% CI 0.40-0.76], p<0.001). The no sex-differences in localization of DAG or sphingolipids in skeletal muscle. inclusion of betatrophin to a basic prediction model for the cardiovascular Men had higher total acylcarnitines in muscle tissue (p<0.05) and higher serum event risk significantly increased model performance (NRI=0.188, p<0.01). lysophosphatidylcholines (LPCs; p<0.01), and both were correlated with insulin In conclusion, this study for the first time shows that betatrophin predicts sensitivity (r=-0.42 and -0.43, respectively; p<0.05). After correcting for mul- cardiovascular events independently from conventional risk factors includ- tiple comparisons, the linoleoylcarnitine species (18:2AC) was significantly ing the presence of T2DM. elevated in men during insulin stimulation (p=0.001), and was negatively associated with insulin sensitivity (r=-0.43; p<0.05). Combined, these data suggest 161‑OR differences in content of acylcarnitines between sexes consistent with dif- Predictive Utilities of Blood Pressure Variables on Coronary Artery ferences in fatty acid processing and oxidation that may impact insulin sen- Disease in Type 1 Diabetes sitivity. Further, greater serum LPC species may play an important role in the JINGCHUAN GUO, TREVOR J. ORCHARD, Pittsburgh, PA decreased insulin sensitivity associated with male sex. The relative importance of different blood pressure indices on cardio- Supported By: National Institutes of Health (RR00036, R01DK089170, vascular risk in type 1 diabetes (T1D) has not been established. We thus T32DK007658, P30DK048520) compared the strengths of associations between different baseline and most recent blood pressure measures (systolic pressure [SBP], diastolic 159‑OR pressure [DBP], pulse pressure [PP], mean arterial pressure [MAP], and IRAK-1/IRS-1 Signaling Cross Talk in Human Skeletal Muscle— mid-blood pressure [Mid-BP, the average of SBP and DBP]) and the 25-year Role in Insulin Resistance risk of coronary artery disease (CAD) in the Pittsburgh Epidemiology of Dia- THEODORE P. CIARALDI, SUNDER MUDALIAR, LIWU LI, ROSARIO SCALIA, XIAO betes Complications (EDC) study of childhood onset T1D (N=605, without JIAN SUN, ROBERT R. HENRY, MICHAEL QUON, La Jolla, CA, San Diego, CA, baseline CAD). CAD was defined as fatal coronary artery event, non-fatal Blacksburg, VA, Philadelphia, PA, Baltimore, MD myocardial infarction, angina, revascularization, or ischemic ECG. Baseline Interleukin-1 receptor-associated kinase 1 (IRAK-1) is downstream from mean age and diabetes duration were 27 and 19 years, respectively. In Cox IL-1 receptors and TLRs. We identified IRS-1 (pSer24) as the first physiologi- Proportional Hazards modes, the baseline age-adjusted hazard ratios (HR) cal substrate for IRAK-1. Decreased binding of IRS-1 (pS24) to PI 3-kinase associated with one increment in SD for the risk of CAD events (n=219) were impairs insulin action. IRAK-1 k/o mice have improved metabolic insulin 1.49 (95% CI: 1.31, 1.70) for SBP; 1.46 (1.28, 1.67) DBP; 1.26 (1.09, 1.46) PP; action. Currently unknown is the relevance of IRAK-1/IRS-1 interactions to 1.50 (1.32, 1.71) MAP and 1.51 (1.32, 1.71) for Mid-BP. There was an interac- human pathophysiology. To investigate this topic, we examined human skel- tion between PP and HbA1c that the age-adjusted HR of PP was 1.20 (0.99, etal muscle (SkM) from NGT (n=10), IGT (n=6), and T2D (n=12) (1st cohort). 1.45) and 1.38 (1.07, 1.77) in those with HbA1c ≤ 9 and > 9%, respectively. Fasting protein expression of IRAK-1 and phospho-IRAK-1 (p-IRAK-1, activity Though area under the receiver operating characteristic curve (AUC) was proxy) and IRS-1 (pS24) did not differ significantly across groups. Insulin infu- significantly lower for PP compared with the other four BP indices in over- sion during hyperinsulinemic glucose clamp (HGC), caused acute reductions all subjects, the predictive utility of PP for CAD becomes similar to that in IRS-1 (pS24) in NGT that was diminished in IGT, and absent in T2D (p < 0.05, of other indices in those with higher HbA1c levels (>9%). Using the most vs. NGT). p-IRAK-1 was unaltered during HGC in 1st cohort. Acute insulin- recent data and adjusting for age, the predictive value of SBP was superior induced reduction of IRS-1 (pS24) in SkM was also found in women with PCOS to DBP (∆ AUC=0.037, p=.006), MAP (∆ AUC=0.021, p=.018), and Mid-BP (∆ (2nd cohort, n=12). Insulin sensitivity improved after pioglitazone treatment AUC=0.014, p=.031), but not PP (∆ AUC=0.007, p=.552). In sum, SBP appears (pio, 6 mo). This was associated with reduction in IRS-1 (pS24) (both fasting to be the strongest determinant of CAD risk in T1D patients overall but PP and HGC (acute insulin stimulation); (vs. pio pre-treatment). In a 3rd cohort, becomes a determinant as strong as SBP in older age and in those with nondiabetic (n=13) and T2D subjects (n=13), associations between IRAK-1 worse glycemic control, consistent with PP reflecting vascular stiffness. phosphorylation in the fasting state and measures of insulin sensitivity were Supported By: National Institutes of Health noted: HOMA-IR (r=0.57, p=0.01; consistent with HGC data from PCOS pre- and post-pio, and trend in 1st cohort). 162‑OR Summary: 1.) p-IRAK-1 in SkM (activity surrogate), predicts insulin resis- A Cost Analysis of Intensified vs. Conventional Multifactorial Ther‑ tance (IR) in human subjects. 2.) Ability of acute insulin stimulation to reduce apy of Patients with Type 2 Diabetes—The Steno 2 Study 24 IRS-1 (pS ) is absent in T2D and improved after pio intervention in PCOS. JOACHIM GAEDE, JENS OELLGAARD, RIKKE IBSEN, PETER GÆDE, EMIL NOER- 24 3.) Changes in IRAK-1 phosphorylation of IRS-1 (S ) represents cross-talk TOFT, JAKOB KJELLBERG, OLUF PEDERSEN, SR., Copenhagen, Denmark, Slagelse, between immune signaling and insulin signaling that may cause IR in human Denmark, Aarhus, Denmark, Søborg, Denmark 24 SkM. Our findings suggest that IRS-1 (pS ) and p-IRAK-1 are biomarkers of IR Introduction: Follow-up at 21.2 years after the initiation of the Steno-2 that may be molecular targets for therapy of IR in human metabolic diseases. study, demonstrated that intensified multifactorial intervention increases Supported By: U.S. Department of Veterans Affairs median life-span with 7.9 years and delays incident cardiovascular disease (CVD) with a median of 8.1 years compared to conventional multifactorial intervention. Here we aimed to analyse the direct medical costs in the two PREDICTION AND PREVENTION OF

original treatment groups during 21.2 years of follow-up. ORALS CARDIOVASCULAR DISEASE Methods: In 1993, 160 Danish patients with type 2 diabetes and microalbuminuria were randomised to receive either conventional or intensified and 160‑OR target-driven multifactorial intervention for 7.8 years. Information on direct Betatrophin Predicts Cardiovascular Events Independently from the health costs was gathered from health registers and any difference of costs Presence of Type 2 Diabetes and Coronary Artery Disease in the two groups was assessed by non-parametric bootstrap t-test analysis. ANDREAS LEIHERER, AXEL MUENDLEIN, KATHRIN GEIGER, CHRISTOPH H. Results: Intensified treatment was on average more expensive regarding SAELY, EVA-MARIA BRANDTNER, JANINE EBNER, BARBARA LARCHER, ARTHUR drug prescriptions, but less expensive in primary health sector services (both MADER, PETER FRAUNBERGER, HEINZ DREXEL, Triesen, Liechtenstein, Feldkirch, p<0.0001) and in-patient admission costs (p=0.02), specifically related to CVD Austria, Berne, Switzerland, Philadelphia, PA (p<0.0001) during the entire follow-up period. There was no significant differ- Betatrophin, also known as ANGPTL8 or lipasin is a nutritionally-regulated ence in total costs between the intensified treatment group, $13.0M and the protein secreted by the liver and adipose tissue. It is associated with type 2 conventional treatment group, $12.3M (p=0.19). When further assessing the diabetes mellitus (T2DM) and lipid metabolism. Whether betatrophin is asso- cost per patient year there was no significant difference between the intensi- ciated with the risk for cardiovascular events is unknown and is addressed fied group, $9,648, and the conventional treatment group, $10,681 (p=0.13). in the present study. We measured betatrophin in 553 patients undergoing Conclusion: Over an average follow-up of 21.2 years we found no signifi- coronary angiography for the evaluation of established or suspected stable cant increase in total costs or in costs per person year associated with inten- coronary artery disease (CAD) and prospectively recorded cardiovascular sified multipronged treatment for 7.8 years when compared to conventional events in these patients during a follow-up period of up to 8 years. During multipronged treatment. Considering the substantial gain of years of life and follow-up, 301 cardiovascular events occurred. The incidence of cardiovas- health benefits achieved with intensified treatment we conclude that inten- cular events was significantly higher in patients with T2DM (n=161) than in sified multifaceted intervention in high-risk patients with type 2 diabetes is those who did not have diabetes (47.2% vs. 34.4%; p=0.005). Betatrophin highly cost-effective in a Danish health care setting. was significantly and inversely associated with cardiovascular events both Supported By: Novo Nordisk A/S univariately (HR 0.64 [95% CI 0.47-0.87], p=0.004) and after full adjustment

ADA-Supported Research

A43 PREDICTION AND PREVENTION OF CARDIOVASCULAR DISEASE

163‑OR 165‑OR Both Diabetes Duration and Cumulative Hyperglycemic Exposure SGLT2 Inhibitors and Stroke Risk in Patients with Type 2 Diabetes— Promote Myocardial Fibrosis and Increase Coronary Microvascular A Systematic Review and Meta-analysis Resistance Evaluated by Comprehensive Myocardial CT Perfusion MAN GUO, YONG XU, CHENLIN GAO, Luzhou, China SHENGPU CHOU, MIKIKO HARAGUCHI, YUSUKE FUJINO, NOBUO TOMIZAWA, Aims: SGLT2 inhibitors are a novel drug used to treat diabetes. They TAKESHI NOJO, SUNAO NAKAMURA, Chiba, Japan also have positive cardiovascular benefits. However, there is some debate Background: Diabetes cardiomyopathy is raising attention recently, how- regarding possible effects of SGLT2 inhibitors on stroke risk. Here, we ever, there is few clinical study to evaluate changes in the early stage before conduct a meta-analysis of relevant RCTs to evaluate the effects of SGLT2 clinical signs appear. The present study investigated the risk of diabetes inhibitors on stroke risk with T2DM. duration and cumulative hyperglycemic exposure (CHE) to myocardial extra- Materials and Methods: We searched Pubmed, Embase, CENTRAL and cellular volume (ECV), a marker of myocardial fibrosis, and coronary micro- ClinicalTrials.gov from database inception up to September 19, 2017. We vascular resistance (CMVR) assessed by dynamic myocardial CT perfusion included RCTs that enrolled patients with T2DM, compared SGLT2 inhibitors (DMCTP). vs. placebo or other comparators for at least 24 weeks, and reported the Material and Methods: We assessed 192 patients who had undergone observed effects of SGLT2 inhibitors on stroke events. Subgroup analyses DMCTP from April 2016 to October 2017. Patients with significant coronary were performed for gender, age, duration of diabetes, race, BMI, and HbA1C stenosis and previous stent intervention were excluded. ECV of myocardium levels. A random-effects meta-analysis was performed to calculate the RR was calculated from CT delay enhancement subtraction image. CMVR was with 95% CI. derived from the diastolic pressure gradient divided by myocardial blood Results: We identified 32 eligible trials enrolling 75,540 participants that flow. We defined HbA1c debt as sum of elevated HbA1c in recent 10 years compared three SGLT2 inhibitors (canagliflozin, dapagliflozin, and empa- as the indicator of CHE. The impacts of diabetes duration and HbA1c debt to gliflozin) to placebo and other active antidiabetic treatments. The incidence ECV and CMVR were analyzed by multivariable analysis. of stroke in groups with SGLT2 inhibitor monotherapy or combination ther- Results: Diabetic patients showed significant increase of ECV (30.4±4.4% apy was not significantly different from that in control groups, with RR=1.01 vs. 25.9±3.8%, p <0.05) and CMVR (32.2±22.6 vs. 18.6±7.0, p <0.01) com- (95% CI 0.93-1.10, p=0.972) and RR=1.0 (95% CI 0.92-1.09, p=0.846), respec- pared with those without diabetes. Diabetes duration was positively related tively. There were no significant differences in RRs between three SGLT2 to ECV (r=0.313) and CMVR (r=0.411) respectively. A positive correlation inhibitors; canagliflozin, RR=0.91; dapagliflozin, RR=0.99; and empagliflozin, also existed between HbA1c debt and ECV (r=0.373), and CMVR (r=0.442) RR=1.03. Subgroup analyses showed that the RR values were not affected respectively. Grouping patients in relation to diabetes duration presented by gender, age, duration of diabetes, BMI, and HbA1C levels; however, there that longer duration elevated ECV and CMVR significantly (p<0.01). Elevation was a small racial disparity, with black participants having a lower incidence of HbA1c debt was also related to increase of ECV and CMVR (p<0.01). of stroke than white or Asian participants. Conclusion: Diabetes induces myocardial fibrosis and increases microvas- Conclusions: SGLT2 inhibitor therapy does not increase stroke incidence, cular resistance since the very early stage. Both longer diabetes duration and no significant differences in the RR for stroke were observed among and CHE are risk factors to the progression of microcirculatory disorders, three SGLT2 inhibitors (class effect). However, black patients did have a suggesting that good glycemic control is warranted to reduce risk of diabe- lower stroke incidence than white or Asian patients. tes cardiomyopathy. 166‑OR 164‑OR Serum Fibroblast Growth Factor-21 Is Related to Atherosclerosis SGLT2 Inhibitors Suppress NLRP3 Inflammasome Activity via Independent of Nonalcoholic Fatty Liver Disease and Predicts Inci‑ Changes in Ketones and Insulin in Type 2 Diabetes and Cardiovas‑ dent Atherosclerotic Cardiovascular Diseases cular Diseases LIANG WU, LINGLING QIAN, HUATING LI, WEIPING JIA, Shanghai, China YONG-HO LEE, SO RA KIM, JAEHYUN BAE, BYUNG-WAN LEE, EUN SEOK KANG, Fibroblast growth factor 21 (FGF-21), a key hepatokine regulating lipid CHUL WOO AHN, BONG-SOO CHA, Seoul, Republic of Korea, Goyang, Republic metabolism, is related to several atherosclerotic diseases. But whether this of Korea relationship is mediated by nonalcoholic fatty liver disease (NAFLD) is not SGLT2 inhibitors significantly reduce cardiovascular events in humans clear. Here we assessed the association of serum FGF-21 with atheroscle- with T2D; however, the underlying mechanism remains unclear. Activation rosis in non-NAFLD subjects and further investigated prospectively whether of NLRP3 inflammasome and subsequent IL-1β release induces atheroscle- baseline FGF-21 could predict incident atherosclerotic cardiovascular dis- rosis and heart failure. Recently, it was revealed that ketone bodies, i.e., ease (ASCVD) in a 7-year community study. Serum FGF-21 were measured β-hydroxybutyrate (BHB) suppresses activation of NLRP3 inflammasome in in a cross-sectional cohort of 371 type 2 diabetic patients without NAFLD as macrophages. As SGLT2 inhibitors cause increases in serum BHB by phar- determined by hepatic magnetic resonance spectroscopy, and a population- macologic profile, we assessed the effect of SGLT2 inhibitor on NLRP3 based prospective cohort of 705 subjects from the Shanghai Diabetes Study. inflammasome activity. In a randomized, active-controlled study, a total of In the cross-sectional study, serum FGF-21 was significantly higher in those 61 patients with T2D and high cardiovascular risk (mean age and HbA1C were with subclinical carotid atherosclerosis (268.8 [139.0-415.2] vs. 188.3 [100.2- 64.4 years and 7.32%, respectively) received SGLT2 inhibitor or sulfonylurea 376.4] pg/ml, P=0.005). Multiple logistic regression analysis showed that ORALS for 30 days. NLRP3 inflammasome activation was analyzed in macrophages serum FGF-21 was independently associated with atherosclerosis. In the and the serum levels of glucose, BHB, and insulin from baseline to the end of prospective study, baseline serum FGF-21 was significantly higher in those treatment were tested. While SGLT2 inhibitor’s glucose-lowering capacity who developed ischemic heart disease (479.5 [302.4-627.0] pg/ml, P=0.004) was similar to sulfonylurea, it significantly decreased IL-1β secretion com- or cerebral infarction (401.6 [238.3-616.4] pg/ml, P=0.021) than those who pared to baseline (2,394 ± 236 to 1,748 ± 295 pg/mL, p <0.001), whereas did not (325.2 [189.0-498.9] pg/ml) during the follow-up. In multivariable Cox sulfonylurea had no effect on IL-1β secretion (2,273 ± 279 to 2,755 ± 331 regression analysis, baseline serum FGF-21 independently predicted incident pg/mL, p = 0.05) (time × group interaction p <0.001). SGLT2 inhibitor caused ASCVD events and significantly improved discriminatory and reclassifying a significant increase in fasting serum BHB and decrease in serum insulin, abilities of the prediction model after adjustment for established cardio- while sulfonylurea had no significant effects on these measurements. We vascular risk factors. This study provides the first evidence that FGF-21 is performed ex vivo experiments using human macrophages to investigate elevated, independent of NAFLD, in subjects with subclinical atherosclero- whether BHB and insulin could affect NLRP3 inflammasome activity. BHB sis. Baseline FGF-21 is an independent predictor of incident ASCVD and could dose-dependently inhibited IL-1β secretion from macrophages. However, be utilized as a novel biomarker for primary prevention in general population. co-treatment with insulin attenuated the inhibitory effect of BHB on NLRP3 Supported By: National Natural Science Foundation of China/National Health inflammasome activation. and Medical Research Council of Australia (81561128016); National Natural In conclusion, SGLT2 inhibitors attenuate NLRP3 inflammasome activa- Science Foundation of China (81220108006, 81200292); Hong Kong Scholars tion, in part, via increased serum BHB and decreased serum insulin and Program (XJ2013035); Shanghai Pujiang Program (17PJ1407500); Municipal Human glucose, which might help to explain the cardioprotective effects of SGLT2 Resources Development Program (2017YQ009) inhibitor (clinicaltrials.gov NCT02964572). Supported By: National Research Foundation of Korea (2016R1A5A1010764); Korea Health Technology R&D Project (HI17C0913)

ADA-Supported Research

A44 ENHANCING THE USE OF DIABETES PREVENTION AND CARDIOVASCULAR RISK FACTOR CONTROL

167‑OR decision making in prediabetes may be a promising approach to enhance Metformin Regresses Left Ventricular Hypertrophy in Normoten‑ prevention efforts among patients at highest risk. sive Patients with Coronary Artery Disease without Type 2 Diabetes Supported By: National Institutes of Health (R18DK105464) Mellitus—The MET-REMODEL Trial MOHAPRADEEP MOHAN, SHAWEEN AL-TALABANY, ANGELA M. MCKINNIE, 169‑OR IFY MORDI, JAGDEEP S. SINGH, STEPHEN J. GANDY, ANNAMARIA CHOY, JOHN Medicare Diabetes Prevention Program Final Rules May Limit Ben‑ G. HOUSTON, JACOB GEORGE, ALLAN D. STRUTHERS, CHIM C. LANG, MET- eficiary Access and Widen Health Disparities REMODEL INVESTIGATORS, Dundee, United Kingdom NATALIE RITCHIE, MARK GRITZ, Denver, CO, Aurora, CO Background: Left ventricular hypertrophy (LVH) is highly prevalent in Introduction: The Centers for Medicare and Medicaid Services recently patients with coronary artery disease (CAD) and is an independent predictor issued final rules for the Medicare Diabetes Prevention Program (MDPP), of cardiovascular mortality. Metformin has been shown to regress LV mass offering an unprecedented opportunity to provide lifestyle intervention to (LVM) in animal models of LVH. We hypothesize that metformin may regress the estimated 48.3% of seniors with prediabetes. The MDPP is based on the LVH in nondiabetic and normotensive CAD patients with prediabetes and/or widely disseminated National Diabetes Prevention Program (NDPP), which insulin resistance. has lesser but still beneficial risk-reduction outcomes among racial/ethnic Methods: In this randomized double-blind placebo controlled trial, 68 minority participants. We assess the extent to which the MDPP’s pay-for- patients with prediabetes (HbA1c ≥39 mmol/mol and less than 48 mmol/ performance reimbursement model may cover costs of service delivery to mol) and/or insulin resistance (fasting insulin resistance index ≥ 2.7) were diverse Medicare beneficiaries. assigned to receive either metformin (2g daily dose) or placebo for 12 months. Methods: Denver Health Medical Center is a safety net healthcare sys- An intention-to-treat (ITT) and per-protocol analysis was designed to deter- tem that provided the yearlong NDPP to 232 diverse Medicare beneficiaries mine the effect of metformin on the following study endpoints: Primary between 2013-2017, primarily through grant funding. Based on their NDPP endpoint was change in left ventricular mass indexed to height1.7 (LVMI), attendance and weight loss outcomes, we compared projected perfor- assessed by magnetic resonance imaging; other endpoints were changes mance-based-payments (e.g., $160 for ≥5% weight loss) to service costs. in LVM, changes in body weight, office blood pressure (BP) and biomarkers. Results: Participating Medicare beneficiaries were 40.6% Hispanic, Results: In the ITT analysis (n=61), metformin treatment significantly 31.6% non-Hispanic black, and 26.9% non-Hispanic white. Only 4.7% of reduced: LVMI (metformin -2.7 ± 2.3 g/m1.7 vs. placebo -1.4 ± 2.7 g/m1.7; beneficiaries achieved all attendance and weight loss outcomes associated P=0.05), body weight (lowered by 3.6 kgs, p=0.002), office systolic BP (met- with the maximum $470 payment for services in months 1-12. Overall ben- formin -4.8 ± 15.6 mmHg vs. placebo 4.6 ± 15.7 mmHg; P=0.02) and reduced eficiary performance would result in an average reimbursement of $138.52 concentration of thiobarbituric acid reactive substances (TBARs), a bio- (SD=142.58). Program delivery costs were $800.64 per-participant, leaving marker for oxidative stress (p=0.04). In the on-per protocol analysis (n=56), an average gap of $662.12 per beneficiary served. metformin resulted in a greater reduction of LVMI (metformin -3.1 ± 1.9 Discussion: Numerous MDPP suppliers are needed to reach Medicare ben- g/m1.7 vs. placebo -1.2 ± 2.7 g/m1.7; P=0.005), and greater weight reduction eficiaries with prediabetes, yet insufficient reimbursement is a likely deterrent. of 4.2kgs (p=0.001). Health disparities may also widen as suppliers serving diverse populations Conclusions: Metformin treatment significantly reduced LVMI, office SBP, will likely receive especially low payments, threatening their MDPP delivery. body weight and oxidative stress. These results reveal a novel mechanism Higher payments appear needed and are supported by strong return-on- for the cardioprotective effect of metformin and raise the possibility of using investment findings. A risk-adjusted model that accounts for demographic dif- metformin in nondiabetic patients with CAD. ferences in outcomes is also needed to promote health equity. Supported By: British Heart Foundation (PG/14/4/30539) Supported By: Colorado Department of Public Health & Environment; America’s Health Insurance Plans/Centers for Disease Control and Prevention; Denver Health

ENHANCING THE USE OF DIABETES PREVENTION 170‑OR AND CARDIOVASCULAR RISK FACTOR CONTROL Cost-Effectiveness of Structured Lifestyle Programs for Diabetes Prevention in the Medicaid Population 168‑OR MICHAEL LAXY, PING ZHANG, HUI SHAO, BOON PENG NG, MOHAMMED K. ALI, Shared Decision-Making for Diabetes Prevention—One-Year EDWARD W. GREGG, Atlanta, GA Results from the Prediabetes Informed Decision and Education Background: The Diabetes Prevention Program (DPP) showed that struc- (PRIDE) Study tured lifestyle interventions can delay or prevent onset of type 2 diabetes TANNAZ MOIN, NORMAN TURK, CAROL MANGIONE, YELBA CASTELLON- in high risk individuals. Despite disproportionate diabetes burdens in low- LOPEZ, KIA SKRINE JEFFERS, KEITH C. NORRIS, CHIHONG TSENG, O. KENRIK income individuals, there are no data regarding expected health and eco- DURU, Los Angeles, CA nomic impacts of structured diabetes prevention in the Medicaid population. Lifestyle change (e.g., the DPP) and metformin can reduce diabetes risk. Objective: To examine the net costs and health benefits of extending cov- Real-world uptake of both strategies remains low. In a pragmatic, cluster- erage for structured lifestyle programs to the Medicaid population.

randomized controlled trial, we tested the effectiveness of a shared Methods: We modelled a prevention strategy consisting of triannual ORALS decision-making (SDM) intervention. After an in-person SDM visit with a screening for prediabetes (FPG≥100 mg/dl or A1c≥5.7%) followed by a pharmacist, overweight patients with prediabetes (A1c 5.7-6.4%) chose to DPP-like lifestyle program vs. routine care in non-disabled U.S. Medicaid start DPP, metformin, both strategies, or continue usual care. We random- beneficiaries aged 25-64 years. Medicaid population characteristics were ized 20 primary care clinics to intervention vs. usual care, and enrolled 364 extracted from 2006-2016 NHANES surveys. Costs of diabetes and its com- study patients with prediabetes from the 10 intervention clinics. We used plications were estimated from Medicaid administrative claims data of 8 propensity score matching to identify 1,086 comparison patients within the states comprising 52% of the U.S. adult Medicaid population. Based on 10 usual care clinics. The primary outcome was uptake of any diabetes pre- DPP-like studies in Medicaid populations, we assumed the intervention will vention strategy (DPP +/- metformin) within 8 months of the SDM visit. A reduce diabetes incidence by 32% and will be delivered at costs defined in secondary outcome was change in weight at 1 year after the SDM visit. We the Medicare Payment Scheme. We estimated incremental health benefits used data from the electronic health record (EHR) together with data from 2 (in quality-adjusted life-years (QALYs)) gained and net costs (in 2016 U.S. local DPP vendors to assess DPP attendance. We used data from the EHR to dollars) of screening and delivering lifestyle programs and subtracted sav- track metformin uptake and weight change, and ran generalized estimating ings through prevention of diabetes from a Medicaid perspective over a 10 equation models that controlled for clustering by clinic. Among 364 inter- year time horizon. vention patients, 57% chose DPP, 17% chose DPP + metformin, 9% chose Results: Given a 25% screening and intervention uptake in approximately metformin, and 17% chose to continue usual care. Intervention patients 27 million non-disabled Medicaid beneficiaries across 50 states, extending were more likely to join the DPP and/or take metformin than matched coverage could prevent or delay 135,000 cases of diabetes, gain 243,000 controls, 43% (n=155) vs. 3% (n=36; p<.001). In a complete-case analysis QALYs, and cost $1.35 billion (incremental cost effectiveness ratio $55,000 among patients with follow-up weight available in the EHR, weight loss was per QALY gained). greater for all intervention patients who had an SDM visit (n=312) vs. control Conclusions: Extending Medicaid coverage for life-style diabetes preven- patients (n=841) at 12 months (-5.2 lbs vs. -0.29 lbs, p<.001). A prediabetes tion programs might be a good use of health care resources and will poten- SDM intervention can enhance patient engagement in evidence-based ther- tially diminish the diabetes burden in low income individuals. apies to prevent diabetes and can promote weight loss at 12 months. Shared Supported By: Commonwealth Fund

ADA-Supported Research

A45 ENHANCING THE USE OF DIABETES PREVENTION AND CARDIOVASCULAR RISK FACTOR CONTROL

171‑OR Table. Cost-Effectiveness of Structured Lifestyle Intervention in Over‑ weight and Obese Adults with Type 2 Diabetes—Results from the Action for Health in Diabetes (Look AHEAD) Study PING ZHANG, HAIYING CHEN, MARK ESPELAND, RENA R. WING, MARIA G. MONTEZ, PETER J. HUCKFELDT, MARY EVANS, EDWARD W. GREGG, WILLIAM C. KNOWLER, Atlanta, GA, Winston-Salem, NC, Providence, RI, San Antonio, TX, Minneapolis, MN, Washington, DC, Phoenix, AZ Look AHEAD is a multicenter randomized clinical trial designed to examine the effects of an intensive lifestyle intervention (ILI), compared with diabetes support and education (DSE), for reducing cardiovascular events in 5,145 adults with overweight/obesity and type 2 diabetes. Here we reported the cost-effectiveness of the ILI, measured in costs per quality-adjusted life year (QALY), relative to DSE as implemented during the 10-year intervention period of the trial. Health utility (HU) values were measured using HU Index-2, HU Index-3, SF-6D and Feeling Thermometer. Cost were measured in 2012 U.S. dollars. All data were collected prospectively. QALYs were calculated as the product of predicted survival probability and predicted health utility values. Cumu- lative total cost was calculated as a sum of intervention cost and health expenditures, each of which was calculated as the product of predicted sur- Supported By: National Heart, Lung, and Blood Institute vival probability and predicted intervention cost and health expenditures. The estimated total and incremental QALYS and costs, and cost-effec- 173‑OR tiveness ratios (CER) are presented in the Table. ILI was likely to be not cost New 2017 ACC/AHA Hypertension Guidelines—Cost-Effectiveness effective over the 10 year intervention period. Long term health benefits among U.S. Adults with Type 2 Diabetes from the follow-up of the study participants can provide important informa- HUI SHAO, MICHAEL LAXY, DEBORAH B. ROLKA, EDWARD W. GREGG, PING tion regarding the long-term cost-effectiveness of ILI. ZHANG, Atlanta, GA, Munich, Germany Introduction: The new 2017 American College of Cardiology (ACC) and Table. American Heart Association (AHA) hypertension guideline defines hyperten- Measurements DSE ILI ILI-DSE CE Ratio sion as either systolic blood pressure (SBP) > 130 mmHg or diastolic blood (95% CI) (95% CI) (95% CI) (95% CI) pressure (DBP) > 80 mmHg, compared with the old guideline of SBP >140 HUI-2 (QALYs) 6.08 6.10 0.03 — mmHg or DBP > 90 mmHg. The new definition would increase number of (6.03, 6.13) (6.05, 6.15) (-0.04, 0.09) persons eligible for hypertension treatment. Our objectives were to estimate HUI-3 (QALYs) 5.94 5.94 0 — how many more persons with type 2 diabetes would be eligible for hyperten- (5.88, 5.99) (5.87, 5.99) (-0.08, 0.07) sion treatment under the new guideline and to assess the cost-effectiveness SF-6D (QALYs) 5.92 5.99 0.07 101,635 (CE) of treating this newly eligible population. (5.88, 5.96) (5.95, 6.03) (0.02, 0.12) (42567, 320360) Method: Data from the 2011-2016 National Health and Nutrition Exami- nation Survey and the 2015 Census were used to estimate the numbers of Feeling 6.03 6.18 0.15 45,487 thermometer (5.98, 6.07) (6.67, 6.77) (0.10, 0.21) (23270, 81851) persons with diabetes who would be eligible for hypertension treatment (QALYs) under the new and old guidelines. The CDC-RTI cost-effectiveness diabetes simulation model was used to project the lifetime health and cost (2017 Total cost ($) 78,466 85,490 7,024 — (76324, 80708) (83341, 87666) (4121, 9932) USD) consequences of treating newly eligible persons. Two hypertension treatments were modeled: moderate BP treatment as defined in the guide- Supported By: National Institutes of Health lines and intensive BP treatment using multiple antihypertensive drugs as implemented in the UK Prospective Diabetes Study trial. The CE of the new guideline was measured using costs per quality-adjusted life year (QALY). 172‑OR Results: The number of persons with diabetes eligible for hypertension A Comparison of the Impact of the 2017 ACC/AHA Hypertension treatment would increase from 8.4 (27.7%) to 15.7 million (51.9%) under the Guidelines on Primary Care Populations With and Without Diabetes new ACC/AHA guideline. Treating hypertension persons defined by new JOANN M. SPERL-HILLEN, JEFFREY P. ANDERSON, JAY R. DESAI, KAREN L. guideline would result in $3038/QALY if the moderate BP treatment were MARGOLIS, A. LAUREN CRAIN, PATRICK J. O’CONNOR, Bloomington, MN, implemented and would be cost-saving if intensive BP treatment were Minneapolis, MN implemented, compared to treating persons defined through old guideline. ORALS Background: Our objective was to assess the impact on primary care of Conclusion: The new ACC/AHA hypertension guideline would nearly dou- the 2017 ACC/AHA lowering of the threshold for diagnosing HTN to a BP ble the number of persons with diabetes who would be eligible for hyperten- > 130/80 mm Hg, and pharmacologic treatment intensification recommen- sion treatment. Treating those newly eligible is likely to be cost-effective or dations for patients with high CV risk and/or DM who have BP levels 130- even cost-saving. 139/80-89 mm Hg. Discussion: 62% of patients with DM and 57% without DM had a BP > 130/80 mm Hg, and over half of these will require reclassification of HTN 174‑OR due to BP 130-139/80-89 mm Hg. Of patients with Stage 1 HTN, 76.8% of Are the Favorable Cardiovascular Outcomes of Empagliflozin Treat‑ patients with DM and 39.4% without DM are already treated (but above ment Explained by Its EffectsWITHDRAWN on Multiple Cardiometabolic Risk goal). 25% of all DM patients (7755/30,608) and 13% of all non-DM patients Factors? A Simulation of the Results of the EMPA-REG OUTCOME (22963/176,555) are currently treated with Stage 1 HTN and will require an Trial increase in BP medication to achieve BP < 130/80 mm Hg.7.7% of all DM SHIHCHEN KUO, WEN YE, JUSTIN N. DUONG, WILLIAM H. HERMAN, Ann Arbor, patients (2344/30,608) and 2.3% of all non- DM patients with CV risk > 10% MI (3960/176,555) are currently untreated with Stage 1 HTN and medication Background: It is unclear whether the improved cardiovascular outcomes initiation will be recommended. If pharmacologic treatment initiation recom- (CVOs) observed in the EMPA-REG OUTCOME trial was due to the effects of mendations were limited to those with CV risk > 10% for DM patients, BP empagliflozin on multiple cardiometabolic risk factors (CRFs). medication initiation recommendations would decrease from 7.7% to 2.9% Methods: We used the Michigan Model for Diabetes, a validated com- (896/30,608) of the DM population. puter simulation model, and published data from the EMPA-REG OUTCOME Conclusion: The 2017 ACC/AHA guideline will result in a significantly trial to estimate three-year CVOs in the placebo and pooled empagliflozin increased need for primary care to address BP medication intensification, treatment groups to assess the proportion of the observed benefits that with a much greater proportional impact on those with DM compared to might be attributable to differences in multiple CRFs. those without. Results: When we programmed the model to match the baseline characteristics of the trial population and the reported trajectories of five CRFs

ADA-Supported Research

A46 DIABETES COMPLICATIONS AND RISK FACTORS

(weight, HbA1c, systolic blood pressure, and low- and high-density lipo- to factor in the significant correlation among macronutrient intake. In the protein cholesterol), the simulated hazard ratio (HR) did not differ from the UK Biobank, dietary data were collected from over 210,000 individuals aged reported result for the primary composite CVO. The simulated HRs for fatal/ 37-74 using the Oxford WebQ, a web-based 24 hour diet recall. For multi-trait nonfatal myocardial infarction (MI) and coronary revascularization proce- GWAS, we combined GWAS association evidence of ~11m imputed variants dure also fell within the reported 95% confidence intervals (CIs), but the in participants of European ancestry (n =192,005) for percent of carbohydrate, simulated HRs for fatal/nonfatal stroke, hospitalization for heart failure, fat, and protein adjusted for age and sex by applying the CPASSOC package. cardiovascular death, and all-cause mortality fell outside the reported 95% We identified 14 independent genome-wide significant (P<5 x 10-8) loci for CIs. The beneficial effects of empagliflozin on CRFs accounted for 43%, 62%, macronutrient intake. We discovered loci associated with lipid metabolism and 50% of the observed relative risk reductions (RRRs) for the primary com- (APOE, GCKR, SLC39A8, PPP1R3B, MLXIPL), alcohol intake (ADH1B, KLB), posite CVO, fatal/nonfatal MI, and coronary revascularization procedure, but and new candidate genes (CCDC171, PSMD7, ARL17B, METTL4), and repli- explained smaller proportions of the observed RRRs for hospitalization for cated previously associated loci with dietary intake (FGF-21 and FTO). Of the heart failure, cardiovascular death, and all-cause mortality. 14 signals, 12 replicated (P<0.0036) in multi-trait analyses using previous Conclusion: The effects of empagliflozin on multiple CRFs account for GWAS on dietary intake from the CHARGE Consortium (n =91,114). Tissue some but not all of the reduced risk of CVOs in the EMPA-REG OUTCOME enrichment analysis across 53 tissues in GTEx revealed enrichment of gene trial. More comparable control of established CRFs in type 2 diabetes CVO expression in the cerebellum. Genome-wide genetic correlations across 234 trials of antidiabetic agents with pleiotrophic effects would facilitate the traits with publicly available GWAS data indicated shared biological links interpretation of the observed outcomes. between dietary intake and lifestyle factors (smoking and education) as well Supported By: National Institute of Diabetes and Digestive and Kidney Diseases as type 2 diabetes and coronary artery disease (P<2.14x10-4). The present (P30DK092926) findings expand our understanding of the genetic architecture of dietary intake and the shared genetic links with related traits. 175‑OR Ten-Year Trends in Consumer-Directed Health Plan Enrollment and 177‑OR Out-of-Pocket Costs among Commercially Insured Members with Use of Oral Contraceptives at Child-Bearing Age Are Associated Diabetes with the Prevalence of Diabetes in Postmenopausal Women LAURA F. GARABEDIAN, DENNIS ROSS-DEGNAN, FANG ZHANG, MATTHEW SUNG-WOO KIM, REBECCA KIM, EONJU JEON, JI-HYUN LEE, HO-SANG SHON, X. CALLAHAN, ROBERT LECATES, JAMIE WALLACE, STEPHEN B. SOUMERAI, Daegu, Republic of Korea, Gumi, Republic of Korea JAMES F. WHARAM, Boston, MA, Waban, MA Objective: The effect of past use of oral contraceptives (OCs) at child- Background: Diabetes is a major cause of morbidity and mortality and a bearing on the incidence of diabetes and insulin resistance (IR) after meno- driver of escalating health care costs in the U.S. In attempts to reduce costs, pause is not clearly elucidated. This study aimed to evaluate the association U.S. employers are increasingly offering consumer-directed health plans of past use of OCs with the development of diabetes and IR in post-meno- (CDHPs) that include Health Savings Accounts (HSA) or Health Reimburse- pausal women. ment Arrangements (HRA) and high annual deductibles. Recent research Methods: This cross-sectional study was based on data from the Korea has found that CDHPs are associated with concerning impacts on diabetes National Health and Nutrition Examination Survey from 2007 to 2012. Of patients, but growth in CDHP enrollment and their out-of-pocket (OOP) cost the 50,405 participants, 6554 post-menopausal women were selected and trends in this vulnerable population are unknown. included in the analysis. The long-term effects of OCs use on the prevalence Methods: We used a time series design to assess 2005-2014 annual of diabetes in post-menopausal women were examined using multivariate trends among ~70 million unique members of all ages insured by a large logistic analysis. In addition, fasting glucose and insulin levels were mea- national commercial insurer from 2005-2014. We analyzed mutually exclu- sured in 3338 nondiabetic post-menopausal women, and the association sive populations of interest, identified by ACG, comprising: (1) diabetes between IR and OCs was examined by analysis of covariance. mellitus with no cardiovascular disease (DM), (2) diabetes with co-morbid Results: The prevalence of diabetes was significantly higher in post-meno- cardiovascular disease (DM+CVD), and (3) “healthy” (i.e., ACG score <=1). pausal participants who had taken OCs for more than 6 months than in those We used linear models, t-tests and chi-square tests to assess (a) CDHP and who had never taken OCs. The association remained significant after adjust- non-CDHP prevalence and (b) total annual OOP costs in 2014 dollars. ing for multiple confounding factors (odd ratio, 1.379; 95% CI, 1.115-1.707; Results: CDHP prevalence among DM members increased by 2.5% per P=0.003). The duration of OCs use was also positively associated with the year (p<0.001), from 1.3% in 2005 to 23.2% in 2014. DM+CVD members had prevalence of diabetes. Furthermore, taking OCs for more than 6 months led a similar increase. In 2014, 19.5% of DM+CVD members and 28.7% of health- to a significant increase in fasting insulin levels and HOMA-IR in nondiabetic ier members were in CDHPs (p<0.001 for both compared to DM). Annual OOP participants. costs for non-CDHP members were significantly lower than for CDHP mem- Conclusion: Past use of OCs for more than 6 months led to a significant bers throughout the study period. For example, in 2014, CDHP DM members increase in the prevalence of diabetes in post-menopausal women, and had average OOP costs of $2103.62 compared to $1379.87 for non-CDHP DM increased IR in nondiabetes participants. These results suggested that members (p<0.001). prolonged use of OCs at a reproductive age is an important risk factor for Conclusions: CDHP enrollment has increased markedly over the last 10 developing diabetes in post-menopausal women. years among members with and without diabetes. However, diabetes mem- ORALS bers were less likely than healthier members to be in CDHPs, particularly if 178‑OR they had co-morbid CVD. Among diabetes patients, OOP costs were signifi- Diabetes and Liver Cancer Risk—A Stronger Effect in Whites than cantly higher for CDHP than non-CDHP members. Blacks? Supported By: Centers for Disease Control and Prevention; National Institute of BAQIYYAH CONWAY, WILLIAM J. BLOT, Tyler, TX, Nashville, TN Diabetes and Digestive and Kidney Diseases (1U18DP006122) Both diabetes and liver and biliary tract cancer are overrepresented among African Americans, but limited information is available on the inter- relationship of these two diseases among African Americans. We tested DIABETES COMPLICATIONS AND RISK FACTORS the relationship of diabetes with the incidence of liver cancer and whether this relationship varied between blacks and whites. Using the Southern 176‑OR Community Cohort Study, we conducted a cancer follow-up (2002-2015) of a Multitrait Genome-Wide Association Analysis of Macronutrient cohort of mostly low income black and white participants aged 40-79 with Intake Identifies 12 Novel Loci for Dietary Intake and Unravels (n=17,644) and without diabetes (n=64,870) at cohort entry. Mean age and Genetic Overlap with Lifestyle Traits and Cardiometabolic Diseases diabetes duration of those with diabetes was 55.0 and 9.5 years, respec- HASSAN S. DASHTI, JORDI MERINO, JACQUELINE M. LANE, JOSE C. FLOREZ, tively. Mean age of those without diabetes was 51.6 years. Logistic regres- MARTIN K. RUTTER, RICHA SAXENA, Boston, MA, Manchester, United Kingdom sion was used to compute ORs (95% CIs) for the risk of incident liver and Suboptimal diets are associated with increased risk of type 2 diabe- biliary tract cancer. There were 429 incident cases of these cancers. In uni- tes, cardiovascular disease and cancer. Genome-wide association studies variate analyses, diabetes was associated with an increased risk of liver and (GWAS) for individual macronutrients have facilitated the discovery of two biliary tract cancer (OR=1.73, 95% CI=1.36-2.21). Upon further controlling for relevant loci (FGF-21, FTO), however a single-nutrient approach may preclude age, sex, race, BMI, current and former smoking, total alcohol consumption, the discovery of additional relevant loci. Here, we conducted a multi-trait and any hepatitis infection, diabetes remained a significant risk factor for macronutrient GWAS for carbohydrate, fat, and protein in the UK Biobank liver and biliary tract cancer (OR=1.49, 95% CI= 1.18-1.89). However, when

ADA-Supported Research

A47 DIABETES COMPLICATIONS AND RISK FACTORS

stratified by race, risk associated with diabetes was significantly greater Studies were selected if they had a prospective design. Studies that com- among whites (OR = 2.67, 95% CI = 1.71-4.19) than blacks (OR= 1.28, 0.98- pared BC incidence in women with diabetes to the incidence in the general 1.68) (pinteraction=.002). Furthermore, controlling for diabetes greatly attenu- population were excluded. Summary relative risks (SRR) were computed ated the higher risk of liver and biliary tract cancer among blacks; indeed, using a random-effect model. Eighteen studies were included in the meta- while the cancer risk among those without diabetes was twice as high analysis, including 28,230,143 person-years of follow-up and 320,111 BC among blacks than whites (OR=2.01, 95% CI = 1.50-2.69), no racial disparity cases. A significantly increased risk of BC among diabetic patients compared was observed among those with diabetes (OR = 0.95, 95% CI= 0.62-1.45). to nondiabetic patients was found: SRR=1.13 (95% CI: 1.04, 1.24). There The findings raise the possibility of markedly different impacts of diabetes was a large amount of unexplained heterogeneity of results across stud- on hepatic carcinogenesis between blacks and whites. ies (I²=95%), but no indication of publication bias. The SRR remained similar Supported By: National Institutes of Health; National Cancer Institute when the analysis was restricted to post-menopausal women (SRR=1.12 (95% CI: 0.99, 1.26)). Only two studies reported data in pre-menopausal 179‑OR women. In the 9 studies that adjusted for adiposity, both the SRR and the Adiposity and Cardiovascular Markers in Offspring of Women with heterogeneity of results across studies substantially decreased (SRR=1.05 Diabetes during Pregnancy—The EPOCH Study (95% CI: 0.97, 1.14), I²=21%). In contrast, in the 9 studies that did not adjust CHRISTINE HOCKETT, ANNE P. STARLING, ANNA BELLATORRE, JENNY AAL- for adiposity, both the SRR and the heterogeneity increased (SRR=1.19 (95% BORG, KATHERINE A. SAUDER, KAVITA GARG, BRANDY RINGHAM, DEBORAH CI: 1.01, 1.39), I²=98%). Only two studies included 1,723 and 14 BC cases H. GLUECK, DANA DABELEA, Aurora, CO among type 1 diabetic women. The RR for BC were 0.90 (95% CI: 0.85, 0.94) We previously showed that exposure to maternal diabetes mellitus (DM) and RR=1.01 (95% CI: 0.60, 1.71), respectively. This analysis provides evi- affects adiposity and cardiovascular (CV) markers in pre-pubertal children. dence for a moderately increased risk of BC in diabetic patients. The effect After further follow-up, we are now testing whether DM exposure continues of the adjustment for BMI on the SRR and on the heterogeneity suggests to affect these markers throughout adolescence. Data on 381 youth (313 that the increased BC risk appears to be linked to adiposity, and not to being unexposed, 68 exposed) were collected at two research visits, on average at diabetic. New studies should collect data allowing the assessment of Breast 10.4 and 16.6 years, respectively. Measures included BMI, waist circumfer- Cancer risk according to the duration of diabetes and treatments that favour ence, height, visceral (VAT) and subcutaneous adipose tissue (SAT), systolic weight loss. and diastolic blood pressure and fasting lipid levels. Linear mixed models were used to assess the longitudinal relationship between DM exposure and 181‑OR offspring outcomes, adjusting for child age, sex, race/ethnicity, and Tanner Impact of Diabetes Age of Onset and Duration on Outcomes stage. BMI, VAT and SAT were log-transformed. We tested for effect modifi- JUDY ZHONG, JINKYUNG HA, CAROLINE BLAUM, MOHAMMED KABETO, cation of exposure by visit. Exposure to DM was associated with markers of CHRISTINE CIGOLLE, New York, NY, Ann Arbor, MI adiposity and CV risk throughout adolescence (Table), and for most outcomes Older adults vary in age of onset and duration of diabetes (DM) but are effects were not different across visits. Youth exposed to DM had higher treated similarly. We hypothesized that age of DM-onset has differential non-HDL cholesterol at the second visit (β=0.30, p<0.001), but not at the first impact on distal outcome risk. visit (β=0.12, p<0.23) (interaction p<0.045). Adjustment for SES, child’s total We analyzed waves 1993-2014 of the Health and Retirement Study, a calories and physical activity did not influence the results. Our data provides nationally-representative health survey where we can study people before novel evidence that exposure to maternal DM has persistent effects on off- and after DM diagnosis for ~ 20 years. Our sample included adults >50 years spring adiposity and CV markers throughout adolescence. (n=27,975) without DM at entry. Age of DM-onset was defined as age when Table. respondent first reported DM diagnosis. We classified adults who developed DM into 3 groups: <65, 65-75, >75. For each group, we constructed a propensity-score-matched control group of respondents who never develop DM. We compared risks of outcomes for each onset-age group and control: cardiac disease, stroke, mild disability (1-2 ADL/IADL dependencies), severe disability (>2 ADL/IADL dependencies), death. At <65 years 1,283 respondents developed diabetes, 1,440 at 65-75, and 1,149 at >75; 21,994 never developed DM. Adults diagnosed with DM at <65 years had substantial increased relative risk of: cardiac 1.3, stroke 1.5, mild disability 1.5, severe disability 1.7, death 1.5, (p<0.001 for each). Respondents diagnosed at older ages had moderate increases with similar duration (Table). Age of DM diagnosis differentially impacts key outcome risks in older adults and has more impact than duration. Findings reinforce clinical heterogeneity in DM and the focus to improve DM management in middle aged adults.

ORALS Table.

Supported By: National Institutes of Health

Supported By: National Institutes of Health 182‑OR Population Screening for T1D and Celiac Disease—Autoimmunity 180‑OR Screening for Kids (ASK) The Risk of Breast Cancer in Women with Diabetes CRISTY R. GENO RASMUSSEN, MARIAN REWERS, JUDITH BAXTER, KATHLEEN MARIA BOTA, PHILIPPE AUTIER, PETER BOYLE, Écully, France, Lyon, France WAUGH, ANDREA STECK, BRIGITTE I. FROHNERT, LIPING YU, EDWIN LIU, Aurora, Some observational studies have suggested an increased risk of breast CO, Denver, CO cancer (BC) among diabetic patients. A meta-analysis was performed to Early detection can prevent morbidity associated with type 1 diabetes assess the risk of BC in diabetic patients compared to nondiabetic patients. (T1D) and celiac disease (CD). ASK is a 4-year program with the goal to screen

ADA-Supported Research

A48 MOLECULAR MECHANISMS OF INSULIN ACTION AND RESISTANCE IN PERIPHERAL TISSUES

Denver metro-area children for pre-symptomatic T1D and CD, increase public MOLECULAR MECHANISMS OF INSULIN ACTION awareness of these diseases, and provide evidence for universal screening. AND RESISTANCE IN PERIPHERAL TISSUES We are reporting first-ever prevalence data for pre-symptomatic T1D and CD in the U.S. general population children aged 2-17 years. In 2017, ASK approached ~15,000 children for consent, screened 5090 184‑OR with results available for 4915. Study participant age, sex, and race/ethnic- Hepatic Insulin Signaling Regulates Hepatokines to Coordinate ity closely reflected Denver’s general population; 4% had a T1D first-degree Energy Expenditure and Weight Gain through Irs2 in Sim1 Neurons relative (FDR). Standard radiobinding assays (RBA) and more specific elec- RONGYA TAO, KYLE D. COPPS, OLIVER STOEHR, MORRIS F. WHITE, Boston, MA trochemiluminescence (ECL) assays for autoantibodies to insulin, GAD, IA-2, Insulin resistance is the underlying cause of type 2 diabetes; however, ZnT8 and transglutaminase (TGA) were used for screening and confirmation. insulin resistance might also be a feed-back mechanism to protect cells and tissues from acute effects of over nutrition. Based upon our results with Children with confirmed persistent islet autoantibodies received follow-up L/L L/L Albumin with education to prevent DKA, psychological support, and referrals to pre- LDKO (hepatic specific Irs1 Irs2 Cre ) mice, we found that hepatic vention trials or clinical services. insulin resistance elevated systemic oxygen consumption and energy Multiple islet autoantibodies, predicting a 44% 5-year risk of T1D, were expenditure, which was associated with a dramatic reduction of the RER found in 32 (0.7%) children; of the 30 children retested to date, all remained (respiratory exchange ratio) and resistance to diet-induced obesity. Deletion persistently positive, six developed dysglycemia and one progressed to T1D. of hepatic FoxO1 in LDKO-mice restored energy homeostasis and oxygen A single islet autoantibody, confirmed as high-affinity by ECL (29% 5-year consumption, and increased the RER into the normal range. Contrary to the risk of T1D), was found in 27 (0.5%); 18/19 children retested remained progressive peripheral insulin resistance of the LDKO-mice, Irs2 expression positive, 5 developed dysglycemia and one T1D. Over 86% (50/59) of the and insulin sensitivity increased in the PVH (paraventricular nucleus of hypothalamus). Interestingly, deletion of Irs2 in the PVH (Irs2L/L-mice intercrossed screening-detected children at risk for T1D did not have an FDR with T1D. The Sim1 prevalence of TGA by both RBA and ECL was 2.2% and persistent in 74/76 with Cre -mice) increased body weight and promoted obesity. To iden- (97%) children retested so far. tify the molecular cross-talk between liver and brain, we used a neuronal This novel population-based screening program for the two most common cell line to screen for the effects of hepatokines that were dysregulated autoimmune diseases of childhood reports high prevalence of pre-symp- in LDKO-mice—but normalized in LTKO-mice. Among these dysregulated tomatic T1D and CD in Denver children. Prospective follow-up of screening- hepatokines, excess circulating NTF3 and GPX3 led to increased Irs2 expres- detected cases for clinical outcomes and cost-effectiveness analysis will sion and insulin sensitivity in neurons. Thus, our study suggests that hepatic inform potential future universal screening. FoxO1-dependent hepatokines—NTF3 or GPX3—modulate systemic energy Supported By: JDRF International; The Leona M. and Harry B. Helmsley Chari- expenditure and body weight through Irs2 expression in the PVH. table Trust; Janssen Research & Development, LLC 185‑OR 183‑OR Hepatic Insulin/Snail1 Axis Protects against Liver Steatosis and Risk Factors for Major Atherosclerotic Cardiovascular Events Insulin Resistance through Epigenetically Suppressing Lipogenesis (MACE) in Type 1 Diabetes (T1D)—The Pittsburgh Epidemiology of YAN LIU, NICOLE M. IRELAND, LIN JIANG, LIANGYOU RUI, Ann Arbor, MI Diabetes Complications (EDC) Study Insulin stimulates hepatic lipogenesis that drives nonalcoholic fatty RACHEL G. MILLER, TREVOR J. ORCHARD, Pittsburgh, PA liver disease (NAFLD). Paradoxically, insulin resistance is associated with The Diabetes Control and Complications Trial (DCCT)/Epidemiology of increased hepatic lipogenesis in obesity, and the underlying mechanism Diabetes Interventions and Complications follow-up study (EDIC) recently remains poorly understood. Here, we delineate an insulin-PI 3-kinase-Snail1 reported that after age mean HbA1c was the strongest predictor of MACE pathway that links metabolic signals to epigenetic reprogramming of de over 27 year of follow-up. The DCCT/EDIC cohort had a short T1D duration novo lipogenesis in the liver. We found that insulin robustly upregulated at baseline (mean 6 year) and those with high blood pressure or cholesterol Snail1 in both primary hepatocytes and liver in a PI 3-kinase-dependent man- were excluded so it is unclear if these results apply to longer T1D durations ner. Notably, the hepatic insulin-PI 3-kinase-Snail1 pathway was severely and without risk factor exclusion. We thus replicated these analyses in the impaired in obesity. Overexpression of Snail1 blocked, whereas deletion of Pittsburgh EDC study a prospective cohort study of childhood onset (<17 endogenous Snail1 enhanced, insulin-stimulated lipogenesis in hepatocytes. year) T1D with 25 year of follow-up and similar age (mean 27 year in both Hepatocyte-specific deletion of Snail1 exacerbated, whereas liver-specific studies) but longer T1D duration (mean 18 year) at baseline. EDC participants overexpression of Snail1 ameliorated, dietary NAFLD in mice. Ablation of were diagnosed 1950-1980 (n=658 49% women) and examined at 1986-88 hepatic Snail1 also attenuated NAFLD-associated insulin resistance and baseline. Follow-up exams occurred at 2, 4, 6, 8, 10, 18 and 25 years. MACE glucose intolerance; conversely, liver-specific overexpression had the oppo- incidence (CVD death, MI, stroke) was ascertained by death certificate or site effect. Mechanistically, Snail1 bound to the fatty acid synthase (Fasn) self reported and confirmed with medical records. Participants with preva- promoter and repressed Fasn promoter activity through histone modifica- lent CVD were excluded (n=54). Baseline (BL), time varying updated mean tions. Our data suggest that insulin pathways bifurcate into the canonical (UM) and time varying most recent (MR) risk factors were assessed in Cox lipogenic and the noncanonical Snail1 two arms wherein the Snail1 arm puts models as in DCCT/EDIC. In EDC 18% (107) developed MACE vs. 6% in DCCT/ a brake on the lipogenic arm, curbing excessive lipogenesis, liver steatosis, EDIC. The final multivariable model for MACE comprised BL T1D duration and lipotoxicity. Thus, insulin-resistance-induced impairment in the Snail1 ORALS (HR 1.1, p<.001), MR albumin excretion rate (AER) (HR 1.3, p<.001), UM SBP arm contributes to increased lipogenesis in obesity. (HR 1.03, p<.001), BL smoking (HR 1.9, p=0.003), UM LDLc (HR 1.01, p=0.03) Supported By: National Institutes of Health; American Heart Association; and UM HbA1c (HR 1.2, p=0.03). In DCCT/EDIC the HbA1c effect size was National Natural Science Foundation of China larger while T1D duration, UM SBP, MR (not UM as in EDC) LDLc and MR (not BL as in EDC) smoking predicted with similar effect sizes. ACE inhibitors 186‑OR (protective) and pulse rate also predicted MACE in DCCT/EDIC. While AER Characterization of the G2L1 Interactome Leads to the Discovery was a strong predictor in EDC no renal markers were significant in DCCT/ That CLIP2, G2L1, and EB1 Undergo Insulin-Stimulated Phosphory‑ EDIC. These results suggest that at longer T1D duration much of the HbA1c lation effect on MACE risk is mediated through other factors as recently reported NATALIE K. BARKER, JAMES L. KRANTZ, SARA PARKER, GHASSAN MOUNEI- by DCCT/EDIC. MNE, PAUL R. LANGLAIS, Tucson, AZ Supported By: National Institute of Diabetes and Digestive and Kidney Diseases G2L1 is a member of the plus-end tracking (+TIP) microtubule (MT)-asso- (R01DK034818); Rossi Memorial Fund ciated protein family. G2L1 coordinates cooperativity between MTs and actin and was recently detected in an adipocyte CLASP2 interactome. Since CLASP2 has been linked to insulin action, and both actin and MTs play an unknown role in insulin action, we chose to study G2L1 within 3T3-L1 adipocytes by performing the first-ever G2L1 interactome. Using a commercially available antibody for G2L1 and two antibodies for epitope-tagged, overexpressed G2L1, we performed multiple affinity purification coupled with mass spectrometry experiments in combination with label-free quantitative proteomics and analysed the data with the bioinformatics tool Significance Analysis of Interactome. In concordance with findings in other cell types, we discovered that G2L1 co-IPs the master integrators of +TIP assembly,

ADA-Supported Research

A49 NOVEL SIGNALING PATHWAYS IN THE ISLET

the end-binding (EB) proteins, with a strong preference for EB1 over EB3 in of a 5-LO inhibitor reduced plasma levels of LTB4 and ameliorated insulin adipocytes. Subsequent EB1 interactome analysis confirmed reciprocal co-IP resistance and glucose intolerance in A-PDK1KO mice. Furthermore, insulin of G2L1. We discovered that G2L1 co-IPs the interaction partners CLASP2 was found to inhibit LTB4 production through downregulation of 5-LO expres- and CLIP2, both of which are also +TIP family members, data which was sion via the PDK1-FoxO1 pathway in isolated adipocytes. These results thus confirmed by IP and western blot. Real-time total internal reflection fluores- indicate that insulin signaling via the PDK1-FoxO1 pathway negatively regu- cence microscopy revealed colocalization of G2L1 and CLASP2 in adipocytes lates the production of LTB4 through the suppression of 5-LO in adipocytes at growing MT ends. Since CLASP2 undergoes insulin-regulated phosphory- and thereby maintains systemic insulin sensitivity. lation in adipocytes, we hypothesized that the G2L1/CLIP2/EB1 network of +TIPs are also candidates for involvement in insulin-regulated control of MT 189‑OR dynamics. Using targeted quantitative phosphoproteomics, we discovered Mfge8 Binding of the αvβ5 Integrin Promotes Insulin Resistance in that insulin stimulates the phosphorylation of G2L1 at Ser597/599, CLIP2 at Mice Ser552, and EB1 at Ser155. This now expands the novel network of insulin- RITWIK DATTA, AMIN KHALIFEH-SOLTANI, ARNOLD HA, KAMRAN ATABAI, San affected +TIP family members to CLASP2, G2L1, CLIP2, and EB1 and intro- Francisco, CA duces the new hypothesis that a coordinated network of MT-associated Milk fat globule EGF factor 8 (Mfge8) promotes obesity by inducing fatty proteins cooperate to mediate insulin-regulated MT dynamics. acid uptake through ligation of the αvβ5 integrin. Global deletion of Mfge8 Supported By: National Institutes of Health/National Institute of Diabetes and in mice increases insulin sensitivity through effects on lean body mass. The Digestive and Kidney Diseases primary objective of the present study was to assess whether acute disruption of the Mfge8 signaling pathway in wild type mice modulates insulin 187‑OR sensitivity independent of body composition. We therefore evaluated insulin Loss of Insulin/IGF-1 Receptors in Muscle Coordinately Downregu‑ sensitivity in age and sex-matched wild type mice with insulin (ITT) and glu- lates Mitochondrial Metabolism and Alters Mitophagy via Foxo cose tolerance tests (GTT) in the presence of a blocking antibody (ALULA) Transcription Factors targeting the β5 integrin. Intraperitoneal administration of 5mg/kg ALULA GOURAV BHARDWAJ, CHRISTIE M. PENNIMAN, PABLO A. SUAREZ BELTRAN, 1h prior to ITT resulted in significantly lower blood glucose levels compared COLLIN M. FOSTER, BRIAN T. O’NEILL, Iowa City, IA to control antibody treated mice. ALULA treated mice had a significantly Diabetes is associated with decreased muscle strength and energy pro- lower blood glucose levels in GTT. Serum insulin level measured during GTT duction. There is a strong association between muscle insulin resistance in ALULA treated mice was lower compared to control group, suggesting and mitochondrial abnormalities, but cause/effect remains hotly debated. enhanced sensitivity to insulin. We validated our findings in vitro using Recently, we showed that insulin receptor (IR) and IGF-1 receptor (IGF1R) differentiated 3T3L1 adipocytes and hepG2 liver cells. Both cell types had signaling controls muscle atrophy by FoxO1/3/4-dependent regulation of significantly higher uptake of the glucose analog 2NBDG after insulin treat- autophagy. To directly test the roles of IR, IGF1R, and FoxO signaling on mus- ment in presence of ALULA as compared with insulin treatment with control cle metabolism and mitochondrial autophagy (mitophagy) we measured mito- antibody. Insulin is known to induce glucose uptake in adipocytes through a chondrial function in mice lacking IR (M-IR-/-), both IR/IGF1R (MIGIRKO), or IR/ PI3K/Akt signaling pathway. β5 integrin blockade followed by insulin admin- IGF1R/FoxO1/3/4 (QKO) in muscle. RNA seq analysis revealed decreases in istration augmented insulin mediated phosphorylation of Akt and As160 in multiple mitochondrial pathways from M-IR-/- and MIGIRKO muscle, includ- white adipose tissue (WAT) and skeletal muscle (SM) in mice indicating that ing OXPHOS. Respiratory capacity and ATP production were decreased in the β5 integrin normally dampens insulin signaling. In preliminary data we mitochondria and soleus fibers isolated from M-IR-/- and MIGIRKO mice have found that after insulin treatment, the β5 integrin associates with the using complex I substrates. These decreases in OXPHOS and mitochondrial mature insulin receptor β (IRβ) in WAT and SM as shown in coimmunopre- function were normalized when FoxOs were deleted in QKO mice. MIGIRKO cipitation studies. Further mechanistic insight to this novel regulation of IRβ muscle showed decreased PINK1 (mitophagy protein) and increased LC3II/I by β5 integrin will direct us towards testing the therapeutic potential of ratio, indicating changes in respiration and mitophagy occurred together. To blocking β5 integrin producing insulin resistance in diabetes. determine if this was cell autonomous, C2C12 myotubes were pre-treated Supported By: National Institutes of Health; American Heart Association with insulin and/or CCCP (an uncoupler and mitophagy-inducer), and assayed for respiration. Insulin increased respiration and PINK1 levels after 24 hours. Four hour CCCP pre-treatment reduced basal respiration and increased NOVEL SIGNALING PATHWAYS IN THE ISLET LC3II/I which were partially, but significantly, rescued with insulin in C2C12 myotubes. PINK1 was also decreased in primary myotubes with acute dele- 190‑OR tion of IR/IGF1R. Thus, loss of IR/IGFR signaling in skeletal muscle directly Sensory Innervation of the Pancreatic Islet reduces mitochondrial respiratory capacity and alters mitophagy, which is MADINA MAKHMUTOVA, JONATHAN WEITZ, RAYNER RODRIGUEZ DIAZ, regulated by FoxO transcription factors. JOANA ALMACA, ERNESTO BERNAL-MIZRACHI, ALEJANDRO CAICEDO, Miami, FL 188‑OR Sensory innervation of viscera is an important component for proper

ORALS Insulin Signaling in Adipocytes Controls Systemic Insulin Sensitiv‑ maintenance of body homeostasis. Visceral stimuli are detected by free ity through the 5-LO-LTB4 Axis nerve endings of the vagus nerve and are transmitted to the hindbrain via TETSUYA HOSOOKA, WATARU OGAWA, Kobe, Japan sensory neurons. However, the signals that activate vagal sensory neurons Although adipocytes are major targets of insulin, the influence of impaired in the periphery are not known. The objective of this study is to identify insulin action in adipocytes on metabolic homeostasis has been unclear. To and characterize the population of vagal sensory neurons that innervates provide insight into the role of insulin action in adipocytes, we generated the pancreas. Using immunohistochemistry for sensory neuronal marker mice that lack 3’-phosphoinositide-dependent kinase1 (PDK1) specifically in substance P, we show that sensory innervation targets islet periphery. Upon adipocytes (A-PDK1KO mice). These mice manifest impairment of metabolic islet damage (in NOD model or following streptozotocin (STZ) treatment), actions of insulin in adipose tissue and a reduction in adipose tissue mass. density of islet sensory innervation increases. Expression of the immedi- The mice developed insulin resistance, glucose intolerance, and hepatic ate early gene c-Fos, shows that hindbrain neurons in the nucleus of the steatosis, all of which were markedly attenuated by additional ablation of solitary tract (NTS) respond to severe islet inflammation induced by STZ. FoxO1 specifically in adipocytes without an effect on adipose tissue mass, We assessed response profile of pancreatic sensory neurons using in-vivo indicating that the PDK1-FoxO1 axis in adipocytes plays an important role in calcium imaging of mouse vagal sensory ganglion (nodose). This method systemic metabolic homeostasis through a mechanism independent of the allows us to monitor in real time the response profile of sensory neurons regulation of adipose tissue mass. Neither circulating levels of adiponectin and identify pancreas-specific subpopulations by chemical stimulation of and leptin nor inflammation in adipose tissue differed between A-PDK1KO the exteriorized pancreas. We have identified sensory neurons that respond mice and adipocyte-specific PDK1/FoxO1 deficient mice (A-PDK1/FoxO1KO to pancreatic application of serotonin (5HT) and cholecystokinin (CCK). The mice), indicating that those factors did not account for the difference in met- observed response profile suggests that these neuronal populations are abolic phenotypes of the two genotypes of mice. Microarray and lipidomics tuned to sense differentially endocrine and exocrine pancreas. While endo- analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose crine sensory neurons are sensing local 5HT that is co-released with insulin, tissues as well as the expression of 5-lipoxygenase (5-LO), a rate limiting exocrine neurons sense CCK a hormone that stimulates release of diges- enzyme for LTB4 production, in adipose tissues were increased and normal- tive enzymes. Altogether our data suggest that pancreatic sensory neurons ized in A-PDK1KO and A-PDK1/FoxO1KO mice, respectively. Administration could be important regulators of pancreatic physiology. By taking a snapshot

ADA-Supported Research

A50 NOVEL SIGNALING PATHWAYS IN THE ISLET of endocrine and exocrine pancreatic microenvironments and sending this 193‑OR information to the brain, sensory neurons might trigger feedback loops and Assessment of Proinflammatory Mediators of Beta-Cell Circadian help the organ to be tuned to physiological needs. Clock Dysfunction in Diabetes Supported By: National Institutes of Health (R01DK113093, 01F31, DK11259602) NAUREEN JAVEED, KUNTOL RAKSHIT, ALEKSEY MATVEYENKO, Rochester, MN Both type 1 and type 2 diabetes are characterized by a decline in β-cell 191‑OR function and mass partly attributed to exposure of β-cells to proinflamma- Foxo1 Corepressor (FCoR) and Foxo1 Regulate α-Cell and β-Cell tory cytokines. The molecular mechanisms underlying this process are not Identity well understood, however several lines of evidence have linked the circadian NORIKO KODANI, JUN NAKAE, HIROSHI ITOH, Tokyo, Japan clock with regulation of the deleterious proinflammatory effects in diabetes. We previously identified a Foxo1 binding protein; Foxo1 CoRepressor Therefore, in this study, we assessed the effect of known proinflammatory (FCoR), in adipocytes. FCoR inhibits Foxo1 activity by enhancing its acety- mediators of β-cell failure in diabetes (IL-1β, TNFα, IL-6, and IFNγ) on the lation. FCoR is expressed in pancreatic α- and β-cells from the embryonic β-cell circadian clock. Through real-time bioluminescence tracking of islets stage. Fcor knockout mice (FcorKO) show impaired glucose tolerance, from Per2:luciferase (a key circadian clock gene reporter) knock-in mice, our decreased insulin secretion, increased glucagon secretion and increased data revealed that IL-1β (2 ng/ml) diminished β-cell circadian rhythmicity α-cell mass. We have shown that FCoR suppresses the expression of mas- via impairment of the amplitude and the phase of oscillations (p<0.05 vs. ter α-cell regulatory transcription factor Aristaless-related homeobox (Arx) vehicle). This was in contrast to TNFα, IL-6, and IFNγ, which showed either by increasing the methylation of the CpG-rich promoter region, while Foxo1 mild suppression or no effect on the β-cell clock. We next assessed the abil- induces Arx expression by hypomethylation of this area. These results sug- ity of proinflammatory cytokines to modulate Bmal1 and Clock (two main gest that FCoR and Foxo1 have an opposing action in the regulation of Arx transcription factors in the circadian pathway) gene and protein expression. expression and α-cell mass. We studied how Foxo1 regulates Arx expres- Interestingly, studies using INS-1 832/13 cells and cadaveric human islets sion. Arx promoter region contains a conserved forkhead responsive ele- revealed that IL-1β addition (0.2-5 ng/ml) for 24 hours significantly reduced ment (FRE). Foxo1 binds to the FRE and induces Arx expression. Binding of Bmal1 (but not Clock) at both the mRNA (~40-50%, p<0.05) and protein (10- Foxo1 to the Arx promoter dissociates binding of DNA methyltransferase 65%, p<0.05) level in a dose dependent manner. In contrast, other cytokines 3a (Dnmt3a), and this may result in hypomethylation of the CpG rich region. (IL-6, TNFα, and IFNγ) only modestly diminished Bmal1 expression after 24 Our results suggest that Arx is the target gene of Foxo1. Immunohisto- hours. In assessing a molecular link between IL-1β and the β-cell clock, chemical staining of FcorKO islets showed Foxo1 nuclear localization and Bmal1 promoter activity was determined using a BMAL1:luciferase express- decreased Foxo1 in the cytosol. In isolated islets from FcorKO, we observed ing INS-1 832/13 stable-transfected cell line. Importantly, IL-1β (0.01-5 significant deacetylation of Foxo1, and decreased endogenous Foxo1 protein ng/ml) showed a dose dependent decrease in Bmal1 promoter activation (up compared to control. These results are consistent with the previous finding to 90%). This body of work provides the first novel connection between dia- that deacetylated Foxo1 is the active form, and is quickly ubiquitinated and betogenic inflammatory cytokines, specifically IL-1β, as the dominant media- degraded. To study the relationship between FCoR and Foxo1, we gener- tor of β-cell clock dysfunction in diabetes. ated β-cell-specific Foxo1 knockout mice in the background of FcorKO (DKO). Supported By: National Institute of Diabetes and Digestive and Kidney Diseases DKO showed further aggravation of glucose tolerance and insulin secretion (DK098468); National Institutes of Health (2T32HL105355-06) compared to FcorKO. Foxo1 single knockout has normal glucose metabolism, thus, our results suggest that Foxo1 is activated to maintain β-cell function 194‑OR in FcorKO. We provide evidence that FCoR and Foxo1 function in harmoniza- Reduced β Cell SPCA1 Leads to Impaired Calcium Oscillations and tion to regulate pancreatic α-cell and β-cell identity. Decreased Autophagy Supported By: Japan Society for the Promotion of Science ROBERT N. BONE, TATSUYOSHI KONO, CARMELLA EVANS-MOLINA, Indianapo- lis, IN 192‑OR The β-cell Golgi, an important site of proinsulin maturation, is a significant 2+ 2+ Localization of ChREBPα and ChREBPβ in Pancreatic β Cells store of intracellular Ca . However, the contribution of Golgi Ca to diabe- LIORA S. KATZ, ANIL K. LAXMAN, PILI ZHANG, DONALD SCOTT, New York, NY, tes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Salt Lake City, UT Pathway Ca2+ ATPase (SPCA1) to maintain intra-Golgi Ca2+ stores. SPCA1 Glucose is a key physiological driver of adaptive β-cell mass expansion, expression was measured in islets from Akita and db/db mice and cadav- promoting β-cell proliferation both in vitro and in vivo. Carbohydrate response eric human islets from donors with T1D and T2D. In all cases, SPCA1 lev- element binding protein (ChREBP) is required for glucose-stimulated β-cell els were significantly lower compared to nondiabetic controls. Proteomics proliferation in both rodent and human β-cells. ChREBP has two splice iso- and Reverse Phase Protein Array performed in INS-1 β-cells lacking SPCA1 forms - α and β. Translation of the ChREBPβ mRNA generates a truncated (SPCA1KO) indicated dysregulated cell death, cell growth, and proliferation protein, resulting in a potent, constitutively active isoform. To study the role pathways compared to WT INS-1 cells. Consistent with this, SPCA1KO cells of the 2 splice isoforms in β-cell biology, we performed numerous experi- exhibited increased caspase-3/7 following tunicamycin treatment. SPCA1KO ments including immunostaining and chromatin immunoprecipitation with cells also had elevated basal cytosolic Ca2+ levels, decreased organelle Ca2+ reuptake following organelle Ca2+ store depletion, and accelerated Golgi antibodies that recognize only ChREBPα or both isoforms. We found that ORALS ChREBPα is only transiently expressed in the nucleus. By contrast, ChREBPβ, collapse. Despite a significant reduction in GSIS, SPCA1KO cells exhibited once expressed at high enough levels to detect, is constitutively nuclear. a 12-fold insulin content increase. Autophagy has been reported as a key Our data indicate that mice fed on high fat diet and diabetic db/db mice regulator in maintaining β-cell insulin homeostasis. Immunoblot revealed have abundant ChREBPβ localized in the nucleus while control mice have reduced expression of the autophagy marker LC3-II and the autophago- mostly cytoplasmic ChREBPα. We generated Ins-1 cell lines where we can some Atg5/Atg12 complex in SPCA1KO cells, following autophagy induction distinguish between the splice isoforms using CRISPR/Cas9: 1.) The C-termi- with Torin. Islets from mice haploinsufficient for SPCA1 (SPCA1+/-) exhibited nus of ChREBPα and ChREBPβ was labeled with eGFP; 2.) The N-terminus elevated cytosolic Ca2+ levels and impaired Ca2+ oscillations as reflected by of ChREBPα was labeled by mCherry; and 3.) ChREBPα was labeled with increased phase 1 amplitude and period, compared to WT littermates. In mCherry and eGFP on the N- and C- terminus, respectively and ChREBPβ is islets, restoration of SPCA1 to normal levels rescued the oscillatory defect. labeled with eGFP on the C-terminus. We validated the three cell lines by Interestingly, islets from SPCA1+/- mice did not exhibit increased insulin con- sequencing, ChIP, qRT-PCR and also showed that the glucose response is not tent, however, SPCA1+/- islets had decreased insulin secretion and increased altered in these cells. We found that in low glucose, ChREBPβ is present at numbers of immature insulin granules. Taken together, we find thatβ -cell very low levels in the nucleus. By contrast, in high glucose, ChREBPβ is pres- SPCA1 plays an important role in the maintenance of Golgi Ca2+ levels, while ent at high levels in the nucleus. Our study suggests that in response to glu- loss of SPCA1 led to dysregulated β-cell Ca2+ homeostasis. cose, ChREBPα, which is mostly cytoplasmic, transiently enters the nucleus, Supported By: National Institutes of Health; JDRF binds to a carbohydrate response element (ChoRE), and induces ChREBPβ expression, which is mostly nuclear. We propose that while the induction of ChREBPβ drives adaptive beta cell expansion, ChREBPβ contributes to glucose toxicity after prolonged exposure to high glucose and in diabetes. Supported By: National Institutes of Health (R01DK108905)

ADA-Supported Research

A51 NEW INSIGHTS INTO THE MECHANISMS OF DEFECTIVE HYPOGLYCEMIA COUNTERREGULATION IN DIABETES

195‑OR contacted by local cholinergic nerves, with an innervation density that was Deletion of OGlcNAc Transferase in Pancreatic Progenitors Causes equivalent to that of beta cells. To study the impact of local cholinergic Partial Pancreas Agenesis and Diabetes Independent of p53 in Mice nerves on resident macrophages, we used living tissue pancreatic slices, EMILYN ALEJANDRO, DANIEL BAUMANN, SEOKWON JO, BRIAN AKHAPHONG, where activity of islet cells, nerves, and macrophages can be visualized in a AMBER D. LOCKRIDGE, RAMKUMAR MOHAN, Minneapolis, MN, Saint Paul, MN preserved local microenvironment. We determined that Ca2+ responses to Deletion of the only nutrient-sensor enzyme, O-GlcNAc Transferase ATP were inhibited in islet macrophages by nicotine, an agonist of nicotinic (OGT), catalyzing O-GlcNAc addition onto target proteins in pancreatic acetylcholine receptors. These results indicate that macrophages receive β-cells induces diabetes through β-cell failure. We hypothesized that OGT, cholinergic input from parasympathetic nerves. We expect our studies to expressed at a very high level in β-cells, has key developmental regulatory identify parasympathetic nerves as a source of anti-inflammatory signals properties. In this study, we tested the hypothesis that OGT is required for that efficiently dampen the potentially pro-inflammatory environment in the pancreas and β-cell development by deleting OGT in pancreatic (Pdx1Cre, islet. OGTKOPanc) or endocrine (Ngn3Cre, OGTKOEndo) progenitors. We show that mice lacking OGT in pancreatic progenitors (Pdx1+, OGTKOPanc) show near pancreas agenesis and loss of β-cell mass at birth. Few mice that grow into NEW INSIGHTS INTO THE MECHANISMS OF adulthood develop overt diabetes due to functional β-cell mass failure. OGT DEFECTIVE HYPOGLYCEMIA COUNTERREGULATION ablation in endocrine progenitors (Ngn3+, OGTKOEndo) resulted in normal IN DIABETES pancreas mass but reduced β-cell mass and diabetes early in life, strongly revealing the temporal and importance of OGT for β-cell development. We 198‑OR observed loss of Pdx1 staining at birth and on embryonic (e) day 14.5 in OGT- Effects of Epinephrine on Atherothrombotic Responses and In Vivo KOPanc. Transcriptome analysis of e14.5 progenitor single cells show distinct Endothelial Function in Healthy Humans cell clustering populations in OGTKOPanc compared to littermate controls. MAIA MIKELADZE, MAKA HEDRINGTON, SOFIO DUMBADZE, VANESSA J. BRIS- Upstream regulator analysis of altered genes show enhanced activation of COE, DONNA TATE, EVAN THAYER, STEPHEN DAVIS, Baltimore, MD p53, also known as TP53 or tumor protein. Next we identified p53 to be In this study we have tested the hypothesis that levels of epinephrine O-GlcNAc-modified in -cells. Concomitant deletion of p53 in the OGTKOPanc β simulating those occurring during hypoglycemia of 70, 60, or 50 mg/dl will background (p53, OGTKOPanc) does not rescue pancreas agenesis in OGTKO- result in pro-coagulant and pro-atherothrombotic responses and endothelial Panc newborn, and adult p53, OGTKOPanc mice develop severe hyperglycemia dysfunction. and diabetes. These data highlight the novel role O-GlcNAc-modifications in 32 healthy humans (16M/16F) participated in 4 separate, single blind, pancreas and -cell development and provide a new model to characterize β randomized, 1-day studies: Placebo (Protoco1 1) and epinephrine infusions the developmental defects associated with pancreatic agenesis. of 0.015, 0.03 and 0.06µg/kg/min (Protocols 2-4) respectively. Studies Supported By: National Institutes of Health consisted of 2-hour hyperinsulinemic (133±7µU/ml) euglycemic clamps of 92±1mg/dl. 2D Doppler imaging determined flow mediated dilation (FMD) of 196‑OR the brachial artery. Role of ROCK1 in Pancreatic β Cells Increases of IL-6, P-selectin, E-selectin, ICAM-1, and VCAM-1 were higher BYUNG-JUN SUNG, KYU YEON HUR, JAE HYEON KIM, YOUNG-BUM KIM, and reductions in FMD and ET-1 greater during all epinephrine infusions com- MOON-KYU LEE, Seoul, Republic of Korea, Boston, MA pared to Placebo (p<0.05) (Table 1). Insulin signaling in pancreatic β-cell is critical for normal insulin secre- Epinephrine activated pro-coagulant, inflammatory and pro-atherothrom- tion. ROCK1 plays an important role in the regulation of insulin and leptin botic mechanisms while impairing endogenous and exogenous nitric oxide signaling in the context of glucose and energy metabolism. However, the mediated endothelial function. role of ROCK1 in pancreatic β-cell remains unknown. The current study was In conclusion, during hyperinsulinemic euglycemia, epinephrine levels to determine whether deleting ROCK1 from β-cell affects insulin secretion simulating those occurring during hypo of only 70mg/dl produced significant and glucose metabolism in mice and cultured cell lines. Here we show that inflammatory effects. Increasing doses of epinephrine resulted in greater β-cell-specific deletion of ROCK1 results in a significant decrease insulin inflammatory responses and in-vivo endothelial dysfunction (p<0.001). Epi- secretion in mice fed a normal chow diet, leading to hyperglycemia. These nephrine may be an important mechanism responsible for adverse vascular effects were independent of changes in adiposity. Consistent with this, in biologic effects during hypo in healthy humans. vitro studies with INS-1 and MIN6 cells indicated that inhibition of ROCK1 decreased insulin secretion in response to glucose without changes in β-cell Table 1. Changes from Baseline to Final 30 Min. proliferation and apoptosis. In addition, β-cell-ROCK1 deficient mice dis- Protocol 1 Protocol 2 Protocol 3 Protocol 4 played glucose intolerance, as evidenced by the fact that the area under the Δ IL-6 (pg/ml) -3.0±1.2 2.8±2.9* 3.6±2.7* 8.1±2.9* glucose curve was increased 53% in β-cell-ROCK1 deficient mice compared with control mice. However, neither body weight, food intake nor insulin sen- Δ P-selectin (pg/ml) -13.7±6.5 1.3±0.8* 2.0±2.0* 5.6±1.8* sitivity was changed in theses mice. Upon insulin stimulation, proximal insu- Δ ICAM-1 (ng/ml) -18.7±12.3 24.1±13.3* 65.5±36.9* 114.3±69.6* lin signaling components, including p-IRS2, p-PI3K and p-Akt were impaired ET-1 (pg/ml) -0.2±0.05 -0.4±0.05* -0.5±0.12* -0.6±0.09* ORALS Δ when ROCK1 expression was inhibited in INS-1 cells, suggesting a role for Δ FMD (mean maximum%) 0.8±0.7 -2.0±0.5* -2.6±0.6* -3.7±0.7* ROCK1 in insulin signaling. These data indicate that activation of ROCK1 in pancreatic β-cell is required for the maintenance of normal insulin secretion. Δ Epinephrine (pg/ml) 18±2 299±38* 532±41* 1011±12* Thus, pancreatic ROCK1 might be a key regulator of glucose homeostasis. Mean±SEM. * p<0.0007-0.05 compared to Protocol 1.

197‑OR Anti-inflammatory Cholinergic Signals Inhibit Islet Resident Macro‑ 199‑OR phage Responses to ATP in Living Pancreatic Tissue Slices The Vagus Nerve Mediates Severe Hypoglycemia-Induced Fatal JONATHAN WEITZ, RAYNER RODRIGUEZ DIAZ, JOANA ALMACA, MADINA Cardiac Arrhythmias MAKHMUTOVA, ALEJANDRO CAICEDO, Miami, FL CANDACE M. RENO, ALLIE J. SKINNER, JUSTIN BAYLES, SIMON J. FISHER, Salt When stimulated by high glucose concentrations, beta cells release ATP Lake City, UT together with insulin. Under these conditions, the ATP concentration in the Severe hypoglycemia causes fatal cardiac arrhythmias. The extent islet reaches levels that would induce inflammatory responses in other tis- to which the parasympathetic nervous system regulates hypoglycemia- sues. Yet inflammation does not occur during normal islet physiology. We induced fatal arrhythmias remains unknown. To investigate the role of vagal therefore postulated that there is a local anti-inflammatory circuit that innervation in regulating arrhythmias during severe hypoglycemia, Sprague prevents pathological immune responses. Our studies focused on resident Dawley rats underwent vagotomy (left vagus nerve transection adjacent to macrophages, which are the local immune cells in the pancreatic islet. We the carotid artery; n = 15) or sham surgery (control; n = 13). One week after recently showed that islet macrophages detect ATP to gauge the level of surgery, all rats underwent hyperinsulinemic (0.2 U/kg/min) severe hypogly- beta cell activation. We hypothesized that the potentially inflammatory cemic (10-15 mg/dl) clamps with electrocardiogram recordings. Rats with responses to ATP are inhibited in macrophages by cholinergic input from vagotomy had increased heart rate at baseline and throughout the clamp, parasympathetic nerves, as reported for other organs. Using immunohis- indicating successful vagotomy. Glucose infusion rates during hypoglycemia tochemistry, we found that most islet resident macrophages were indeed were increased 1.7-fold in rats with vagotomy, but epinephrine values were

ADA-Supported Research

A52 NEW INSIGHTS INTO THE MECHANISMS OF DEFECTIVE HYPOGLYCEMIA COUNTERREGULATION IN DIABETES similar between the groups. Vagotomy resulted in fewer cardiac arrhythmias resonance spectroscopy at 7 T to measure (Glucose+Taurine)/ total Creatine including a 12-fold decrease in 2nd degree heart block. Reduction in arrhyth- in a 13x12x10 mm3 hypothalamic voxel in such patient groups while they mias was associated with a 7-fold decrease in severe hypoglycemia-induced were maintained at euglycemia and then hypoglycemia using a glucose mortality in rats with vagotomy (7%) compared to controls (46%). clamp. Since taurine and creatine do not change with glycemia, this is a valid In summary, vagal transection reduces mortality and cardiac arrhythmias measure of brain glucose concentrations. IAH was defined using the Clarke during severe hypoglycemia. Since vagal innervation mediates severe hypo- questionnaire with subjects found indeterminate further classified using the glycemia-induced sudden death, targeting the parasympathetic nervous Gold questionnaire. We found that hypothalamic (Glucose+Taurine)/ total system may be a logical approach to prevent sudden death for at-risk people Creatine was significantly higher in T1D with IAH at both euglycemia and with insulin treated diabetes. hypoglycemia when compared to T1D without IAH. These data support the Figure. hypothesis that a change in glucose transport may contribute to the pathogenesis of IAH. Table. T1D with IAH T1D without IAH (n=9) (n=9) Age (years) 47.6 ± 17 29.2 ± 8 Hemoglobin A1c (%) 7.3 ± 0.9 7.3 ± 0.7 Glycemia at euglycemia (mg/dl) 98 ± 4 95 ± 7 Glycemia during hypoglycemia (mg/dl) 48 ± 3 48 ± 5 Epinephrine during hypoglycemia (pg/ml) 250 ± 155 711 ± 442* Symptom score during hypoglycemia 8 ± 9 36 ± 14** Hypothalamic glucose at euglycemia 0.43 ± 0.11 0.27 ± 0.08** (Glucose+Taurine)/ total Creatine (mM) Supported By: National Institutes of Health (5T32DK091317, R01NS070235); Hypothalamic glucose at hypoglycemia 0.23 ± 0.04 0.15 ± 0.02** JDRF (3-APF-2017-407-A-N) (Glucose+Taurine)/ total Creatine (mM) *p=0.03, **p < 0.001. 200‑OR Carvedilol Prevents Counterregulatory Failure in Recurrently Hypo‑ Supported By: National Institutes of Health glycemic Rats RAWAD FARHAT, GONG SU, OWEN CHAN, Salt Lake City, UT 202‑OR We showed previously that recurring activation of β2-adrenergic recep- Identification of VMNCckbr Neurons as Crucial Mediators of the tors in the ventromedial hypothalamus (VMH) leads to the development of Counterregulatory Response to Hypoglycemia counterregulatory failure, in part by increasing local lactate levels. The cur- JONATHAN N. FLAK, AHSAN ANSARI, PAUL SABATINI, PAULETTE B. GOFORTH, rent study evaluates whether the non-selective β-blocker, carvedilol, can be CHIEN LI, DARLEEN A. SANDOVAL, DAVID OLSON, MARTIN G. MYERS, JR., Ann used to prevent the development of counterregulatory failure and improve Arbor, MI, Seattle, WA hypoglycemia awareness (HA) in recurrently hypoglycemic (RH) rats. While the goal of diabetes treatment is normoglycemia, most therapeu- Sprague-Dawley rats (n=6-10 per group) underwent surgery for the implan- tics (especially insulin and insulin secretagogues) carry with them a sig- tation of vascular catheters and intracranial guide cannulas that targeted nificant risk of potentially life-threatening hypoglycemia; this risk increases the VMH. These animals then received either 3 bouts of insulin-induced with the intensity of therapy. While neurons of the ventromedial hypotha- hypoglycemia (RH) over the course of 3 days or they received 3 injections of lamic nucleus (VMN) play an essential role in the CRR, the VMN contains saline (Controls). A subgroup of RH rats was treated with carvedilol (3mg/kg several populations of neurons that have distinct or even opposing effects, QID; IP) beginning one day prior to the start of RH and continuing through to and the neurons that mediate the CRR have not previously been molecularly the last episode of hypoglycemia. Following the last bout of hypoglycemia, defined. Since we previously showed that cholecystokinin (CCK) neurotrans- the animals underwent a hypoglycemic clamp with microdialysis to evaluate mission plays an important role in the VMN-mediated CRR, we generated changes in central lactate and hormone levels. Compared to controls, RH CCK receptor b (Cckbr)-cre mice to study the potential role of VMNCckbr increased VMH lactate by 1.7-fold (P<0.03) and this was associated with neurons in the CRR. In addition to other expected brain areas that contain an impaired counterregulatory response to hypoglycemia (glucagon and epi- Cckbr neurons, CckbrCre reporter mice identified a substantial population of nephrine responses were reduced by ~45% and ~73%, respectively, P<0.04), VMNCckbr neurons in the dorsomedial VMN. Consistent with a potential role as expected. Treatment of RH animals with carvedilol restored VMH lactate in the CRR, VMNCckbr neurons project to regions that control sympathetic levels and improved the counterregulatory responses to hypoglycemia. To outflow, including the BST, PAG, and preoptic area. Furthermore, expres- Cckbr

assess whether carvedilol improved HA, we treated rats that were made sion of tetanus toxin in VMN neurons to prevent their downstream neu- ORALS “hypoglycemia unaware” with repeated 2-deoxyglucose (2DG; 200mg/kg, rotransmission reduces blood glucose at baseline and impairs the CRR to SQ) injections with carvedilol and measured food intake in response to insu- insulin-induced hypoglycemia and glucoprivation in both male and female lin-induced hypoglycemia as a surrogate marker for HA. Compared to Con- mice. This effect is unique to silencing this discrete population of VMN cells; trols, 2DG reduced food intake in response to hypoglycemia and treatment silencing all VMN neurons (SF1cre) does not alter the CRR, but instead results with carvedilol increased food intake in 2DG rats (P<0.01). We conclude that in obesity and hyperglycemia. Thus, VMNCckbr neurons play a crucial role in carvedilol may be a useful therapeutic strategy to prevent counterregulatory the CRR, and their dysfunction may underlie hypoglycemia associated auto- failure and improve HA in RH rats. nomic failure. Supported By: National Institutes of Health; JDRF Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-17- INI-15 to J.N.F.); National Institutes of Health; Novo Nordisk 201‑OR Hypothalamic Glucose Concentrations Are Higher in Subjects with 203‑OR Type 1 Diabetes (T1D) and Impaired Awareness of Glycemia (IAH) Glycogen Is the Major Source of Lactate in the Ventromedial Hypo‑ than in T1D without IAH thalamus of Recurrently Hypoglycemic Rats AMIR MOHEET, DINESH DEELCHAND, ANJALI KUMAR, GULIN OZ, ELIZABETH R. GONG SU, OWEN CHAN, RAWAD FARHAT, KIMBERLY CHAPMAN, Salt Lake City, UT SEAQUIST, Minneapolis, MN Lactate is an important metabolic substrate for sustaining brain energy IAH occurs commonly in patients with T1D but its pathogenesis remains demands when glucose supplies are limited. Recurrent hypoglycemia (RH) uncertain. Previous investigation has suggested that recurrent hypoglyce- leads to increased lactate levels in the ventromedial hypothalamus (VMH) mia leads to an upregulation of brain glucose transport thereby assuring which contributes to counterregulatory failure, but the source of this lactate that the brain will have better fuel delivery during subsequent episodes of remains unclear. It has been shown that astrocytic glycogen can be broken hypoglycemia. If so, subjects with T1D and IAH should have higher hypo- down into lactate in the brain. Therefore, the present study investigates thalamic brain glucose concentrations than subjects with T1D without IHA whether glycogen serves as the major source of lactate in the VMH of RH when studied at the same level of glycemia. Here we used 1H-magnetic rats. To address this question, Sprague-Dawley rats were implanted with

ADA-Supported Research

A53 CLINICAL TRIALS IN TYPE 1 DIABETES

mini-osmotic pumps and bilateral intracranial guide cannulas that targeted 205‑OR the VMH to deliver either artificial extracellular fluid (aECF) or the glycogen Acute Caloric Restriction Leads to Loss of Hypoglycemic Counter- phosphorylase (GP) inhibitor, 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). Sub- Regulation in Mice following Short-Term Refeeding sequently, the rats underwent 3 bouts of hypoglycemia before undergoing DAVID MCDOUGAL, Baton Rouge, LA a hyperinsulinemic-hypoglycemic clamp on day 4 (n=6/group). A separate Background: Hypoglycemia-associated autonomic failure (HAAF) is a mal- group of rats receiving aECF into the VMH were given 3 saline injections as adaptive failure in glucose counter-regulation known to be caused by recur- controls before undergoing a hypoglycemic clamp on day 4 (n=6). During the rent exposure to insulin-induced hypoglycemia. Fasting or caloric restriction clamps, microdialysis was used to measure VMH lactate levels and VMH is the only natural physiological process which induces prolonged moderate- glycogen content was determined at the end of the clamp from liquid nitro- to-severe hypoglycemia similar to the antecedent of HAAF. In this study we gen fixed brain samples. VMH lactate levels were elevated in RH rats com- tested the hypothesis that exposure to fasting-induced hypoglycemia can pared to hypoglycemia-naïve controls (3.6±0.2 vs. 2.3±0.4 mM; P=0.03) and cause HAAF-like symptoms in mice. this was associated with impairments in the counterregulatory responses Methods: Two groups of mice were used, an ad libitum (ad lib) fed group (37% and 45% reduction in glucagon and epinephrine, respectively; P<0.05). (n=6) and a caloric restriction (CR) group (n=6). CR mice were placed on 60% Compared to the RH rats, inhibition of GP with DAB prevented the break- caloric restriction for 6 consecutive days. Ad lib mice were housed in an down of VMH glycogen in RH rats (5.3±0.5 vs. 2.4±0.5 µmol/g tissue; identical manner but fed ad libitum during this same period. Following 6 days P<0.002), prevented the rise in VMH lactate levels (1.5±0.3 vs. 3.6±0.2 mM; of restriction, CR mice were given ad lib access to food for 16 hour. After P<0.001) and prevented the onset of counterregulatory failure (glucagon and the 16 hour period of refeeding, both CR and ad lib mice began a 6 hour fast epinephrine responses were similar to controls, P=NS). Our data suggests which was immediately followed by a hypoglycemic insulin tolerance test that astrocytic glycogen likely serves as the primary source of lactate in the (ITT). ITTs consisted of a variable dose of insulin (1.4-1.75 U/kg, IP) which VMH of RH rats. produced similar levels of hypoglycemia in each group (~45 mg/dL). Supported By: JDRF; National Institutes of Health Results: As expected, mice exposed to 6 days of 60% CR lost ≈30% of their initial body weight, and experienced a gradual fall in blood glucose 204‑OR to an average minimum of 67.9 mg/dL. CR mice displayed a significantly Increased Glycemic Variability Is Associated with Augmented reduced fasting glucose level relative to ad lib mice (102.7 and 171.0 mg/ Brain Glucose Transport amongst Poorly Controlled T1DM Indi‑ dL respectively, p = 0.0002) as well as reduced serum glucagon levels in viduals response to the ITT (6.0 and 30.4 pg/mL respectively, p = 0.0182). JANICE HWANG, LIHONG JIANG, WAI LAM, ELIZABETH SANCHEZ RANGEL, Conclusions: Our results demonstrate that exposure to fasting-induced DOUGLAS L. ROTHMAN, GRAEME F. MASON, ROBERT SHERWIN, New Haven, CT hypoglycemia produces HAAF-like symptoms in mice following refeeding. Both hyperglycemia and hypoglycemia have been shown to modulate These results suggest that HAAF may be a result of an adaptive response to glucose transport into the brain; however, the impact of glycemic variabil- fasting which is inappropriately elicited during exposure to insulin-induced ity remains unknown. This study was undertaken to investigate the impact hypoglycemia. Further characterization of the effects of CR on the induction of glycemic variability on brain glucose transport kinetics amongst T1DM of deficits in glucose counterregulation warrants further investigation. individuals. Nine individuals with poorly-controlled T1DM (age 6F/3M, age 30±4 years, BMI 27±1.2 kg/m2, HbA1C 8.0±0.3%, duration of DM 17±5 years) CLINICAL TRIALS IN TYPE 1 DIABETES underwent 1H magnetic resonance spectroscopy scanning in the occipital lobe to measure the change in intracerebral glucose levels during a 2-hour 206‑OR hyperglycemia clamp (target glucose 220 mg/dl). In addition for 5-6 days Overnight Insulin Delivery and Glucose in Children Using the prior to scanning, individuals wore a continuous glucose monitor (Dexcom MiniMed™ 670G System G4) to assess several measures of glycemic variability (EasyGV). ANIRBAN ROY, BENYAMIN GROSMAN, NEHA PARIKH, DI WU, SCOTT W. LEE, The mean change in brain glucose was 0.99±0.1 mmol/L. There was no FRANCINE R. KAUFMAN, Northridge, CA, Los Angeles, CA significant relationship between HbA1C% and change in intracerebral glu- The Medtronic MiniMed™ 670G hybrid closed-loop system was designed cose (P=0.31), which may be due to the narrow range of HbA1C. However, to automatically adjust basal insulin delivery every 5 minutes based on sen- there were significant positive correlations between measures of glycemic sor glucose (SG) values. Its safety and effectiveness have been recently variability and change in intracerebral glucose (Figure). investigated in a multi-center pivotal trial conducted in 105 T1D children These findings suggest that the degree of glycemic variability can mod- aged 7-13 years. ulate brain glucose transport and highlight the need for future studies to During the 2 week run-in phase, participants used the system in Manual investigate the impact of glycemic variability on brain glucose kinetics. Mode. This was followed by a 3-month at-home study phase in which the Figure. Auto Mode feature was enabled. The SG data and the amount of insulin delivered were analyzed during the overnight period (11:00 p.m.-7:00 a.m.). Figure 1 (top panel) shows overnight SG tracings from the run-in phase and study phase, and that the median [IQR] reduced significantly from run-in ORALS (142 [71]mg/dL) to study phase (126 [34]mg/dL), by 7:00 a.m. This was mainly achieved by the system transferring variability from SG to insulin infusion rate (bottom panel). During the nighttime period, the system suspended insulin for an average 1.3 hours and delivered insulin at the maximum allowable limit for an average 3.6 hours. The remainder of the time, it delivered insulin between 1-99% of the maximum allowable limit. These data suggest that greater variability of basal insulin delivery, such as that achieved with MiniMed™ 670G system automation, compared to fixed pre-set basal rates, improves nighttime glucose control in children.

Supported By: American Diabetes Association (1-17-ICTS-013 to J.H.); National Institutes of Health

ADA-Supported Research

A54 CLINICAL TRIALS IN TYPE 1 DIABETES

pump and Dexcom G5® sensor) and centralised remote monitoring. Adult patients with T1D for ≥ 2 years, pump therapy for ≥ 6 months, HbA1c ≤ 10%, and preserved hypoglycemia awareness were included among 12 investigation centers. Design was a crossover trial with a 2-week run-in period, followed by 2 sequences of 12 weeks each separated with washout. Results of the first 12-week arm are reported. Results: 68 patients (27 men, age 47.2±13.4 years, HbA1c 7.6±0.9%, duration of disease 27.9±13.2 years) were randomised between closed-loop (CL, n=33) and open-loop (OL, n=35) first. During the first 12-week period, time spent in the 70-180 mg/dl range was 69.5% [62.4;75.6] in CL vs. 58.7% 207‑OR [45.2;64.9] in OL. Time in hypoglycaemia <70 mg/dl was 2.1% [1.2;2.6] in CL Safety and Performance of the Omnipod® Hybrid Closed-Loop Sys‑ vs. 4.1% [1.9;6.1] in OL. tem in Adults with Type 1 Diabetes over Five Days Under Free-Living Conclusion: The Diabeloop closed-loop system was efficient regarding Conditions metabolic outcomes. BRUCE A. BUCKINGHAM, JENNIFER SHERR, GREGORY P. FORLENZA, THOMAS A. PEYSER, JOON BOK LEE, JASON B. OCONNOR, BONNIE DUMAIS, LAUREN M. 209‑OR HUYETT, JENNIFER E. LAYNE, TRANG T. LY, Palo Alto, CA, New Haven, CT, Aurora, Real-World Data from the MiniMed™ 670G System Commercial CO, Billerica, MA Launch—Patients Previously Using MDI Therapy ® The safety and performance of the Omnipod hybrid closed-loop (HCL) SHWETA GOPALAKRISHNAN, PRATIK AGRAWAL, MICHAEL STONE, CATHERINE personalized model predictive control algorithm was assessed in adults with FOGEL, SCOTT W. LEE, Northridge, CA type 1 diabetes (T1D) using an investigational device over 5 days in a super- The Medtronic MiniMed™ 670G hybrid closed-loop system with Smart- vised hotel setting under free-living conditions. Eligible participants were Guard™ technology was released for commercial use in the U.S. in March aged 18-65.0 years with A1C <10.0% using CSII or MDI. A 7-day open-loop (OL) 2017. Data from patients previously managing their diabetes with multiple phase of standard therapy (CSII/MDI) plus CGM use at home preceded the 96 daily insulin injections (MDI) therapy who transitioned to the MiniMed™ h HCL phase. Meals during HCL were unrestricted, with boluses administered 670G system are reported. MiniMed™ 670G system data from patients per usual routine. Moderate-intensity exercise was performed for ≥30 min/d. who voluntarily uploaded to CareLink™ Personal software, from 3/17/17 to An adaptive approach was used to update participant parameters after the 12/14/17 (n=158), were de-identified and analyzed if there were≥ 7 days of first 48 hours of HCL. Eleven participants (MDI n=3) studied were (mean ± SD): sensor wear. Comparisons were made of glycemic metrics between 4,529 age 28.8 ± 7.9 years, diabetes duration 14.9 ± 6.9 years, A1C 7.4 ± 1.2% and patient-days in the initial open-loop Manual Mode and 14,106 patient-days TDD 0.67 ± 0.24 U/kg. Glycemic outcomes are reported in the Table. The per- after initiating closed-loop Auto Mode features. The Table shows glycemic centage of time 70-180 mg/dL was 11.2% higher during HCL compared to OL metrics during Manual Mode and after Auto Mode start and that time spent overall (HCL 73.7 ± 7.5 vs. OL 62.5 ± 16.0) and 13.2% higher overnight (HCL 73.9 in target range increased, while time spent in low and high glucose levels ± 21.0 vs. OL 60.7 ± 21.8). A concomitant reduction in the percentage of time decreased. Real-world findings from patients previously using MDI who <70mg/dL during HCL vs. OL occurred both overall (HCL 1.9 ± 1.3 vs. OL 5.1 ± transitioned to the MiniMed™ 670G system demonstrated increased TIR and 4.8) and overnight (HCL 0.7 ± 1.1 vs. OL 5.7 ± 7.4). The Omnipod HCL algorithm reduced time in hypoglycemia and hyperglycemia, consistent with pivotal was safe and performed well over 5 days of use in adults with T1D under free- trial1 results in patients previously using pump therapy +/- CGM. Prior MDI living conditions with unrestricted meals and moderate-intensity exercise. patients also showed greater TIR the more time spent in Auto Mode. Table. Glycemic Outcomes During Hybrid Closed-Loop (Hcl) and Open-Loop Table. (Ol) Phases. Glycemic outcomes HCL OL HCL OL Overall Overall Night Night (23:00-06:59) (23:00-06:59) Mean glucose (mg/dL) 149.7 ± 11.3 156.1 ± 28.6 151.9 ± 31.7 156.3 ± 38.5 Percent time <54 mg/dL (%) 0.2 ± 0.3 1.1 ± 1.6 0.2 ± 0.5 1.1 ± 2.1 Percent time <70 mg/dL (%) 1.9 ± 1.3 5.1 ± 4.8 0.7 ± 1.1 5.7 ± 7.4 Percent time 70-140 mg/dL (%) 49.4 ± 9.2 41.2 ± 17.6 52.7 ± 28.4 43.5 ± 20.9

Percent time 70-180 mg/dL (%) 73.7 ± 7.5 62.5 ± 16.0 73.9 ± 21.0 60.7 ± 21.8

Percent time >180 mg/dL (%) 24.5 ± 7.7 32.3 ± 18.1 25.3 ± 21.2 33.6 ± 23.5

Percent time ≥250 mg/dL (%) 4.5 ± 4.2 8.5 ± 9.1 6.1 ± 10.9 8.6 ± 14.1 ORALS Supported By: Insulet Corporation 210‑OR Insulin-plus-Pramlintide Artificial Pancreas in Type 1 Diabetes— 208‑OR Randomized Controlled Trial Twelve-Week Home Use of Hybrid Closed-Loop Insulin Delivery AHMAD HAIDAR, MICHAEL TSOUKAS, SARAH TWARDY, NATALIA STRAUSS, System vs. Sensor-Assisted Pump Therapy in Adults with Type 1 JEAN FRANCOIS YALE, JOANNA RUTKOWSKI, ANNE BOSSY, EVELYNE PYTKA, Diabetes—Intermediate Results of the Multicenter Randomised HIEU T. NGUYEN, LAURENT LEGAULT, Montreal, QC, Canada, Pincourt, QC, Canada Crossover Diabeloop WP7 Trial We conducted a randomized crossover comparison between i) dual-hor- SYLVIA FRANC, PIERRE Y. BENHAMOU, SOPHIE BOROT, LUCY CHAILLOUS, mone, rapid insulin and amylin (Pramlintide), artificial pancreas (DAP), ii) reg- BRIGITTE DELEMER, BRUNO GUERCI, HÉLÈNE HANAIRE, NATHALIE M. JEAN- ular insulin (Humulin R) and amylin artificial pancreas (R-DAP), and iii) rapid DIDIER, SR., ERIC RENARD, YVES REZNIK, PAULINE SCHAEPELYNCK, CHARLES insulin-alone artificial pancreas (AP) in 12 adults with type 1 diabetes. Insulin THIVOLET, GUILLAUME CHARPENTIER, Corbeil-Essonnes, France, Grenoble, and amylin were delivered simultaneously, in a basal-bolus manner, using a France, Besançon, France, Nantes, France, Reims, France, Vandœuvre-lès-Nancy, novel dosing algorithm, with a fixed ratio (6 µg/u), mimicking a co-formula- France, Toulouse, France, Strasbourg, France, Montpellier, France, Caen, France, tion. Participants were admitted three times for 24 hours and ingested three Marseille, France, Lyon, France meals and a bedtime snack. Prior to closed-loop study, amylin was delivered Objective: Availability of closed-loop insulin delivery systems is expected for two weeks, and basal rates and carb ratios were optimized. Over the by patients with type 1 diabetes. Our main objective was to assess whether 24-hour study, DAP increased time spent in target range [3.9-10.0 mmol/L] patients using the Diabeloop artificial pancreas system could achieve a bet- compared to AP from 71% to 85% (P=0.03) and decreased glucose variability ter glucose control compared to sensor-assisted pump therapy, as measured (CV) from 34% to 25% (P=0.01), without increasing the risk of hypoglycemia by percentage time in range 70-180 mg/dl. (P=NS). Overnight, DAP and AP achieved high and similar time in tight target Methods: A multicenter clinical trial was conducted in a home setting, range [3.9-7.8 mmol/L; 77%, 71%, P=NS]. Moderate nausea was reported using an MPC-based algorithm, a hybrid monohormonal system (CellNovo® by two participants (16%) on DAP compared to 0 (0%) on AP. There was no

ADA-Supported Research

A55 CLINICAL TRIALS IN TYPE 1 DIABETES

benefits associated with R-DAP compared to AP during day or night period. In this double-blind, 52-week North American trial, 793 adults with T1D We conclude that a dual-hormone artificial pancreas delivering rapid insulin treated with multiple daily insulin injections (40%) or pump (60%) were ran- and amylin with fixed ratio improves glucose control and reduces glucose domized 1:1:1 to placebo (n=268), SOTA 200 mg (n=263) or SOTA 400 mg variability compared to insulin-alone, first-generation, artificial pancreas. (n=262) once daily after 6 weeks of insulin optimization. Primary endpoint Table 1. Comparisons Between AP, DAP, and R-DAP (Mean±SD). was change from baseline in A1C at Week 24. Other endpoints included A1C, weight, bolus insulin and FPG changes at Week 52, patient (pt) reported out- Outcome AP DAP R-DAP P-value P-value comes and net clinical benefit (NCB), assessing the proportion of pts with DAP vs. AP R-DAP vs. AP A1C <7.0% without severe hypoglycemia (SH) or diabetic ketoacidosis (DKA). Time spent between 3.9 and 71 ± 19 85 ± 10 72 ± 16 0.03 0.86 Baseline characteristics were similar between groups. Compared with 10.0 mmol/L during 24-hour placebo, SOTA 200 and 400 mg improved A1C and pt satisfaction at Week study period (%) 24 and reduced A1C, weight, bolus insulin, FPG and pt distress at Week 52. Mean glucose level during 24- 8.2 ± 1.3 7.4 ± 1.1 7.7 ± 0.8 0.07 0.38 More pts achieved NCB with SOTA vs. placebo (each P<0.05; Table). The hour study period (mmol/L) highest incidence of SH was in the placebo arm; more genital mycotic infec- Coefficient of variance during 34 ± 11 25 ± 6 35 ± 8 0.01 0.65 tions, diarrhea and DKA events (more with pump than MDI) were in the SOTA 24-hour study period (%) arms (Table). Time spent between 3.9 and 58 ± 26 78 ± 16 66 ± 21 0.02 0.58 In conclusion, SOTA 200 and 400 mg were associated with statistically 10.0 mmol/L during daytime significant A1C reductions that were sustained (P<0.05) at Week 52, with period (%) an increased (but low) rate of DKA, and a lower incidence of SH, when com- Mean glucose level during 9.1 ± 1.9 7.8 ± 1.5 8.4 ± 1.5 0.02 0.34 pared with placebo. daytime period (mmol/L) Table. Efficacy (mITT Population) and Safety (Safety Population) Results. Time spent between 3.9 and 77 ± 20 71 ± 29 65 ± 19 0.47 0.16 Placebo SOTA 200 mg SOTA 400 mg 7.8 mmol/L during overnight n=268 n=263 n=262 period (%) Mean A1C at Baseline, after 6-week 7.54 7.61 7.56 Mean glucose level during 6.6 ± 0.9 6.8 ± 0.9 6.6 ± 1.2 0.51 0.87 insulin optimization, % overnight period (mmol/L) Outcomes at Week 24 Supported By: JDRF A1C LSM difference from placebo, % — -0.36±0.05 -0.41±0.05 ± SE (P-value) (<0.001) (<0.001) 211‑OR Outcomes at Week 52 rtCGM Usage Is Associated with a Significant Reduction of Time Spent in Hypoglycemia in Patients with Type 1 Diabetes Treated A1C LSM difference from placebo, % — -0.25±0.06 -0.31±0.06 with Multiple Daily Injections—Results of the HypoDE Study ± SE (P-value) (P<0.001) (P<0.001) LUTZ HEINEMANN, GUIDO FRECKMANN, DELIA WALDENMAIER, DOMINIC FPG LSM difference from placebo, — -0.68±0.31 -1.08±0.31 EHRMANN, NORBERT HERMANNS, Düsseldorf, Germany, Ulm, Germany, Bad mmol/L ± SE (P=0.028) (P<0.001) Mergentheim, Germany Body weight LSM difference from — -3.14±0.34 -4.32±0.35 In this randomized multi-center study, performed in 12 specialized diabetes placebo, kg ± SE (P-value) (P<0.001) (P<0.001) practices in Germany, we examined the impact of rtCGM use (Dexcom G5) on Mean daily bolus insulin dose at 31.7 30.3 30.8 the duration of time spent in hypoglycemia. In total, 149 MDI-treated patients Baseline, IU with type 1 diabetes and hypoglycemia problems (age 47±20 years, HbA1c Bolus insulin dose mean change from 7.01±3.40 -1.48±3.40 -8.63±3.42 7.5±1.0%, 60.4% with at least one recent severe hypoglycemic event, unaware- Baseline, % ± SE ness score 4.9±1.2) used a masked rtCGM system for 4 weeks. After this base- Bolus insulin dose LSM difference — -5.53±4.59 -15.63±4.60 line phase, 75 patients were randomized to rtCGM use and 74 were randomized from placebo, % ± SE (P-value) (P=0.23) (P<0.001) to the control group with continuation of SMBG for the next 26 weeks. From Net clinical benefit at Week 52 week 22 till 26 after randomization, the control group had a masked rtCGM system again. The CGM group continued their use of rtCGM. Six-month follow-up A1C <7.0% without SH and without 51 (19.0) 69 (26.2) 85 (32.4) data could be obtained from 141 participants. In the CGM group, the duration of DKA, n (%) time per day spent ≤70 mg/dl was lower compared to the control group (42.6 vs. Safety outcomes over 52 weeks 112.0 min/day; adjusted Δ 69.4 min/day, 95% CI 52.4 to 86.4 min/day, p<0.0001). Any TEAE, n (%) 216 (80.6) 215 (81.7) 209 (79.8) The time per day spent in <55 mg/dl showed the same pattern (11.2 vs. 53.2 TEAEs leading to study 11 (4.1) 13 (4.9) 17 (6.5) min/day; adjusted Δ 41.9 min/day, 95% CI 31.4 to 52.2 min/day, p<0.0001). discontinuation, n (%) Time in range per day was longer in the CGM group than in the control group (850.5 vs. 805.6 min/day; adjusted 44.9 min/day, 95% CI -0.3 to 90.0 min/day, Treatment-emergent serious adverse 20 (7.5) 27 (10.3) 29 (11.1) Δ events, n (%)

ORALS p=0.051). Time per day spent in hyperglycemia (>180 mg/dl) was slightly higher in the CGM group than in the control group (544.2 vs. 525.5 min/day; adjusted Δ Death, n (%) 1 (0.4) 0 0 -18.7 min/day, 95% CI -70.3 to 32.9 min/day, p=0.474). HbA1c at follow-up was DKA, n (%)1 1 (0.4) 9 (3.4) 11 (4.2) comparable between CGM and control group (7.3% vs. 7.3%; adjusted Δ 0.0%, Severe hypoglycemia, n (%)1 26 (9.7) 17 (6.5) 17 (6.5) 95% CI -0.1 to 0.2%, p=0.665). The use of rtCGM in MDI-treated type 1 dia- Diarrhea2, n (%) 18 (6.7) 22 (8.4) 27 (10.3) betic patients with hypoglycemia problems can significantly reduce exposure to hypoglycemic glucose values without compromising glycemic control. Given Genital mycotic infection, n (%) 9 (3.4) 24 (9.1) 34 (13.0) the rather higher costs of CSII treatment compared to MDI, these results are of Patient reported outcomes high clinical as well as health-economic relevance. DTSQ score LSM difference from — 2.5±0.40 2.5±0.40 Supported By: Dexcom, Inc. placebo at Week 24 ± SE (P-value) (P<0.001) (P<0.001) DDS2 score LSM difference from — -0.4±0.15 -0.5±0.15 212‑OR placebo at Week 52 ± SE (P-value) (P=0.003) (P<0.001) Fifty-Two-Week Efficacy and Safety of Sotagliflozin, a Dual SGLT1 DDS2, two-item Diabetes Distress Screening Scale (positive scores indicate and SGLT2 Inhibitor, as Adjunct Therapy to Insulin in Adults with improvement); DKA, diabetic ketoacidosis; DTSQ, diabetes treatment Type 1 Diabetes (inTandem1) satisfaction questionnaire; FPG, fasting plasma glucose; LSM, least squares JOHN B. BUSE, SATISH K. GARG, JULIO ROSENSTOCK, TIMOTHY S. BAILEY, mean; mITT, modified intent-to-treat; SD, standard deviation; SE, standard PHILLIP L. BANKS, BRUCE W. BODE, THOMAS DANNE, JAKE A. KUSHNER, error; SH, severe hypoglycemia; SOTA, sotagliflozin; TEAE, treatment emergent WENDY LANE, PABLO LAPUERTA, DARREN K. MCGUIRE, ANNE L. PETERS, adverse events. 1Positively-adjudicated events (defined as an adjudicator JOHN H. REED, SANGEETA SAWHNEY, PAUL STRUMPH, Chapel Hill, NC, Aurora, assessment of yes/certainly or yes/probably); 2Discontinuation of drug due to CO, Dallas, TX, Escondido, CA, The Woodlands, TX, Atlanta, GA, Hannover, Germany, diarrhea was: 0.4% placebo, 0% SOTA 200 mg, and 0.4% SOTA 400 mg. Houston, TX, Asheville, NC, Basking Ridge, NJ, Los Angeles, CA, Roswell, GA Sotagliflozin (SOTA) is a dual SGLT1 and SGLT2 inhibitor in development as adjunct therapy to insulin in T1D.

ADA-Supported Research

A56 HIGH RISK BEHAVIORAL AND PSYCHOSOCIAL ISSUES IN DIABETES

213‑OR Safety data (patients with an event) Efficacy and Safety of Dapagliflozin in Patients with Inadequately ≥1 AE, n (%) 197 (72.7) 181 (67.0) 172 (63.2) Controlled Type 1 Diabetes—DEPICT-2 Study ≥1 SAE, n (%) 18 (6.6) 7 (2.6) 5 (1.8) CHANTAL MATHIEU, PARESH DANDONA, PIETER GILLARD, PETER A. SENIOR, ≥1 related SAE, n (%) 13 (4.8) 3 (1.1) 2 (0.7) CHRISTOPH HASSLACHER, EIICHI ARAKI, MARCUS LIND, STEPHEN C. BAIN, ≥1 event of hypoglycemia, n (%) 223 (82.3) 231 (85.6) 234 (86.0) SERGE JABBOUR, NIKI ARYA, FREDRIK A. THOREN, ANNA MARIA LANGKILDE, ≥1 event of severe hypoglycemia, n (%) 17 (6.3) 23 (8.5) 21 (7.7) ON BEHALF OF THE DEPICT-2 INVESTIGATORS, Leuven, Belgium, Buffalo, NY, Definite DKA, n (%) 7 (2.6) 6 (2.2) 0 Edmonton, AB, Canada, Heidelberg, Germany, Kumamoto, Japan, Gothenburg, Supported By: AstraZeneca Sweden, Swansea, United Kingdom, Philadelphia, PA, Gaithersburg, MD, Mölndal, Sweden The DEPICT-2 trial (NCT02460978) evaluated the efficacy and safety of dapagliflozin (DAPA) as add-on to adjustable insulin (INS) in patients HIGH RISK BEHAVIORAL AND PSYCHOSOCIAL (pts) with inadequately controlled T1D (A1c 7.5-10.5%) over 24 weeks. This ISSUES IN DIABETES phase 3 study randomized pts 1:1:1 to DAPA 5 mg (n=271), 10 mg (n=270) or placebo (PBO; n=272) plus INS. INS dose could be adjusted by the investi- 214‑OR gator according to self-monitored blood glucose readings, local guidance Youth with Avoidable DKA—Going from Bad to Worse without NICH and individual circumstances. At Week 24, DAPA 5 and 10 mg significantly RAJKARAN SACHDEJ, NATALIE C. KOSKELA, DAVID V. WAGNER, SAMANTHA decreased A1c (0.37% and 0.42% reductions in the DAPA 5 mg and 10 A. BARRY, CELESTE JENISCH, MICHAEL A. HARRIS, Portland, OR, Worcester, MA mg groups respectively), total daily insulin dose (TDD), and body weight Objective: Novel Interventions in Children’s Healthcare (NICH) was devel- (Table). As measured by masked continuous glucose monitoring (CGM), oped for youth with T1D who experience avoidable DKAs. Previous findings mean interstitial glucose, mean amplitude of glucose excursion (MAGE) suggest that NICH participation is associated with fewer PICU visits, hospi- and mean percent of readings within target glycemic range (>70-≤180 mg/ tal admissions, and DKA events, but interpretation is limited due to possibil- dL) vs. PBO were improved. There was an increase in pts who reduced their ity of regression to the mean. This study examines whether youth experi- A1c by ≥0.5% without severe hypoglycemia (odds ratios [95% CI]: 2.71 ence fewer acute events during the wait period following referral to NICH. [1.81, 4.06] and 3.07 [2.05, 4.60] for DAPA 5 and 10 mg respectively). Hypo- Methods: Retrospective chart reviews were conducted for youth with T1D glycemic events, including severe hypoglycemia were balanced between (n=63) who participated in NICH services. Youth mean age was 14.8 years; treatment groups. There were more adjudicated definite diabetic ketoaci- 57% were female; and 86% were caucasian. Mean days prior to referral, dosis (DKA) events on DAPA. on wait list, and during NICH were 365, 129, and 353, respectively. Results In conclusion, DAPA vs. PBO as add-on to INS in pts with T1D was well were normalized across time points to reflect rate of likely event occurrence tolerated, improved glycemic control and decreased variability without during 1 year period. increasing hypoglycemia but with more DKA events. Results: Youth were significantly more likely to experience a PICU stay Table. (p=.03) while waiting for NICH compared to baseline and were significantly less likely to experience a DKA (p=.02) or PICU stay (p=.01) during NICH Week 24 outcomes DAPA 5 mg DAPA 10 mg Placebo involvement compared to the wait period. + INS + INS + INS Conclusions: Instead of spontaneously improving after baseline (i.e., (n=271) (n=270) (n=272) regression to the mean), youth referred to NICH experience an increase in HbA1c, % acute complications while waiting for services, then improvement during Mean (SD) at baseline 8.45 (0.69) 8.39 (0.67) 8.40 (0.63) NICH involvement. Perhaps referring providers are identifying factors that Week 24 adjusted mean change from baseline (SE) −0.34 (0.05) −0.39 (0.05) 0.03 (0.05) suggest further deterioration of health without more intensive services. Difference vs. PBO (95% CI) −0.37 −0.42 — (−0.49, −0.26) (−0.53, −0.30) Figure. p value <0.0001 <0.0001 — TDD, IU Mean (SD) at baseline 59.09 (28.05) 59.28 (28.21) 56.45 (25.23) Week 24 adjusted mean change from baseline (SE) −8.73 (1.22) −9.05 (1.23) 2.29 (1.39) Difference vs. PBO (95% CI) −10.78 −11.08 — (−13.73, −7.72) (−14.04, −8.02) p value <0.0001 <0.0001 — Body weight, kg Mean (SD) at baseline 79.22 (17.21) 80.39 (18.51) 79.03 (19.05) Week 24 adjusted mean change from baseline (SE) −3.22 (0.27) −3.76 (0.27) −0.02 (0.28) Difference vs. PBO (95% CI) −3.21 −3.74 —

(−3.96, −2.45) (−4.49, −2.99) ORALS p value <0.0001 <0.0001 — 24-hour CGM glucose reading, mg/dL Mean (SD) at baseline 192.67 (28.68) 191.53 (28.09) 190.89 (28.95) week 24 adjusted mean change from baseline (SE) −6.46 (1.83) −10.54 (1.83) 9.20 (1.85) Difference vs. PBO (95% CI) −15.66 −19.74 — (−20.26, −11.05) (−24.34, −15.14) p value <0.0001 <0.0001 — Supported By: The Leona M. and Harry B. Helmsley Charitable Trust MAGE during 24-hour CGM, mg/dL Mean (SD) at baseline 169.35 (29.60) 171.02 (29.85) 168.38 (29.29) week 24 adjusted mean change from baseline (SE) −10.17 (1.90) −9.68 (1.91) −0.33 (1.93) 215‑OR Difference vs. PBO (95% CI) −9.85 −9.36 — Suicidal Screening in Youth with T1D—Risk or Benefit? (−14.66, −5.03) (−14.16, −4.55) HARPREET NAGRA, DANNY C. DUKE, MELINDA JONES, KIMBERLY KRAUS, p value <0.0001 0.0001 — MICHAEL A. HARRIS, Portland, OR 24-h interstitial glucose values within >70-≤180 mg/dL, % Objectives: Insulin access with intent to overdose coupled with limited forethought and higher impulsivity during childhood and adolescence, makes Mean (SD) at baseline 43.50 (12.43) 43.68 (11.83) 43.53 (12.55) identification of suicidal ideation (SI) among youth with type 1 diabetes (T1D) week 24 adjusted mean change from baseline (SE) 5.92 (0.82) 7.60 (0.82) −3.10 (0.83) Difference vs. PBO (95% CI) 9.02 10.70 — of high importance. Correlations between SI and poor diabetes self-manage- (6.97, 11.06) (8.66, 12.74) ment have been identified, with 13% of adolescents with T1D endorsing SI p value <0.0001 <0.0001 — in the last 12 months. The present study aimed to determine feasibility and effectiveness of screening for SI in youth with T1D. Methods: Data were obtained during routine behavioral health screening of youth (ages 11-18) with T1D at a regional diabetes center. SI data

ADA-Supported Research

A57 HIGH RISK BEHAVIORAL AND PSYCHOSOCIAL ISSUES IN DIABETES

were derived from question 9 of the Patient Health Questionnaire-9 (PHQ-9), “Thoughts that you would be better off dead or of hurting yourself in some way.” Results: PHQ-9 was completed by a total of 587 pediatric patients (Males = 289; Females =298) with a mean HbA1c of 9.1% (SD= 2.2). Findings suggest a significant relationship between gender and endorsement of SI with more females endorsing SI (n=52) than males (n = 26). SI was significantly and positively associated with HbA1c. Of the 13% of youth who screened positive for SI, ¾ indicated SI for several days in the last 7 days and received an on-site behavioral health consultation. Approximately, 1/5 indicated SI for more than ½ of the days in the last week and received an onsite referral and a telephone follow-up, and 5% indicated SI nearly every day in the last week and received an on-site clinical assessment with one youth requiring emergency services. Conclusions: SI screening in youth with T1D is necessary to address this concern. Results suggest our protocol was effective in identifying SI, allowed for rapid clinical assessment, and referral for additional care. In addition, SI rates in youth with T1D were consistent with the general population, suggesting that diabetes does not put youth at higher risk for SI, Supported By: The Leona M. and Harry B. Helmsley Charitable Trust but imposes greater concern for suicide given the immediate availability of insulin. 217‑OR Diabetes-Specific Risk Taking—Psychometric Properties of a 216‑OR Measure for Adolescents with Type 1 Diabetes (T1D) Substance Use in Adults with Type 1 Diabetes in the T1D Exchange RACHEL M. WASSERMAN, SUSANA R. PATTON, MARK A. CLEMENTS, JONA- NANCY PETRY, NICOLE C. FOSTER, EDA CENGIZ, WILLIAM V. TAMBORLANE, THAN H. FINCH, KELSEY BOSCHERT, DAVID D. SCHWARTZ, BARBARA ANDER- JULIE WAGNER, SARIT POLSKY, Farmington, CT, Tampa, FL, New Haven, CT, SON, Orlando, FL, Kansas City, KS, Kansas City, MO, Leawood, KS, Houston, TX, Aurora, CO Charlottesville, VA Substance use can have adverse effects on many health conditions, yet Among people with T1D, adolescents have the poorest adherence and little is known about the prevalence of use in individuals with type 1 diabe- worst glycemic control of any other age group, despite recent advances in tes (T1D). We evaluated the frequency of use and problem use of psychoac- treatment and technology. Clinically, adolescents are known to take risks tive substances in adults with T1D. Standardized instruments for assessing with their diabetes care (e.g., driving without first checking blood glucose), tobacco, alcohol, and psychoactive substance use were emailed to 4311 but these risky diabetes behaviors have not been empirically studied. We adult participants at 69 T1D Exchange Clinic Registry centers. A total of 936 report psychometric properties of the new 31-item Diabetes-Specific Risk- respondents (61% female, 90% non-Hispanic white, age 38±16 years) com- Taking Inventory (DSRI) for youth 15-18 years old. On the DSRI, youth report pleted the survey. In the sample, tobacco use was reported by 166 (18%) par- the frequency they engaged in 31 diabetes-specific risk-taking behaviors ticipants in the past year and 51 (5%) daily (Figure 1). Past year alcohol use over the past year. We surveyed a national sample of 224 adolescents (par- was reported by 742 (79%) participants, past month use by 592 (63%) and ticipants in the T1Dx registry) (M age=16.4±1.1, 47% female, M A1c=8.3±1.3, daily or near daily use by 87 (9%). Use of any other psychoactive substance 76.8% on insulin pumps) in a cross-sectional design. Participants completed in the past year was reported by 228 respondents (24%), with marijuana the DSRI as well as measures of adherence, general risk-taking, executive being the most commonly used substance (Figure 2). Past year problem use functioning, depressive symptoms, and incidence of severe hypoglycemia of these substances was noted in 31 (3%) respondents. Adults with T1D in and DKA over the past year, using a secure web portal, RedCap. The DSRI the U.S. use substances at rates that meet or exceed that of the general demonstrated excellent internal reliability (internal consistency: α=.90; population; problematic use occurs at rates similar to the general popula- item-total correlations: r range=.21-.64). Construct validity was demon- tion. These data delineate the need to inquire about regular, intermittent strated through significant associations (all p<.01) with validated measures: and problematic use of nicotine, alcohol, and other substances in individuals more frequent diabetes-specific risk-taking was related to poorer adherence with T1D. A better understanding of the impact of moderate and occasional (r=-.75), greater general risk-taking (r=.57), greater executive dysfunction use of substances on T1D management and clinical outcomes is also needed. (r=.34), more depressive symptoms (r=-26), and report of severe hypoglycemia over the past year (r=.22). The DSRI was not associated with DKA. Over- all, initial psychometrics suggest the DSRI reliably and validly captures risks that adolescents take with their diabetes care. This self-report measure has the potential to be an innovative and actionable clinical tool that can be used to screen for high risk behaviors that are not routinely assessed in clinical

ORALS care and inform transition readiness from pediatric to adult care. Supported By: National Institutes of Health; The Leona M. and Harry B. Helms- ley Charitable Trust; Type 1 Diabetes Exchange

218‑OR The Association of Dispositional Mindfulness and Adverse Child‑ hood Experiences with Glycemic Control among Young Adults with Type 1 Diabetes KATHRYN NAGEL, TRACY DEARTH-WESLEY, ALLISON N. HERMAN, HANNAH G. SMITH, RACHELLE GANDICA, LAUREN GOLDEN, HENRY F. WEIL, ROBERT C. WHITAKER, Cooperstown, NY, Philadelphia, PA, New York, NY To assess the potential of mindfulness practices to improve glycemic control in a diverse population of young adults with type 1 diabetes (T1D), we examined the cross-sectional association of hemoglobin (Hb)A1c levels with dispositional mindfulness and adverse childhood experiences (ACEs). In 2017 we conducted an online survey of all 19-31 year olds (y/o) with T1D receiving care at Columbia’s Naomi Berrie Diabetes Center. Responses were received from 423 of 752 (56%) patients and linked to medical record HbA1c. After adjusting for sociodemographic factors, HbA1c levels were compared between those with high and low mindfulness (median split) and those with (62%) and without ACEs (38%). The sample was 59% female and 69% non-Hispanic white, with a mean (SD) HbA1c of 8.0% (1.7). The relationship between HbA1c and mindfulness was moderated by age and ACEs. Among

ADA-Supported Research

A58 HIGH RISK BEHAVIORAL AND PSYCHOSOCIAL ISSUES IN DIABETES

27-31 y/o, those with high mindfulness had 0.7% lower HbA1c (95% CI:-1.2%, emerge during adolescence. We explored associations between EF and risk -0.2%) and this relationship was stronger in those with ACEs. The relation- of DEBs in teens, ages 13-17, with T1D. ship between HbA1c and ACEs was also moderated by age. Among 23-26 Methods: EF was assessed by the Behavior Rating Inventory of Execu- y/o, those with ACEs had 0.4% higher HbA1c (-0.1%, 0.9%). Among 19-22 y/o, tive Function (BRIEF), both teen self-report and parent proxy-report versions. HbA1c levels were 0.5% higher in those with high mindfulness (-0.2%, 1.2%) Scores ≥60, as total (Global Executive Composite [GEC]) score or on 2 indices and 0.8% lower in those with ACEs (-1.53%, -0.04%). The potential impacts (Behavioral Regulation [BRI] and Metacognition [MI]) or 8 subscales, indicate of mindfulness practices for young adults with T1D may differ by age and EF problems. DEB risk was assessed by the Diabetes Eating Problem Survey history of ACEs. Revised (DEPS-R). DEB risk was categorized by DEPS-R scores: low risk <10; Figure. moderate risk 10-19; high risk ≥20. Results: Teens (N=169, 46% female) were aged 15.9±1.3 (M±SD) years and had T1D for 8.4±3.7 years; A1c was 8.5±1.2% and 44% were overweight/ obese. Based on teen self-reported DEPS-R score, 59% had low DEB risk, 29% moderate risk, and 12% high risk. Higher DEB risk was significantly associated with EF problems by teen self-report on GEC, BRI, and MI and by parent report on BRI (Figure). EF subscales associated with DEB risk included emotional control, organization, task completion, goal setting, and problem- solving. Conclusion: In teens with T1D, executive dysfunction was associated with DEB risk, suggesting need for future study of the relationship between EF and DEBs. Figure.

219‑OR Few Pediatric Diabetes Patients Access Outpatient Psychotherapy following Screening and Referral JOANNA TSIKIS, JESSICA M. VALENZUELA, ARETI VASSILOPOULOS, CHELSEA WILCOCKS, LITAL REITBLAT, ERNESTO J. BLANCO, SHELLEY NICHOLLS, RISA WOLF, Davie, FL, Fort Lauderdale, FL, Baltimore, MD Introduction: Adolescents and young adults (AYAs) with diabetes are at risk for suboptimal psychological functioning. This study aims to describe the prevalence of elevated depression scores, outpatient psychotherapy referrals, and individuals receiving follow-up care in a clinic with an integrated psychologist. Further, we examine whether having an integrated psychologist leads to better access to mental health services for AYA. Methods: Participants were seen in a multidisciplinary diabetes clinic from September 2016 to 2017. Assessment included a semi-structured interview and depression screener (PHQ). Families with concerns related to diabetes management were offered on-site outpatient psychotherapy, others given community referrals. Follow-up with referrals was assessed at next diabetes visit. Results: Participants included 100 ethnically diverse AYAs, 11-21 years (M=16.06±2.42; 56% female), diagnosed with type 1 (79%), type 2 (19%), Supported By: National Institutes of Health (R01DK095273, K12DK094721, or MODY (2%), with a range of glycemic control (HbA1c 5.5 to >14%). PHQ P30DK036836); JDRF (2-SRA-2014-253-M-B); Fundación Alicia Koplowitz

scores were: no/minimal (58%), mild (26%), moderate (11%), moderately ORALS severe (3%), and severe (2%). 59% of patients were referred for outpatient 221‑OR psychotherapy. Referral reasons included depression, anxiety, behavioral Key Factors for Overcoming Psychological Insulin Resistance— difficulties, family conflict, and diabetes management challenges. At follow- An Examination of a Large International Sample through Content up, only 9% were receiving services. Barriers included transportation, orga- Analysis nization (e.g., losing referral), and stigma. HEATHER L. STUCKEY, WILLIAM POLONSKY, LAWRENCE FISHER, DANIELLE M. Conclusions: Pediatric psychologists within integrated diabetes teams HESSLER, FRANK J. SNOEK, TRICIA S. TANG, NORBERT HERMANNS, XAVIER offer effective mental health screening and referrals. Almost half (42%) MUNDET, MARIA ELIZABETH R. SILVA, JACKIE A. STURT, KENTARO OKAZAKI, of AYA in our cohort had ≥5 depressive symptoms, and while 59% were DACHUANG CAO, IRENE HADJIYIANNI, JASMINA I. IVANOVA, URVI DESAI, referred for outpatient therapy, only 9% received these services. Despite MAGALY PEREZ-NIEVES, Hershey, PA, Del Mar, CA, San Francisco, CA, Amster- patient access to a pediatric psychologist in clinic, a large discrepancy dam, Netherlands, Vancouver, BC, Canada, Bad Mergentheim, Germany, Barcelona, between referral and follow-up rates remains. This disparity highlights the Spain, São Paulo, Brazil, London, United Kingdom, Nagoya, Japan, Charlotte, NC, Bad importance of identifying and reducing barriers to follow-up care for AYAs Homburg, Germany, New York, NY, Boston, MA, Indianapolis, IN with diabetes. Treatment with insulin is often associated with higher rates of achieving glycemic control. This qualitative analysis identified the key patient-reported 220‑OR behaviors of health care providers (HCP) that motivated reluctant patients Associations between Executive Dysfunction and Disordered Eat‑ from 7 countries (n=40) to start insulin treatment. As part of a larger mixed ing Behavior (DEB) Risk in Teens with Type 1 Diabetes (T1D) methods study (EMOTION), which included a quantitative survey, explana- RAQUEL CECILIA-COSTA, MEREL HANSMANN, KARA R. HARRINGTON, LISA tory interviews with a subset of surveyed patients were conducted, tran- VOLKENING, LORI M. LAFFEL, Boston, MA scribed, and coded (.992 kappa) with the purpose of clarifying responses to Background: The adolescent brain remains immature with ongoing the survey. Frequently reported HCP behaviors that helped patients over- development in areas such as executive function (EF). DEBs also commonly come insulin resistance included: (1) explaining that insulin would help with diabetes control and/or avoid complications in the future [We have tried all

ADA-Supported Research

A59 BASIC AND TRANSLATIONAL MECHANISMS IN GESTATIONAL DIABETES MELLITUS

the best you could do with oral treatment; insulin might now be a better epsilon (IKKe) expression measured by immunofluorescence and Real-Time option]; (2) establishing trust with the patient [If I don’t trust a physician, I PCR. Knocking down IKKe, partially reversed this phenotype. PM2.5 exposure go somewhere else, because that’s very important]; (3) being available for during pregnancy and lactation increased body weight and fat mass of male support or questions [She always talks to me about my medications and she offspring, observed by a MicroPet CT analysis, accompanied by decreased FI wants to make sure that if I have any questions that I ask them]; (4) having and increased respiratory exchange ratio. Male offspring presented higher the patient give an insulin injection while in the office [He showed me how serum insulin levels and glucose intolerance measured by GTT despite nor- to stick the needle in and I didn’t even feel anything]; (5) explaining that mal insulin tolerance measured by ITT. Female offspring had no phenotype injecting insulin was easy [He let me know that I wouldn’t even notice it if I alterations, except by the light reduced food intake. injected the way he told me about]; and (6) referring the patient to a special- In conclusion, PM2.5 exposure during adulthood induced obesity, partially ist [First it was the GP, but he referred me to the endocrinologist who told due to a marked reduction in energy expenditure caused by hypothalamic me about starting on insulin]. Many of the patients understood that the path leptin resistance. Elevated inflammation may explain that with a significant of the disease was leading toward insulin, but it was the HCP who made the role of IKKe in the hypothalamus. Air pollution exposure during pregnancy recommendation based on hemoglobin A1C results. Reluctance to insulin ini- and lactation had a remarkable effect inducing obesity in the male offspring tiation can be alleviated by HCPs by preparing patients for insulin initiation due to an energy unbalance, which may lead to increased insulin levels and by explaining the benefits of insulin, establishing trust, being available for glucose intolerance. support, and demonstrating the insulin injection process. Supported By: Eli Lilly and Company; Boehringer Ingelheim 224‑OR Activation of Browning Adipose Tissues by Placental Growth Factor RUILI YIN, JING ZHOU, CHUANHAI ZHANG, CEN YAN, WEI MA, NANNAN WU, BASIC AND TRANSLATIONAL MECHANISMS IN DONG ZHAO, YINGMEI FENG, Beijing, China GESTATIONAL DIABETES MELLITUS Background: Gestational diabetes mellitus (GDM) is a type of metabolic disorders. Browning adipose tissue (BAT) activity is reduced which strength- 222‑OR ens insulin resistance in GDM. Placenta growth factor (PlGF) is one of the Maternal High-Fat Diet Persistently Alters Bone Marrow Immune main factors produced by placenta during pregnancy. Once secreted into Cell Proportions and Function in a Nonhuman Primate Model circulation, it participates in angiogenesis in peripheral tissues. However, MICHAEL J. NASH, TAYLOR K. SODERBORG, RACHEL C. JANSSEN, ERIC M. whether and how PlGF modulates BAT function is yet unknown. PIETRAS, JACOB E. FRIEDMAN, Aurora, CO Methods: We recruited 91 nondiabetic pregnant participants and 73 GDM Maternal obesity is rapidly increasing in prevalence and predisposes patients. Serum levels of PlGF were quantified by ELISA. Skin temperature offspring to many serious health risks such as cardiovascular disease, obe- was measured by Far infrared thermography in the supraclavicular region sity, and nonalcoholic fatty liver disease. Maternal high-fat diet (MHFD) where classical BAT was located. Primary human preadipocytes isolated can alter offspring metabolism and predispose offspring to obesity through from intercapsular BAT were differentiated under different concentrations many mechanisms, including alterations in immune cell function. However, of PlGF in vitro. Terminally differentiated adipocytes were characterized by whether maternal diet impacts development of the neonatal immune sys- qPCR, western blot and oxygen consumption rates by respirometry. Phos- tem remains unclear. To investigate the impact of MHFD on offspring bone phorylation signaling pathways in differentiated cells were dissected using marrow, a major site of immune cell generation via hematopoiesis, we Bio-Plex target assay (Bio-Rad). Cells were exposed with 1 ng PlGF in the utilized two distinct cohorts of non-human primates (NHPs). Bone marrow presence or absence of 1 nM AMPK inhibitor Compound C to assess UCP-1 from a cohort of early third trimester fetuses was examined to determine expression. the effects of MHFD vs. chow diet on bone marrow cellularity and func- Results: Serum levels of PlGF were lower in GDM patients than non GDM tion. The second cohort, 3-year-old juvenile NHPs exposed to MHFD early controls, which was accompanied by decreased skin temperature in the in life, then shifted to a chow diet at weaning for the remaining 2.5 years, supraclavicular region. By qPCR, PlGF dose-dependently induced BAT marker were studied to see whether bone marrow phenotypes resulting from MHFD expression in differentiated brown adipocytes (BAC) and peaked at 1 ng as exposure were reversible upon altering diet later in life. Colony-forming evidenced by the increased mRNA expression of UCP-1 and Oxphos. The assays of plated fetal bone marrow cells showed a significant 34.5% relative increased BAT marker expression was further confirmed by western blot in increase in innate immune system cells at the expense of erythroid (-78.9%) differentiated BAC exposed to 1 ng PlGF. Accordingly, PlGF-exposed BAC and multilineage (-53.8%) progenitors, and a decrease in total numbers of showed higher oxygen consumption rates than control and stimulated AMPK all cell types. Bone marrow stem cells differentiated to macrophages were phosphorylation. Blockade of AMPK phosphorylation abrogated UCP-1 and treated with LPS and showed significantly lower IL1B expression and trend- Oxphos expression in BAC induced by PlGF. ing decreased IL10 and TNF via qPCR, suggesting hyporesponsivity to inflam- Conclusions: Physically, PlGF improves preadipocyte differentiation into matory LPS stimuli. Similar phenotypes were found in the cohort of juveniles active BAC. Decreased PlGF levels might contribute to the reduced BAC exposed to MHFD then shifted onto a chow diet, suggesting that exposure activity in GDM. to MHFD has long lasting immunological consequences in offspring not cor- rectable by dietary changes later in life. Further, our results suggest remod- ORALS 225‑OR eling in macrophage function, potentially implicating an epigenetic program- Free Fatty Acids-Mediated Inflammation and Insulin Resistance/ ming event early in life driven by maternal diet. Insulin Production in Healthy and Gestational Diabetes Pregnant Supported By: National Institutes of Health Women XINHUA CHEN, THOMAS P. STEIN, THERESA O. SCHOLL, ROBERT A. STEER, 223‑OR Stratford, NJ Air Pollution Exposure during Pregnancy and Lactation Induces We investigated the relationship of individual free fatty acid (FFA, GCMS), Obesity and Glucose Intolerance in the Offspring inflammatory biomarkers (multiplex assay) with insulin resistance (HOMA- OLIVIA P. ZORDÃO, LAÍS W. CLARO, PAULO SALDIVA, JOSE DONATO, JR., MARI- IR) and production (C-peptide) levels during pregnancy from the Camden ANA VERAS, PATRICIA O. PRADA, Campinas, Brazil, São Paulo, Brazil Study, a prospective longitudinal cohort (n=1,784, African-American 36%, Air pollution has been a very significant environmental cause of diseases Hispanic 48%, caucasian 16%, age 22 ± 5.2 year., BMI 25.6 ± 6.1 kg/m2). Multivariate analyses were performed along with separate analyses for globally. Fine particulate matter (PM2.5) is one of the most toxic substances in air pollution and is associated with type 2 diabetes mellitus risk. However, each individual FFA. All of the analyses were adjusted for maternal age, there is a gap in knowledge regarding the effects of air pollution exposure pre-pregnancy BMI, parity, cigarette smoking and ethnicity. on insulin resistance and obesity development. We aimed to investigate The results obtained at entry to care (16 weeks gestation) are summarized: (i) Elevated HOMA-IR and C-peptide levels were associated with a whether 3 months of PM2.5 exposure induces obesity, hypothalamic leptin resistance and inflammation in adult mice. Also, we aimed to determine 50% to 2-fold (p<0.01 to p<0.0001) increased risk for developing gestational diabetes mellitus (GDM); (ii) Specific FFAs (%) (palmitoleic, oleic, linolenic whether PM2.5 exposure during pregnancy and lactation changes food intake (FI), energy expenditure, body composition and weight, glucose, and insu- acids) were significantly associated with decreased HOMA-IR and C-peptide levels (p<0.01 to p<0.0001); In contrast, palmitic, stearic, arachidonic lin metabolism in the offspring. Three months of PM2.5 exposure decreased acids and dihomo-γ-linolenic acid were associated with increased HOMA- O2 consumption measured by CLAMS, and increased body weight and fat mass in adult mice without FI changes. These mice presented hypothalamic IR and C-peptide (p<0.01 to p<0.0001); (iii) TNF-α, IL-8 and resistin levels leptin resistance with decreased STAT3 phosphorylation and enhanced IKK were positively and adiponectin was negatively correlated with HOMA-IR

ADA-Supported Research

A60 BASIC AND TRANSLATIONAL MECHANISMS IN GESTATIONAL DIABETES MELLITUS and C-peptide (p<0.001 to p<0.0001); and (iv) FFAs predicted cytokine levels. ing for the same covariates. Our results suggest that exposure to maternal For example, women with elevated palmitic acid were 50% (p<0.01) more obesity adversely impacts hippocampus volume in children, and this impact likely to have higher IL-8 and TNF-α (highest quartile), whereas women with is uniform across subfields. higher palmitoleic, oleic, linolenic acids levels had a reduced risk (~2-fold, Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-14- p<0.01 to p<0.001) of having higher IL-8, IL-6 and TNF-α levels. ACE-36 to K.A.P.) Our data suggest that different FFAs are involved in the regulation of insulin resistance and production during pregnancy. The effects are either 228‑OR direct or indirect via modulating inflammatory responses. Thus, recognition Maternal Diabetes Induces Congenital Heart Defects by Suppress‑ and modifying individual FFAs are important in lowering the risk for impaired ing Mitochondrial Fusion through the miR140 Mitofusin 1 Circuit glucose tolerance and the development of GDM. XI CHEN, WEI-BIN SHEN, PEIXIN YANG, Baltimore, MD Supported By: National Institutes of Health Pregestational maternal diabetes is a significant risk factor for congenital heart defects (CHDs) in the offspring. Increased apoptosis is implicated in 226‑OR CHD formation, yet the underlying mechanism is still unclear. Mitochondrial The Mitochondrial Cholesterol Transporter TSPO—Gatekeeper of fusion and fission dynamics play a key role in the regulation of cell viability. Gestational Progesterone Production Embryonic cardiomyocytes exposed to maternal diabetes display impaired MARICELA HAGHIAC, JUDI MINIUM, YELENNA SKOMOROVSKA-PROKVOLIT, fusion and enhanced fission, suggesting that maternal diabetes represses PATRICK CATALANO, SYLVIE HAUGUEL DE MOUZON, Cleveland, OH mitofusion gene expression. Here, we demonstrated that maternal diabe- During pregnancy, progesterone is exclusively produced by the placenta. tes in vivo and high glucose in vitro significantly increased microRNA-140 As in all steroidogenic tissues, the first and rate-limiting step for the bio- (miR140) expression whereas down-regulated its putative target gene synthesis of placental progesterone is the import of cholesterol into the Mitofusin 1 (Mfn1) expression in cardiomyocytes. We found that miR140 mitochondria via specific transporters. Plasma progesterone is 20% lower bound to the 3’ untranslated region of Mfn1 mRNA, leading to Mfn1 mRNA in pregnant obese women despite increased cholesterol availability sug- degradation. A miR140 mimic induced mitochondrial fragmentation, Mfn1 gesting an impairment in cholesterol import mechanisms. We investigated reduction and cell apoptosis, whereas a miR140 inhibitor abrogated high the role of the outer mitochondrial translocator protein (TSPO), the placental glucose-repressed Mfn1 expression and mitochondrial fusion. Deletion of analog of StAR, as a potential regulator of placental progesterone produc- the miR140 gene in vivo reversed diabetes-suppressed Mfn1 expression, tion. restored mitochondrial fusion, blocked cardiac cell apoptosis leading to a Study Design: Villous fragments of placentas were collected at term significant reduction of CHDs in diabetic pregnancy. Analysis of the miR140 scheduled Cesarean delivery of lean and obese women with normal glucose gene promoter revealed several putative binding sites of the transcription tolerance. Trophoblast cells were isolated and plated for primary culture. In factor, c-Jun, which is downstream of JNK2. Deletion of the Jnk2 (c-Jun vitro silencing of TSPO was performed by transfecting primary trophoblast NH2-terminal kinase 2) gene using the JNK2-/- mice mimicked the effect of cells with siRNA. Progesterone production was measured in lysates of tro- miR140 deficiency in suppressing diabetes-induced CHDs. JNK2 deletion phoblast cells before and after TSPO silencing. abolished the increase of miR140 and restored Mnf1 expression and mito- Results and Discussion: TSPO mRNA and protein expression were 2 to chondrial fusion. Our findings support the hypothesis that the JNK2-miR140- 3-fold lower in placentas of obese vs. lean women. TSPO siRNA silencing in Mfn1 circuit is critically involved in maternal diabetes-induced mitochondrial trophoblast cells induced a 90% (p<0.001) decrease in TSPO protein expres- fusion impairment and CHD formation. sion, with no change in other proximal translocators: the voltage anion Supported By: National Institutes of Health (1R01HL134368-01A1) dependent channel (VDAC) and the adenine nucleotide translocator (ANT). Progesterone concentration dropped by 85% (p< 0.001) upon TSPO silencing. 229‑OR These results demonstrate that the cholesterol translocator TSPO directly Survivin from Flk1+ Endothelial Progenitor Cells in the Develop‑ regulates the production of placental progesterone. Together, our data point ing Embryo Ameliorates Maternal Diabetes-Induced Neural Tube to mitochondrial dysfunction as a component of progesterone deficiency in Defects through Exosome obese pregnant women. SONGYING CAO, WEI-BIN SHEN, PEIXIN YANG, Baltimore, MD Supported By: National Institutes of Health Neural tube defects (NTDs) are among the most common and severe structural birth defects and maternal diabetes induces NTDs. During mouse 227‑OR embryonic development, blood vessel formation initially occurs in the yolk Maternal Obesity Is Associated with Reduced Hippocampal Volume sac, preceding neurulation which takes place from embryonic day 8 (E8) in Children to E10.5. Our previous studies have demonstrated that maternal diabetes KATHLEEN A. PAGE, SHAN LUO, XINHUI WANG, JASMIN ALVES, MAYRA P. induces embryonic vasculopathy at early embryonic developmental stage. MARTINEZ, ANNY XIANG, Los Angeles, CA, Pasadena, CA We hypothesize that early vasculopathy accounts for late failed neural Offspring exposed to maternal obesity in utero are at increased risk for tube closure in diabetic pregnancy. To test this hypothesis, we created the the development of metabolic disorders. Recent evidence also links expo- Flk1 promoter driven survivin (the cell cycle-regulated apoptosis inhibi- +

sure to maternal obesity with impaired cognition during childhood. While the tor) transgenic mice. Maternal diabetes induced the loss of Flk1 progeni- ORALS underlying mechanisms are unclear, studies in animals have shown that fetal tors, impaired blood island formation and reduced blood vessel densities. exposure to maternal obesity causes decreased neurogenesis in the dentate Expression of survivin in the Flk1+ progenitors ameliorated diabetes-induced gyrus and impaired hippocampal learning. The hippocampus is important for NTDs. Under nondiabetic conditions, survivin overexpression did not affect memory, and its development is sensitive to the in utero metabolic environ- embryonic development. Overexpression of survivin significantly reduced ment. We tested the hypothesis that in utero exposure to maternal obesity diabetes-induced NTDs. Flk1+ progenitors produced survivin-containing exo- would be associated with reduced hippocampal volume in children. Sixty- somes. Immunocytochemistry staining indicated that survivin-containing seven children (41 girls, 26 boys) ages 7 to 11 years participated in the study. exosomes were transported and incorporated into cells of the develop- Children were born at Kaiser Permanente Southern California. Maternal pre- ing neuroepithelium, leading to blockage of neuroepithelial cell apoptosis pregnancy BMI was obtained from electronic medical records. Children’s induced by maternal diabetes. Furthermore, we isolated exosomes from height and weight were measured, and a high-resolution anatomical scan Flk1+ progenitors derived from E7.5 embryos. In utero delivery of the survivin- was performed using a 3 Tesla MRI. Total hippocampus volume and hippo- containing exosomes by microinjection into the amniotic cavity of diabetic campus subfield volumes were analyzed using FreeSurfer 6.0. Linear regres- mice at E9 significantly inhibited maternal diabetes-induced neuroepithelial sion was used to investigate relationships between maternal pre-pregnancy cell apoptosis and reduced NTDs. Taken together, these data revealed that BMI and child hippocampus volume. Maternal pre-pregnancy BMIs ranged the cross-talk between the extra-embryonic structures and the developing from 19.0 to 50.4 kg/m2. Children’s BMI z-scores ranged from -1.8 to 2.6. neuroepithelium through the survivin-containing exosome plays a critical Maternal pre-pregnancy BMI was negatively correlated with total hippo- role for the proper neural tube closure. campus volume, adjusting for child age, sex, BMI-z score, intracranial volume (P=0.05). Results remained after further adjusting for maternal diabetes exposure (P=0.04). A 5-unit increase in maternal pre-pregnancy BMI was associated with 78 mm3 (95% CI:3.3-152.8 mm3) reductions in total hippocampus volume. The inverse correlation was also observed in CA3 (P=.04), CA4 (P=.03), dentate gyrus (P=.03) and subiculum (P=.01) subfields, adjust-

ADA-Supported Research

A61 FROM PROGRESSION TO MANAGEMENT IN TYPE 1 DIABETES—WHAT IS NEW?

FROM PROGRESSION TO MANAGEMENT IN TYPE 1 232‑OR DIABETES—WHAT IS NEW? The Pathological Evolution of Glucose Response Curves (GRCs) dur‑ ing the Progression to Type 1 Diabetes (T1D) in the TrialNet Pathway 230‑OR to Prevention Study Gut Microbiome Dysbiosis and Increased Intestinal Permeability in HEBA M. ISMAIL, PING XU, DOROTHY J. BECKER, INGRID LIBMAN, JENNIFER B. Australian Children with Islet Autoimmunity and Type 1 Diabetes MARKS, JAY S. SKYLER, JERRY P. PALMER, JAY SOSENKO, Pittsburgh, PA, Tampa, FL, Miami, FL, Seattle, WA JESSICA E. HARBISON, ALEXANDRA J. ROTH-SCHULZE, SIMON C. BARRY, Individuals with a biphasic (BP; two peaks) GRC during a 2-hour oral glu- CUONG D. TRAN, KATRINA NGUI, MEGAN A. PENNO, JOHN WENTWORTH, cose tolerance test (OGTT) are at lower risk of T1D than those with mono- PETER G. COLMAN, REBECCA THOMSON, MARIA E. CRAIG, ANTHONY T. PAPEN- phasic (MP; one peak) and monotonic (MT; continuous increase) GRCs. We FUSS, LYNNE GILES, LEONARD C. HARRISON, JENNY COUPER, Adelaide, Austra- thus performed a longitudinal analysis to examine whether: 1.) the different lia, Melbourne, Australia, Parkville, Australia, Westmead, Australia We aimed to characterise the gut microbiome and small intestinal perme- GRC risks are indicative of changes in GRC shapes during the progression to ability in children with islet autoimmunity and type 1 diabetes (T1D). 88 partici- T1D, and 2.) changes in GRCs are related to a deterioration in β-cell function. pants, median age 11.8 (IQR 8.1-13.3) years at baseline were followed 6 monthly Changes in shapes of GRCs were compared between progressors [n=360; for a median of 13 months (range 2-34). 18 had islet autoimmunity, 29 recent- mean age 13.8±10.9 years; 47.3% male] and non-progressors [n=2404; mean onset T1D, and 41 were age and gender-matched unrelated or sibling controls. age 18.1±13.1 years; 52.1% male] from first OGTTs to last OGTTs (before Stool samples were collected (Omnigene kit) for 16S rRNA gene sequencing. diagnosis for progressors). Median intervals from first to last OGTTs for Small intestinal permeability was measured by blood lactulose:rhamnose ratio progressors and non-progressors were 1.6 (0.9, 3.0) and 2.1 (1.0, 4.4) years, (L/R), 90 minutes after drinking a 5gm lactulose/1gm rhamnose solution. Anti- respectively. Logistic regression was used with adjustments for baseline bodies to insulin, GAD, IA2 and ZnT8 were assayed using IASP standardized age, sex, BMIZ and the interval between OGTTs. Progressor GRCs changed radiobinding assays. Statistical analysis used linear mixed models. Differential significantly more frequently than non-progressor GRCs from BP to MP abundance analysis used limma with total-sum scaling. [70.7% (29/41) vs. 46.3% (271/585); adjusted OR 3.00 (1.42-6.33); p=0.004)], Children with islet autoimmunity or T1D had gut microbiome dysbiosis and from MP to MT [12.4% (31/251) vs. 1.7% (19/1119); adjusted OR 8.85 (4.60-17.00); p<0.001)]. Overall, progressors had a higher frequency of MT with reduced α diversity and different β diversity compared with controls. Progressors from islet autoimmunity to T1D had lower observed richness than non-progressors at the last visit [12.8% (46/360) vs. 1.3% (31/2404); p<0.001)]. Of progressors with OGTTs at diagnosis, 39.7% (91/229) had MT (OTUs) (p=0.01) and different β diversity (Adonis, P=0.004), compared with non-progressors. Genus Prevotella, with anti-inflammatory properties, was GRCs. Among progressors, the early (30-0 min) C-peptide response (ng/ml) decreased in those who changed from BP to MP (3.27±1.78 vs. 2.44±1.63, less abundant in children with T1D (p<0.005) or ≥ 2 islet antibodies (p<0.005) than in controls. Diet did not explain this difference. L/R was higher in T1D than p=0.003) and from MP to MT (2.31±1.31 vs. 0.96±0.71, p<0.001). sibling controls (mean difference +3.03±1.02, p=0.003). Progressors had higher In conclusion, GRC risk differences for T1D result from characteristic L/R than non-progressors (+4.74±1.97, p=0.01) and sibling controls (+4.62±1.69, changes in GRCs during progression. A substantial proportion of progressors p=0.006). There was a trend towards a relationship between higher L/R and to T1D evolve to MT GRCs at diagnosis. Reductions in the early C-peptide response correspond to changes in GRCs from BP to MP and from MP to lower observed richness in the microbiome over time in children with ≥ 2 islet antibodies or T1D (coeff -0.0617, 95% CI -0.1254, 0.002 P=0.058). MT, which suggests that the changes in GRCs reflect a pathological decline Australian children progressing from islet autoimmunity to T1D have gut in β-cell function. dysbiosis and increased intestinal permeability. This observation supports a Supported By: National Institutes of Health mechanism underlying progression to T1D. Supported By: Channel 7 Children’s Research Foundation of Australia; JDRF; 233‑OR National Health and Medical Research Council of Australia Pancreas Volume Declines over the First Year after Diagnosis with Type 1 Diabetes (T1D) 231‑OR JACK VIROSTKO, MELISSA A. HILMES, JONATHAN M. WILLIAMS, JORDAN Comparison of Novel MicroRNA and Cell-Free DNA Biomarkers of WRIGHT, LIPING DU, HAKMOOK KANG, WILLIAM E. RUSSELL, DANIEL J. Beta-Cell Death and Diabetes Progression MOORE, ALVIN C. POWERS, Austin, TX, Nashville, TN The pancreas is smaller in individuals with T1D, but it is unclear if these MUGDHA JOGLEKAR, RYAN FARR, WILSON WONG, KRISTINA ROTHER, ALICIA individuals are born with a smaller pancreas or whether their pancreatic JENKINS, CHITTARANJAN S. YAJNIK, RONALD C. MA, MARIA E. CRAIG, TIM volume declines as part of disease progression. To determine the dynamics JONES, ANAND HARDIKAR, PREDICT T1D STUDY GROUP, Sydney, Australia, Gee- of pancreas volume in new-onset T1D, we performed longitudinal magnetic long, Australia, Bethesda, MD, Pune, India, Hong Kong, China, Westmead, Australia, resonance imaging (MRI) of the pancreas in individuals with recent-onset Perth, Australia Type 1 diabetes (T1D) is characterized by the loss of insulin-producing beta- T1D (n = 51, mean age 13.7 years, range 8-24 years) within the first 100 days cells. We currently lack diagnostic tools to measure T1D progression. Using after diagnosis (mean 67 days), six months after diagnosis, and one year systematic approaches involving unprejudiced discovery analyses in human post diagnosis. In agreement with and extension of our results presented at 2017 ADA Scientific Sessions, we found that at disease onset, young indi- ORALS tissues and clinical samples, we identify microRNAs that are: i) associated with insulin expression during embryonic development (N>60); ii) predictive of viduals with T1D had a smaller pancreas (median volume = 29.6 ml) than and necessary for insulin transcription (N=698); and iii) dysregulated in indi- similarly aged controls (median volume = 49.6 ml, n = 51, p < 0.001). Pancreas viduals with (vs. without) T1D (N=200). Insulin cfDNA (assessed by ddPCR) and volume was lower in T1D individuals when normalized by subject weight microRNA (assessed by TaqMan qPCR) biomarkers were measured in around; i) (p < 0.001), as well as when adjusted for age, gender and body mass index N=400 Australian T1D individuals; ii) N=30 longitudinal samples from children (p < 0.001). Longitudinal measurements of pancreas volume increased in at risk of T1D; iii) N=100 clinical trial samples from individuals receiving IFN-a controls over the year, corresponding with adolescent growth, but pancreas or Exenatide vs. placebo; iv) N=400 Hong Kong Chinese individuals with T1D; volume declined over the first year after T1D diagnosis (p < 0.001). Pancreas v) N=700 Danish individuals at diagnosis of T1D and vi) N=400 Asian Indian volume measurements normalized by body weight were stable in controls, T1D subjects. Among N=600 Australian samples, we identified miRNAs (N=5) but declined in subjects with T1D (p < 0.001). We also performed MRI in 14 that were significantly increased in the circulation of T1D individuals (vs. con- individuals enrolled in TrialNet with multiple autoantibodies portending the trols), decreased (at least 2 Fold Change/FC, P<0.05, N=7miRNAs) in T1DCx+ development of T1D, and found that pancreas volume was similar to con- vs. T1DCx- (no T1D complications) or significantly increased (2 FC, P<0.05, N=5) trols (median volume = 59.1 ml), but significantly greater than the T1D cohort in T1D individuals with detectable C-peptide vs. non-detectable C-peptide. Our (p < 0.001). To interrogate microstructural changes in the pancreas, we studies highlight the potential of circulating microRNAs and cfDNA in quanti- performed diffusion-weighted MRI and found that individuals with recent- fying beta cell death before clinical diagnosis of T1D. These data demonstrate onset T1D had a higher apparent diffusion coefficient (p = 0.023), indicating that microRNA/cfDNA signatures not only facilitate the potential prediction reduced restrictions on water diffusion in the pancreas in T1D. These results of diabetes progression, but can also help in stratifying individuals at risk of indicate the existence of ongoing pancreatic atrophy during the first year T1D as well as assess treatment efficacies in trials aiming to retard beta-cell after diagnosis with T1D which may be reflected by microstructural changes death. We also present our preliminary analysis of cross-ethnic variation in the pancreas. in these cfDNA/miRNA biomarkers and present ongoing/future studies to Supported By: JDRF develop a diagnostic test for T1D prediction. Supported By: JDRF; The Leona M. and Harry B. Helmsley Charitable Trust

ADA-Supported Research

A62 FROM PROGRESSION TO MANAGEMENT IN TYPE 1 DIABETES—WHAT IS NEW?

234‑OR 236‑OR Metformin Improves Insulin Resistance (IR) and Vascular Health in Overnight to Early-Morning Glycemic Outcomes in Children Using Youth with Type 1 Diabetes (T1D) the MiniMed™ 670G Hybrid Closed-Loop (HCL) System KRISTEN J. NADEAU, PETTER BJORNSTAD, MICHAL SCHÄFER, LORNA BROWNE, GREGORY P. FORLENZA, DOROTHY I. SHULMAN, MICHAEL A. WOOD, TIMOTHY AMY BAUMGARTNER, YESENIA GARCIA REYES, ARISTIDES MANIATIS, R. PAUL S. BAILEY, BRUCE W. BODE, BRUCE BUCKINGHAM, SUIYING HUANG, JOHN WADWA, SUNIL NAYAK, LAURA PYLE, MELANIE CREE-GREEN, JANE E. REUSCH, SHIN, SCOTT W. LEE, TONI L. CORDERO, FRANCINE R. KAUFMAN, Aurora, CO, Aurora, CO, Denver, CO, Centennial, CO, Greenwood Village, CO Tampa, FL, Ann Arbor, MI, Escondido, CA, Atlanta, GA, Palo Alto, CA, Northridge, CA Cardiovascular disease is the leading cause of mortality in T1D and relates In-home use of the MiniMed™ 670G system with SmartGuard™ technology to IR. We demonstrated that lean and obese T1D youth have IR and that for 3 months improved A1C, 24-hour, and overnight (10PM-7AM) glycemia metformin improves surrogate markers of IR in obese T1D youth. Yet, little is in T1D patients ≥14 years.1 Recently, the system was shown to improve known about vascular health in T1D youth or about the effect of metformin A1C and time in target range (TIR) (>70-180mg/dL, 3.9-10mmol/L) during on directly-measured IR or vascular health. In The Effects of MEtformin on 24-hours and overnight periods in T1D children aged 7-13 years. Effect of the cardiovasculaR function in AdoLescents with type 1 Diabetes (EMERALD) system on insulin delivery and early morning glycemia was also examined. study, we hypothesized that T1D youth have impaired vascular function and During a 2 week run-in phase in Manual Mode and a 3-month study phase that metformin decreases IR and improves vascular health. 49 T1D youth using HCL Auto Mode; 24-hour, 10PM-7AM, and 3AM-7AM insulin delivery ages 12-21 years (40% with BMI ≥ 90%ile; 44% male) and 24 controls of (programmed hourly basal rates during Manual Mode, variable basal from similar age, BMI and sex underwent phase contrast MRI of the ascending 0 units to an individualized daily maximum every 5min during Auto Mode); (AA) and descending aorta (DA) to determine aortic pulse wave velocity time in sensor glucose (SG) ranges; and SG SD were examined in 105 T1D (PWV) and wall shear stress (WSS). T1D youth also had carotid intima-media children (mean±SD age 10.8±1.8 years). The Table shows Auto Mode use thickness (cIMT) by ultrasound, DXA scan, fasting labs following overnight IV across periods. Total insulin delivered increased from baseline run-in (1990 glycemic control, and a hyperinsulinemic-euglycemic clamp (80 mU/m2/min patient-days) through study phase (10,602 patient-days); SG, TIR, and SG SD insulin) to assess IR (glucose infusion rate [mg/kg/min)]/insulin or M/I). T1D improved. During the study phase, there was no severe hypoglycemia, DKA, youth were then randomized 1:1 to 2g of metformin or placebo daily, and or serious device-related AEs. The ability of the MiniMed™ 670G system to all measures repeated at 3 months. At baseline, T1D youth vs. controls had automate basal insulin delivery and safely improve 24-hour and overnight elevated aortic PWV (AA: 3.7±0.2 vs. 2.5±0.4 m/s, p<0.05; DA: 4.2±0.2 vs. glycemic control in children was comparable to that observed in patients 3.1±0.3 m/s, p=0.02) and WSS (AA: 1.1±0.04 vs. 0.9±0.05 N/m2, p=0.003; DA: 14-75 years, over 3 months. Improved early morning glycemia further indi- 1.6±0.06 vs. 1.1±0.07 N/m2, p<0.0001). Compared to the placebo group, T1D cates that patients ≥7 years can benefit from this HCL therapy. youth in the metformin group had no baseline differences but had improve- Table. ment at 3 months in M/I (12.2±3.2 vs. -2.4±3.6 uIU/uL, p<0.01), AA PWV (-1.1±1.1 vs. 4.1±1.6 m/s, p<0.04), AA WSS (-0.03±0.04 vs. 0.2±0.05 N/m2, p<0.03) and far wall diastolic cIMT (-0.04±0.01 vs. 0.00±0.01 mm, p=0.04) in baseline-adjusted models. T1D youth have IR and vascular dysfunction compared to controls of similar age, sex and BMI. Metformin mitigated IR and improved aortic and carotid health in T1D youth, and thus may hold promise as a cardiovascular protective intervention. Supported By: American Diabetes Association (7-11-CD-08 to K.J.N.)

235‑OR Alpha-1 Antitrypsin Therapy in Recent-Onset Type 1 Diabetes YAEL LEBENTHAL, AVIVIT BRENER, ELI HERSHKOVITZ, NAIM SHEHADEH, SHLO- MIT SHALITIN, SHARON GAI-CASTRO, MICHAL STEIN, NAVEH TOV, MARIANNA RACHMIEL, Tel Aviv, Israel, Beersheba, Israel, Haifa, Israel, Petah Tikva, Israel, Rehovot, Israel, Zerifin, Israel Previous phase I studies demonstrated that alpha-1 antitrypsin (AAT) was safe and well tolerated in type 1 diabetes (T1D) patients. This multicenter, randomized, placebo-controlled phase II trial was designed to test whether 237‑OR AAT could preserve C-peptide secretion in new-onset T1D patients aged Timing of CGM Initiation in Pediatric Diabetes—The CGM TIME 8-25 years. Seventy patients (37 males; mean age 13.1 ± 4.1 years) were Trial randomized (1:1:1) to treatment with 22 IV infusions − 60 mg/kg AAT, 120 MARGARET L. LAWSON, JENNILEA COURTNEY, BRENDA J. BRADLEY, KAREN mg/kg AAT or placebo administered over 1 year. The primary end point of MCASSEY, CHERIL CLARSON, SUSAN E. KIRSCH, JACQUELINE CURTIS, FARID the study was the change in C-peptide area under the curve (AUC) from a H. MAHMUD, CHRISTINE RICHARDSON, TAMMY COOPER, KEN TANG, THE 2-hour mixed-meal tolerance test (MMTT) after 1 year. Secondary endpoints CGM TIME TRIAL STUDY GROUP, JDRF CANADIAN CLINICAL TRIAL NETWORK ORALS were HbA1c and safety parameters. Post-hoc sub-group analysis by age CCTN1101, Ottawa, ON, Canada, Hamilton, ON, Canada, London, ON, Canada, was performed. At 1 year, the decrease in C-peptide AUC was not different Markham, ON, Canada, Toronto, ON, Canada in AAT vs. placebo groups (P = 0.66 and 0.96, for 60 and 120 mg/kg AAT). Background: The impact of timing of CGM initiation in pump naïve pediat- Within 12-18 years subgroup (n=35), C-peptide AUC in placebo-treated and ric patients on CGM adherence and hemoglobin A1C remains unclear. 60 mg/kg AAT-treated patients decreased significantly from baseline (-0.34 Methods: A 5-site RCT of pump naïve subjects (aged 5-18 years) with and -0.55 pmol/mL, respectively, P < 0.01) while no significant decrease was type 1 diabetes (T1D) > 1 year was conducted to compare simultaneous observed in the 120 mg/kg AAT-treated patients (-0.18 pmol/mL, P = 0.20). pump and CGM (“SIM” group) vs. standard pump therapy with CGM initia- At 1 year, mean HbA1c levels were higher in placebo-treated patients and tion delayed for 6 months (“DEL” group). A step-wise approach to CGM set- 60 mg/kg AAT-treated patients vs. 120 mg/kg AAT-treated patients (8.24 ± tings was used. Primary outcome was CGM adherence (hours per 28 days 1.42% and 7.85% ± 1.67% vs. 6.66% ± 1.01%, respectively, P = 0.05 for 120 (MiniMed™ Veo™ system and CareLink Professional use)) assessed over six mg/kg AAT vs. placebo); a lower percentage of patients attained HbA1c months after CGM initiation. The main secondary outcome, A1Cs, was mea- ≤ 7% in the placebo and 60 mg/kg AAT groups compared to 120 mg/kg AAT sured centrally at baseline and every 6 months. Linear mixed-models and (25% and 28.6% vs. 70%, P = 0.07). The infusions were well tolerated with ordinary least squares models were fitted to further estimate the effect of a similar safety profile in the AAT and placebo groups. Two patients in the intervention, and covariates of baseline age, T1D duration, and A1C, as well 60 mg/kg treatment group had allergic reactions after 4 and 17 infusions. as gender and clinical site. Although AAT intervention shows promise with a favorable safety profile, Results: We randomized 144 of 152 (95%) eligible subjects into the SIM the efficacy of AAT in T1D patients is inconclusive. Further studies should or DEL intervention. Five (3 SIM, 2 DEL) withdrew before visit 1 and 1 (DEL) be undertaken with appropriate patient stratification pre-randomization and a week after pump start, with 70 SIM, 68 DEL eligible for analysis. Base- implementation of biomarkers to determine whether AAT has a beneficial line mean age was 11.5±3.3(SD) years, T1D duration 3.4±3.1 years, and A1C effect on beta cell preservation. 7.9±0.9% (range 5.5-11.2%). During the 6th month, median CGM adherence was 485.6 hours (95% CI: 229.5, 585.7) in SIM vs. 408.2 (95% CI: 134.9, 572.8) in DEL, with this difference in adherence translating into an additional

ADA-Supported Research

A63 NEW INSIGHTS INTO THE PATHOGENESIS OF DIABETIC RETINOPATHY

2.8 hours of CGM per day. Median 6-month A1C was 7.9 [IQR: 7.5, 8.4] for Results: High glucose increased 5mC levels at Mfn2 promoter by over 2 SIM vs. 7.8 [IQR: 7.4, 8.4] for DEL. Fitted models suggest those randomized fold, and decreased Mfn2 expression. Dnmt inhibition ameliorated glucose- to SIM had better CGM adherence (main effect = 74.4 [95% CI: 26.8, 121.9], induced increase in 5mC at Mfn2 and mitochondrial membrane permeability, p=0.002), with higher adherence among females (p=0.03). Overall, there was and decrease in Mfn2 expression, complex III activity and Cytb transcripts. no difference in A1C 6 months after CGM initiation (-0.20 [95% CI: -0.45, Similarly, in mouse retinal microvessels, inhibition of Dnmt prevented dia- 0.06], p=0.13). betes-induced increase in 5mC at Mfn2 promoter and decrease in Mfn2 Conclusion: CGM adherence was higher when initiated at the same time expression. as pump therapy however there was no significant effect on A1C. More Conclusions: In diabetes, epigenetic modifications of Mfn2 promoter research is required to understand why CGM did not have an effect on A1C impair mitochondrial functional and genomic stability. Thus, targeting DNA and examine how CGM may affect other measures of glycemic control. methylation could help maintain mitochondrial homeostasis, and halt/retard Supported By: JDRF Canadian Clinical Trials Network the development of diabetic retinopathy. Supported By: National Institutes of Health

NEW INSIGHTS INTO THE PATHOGENESIS OF 240‑OR DIABETIC RETINOPATHY MALAT1 and HOTAIR—Key Epigenetic Regulators in Diabetic Reti‑ nopathy 238‑OR SAUMIK BISWAS, ANU A. THOMAS, BIAO FENG, SHALI CHEN, ERFAN AREF- Loss of Hypothalamic Somatostatin (SST) Contributes to Bone Mar‑ ESHGHI, JOHN GONDER, BEKIM SADIKOVIC, SUBRATA CHAKRABARTI, London, row (BM) Dysfunction and Diabetic Retinopathy (DR) in Type 2 Dia‑ ON, Canada betic Rats Introduction: With the increasing incidence of diabetic retinopathy (DR), ANA LEDA LONGHINI, JEFFREY THINSCHMIDT, MICHAEL BOULTON, MICHAEL A. the need for novel and specific therapy is essential. Genomic studies have KING, MARIA B. GRANT, Birmingham, AL, Gainesville, FL identified several long non-coding RNAs (lncRNAs). However, majority Purpose: Since loss of retinal SST and circulating SST occurs in DR, we of lncRNAs have not been characterized in DR. We examined the roles of asked if loss of hypothalamic SST production would impact the innervation lncRNAs MALAT1 and HOTAIR in DR. and function of the BM and influence DR in T2D rat model. Methods: Human retinal endothelial cells (HRECs) were cultured in 5 mM/L Methods: Brain sections of BBZ/Wor rats with 4 months of diabetes and (NG) or in 25 mM/L (HG) glucose. RNA expressions of MALAT1, HOTAIR, and age-matched nondiabetic lean BBZ rats were immuno-stained for SST, Iba-1 inflammatory and angiogenic cytokines were tested. HRECs were similarly and Neu-N. To examine the neuronal connection between the BM and pre- examined following siRNA-mediated MALAT1 or HOTAIR knockdown, or sympathetic brainstem and hypothalamic areas, both control and diabetic treatment with histone (DZNep) and DNA methylation (5’-aza-dC) blockers. rats were injected in a blinded manner with GFP-transgenic pseudorabies DNA methylation patterns and RNA-protein interactions were analyzed. virus (PRV-152) into their femurs. Four days later rats were euthanized and Retinal tissues from Malat1 knockout (KO) and wild type (WT) mice with or brain sections prepared for colocalization of GFP with SST. DR was assessed without diabetes were examined. We further assessed MALAT1 and HOTAIR by enumeration of acellular capillaries in trypsin-digested retinas. in human diabetic and nondiabetic vitreous. Results: Injection of PRV into femur showed the presence of active virus Results: HG caused upregulations of MALAT1, HOTAIR, IL-6, TNF-α, and in neurons of the paraventricular nucleus of the hypothalamus (PVN), ros- VEGF-A transcripts in HRECs. HG increased binding of EZH2 (a PRC2 com- tral ventrolateral medulla and nucleus tractus solitarius of the brain of con- ponent) with MALAT1 and HOTAIR and evoked unique DNA methylation trol rats. Colocalization studies showed that periventricular hypothalamic patterns in the MALAT1 and HOTAIR CpG regions. DZNep, 5-aza-dC and SST neurons were also GFP+ and directly innervated the BM. Quantitation knockdown of MALAT1 or HOTAIR reduced mRNA expressions of IL-6, TNF- showed that diabetic rats had significantly fewer GFP+ neurons in the PVN α, and VEGF-A. Similarly, diabetes-induced elevations of these inflammatory (n=6, *P<0.05), supporting neuronal loss. SST staining of brain sections and angiogenic cytokines in the retina were prevented in the Malat1 KO showed significant reduction in the number of SST+ cells in diabetic rats mice. Furthermore, MALAT1 and HOTAIR levels were elevated in the diabetic (58±5) compared with nondiabetic rats (88±6) (n=6, *P<0.05). The density vitreous. of Iba-1+ microglia increased, while the NeuN+ neurons decreased in the Conclusions: Our findings allude to the importance of lncRNAs in influenc- hypothalamus supporting an increase of regional inflammation and neuronal ing DR through epigenetic mediators. Understanding the role of lncRNAs loss. Diabetic rats showed an increase in acellular capillaries compared to may allow the development of better-targeted therapies in DR. controls (15± 4 vs. 4±1mm2) (p<0.001). Supported By: Diabetes Canada; Heart and Stroke Foundation of Canada; Law- Conclusions: These studies support the notion that periventricular son Internal Research Fund hypothalamic SST neurons directly innervate BM and that preservation of this small, but important population is relevant to prevention of diabetes 241‑OR induced-BM dysfunction and development of DR. Effects of Diabetes on Retinal Protein Lysine Malonylation Supported By: National Institutes of Health (EY028037) STEVEN F. ABCOUWER, SUMATHI SHANMUGAM, HEATHER M. HAGER, CHENG-

ORALS MAO LIN, PATRICE E. FORT, THOMAS W. GARDNER, KELLI SAS, SUBRAMANIAM 239‑OR PENNATHUR, Ann Arbor, MI Epigenetics and Mitochondrial Stability in Diabetic Retinopathy Purpose: The addition of short chain acyl groups to lysines is emerging as RENU KOWLURU, MANISH MISHRA, Detroit, MI key post-translational modifications (PTM) linking metabolite levels to meta- Mitochondria constantly fuse and divide to form a cellular network, and bolic enzyme activities. Malonyl-lysine (Kmal) targets and inhibits glycolysis this dynamic fusion-fission controls their function and genome stability. and beta-oxidation enzymes. Recently, formation of Kmal was suggested Although both, mitofusin 1 and 2 (Mfn1 and 2) regulate outer mitochondrial to act as a sink for mitochondrial malate, preventing its inhibition of suc- membrane fusion, Mfn2 itself is sufficient to modulate mitochondrial metab- cinate dehydrogenase in the electron transport chain (ETC). Using the db/ olism. In diabetes, retinal mitochondria are swollen, respiration is impaired, db T2DM mouse model, we recently found that diabetes increases retinal Mfn2 is decreased and mtDNA is damaged. Diabetes also facilitates epigen- glycolysis and beta-oxidation metabolite levels, whereas malate was the etic modifications, altering gene expressions without changing their DNA only TCA cycle metabolite significantly increased. Metabolic flux measure- sequences. This study aims to investigate the role of DNA methylation of ment with 13C-labeled glucose also demonstrated increased enrichment of Mfn2 in the development of diabetic retinopathy. labeled malate. We therefore compared the relative levels of Kmal-modified Methods: Human retinal endothelial cells, incubated in normal (5mmols/L) proteins in retinas from diabetic mice as well as in postmortem retinas from or high glucose (20mmols/L) for four days, in the presence/absence of DNA diabetic patients to determine if this PTM is altered by diabetes. methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), were Methods: Immunofluorescence (IF) with antibodies specific to acetyl- analyzed for methylated cytosine (5mC) at Mfn2 promoter by immunocap- lysine, to succinyl-lysine and to Kmal was used to probe retinal sections turing method. Mitochondrial integrity was evaluated by membrane per- from BKS-db/db mice and BKS-db/+ controls (n=9/group) at 24 weeks of age. meability, coenzyme Q: cytochrome-c reductase (complex-III) activity, and Kmal modifications were also examined in mouse retinas from mice made transcripts of mtDNA-encoded cytochrome b (Cytb). Similar measurements diabetic with streptozotocin (STZ) and controls (n=6/group) and a limited were made in the retinal microvessels from streptozotocin-induced diabetic number of retinal sections from postmortem eyes of diabetic (n=4) and con- mice receiving 5-Aza (2.5mg/kg, i.p.) or Dnmt1-siRNA (2µg/2µl, intravitreal). trol (n=2) patients. Antibodies to markers of retinal ganglion and glial cells were used to co-localize Kmal-modified proteins.

ADA-Supported Research

A64 NEW INSIGHTS INTO THE PATHOGENESIS OF DIABETIC RETINOPATHY

Results: Only Kmal IF intensity was significantly reduced by approximately 244‑OR 30% in both db/db and STZ diabetic mouse retinas. Kmal IF exhibited a 25% Longitudinal Association of Microaneurysm Wall Characteristics reduction in human diabetic retinas. Kmal-modified proteins were concen- with Perfusion and Local Neuroretinal Pathology trated in the ganglion cell layer and largely coincided with Müller glia end- JENNIFER K. SUN, OMAR ABU-QAMAR, KONSTANTINA SAMPANI, WARD FICK- feet and/or astrocytes. WEILER, LLOYD P. AIELLO, Boston, MA Conclusions: The results suggest that protein malonylation in glial cells is Although microaneurysms (MAs) are a hallmark lesion of diabetic reti- diminished in the diabetic retina, which may increase glycolysis and beta- nopathy, it is unclear why some MAs result in local neural retinal pathology oxidation while inhibiting the ETC. while others are benign. To address this issue, longitudinal follow-up of MAs Supported By: National Institutes of Health/National Institute of Diabetes with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral and Digestive and Kidney Diseases (R24DK082841); National Eye Institute domain optical coherence tomography (SDOCT) was performed. AOSLO (R01EY020582) MA images were graded for wall hyperreflectivity (WH), wall deformability (WD), cross-sectional area and perfusion percentage. SDOCT images were 242‑OR graded within a 500µm zone centered on each MA for disorganization of Deletion of REDD1 Prevents Hyperglycemia-Induced Reactive Oxy‑ the retinal inner layers (DRIL). Fluorescein angiography evaluated leakage gen Species Accumulation and Retinal Cell Death in a subset. A total of 457 MAs of 102 eyes (57 subjects: mean±SD age WILLIAM P. MILLER, ALLYSON TORO, MICHAEL D. DENNIS, Hershey, PA 44±12 years, DM duration 23±10 years, 61% male, 73% type 1 DM) were Apoptosis of retinal neurons is accelerated by diabetes and the onset of assessed over 843 visits (154 MAs with >1 visit and mean follow-up of increased cell death occurs early in disease progression. The present study 0.52±0.35 years). At baseline, MAs with WH were larger (p<0.0001), less was designed to evaluate molecular defects that contribute to diabetes- perfused (p<0.0001), and associated with DRIL (p=0.002). There was a bor- induced neuroretinal apoptosis and visual dysfunction. We recently demon- derline trend for such MAs to leak fluorescein (p=0.05). Over time, MAs with strated that the stress response protein REDD1 is induced by hyperglycemia resolved WH had greater likelihood of DRIL improvement. Conversely, MAs in the retina of diabetic mice in a manner that contributes to neuronal cell that developed WH had more DRIL worsening (p=0.005). WH change vs. death and visual dysfunction. One potential mechanism whereby REDD1 stability was associated with decreased perfusion over time (p<0.0001). At promotes neuronal apoptosis is through upregulation of reactive oxygen baseline, WD was associated with smaller MAs (p<0.0001) and increased species (ROS), as the protein forms a pro-oxidant complex. In the retina of perfusion (p<0.0001), but was not related to fluorescein leakage or DRIL. WD streptozotocin-induced diabetic mice, ROS levels, neuronal cell death, and resolution and development were associated with decreased and increased REDD1 expression were increased concomitant with attenuation of contrast perfusion, respectively (p<0.0001), but not with change in DRIL. These data sensitivity thresholds, as assessed by behavioral optomotry. In R28 retinal suggest that whereas WH is a feature of more mature, larger and less per- cells in culture, hyperglycemic conditions increased REDD1 expression, ROS fused MAs associated with local neural pathology, WD may be present in levels, and cell death. However, addition of the antioxidant N-acetyl-L-cys- earlier-stage MAs that are smaller and more fully perfused. Future studies teine (NAC) to culture medium was sufficient to prevent the increase in ROS will determine whether these parameters can be used to predict changes and cell death in response to hyperglycemic conditions. Moreover, REDD1- in MA size, perfusion, leakage or local neural retinal pathology over time. deficient cells did not exhibit increased ROS or cell death in response to Supported By: National Eye Institute (5R01EY024702-04); JDRF (3-SRA-2014- hyperglycemic conditions. Similarly, in REDD1-deficient diabetic mice, ROS 264-M-R); Massachusetts Lions Eye Research Fund; Thomas J. Beatson, Jr. levels and cell death in retina were similar to that observed in nondiabetic Foundation mice. Finally, oral administration of NAC improved contrast sensitivity in diabetic mice relative to vehicle treated diabetic controls, demonstrating a key 245‑OR role for ROS levels in diabetes-induced visual dysfunction. Overall, the find- Diabetic Retinopathy Reversed by Intravitreous Application of Reti‑ ings support therapeutic approaches targeting REDD1 and ROS production nol Binding Protein 3 in the prevention of diabetes-induced visual dysfunction. HISASHI YOKOMIZO, KYOUNGMIN PARK, ALLEN CLERMONT, YASUTAKA Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-14- MAEDA, WARD FICKWEILER, ATSUSHI ISHIKADO, QIAN LI, LIANE J. TINSLEY, INI-04 to M.D.D.) DAVID M. POBER, I-HSIEN WU, LLOYD P. AIELLO, HILLARY A. KEENAN, JENNIFER SUN, GEORGE L. KING, Boston, MA, Takatsuki, Japan 243‑OR The Joslin Medalists study, enrolling people with type 1 diabetes of >50 Epsin Deficiency Promotes Lymphangiogenesis through Regulation years, suggested protective factors exist against diabetic retinopathy (DR), of VEGFR3 Degradation in Diabetes even in the presence of hyperglycemia. Proteomic analysis of retina and HONG CHEN, Boston, MA vitreous suggested that Retinol Binding Protein 3 (RBP3), secreted by photo- Lymphangiogenesis occurs in adult tissues of chronic complex diseases, receptors, to be elevated in the Medalists without vs. those with advanced including diabetes. Whether lymphangiogenesis is altered owing to diabetic DR. This finding was confirmed by ELISA RBP3 assays in replicating vitreous complication remains unknown. VEGF-C/VEGFR-3 signaling axis fuels the samples of Medalists and other diabetic patients. In contrast, vitreous VEGF growth of lymphatic vessels. How VEGF-C/VEGFR3 signaling is regulated levels increased with DR severity; resulting in decreasing RBP3/VEGF with worsening DR. Transgenic mice overexpressing RBP3 targeted to the pho-

under pathological conditions, such as diabetes, however, is poorly under- ORALS stood. Here, we report that mice with inducible lymphatic endothelial cell- toreceptors prevented diabetes induced retinal capillary permeability and specific deficiency of epsins 1 and 2 (LEC-iDKO) display enhanced lymphan- formation of acellular capillaries. To determine whether RBP3 can inhibit DR giogenesis compared to wild type (WT) in diabetic condition. A profound progression, recombinant human RBP3 (rhRBP3) was injected intravitreously increase in VEGF-C-induced lymphatic vessel growth is exhibited in corneas (IVT) in rats with VEGF or after 2 months of diabetes. IVT injection of rhRBP3 and subcutaneous Matrigel implanted in diabetic LEC-iDKO mice compared inhibited VEGF induced increases in retinal vascular permeability both after to diabetic WT mice. Conversely, lymphatic endothelial cells isolated from a few min or 1 day post-VEGF injections (p=0.009 or p=0.01), respectively. diabetic WT mice show marked impairment in proliferation, migration, and After 2 months of diabetes, IVT rhRBP3 reversed capillary permeability to tube formation in response to VEGF-C relative to those from diabetic LEC- levels of nondiabetic rats (p=0.007), whereas boiled hRBP3 was ineffective. iDKO in vitro. Mechanistically, reactive oxygen species (ROS) generated from Surprisingly, IVT rhRBP3 also reduced retinal expression of VEGF and IL-6 in hyperglycemia induces c-Src-dependent VEGFR3 phosphorylation indepen- diabetic rats and normalized retinal function, as measured by B wave ampli- dent of VEGF-C, which parallels c-Src-dependent epsin upregulation through tude of electroretinography. Using cultured retinal endothelial cells and the transcription factor AP-1. Heightened epsin expression induced by ROS Muller cells, the addition of rhRBP3 inhibited VEGF- or high glucose-induced facilitates epsin binding to VEGFR3 within Golgi compartment, promoting cell migration, possibly by binding to VEGF receptors. Surprisingly, rhRBP3 degradation of newly synthesized VEGFR3, and progressively reducing avail- also inhibited high glucose-induced expression of VEGF and IL-6 mRNA lev- ability of VEGFR3 at the cell surface. Consequentially, lymphatic-specific els (p=0.05) similar to in vivo studies. Thus, IVT application of RBP3 can inter- epsin loss strengthens insufficient lymphangiogenesis and improves the vene to delay, stop and reverse diabetes-induced neuro- and vascular-retinal resolution of tail edema in diabetic mice. Collectively, our data indicate that functional and inflammatory abnormalities, strongly supporting a therapeu- inhibiting epsin expression protects VEGFR3 against degradation and ame- tic potential for the use of RBP3 for DR. liorates diabetes-triggered downregulation of lymphangiogenesis, providing a plausible original therapeutic strategy to treat diabetic complication.

ADA-Supported Research

A65 DIABETES SELF-MANAGEMENT EDUCATION AND SUPPORT—EMBRACING DATA AND DIGITAL DIALOGUE ADVANCES

DIABETES SELF-MANAGEMENT EDUCATION 248‑OR AND SUPPORT—EMBRACING DATA AND DIGITAL Enabling Data-Driven Decision Making for Personalized Care in DIALOGUE ADVANCES Type 2 Diabetes with Clinical and Lifestyle Journey Data Using a Digital Therapeutic RAJEEV CHAWLA, MAAZ SHAIKH, ABHISHEK SHAH, BANSHI D. SABOO, BRIJ 246‑OR M. MAKKAR, JOTHYDEV KESAVADEV, SHILPA JOSHI, NEETA DESHPANDE, SAN- Presessions to the National Diabetes Prevention Program Are a JAY AGARWAL, ANUJ MAHESHWARI, ARAVIND R. SOSALE, SR., SV MADHU, Promising Strategy to Improve Attendance and Weight Loss Out‑ New Delhi, India, Mumbai, India, Ahmedabad, India, Delhi, India, Trivandrum, India, comes Belgaum, India, Pune, India, Lucknow, India, Bangalore, India NATALIE RITCHIE, PETER G. KAUFMANN, MARK GRITZ, JODI S. HOLTROP, Den- Background: Lack of data on patient lifestyle between consultation visits is a ver, CO, Aurora, CO major limitation in practice of data-driven clinical decision making in type 2 dia- Introduction: The National Diabetes Prevention Program (NDPP) is a betes (T2DM). We showcase Wellthy Diabetes (WD), a digital therapeutic, as widely-disseminated lifestyle intervention. Retention in the NDPP is prob- an effective tool for the amplification of patient’s clinical and lifestyle journey lematic and leads to suboptimal weight loss, especially among racial/eth- data between visits to aid doctors in practice of data-driven decision-making. nic minority participants. We report results of implementing a novel “pre- Methods: Wellthy Diabetes (WD) smartphone app is a digital therapeutic session” protocol to support engagement among a diverse sample of NDPP that delivers an artificial intelligence (AI) augmented lifestyle modification participants. program for people with T2DM, developed in scientific collaboration with Methods: The NDPP was delivered in a safety net healthcare system. As the Research Society for Study of Diabetes India (RSSDI). The WD app uses of September 2016, pre-sessions were offered prior to NDPP attendance and an AI-based digital persuasion model to encourage users through person- focused on (1) education on diabetes risks, (2) motivational interviewing to alized reminders and nudges to self-report data on blood glucose, weight, participate in the NDPP, and (3) problem-solving barriers to engagement. We meals, and physical activity. The app also tracks the duration of physical compared NDPP attendance and weight loss outcomes between 75 pre-ses- activity through inbuilt sensors on the phone. This study presents data from sion participants and a historical control group of 42 individuals who were a cohort (N=102) of completers of a 16-week program. not offered a pre-session prior to their beginning the NDPP in July 2016. This Results: During the study 121,675 diabetes-related clinical and lifestyle comparator group was selected to reduce potentially confounding effects of data points (including 112,960 mins of sensor-tracked physical activity) were other prior modifications to NDPP dissemination over time. reported by 102 participants over 17,052 person days. The mean duration Results: The majority of participants in this analysis were female (78.3%), of a participant on WD was 167 days during which, on average, 452 clinical low-income (51.1%), and Hispanic (57.6%). Mean age was 46.6 (SD=12.7) and lifestyle data points were tracked per participant at a mean velocity of with a mean baseline BMI of 35.9 (SD=5.7). There were no significant base- 5.6 data points per day. On average each participant reported 68.8, 11.4, 5.1, line differences in characteristics between NDPP participants who received and 1107.7 instances of meals, blood sugar, weight, and activity respectively. a pre-session and historical controls. Pre-session participants attended Conclusion: The results confirm WD amplifies self-reporting of clinically 5.3 (SD=1.4) more NDPP sessions (p<.001) and stayed in the program 96.4 relevant lifestyle data by users during the journey between doctor consulta- (SD=24.6) days longer (p<.001) than historical controls. Pre-session partici- tions. First-hand data on clinical trends and lifestyle between consultations pants achieved 1.8% (SD=0.8) greater weight loss (p=.039) and were 2.4 provide personalized insight into patient behavior enabling doctors to make times more likely to achieve >5% weight loss in the NDPP as historical con- data-driven decisions for personalizing evidence-based care for T2DM. trols; p=.044, 95% CI [1.0-5.8]. Conclusions: By improving engagement and weight loss, findings suggest pre-sessions may be a promising strategy to improve NDPP effectiveness 249‑OR and mitigate health disparities in program outcomes. Innovative Tailored Microlearning Approach to Insulin Self-Man‑ Supported By: Colorado Department of Public Health & Environment; America’s agement Education Shows High Engagement Levels among Diverse Health Insurance Plans/Centers for Disease Control and Prevention; Denver Health Adults MAGDALENA M. BOGUN, NICOLE E. JELESOFF, CARINE M. NASSAR, CONNIE R. THACKER, JOHANNA CAMACHO RIVERA, CLAYTON J. BOURGES III, KARAN 247‑OR SETHI, LISA EGBUONU-DAVIS, ANNE C. BEAL, DEBORAH A. GREENWOOD, Mediating Role of Acculturation and Lifestyle Behaviors on Cardio‑ HERLENE CHATHA, ANJALI KATARIA, DIANA B. MCNEILL, ROBIN GOLAND, metabolic Risks among Asian Indians in the United States MICHELLE F. MAGEE, New York, NY, Hillsborough, NC, Washington, DC, Durham, NITHA MATHEW JOSEPH, RANJITA MISRA, JING WANG, STANLEY CRON, PAD- NC, Boston, MA, Hanover, MD, Bridgewater, NJ, Bethesda, MD MAVATHY RAMASWAMY, Houston, TX, Morgantown, WV nd Self-management education for adults with type 2 diabetes (T2DM) Background: Asian Indians are the 2 largest Asian immigrants in the U.S. improves knowledge, skills and motivation; yet, new strategies to more and have higher rates of diabetes, metabolic syndrome, and cardiovascular effectively educate patients and improve adoption of insulin therapy are disease when compared to the general U.S. population and other immigrant still needed. We conducted a prospective, non-randomized multi-center groups. Acculturation levels influence the lifestyle practices and studies pilot, using a novel Patient Experience Cloud to deliver microlearning edu- have reported acculturated individuals have increased risks for chronic dis- cation focused on survival skills, insulin injection technique, and lifestyle ORALS eases. management. Content was culturally relevant and tailored to minority Aim: Hence, this study explored the mediating role of lifestyle behaviors patients with T2DM who recently initiated insulin treatment to enhance (physical activity and dietary behavior) between acculturation and cardio- engagement. Consented T2DM adults, ages 20 to 70 years, were presented metablic risk factors among Asian Indians using the DIA (Diabetes in Indian an online library of 56 education videos (1-4 minutes each). Pre and post Americans) national study. surveys of self-reported attitudes towards insulin and patient activation Methods: The sample comprised 1038 randomly selected adult Asian measures (using the validated PAM scale) were obtained. The study enrolled Indians in seven U.S. sites (mean age was 48.54 ±12.8 years). Acculturation 201 patients: 78 non-Hispanic whites (NHWs), 74 African Americans (AAs), was assessed using the English proficiency scores from the Acculturation 39 Hispanic whites (HWs) and 10 Other. Of these, 181 (90%) were engaged Scale for Southeast Asians and dietary behavior and physical activity were (defined as completing baseline surveys and watching 4+ videos). The num- calculated from the subscales of Health Promotion Lifestyle Profile II. Path ber of videos viewed differed by race, p=0.012; with median number of 23 analyses with bootstrap methods were used to explore if physical activ- for AAs, 13 for NHWs and 5 for HWs. One week later, 83 participants (41%) ity or dietary behavior mediates the relationship between acculturation and continued engagement via viewing and completion of post questionnaires. cardiometabolic risk factors (HbA1c, fasting blood glucose levels, Body Mass Highest engagement was seen among AAs (51%) vs. 40% of NHWs and Index, systolic and diastolic blood pressure, and lipid profiles [HDL, LDL, total 26% of HWs. Overall attitudes towards insulin improved significantly, with cholesterol, and triglycerides]). a mean change of 0.29 points (on a 6 point Likert scale, 95% CI 0.02 to 0.56, Results: Dietary behavior significantly mediated the relationship between p=0.03). Similarly, the PAM score increased significantly after viewing (by acculturation and HbA1C (β=0.004, p = .047) and physical activity mediated 0.17 point on a 4 point scale, 95% CI 0.08 to 0.26, p<0.001), independent of the relationship between acculturation and HDL (β=0.08, p = .011). All other race. After viewing culturally tailored microlearning delivered via a cloud mediation models were not significant (p>.05) based on the nonsignificant platform, patient attitudes towards insulin and activation measures for self- indirect effects detected in the models. care improved significantly. This novel approach should be further investi- Conclusion: Culturally specific dietary education and physical activity gated for its ability to improve adherence to insulin and glycemic outcomes. interventions for this high risk ethnic group may have potential to reduce Supported By: Mytonomy; Sanofi cardiometabolic risks. Results provide firm basis for educational program.

ADA-Supported Research

A66 DIABETES SELF-MANAGEMENT EDUCATION AND SUPPORT—EMBRACING DATA AND DIGITAL DIALOGUE ADVANCES

250‑OR 252‑OR On Time—An Innovative Online Discussion Tool to Overcome Bar‑ Impacts of a Two-Part Multidisciplinary Diabetes Education Pro‑ riers to Insulin Initiation gram on Glycemic Control and Hospital Use JEREMY GILBERT, GAIL MACNEILL, ELAINE M. COOKE, PIERRE FILTEAU, HEATHER P.Q. WHITLEY, ANGELA STALLWORTH, DEEPTHI DESAI, COURTNEY MICHAEL VALLIS, MÉLANIE GROLEAU, PASHA JAVADI, CYNTHIA LEBOVICS, HANSON, Montgomery, AL, Tuscaloosa, AL Toronto, ON, Canada, Maple Ridge, BC, Canada, Saint-Marc-des-Carrières, QC, Background: Emergency room and hospital use remain high among those Canada, Halifax, NS, Canada, Laval, QC, Canada, Paris, France with diabetes. Interventions need to be developed to reduce the utilization The On Time education program is an innovative, online point-of-care tool of this high-cost care while still improving diabetes management. designed to uncover and address barriers to insulin initiation, and to facili- Purpose: To analyze changes in outcomes for patient with diabetes who tate timely insulin initiation, when appropriate, in insulin-naïve individuals attend a two-part multidisciplinary diabetes education program, which with type 2 diabetes (T2D). A total of 195 health care professionals (HCPs) includes a 1-day DSME class and a ½ day “Speed Dating” session in terms completed online profiles of 1025 insulin-naïve individuals with T2D cur- of ER and hospital use, A1C, and LDL-cholesterol from January 2016-2018. rently treated with noninsulin antihyperglycemic agents (NIAHAs) and with Method: Adult patients with diabetes at a family medicine clinic are a glycated hemoglobin (A1C) above the Diabetes Canada target (for most enrolled and attend an 8-hour DSME class followed by a 3-hour multidisci- individuals ≤7%). After having completed the discussion tool and question- plinary clinic 2 weeks later during which patients rotate every 30 min through naires, participating HCPs were asked to evaluate the program, tool, and 5 centers hosted by a 1.) family medicine physician, 2.) clinical pharmacist questionnaires. Mean age of participants was 61.2 years; 55% were male; (CDE), 3.) nurse or dietician (CDE), 4.) case manager, and 5.) clinical psycholo- mean duration of diabetes was 10.5 years. For the majority of participants gist to address and improve care. Data was systematically extracted from (70%) the recommended A1C target was ≤7.0%. On average, participants the electronic medical record (EMR) 6 months before, 6 and 12 months after were prescribed 2.6 NIAHAs, mainly metformin and dipeptidyl peptidase-4 attending the class to determine impact of the program. Paired t-tests deter- inhibitors. Prior to using the On Time discussion tool, only 23% of individuals mined significance between baseline and post-intervention measures. with diabetes were judged as likely (16%) or extremely likely (7%) willing to Results: Over 24 months, 22 adults attended the program. At baseline initiate insulin. The leading barriers to initiating insulin were apprehension patients had an average A1C of 10.2% and LDL of 115 mg/dL. Six months toward needles/injections (59%), belief that insulin was complicated (56%), before attending the class they presented to the ER 0.68 times, were admit- and psychological insulin resistance (45%). After using the On Time discus- ted to the hospital 0.5 times, and when admitted, had an average length of sion tool, participants’ perceived willingness to initiate insulin increased stay (LOS) of 5.3 days. After attending the program patients’ A1C declined (likely: 34%, extremely likely: 28%). Initiation of insulin was planned in 77% to 9.1% at 6 months (p=0.28) and 8.7% at 12 months (p=0.022). Their LDL-C of participating individuals. The evaluation questionnaire was completed by declined to 110 mg/dl (p = 0.71). Although ER use increased to 0.8, their hos- 149 HCPs (76.4%), and showed that the discussion tool was perceived to pital admission rate and LOS decreased to 0.36 and 3.3 days respectively help HCPs address insulin-related barriers (82%), positively impacted their per patient. practice (79%), and improved their approach when discussing insulin initia- Implications: Incorporation of the DSME plus Speed Dating program tion with individuals with diabetes (76%). Identifying the barriers to initiat- improved care by lower A1C by 1.5%, hospital admission rate by 0.14, and ing insulin and providing educational interventions to address them may help LOS by 2 days over a 6-month window. Ultimately these improvements addi- to improve insulin acceptance. tionally lower healthcare costs. Supported By: Alabama Department of Public Health; Auburn University; Baptist 251‑OR Health Foundation CoMac Communication System—A Feasibility Implementation of Language-Centered Intervention for T2DM 253‑OR ULLA M. CONNOR, ROBERT SANDY, MARY DE GROOT, ROBERT S. MACNEILL, Diabetes Educator Impact in Value-Based Care Models JR., Indianapolis, IN JANICE L. KOSHINSKY, JODI KRALL, KRISTINE RUPPERT, JUSTIN KANTER, The CoMac Communication System is a scientifically tested set of tools to FRANCIS X. SOLANO, JR., LINDA M. SIMINERIO, Pittsburgh, PA individualize Diabetes Self-Management Education and Support (DSMES), Background: Health systems are implementing value-based care models by speaking to persons with type 2 diabetes (T2DM) using their own lan- focused on quality. Organized care teams set the foundation for successful guage and worldviews. models with processes that address patient outreach and engagement. The In this quasi experimental study, conducted in a Midwestern diabetes purpose of this study was to evaluate the impact of “Glucose to Goal (G2G),” care clinic with an accredited DSMES, a tablet was provided for patients a diabetes educator (DE) driven, population-based intervention on glycemic to complete the CoMac segmentation survey. Altogether 72 patients took improvement in reaching quality metrics for diabetes mellitus (DM) care. the survey and received the tailored communication. An unplanned control Methods: A large primary care (PC) network established pod-style group of 48 patients emerged, due to occasional unavailability of the tablet/ patient-centered medical homes (PCMHs) clustered by grouping multiple survey. These patients received the clinic’s standard DSMES, without the PC practices together based on patient panel size, hospital affiliation, and use of the CoMac tools. A1C levels from the first visit to the follow-up visit geographic proximity, and incorporating expanded care teams (DE, behav- for each group were compared. ioral health, social worker, wellness guide, nurse care manager). Specific Interviews with the healthcare providers (HCPs), conducted during the to DM, PCMHs adopted the G2G intervention whereby DEs used popula- ORALS six-month study, provided evidence about the success of the implementation health reports to proactively identify patients with HbA1c levels above tion. Individual psychosocial profiles and language recommendations helped target (>7%). G2G was assessed in 5 pods by comparing HbA1c values for HCPs better align their language. “Words matter” led to more efficient and only those patients with DM with pre/post results who participated over a effective behavioral goal settings with the patients. three-month period (n=284) to those who did not (n=265). The A1Cs of the intervention group decreased from 8.9 to 7.3, the control Results: A total of 80% of patients receiving G2G improved their HbA1c group’s from 8.3 to 7.6. A multiple regression analysis with the A1C level levels with 46.8% reducing HbA1c by > 1% and 24% reducing HbA1c by > 2%. change as the dependent variable showed that the independent variables of Comparatively, 31.3% of patients not receiving G2G improved their HbA1c age, gender, and starting weight were not significant predictors of A1C reduc- with only 6.4% reducing HbA1c by > 1% and only 1.5% reducing HbA1c by tion. Statistically significant were the starting value of A1C and the CoMac 2% or more. This translated into more than 1.5% difference in mean HbA1c intervention. Patients with higher initial A1Cs showed a greater decline in change between groups, with patients receiving DE experiencing a mean A1Cs. The key variable of interest, treated with the CoMac intervention, had reduction in HbA1c of 1.35% (8.61 to 7.26%) compared to increase of 0.35% a t-value of -1.81, with a prob value of 0.037. Although a cause-and-effect (7.96 to 8.30%) for those not participating in DE (p<0.0001). relationship cannot be proven without random assignment, the significantly Conclusions: Study findings demonstrate the relevance of DE services in improved A1Cs of the intervention group are highly encouraging. value-based PC models. As healthcare systems shift focus from volume to As diabetes care teams and delivery models expand, person-centered lan- value, effective approaches to manage diabetes and improve patient out- guage tools are an innovation in effective management of DSMES. comes take on new meaning.

ADA-Supported Research

A67 SGLT2 INHIBITORS—FROM MECHANISMS TO CLINICAL TRIALS

SGLT2 INHIBITORS—FROM MECHANISMS TO 256‑OR CLINICAL TRIALS Research into the Effect of Sodium-Glucose Linked Transporter 2 Inhibition on Left Ventricular Remodeling in Patients with Heart 254‑OR Failure and Diabetes Mellitus Mechanism by Which Dapagliflozin Induces Euglycemic Ketoacido‑ JAGDEEP S.S. SINGH, IFY MORDI, MOHAPRADEEP MOHAN, STEPHEN J. GANDY, sis in Rats EWAN PEARSON, JOHN G. HOUSTON, ALLAN D. STRUTHERS, CHIM C. LANG, Dundee, United Kingdom RACHEL J. PERRY, JOONGYU D. SONG, YONGLIANG WANG, GERALD I. SHUL- Introduction: DM and heart failure (HF) are lethal, with limited diabetic MAN, New Haven, CT Sodium-glucose transport protein 2 inhibitors (SGLT2i) are the most recent therapy options. Sodium-glucose linked transporter 2 (SGLT2) inhibitors have class of antidiabetic agents; however concerns have been raised about their been reported to have CV benefits in at risk DM patients. We are the first to potential to induce euglycemic ketoacidosis by an unknown mechanism. study the CV effects of SGLT2 inhibition in patients with DM and HF. Here we report that dapagliflozin increases rates of hepatic ketogenesis Methods: In this randomized double-blind placebo controlled trial, 56 (35±4 vs. 136±8 µmol/[kg-min], p<0.0001) in normal Sprague-Dawley rats patients (mean age: 67.1 years, males: 66%) with DM and echocardiographi- by increasing plasma catecholamine (epinephrine 0.5±0.1 vs. 7.3±0.5 nM, cally confirmed HF with reduced ejection fraction (HFrEF) on regular diuretic p<0.001, norepinephrine 2.5±0.5 vs. 19.8±5.2 nM, p<0.01) and corticoste- therapy were assigned to dapagliflozin 10mg OD or placebo for one year. rone (115±15 vs. 233±28 ng/mL, p<0.01) concentrations secondary to volume Primary endpoint was the difference in left ventricular (LV) volumes between depletion (weight change -1.9±0.4% vs. -7.1±0.4%), thereby increasing white both groups using cardiac MRI. Secondary endpoints include LV mass, LV adipose tissue lipolysis, hepatic acetyl-CoA content, hepatic glucose pro- ejection fraction (EF), weight, BP and diuretic use. Outcomes were analysed duction and hepatic ketogenesis. Dapagliflozin-induced reductions in plasma using linear regression controlling for baseline values, age, sex and renal insulin concentrations were shown to be necessary but not sufficient to function. induce these changes since the effects of dapagliflozin to increase hepatic Results: There was no difference between dapagliflozin and placebo in ketogenesis were abrogated when extracellular volume depletion was pre- the primary endpoints of LV end diastolic volume (LVEDV) or LV end systolic vented with saline infusion or free access to water. Furosemide treatment volume (LVESV); +4.71mls; 95% CI: -17.08 to 26.50 and +1.52mls; 95% CI: induced similar volume depletion and increases in plasma catecholamine -15.68 to 18.72 respectively. However, when an interaction term for start- and corticosterone concentrations without any associated changes in WAT ing LVEF was added to the model, dapagliflozin significantly lowered LVEDV, LVESV and LV mass in those with starting LVEF ≥ 45%; -15.59mls; p=0.019, lipolysis, hepatic glucose production or hepatic ketogenesis, likely due to 2 its lack of an effect on plasma insulin levels. Furthermore the effects of -15.20mls; p=0.016 and -4.87gm/m ; p=0.026. Patients on dapagliflozin had dapagliflozin to promote increased rates of hepatic ketogenesis and hepatic weight reduction; -1.90kg; 95% CI: -3.83 to +0.04; p=0.054, lower diastolic glucose production were dissociated from changes in plasma glucagon BP; -6.34mmHg; 95% CI: -11.35 to -1.32; p=0.014 and higher hemoglobin; concentrations, which increased (89.5±3.8 vs. 379.2±11.8 ng/mL, p<0.0001) +1.23 g/dl; 95% CI: 0.65 to 1.82; p<0.001. They were also more likely to stop through a central mechanism following ICV injection of dapagliflozin without or reduce loop diuretics; 50.0% vs. 8.7%; p=0.005. any alterations in body weight or ketosis, and hepatic malonyl-CoA. Taken Conclusions: Our data show dapagliflozin treatment resulted in weight together these data identify SGLT2i-induced dehydration as a potential tar- and blood pressure reduction among patients with DM and HFrEF. There was get to prevent SGLT2i-induced euglycemic ketoacidosis. evidence to suggest that dapagliflozin may cause LV reverse remodelling in DM patients with mild, but not with more severe HFrEF. Supported By: U.S. Public Health Service (R01DK-113984, R01DK-40936, P30DK- Supported By: European Foundation for the Study of Diabetes 059635, T32DK-101019, K99CA-215315, R01NS-087568, UL1TR000142, T32DK- 007058) 257‑OR 255‑OR Comparing the Effects of Ipragliflozin and Metformin on Visceral Fat SGLT2 Inhibitor Ipragliflozin Induces Breast Cancer Apoptosis via Reduction in Patients with Type 2 Diabetes Inadequately Controlled Membrane Hyperpolarization and Mitochondria Dysfunction with Sitagliptin—A Prospective, Multicenter, Blinded-Endpoint, Randomized Controlled Study in Japan (PRIME-V Study) SHIHO KOMATSU, TAKASHI NOMIYAMA, TOMOHIRO NUMATA, TAKAKO MASAYA KOSHIZAKA, KO ISHIKAWA, RYOICHI ISHIBASHI, YOSHIRO MAEZAWA, KAWANAMI, YURIKO HAMAGUCHI, TOMOKO TANAKA, RYUJI INOUE, TOSHI- KENICHI SAKAMOTO, DAIGAKU UCHIDA, SUSUMU NAKAMURA, HIDETAKA HIKO YANASE, Fukuoka, Japan Currently, cancer is one of major cause of death in patients with type 2 YOKOH, AKINA KOBAYASHI, SHUNICHIRO ONISHI, KAZUKI KOBAYASHI, JUN diabetes. We have previously reported the anti-prostate and anti-breast OGINO, HIROTAKE TOKUYAMA, FUMIO SHIMADA, EMI OHARA, TAKAHIRO cancer effect of GLP-1R agonist Exendin-4 (Diabetes 2014, Endocrinology ISHIKAWA, MAYUMI SHOJI, KANA IDE, SHINTARO IDE, YUSUKE BABA, TAKURO 2017). In the present study, we examined the anti-cancer effect of SGLT2 HORIKOSHI, RYOTA SHIMOFUSA, SHO TAKAHASHI, KENGO NAGASHIMA, inhibitor ipragliflozin (Ipra) using a breast cancer model. In human breast can- MINORU TAKEMOTO, KOUTARO YOKOTE, Chiba, Japan, Kisarazu, Japan, Asahi, cer cell line, MCF-7 cells, SGLT2 expression was detected using both RT-PCR Japan, Yachiyo, Japan, Narita, Japan and immunohistochemistry. 1-50nM Ipra significantly and dose-dependently Objective: To determine the effects of SGLT2 inhibitor or metformin on reducing visceral fat in Asian patients with type 2 diabetes inadequately ORALS suppressed the growth curve of MCF-7 cells. BrdU assay revealed that Ipra attenuates the proliferation rate of MCF-7 in a dose dependent manner. Fur- controlled with DPP-4 inhibitor, sitagliptin. ther, apoptosis was also induced by Ipra in Tunel assay. Because the anti- Methods: A prospective, multicenter, blinded-endpoint, randomized con- breast cancer effect of Ipra was completely canceled by knocking down of trolled trial was conducted to evaluate the efficacy of treatment with either SGLT2, this effect could be induced by SGLT2 inhibition by Ipra. We next SGLT2 inhibitor (ipragliflozin) or metformin added on to sitagliptin for visceral measured membrane potential and whole cell current using the patch clamp fat reduction and glucose control in patients with type 2 diabetes. Patients technique. When we treated MCF-7 cell with Ipra or glucose free medium, who met the eligibility criteria were randomly selected (1:1) to receive ipra- membrane hyperpolarization was observed. In addition, the replacement of gliflozin (Ipr; 50 mg daily) or metformin (Met; 1000 mg daily). The primary sodium with NMDG and knock-down of SGLT2 by siRNA suppressed glucose outcome is the change rate in visceral fat area, measured using computed induced whole cell current of MCF-7 cell, suggesting that Ipra inhibits sodium tomography, after 24 weeks of therapy. Two radiologists, blinded to the and glucose incorporation through SGLT2. Further, mitochondrial membrane information, analyzed the images. protein Bcl-2 was decreased and Bax was increased in western blotting, Results: A total of 51 patients were in enrolled in the Ipr group and 52 and JC-1 fluorescence was significantly increased, suggesting the change of in the Met group. The reduction rate in visceral fat area was significantly mitochondrial membrane potential. These data suggest that SGLT2 inhibitor greater in the Ipr group (-12.06% vs. -3.65%, group difference of -8.40%; 95% Ipra induces apoptosis in the breast cancer cell via membrane hyperpolariza- CI of -16.4 to -3.38, P<0.05) than in the Met group. Subcutaneous fat area, tion and mitochondria dysfunction. total fat area, body weight, BMI, and waist circumference also decreased significantly in the Ipr group. The Ipr group showed a significant decrease in fasting insulin (-20.73% vs. 0.85%, group difference -21.58%, 95% CI 2.80 to 34.20, P<0.05), while HbA1c decreased in the Met group compared with the Ipr group. The difference in the muscle volume and bone mineral density between the two groups was insignificant. Conclusion: Ipr significantly reduced the visceral fat area compared with Met as the secondary agent used in combination with DPP-4 inhibitors. As

ADA-Supported Research

A68 SGLT2 INHIBITORS—FROM MECHANISMS TO CLINICAL TRIALS the fasting insulin level decreased, the reduction in visceral fat associated gram and the NCS are reported. In the CANVAS Program, incidence of OD- with Ipr administration may have contributed to the improvement of insulin and VD-related AEs and GMI was higher with CANA vs. PBO, consistent with resistance. NCS (Table). There were no imbalances in other labeled AEs including hypo- Supported By: Astellas Pharma Inc. glycemia and renal-related AEs. The rate of serious and discontinuation- related labeled AEs was similar with CANA and PBO. CANA was generally 258‑OR well tolerated for up to 6.5 years in the CANVAS Program with an AE profile Canagliflozin and Cardiovascular (CV) Outcomes in Patients with (excluding amputation and fracture) consistent with NCS, which included a Chronic Kidney Disease more general T2D population. BRENDON L. NEUEN, TOSHIAKI OHKUMA, BRUCE NEAL, DAVID R. MATTHEWS, DICK DE ZEEUW, KENNETH W. MAHAFFEY, GREG FULCHER, MEHUL DESAI, QIANG LI, HSIAOWEI DENG, NORM ROSENTHAL, MEG JARDINE, GEORGE BAKRIS, VLADO PERKOVIC, Sydney, Australia, Oxford, United Kingdom, Groningen, Netherlands, Stanford, CA, Raritan, NJ, Chicago, IL SGLT2 inhibitors are approved for glucose lowering in type 2 diabetes (T2DM) and have proven CV benefits, but are not approved for people with significantly reduced kidney function as glycemic efficacy is dependent on glomerular filtration. This analysis of the CANagliflozin cardioVascular Assessment Study (CANVAS) Program assessed effects of canagliflozin on CV outcomes in T2DM according to kidney function (estimated glomerular filtration rate [eGFR] <45, 45-<60, 60-<90, and≥ 90 ml/min/1.73 m2). The CANVAS Program included 2039 patients (20.1%) with baseline eGFR <60 ml/min/1.73 m2 (mean age 68 years, BP 138/76 mmHg, HbA1c 8.3%, eGFR 49 ml/min/1.73 m2, median urinary albumin:creatinine ratio 22 mg/g). Effects of canagliflozin on HbA1c and body weight were smaller in patients with eGFR <60 vs. ≥60 ml/min/1.73 m2 (-0.43 vs. -0.64%, P-heterogeneity <0.0001, and -1.16 vs. -1.43 kg, P-heterogeneity = 0.0002), but BP effects were similar (-3.89 vs. -4.11 mmHg, P-heterogeneity = 0.21). Relative effects on the primary and most other CV outcomes were similar across four eGFR subgroups, with possible heterogeneity suggested only for the exploratory outcome of stroke (Figure). Results for almost all safety outcomes were also consistent across eGFR subgroups. Despite smaller glycemic effects in people with reduced eGFR, the cardioprotective benefits of canagliflozin are similar across different levels of kidney function. ORALS

Supported By: Janssen Research & Development, LLC.

259‑OR Overall Safety of Canagliflozin (CANA) in the CANagliflozin Cardio‑ Vascular Assessment Study (CANVAS) Program PRISCILLA HOLLANDER, VISWANATHAN MOHAN, VINCENT C. WOO, RICHARD OH, GREG FULCHER, Dallas, TX, Chennai, India, Winnipeg, MB, Canada, Raritan, NJ, Sydney, Australia CANA is an SGLT2 inhibitor that was generally well tolerated in 12 placebo (PBO) and active-controlled phase 3/4 trials (non-CANVAS studies [NCS]) for ≤104 weeks in a broad range of patients with type 2 diabetes (T2D). Here we report the longest-term safety data to date (up to 6.5 years) with CANA Supported By: Janssen Research & Development, LLC. in patients with T2D and high CV risk from the CANVAS Program vs. the NCS. Amputation and fracture were novel safety signals in the CANVAS Program (HR [95% CI]: 1.97 [1.41, 2.75] and 1.26 [1.04, 1.52]) that were not seen in the NCS (RR [95% CI]: 0.23 [0.06, 0.89] and HR [95% CI]: 1.13 [0.74, 1.72]). The incidence of labeled AEs (i.e., osmotic diuresis [OD]- and volume depletion [VD]-related AEs, hypoglycemia, urinary tract infection, genital mycotic infection [GMI], renal-related AEs, hypersensitivity, diabetic ketoacidosis) and those that were serious or led to discontinuation in the CANVAS Pro-

ADA-Supported Research

A69 GENETIC AND ETHNIC HETEROGENEITY AND THE ROAD TO PRECISION MEDICINE IN DIABETES

260‑OR (SAXA) 5 mg/d vs. single add-on of titrated glimepiride (GLIM) 1-6 mg/d in Dapagliflozin plus Saxagliptin Shows Noninferior A1C Reduction vs. patients with T2D (A1C, 7.5-10.5%) receiving stable MET ≥1500 mg/d. The Insulin Glargine in Patients with Type 2 Diabetes Inadequately Con‑ primary end point was change in A1C from baseline to week 52; secondary trolled by Metformin With or Without Sulfonylurea end points were changes in body weight and systolic blood pressure (SBP) TINA VILSBØLL, ELLA EKHOLM, EVA K. JOHNSSON, NALINA DRONAMRAJU, and the proportions of patients achieving A1C <7% or requiring treatment SERGE JABBOUR, MARCUS LIND, Copenhagen, Denmark, Mölndal, Sweden, intensification by week 52. Mean±SD baseline data (N=443) were similar Gaithersburg, MD, Philadelphia, PA, Gothenburg, Sweden in both arms: age, 56.1±9.7 years; A1C, 8.5±0.8%; weight, 89.7±18.5 kg; This international, multicenter, randomized, open-label phase 3b trial duration of T2D, 7.8±6.4 years. DAPA + SAXA + MET resulted in significant (NCT02551874) evaluated the efficacy and safety of dapagliflozin (DAPA) reductions in A1C, body weight and SBP, a greater proportion of patients 10 mg/day plus saxagliptin (SAXA) 5 mg/day vs. titrated insulin glargine achieving A1C <7% and a lower proportion of patients requiring treatment (INS) in patients with type 2 diabetes (T2D) poorly controlled by metformin intensification vs. GLIM + MET (Table). All treatments were well tolerated. (MET) ≥1500 mg/day ± sulfonylurea (SU) ≥50% maximum dose (A1C levels Hypoglycemia incidence was lower for DAPA + SAXA + MET (18.5%) than 8-12%). The primary end point was change in A1C from baseline to week 24; GLIM + MET (44.0%). Add-on of DAPA + SAXA provided greater improve- DAPA + SAXA was tested for non-inferiority vs. INS. Secondary end points ment in glycemic control and reduced body weight vs. GLIM in patients with were: change in body weight; proportions of patients a) with hypoglycemia, T2D poorly controlled by MET. b) achieving A1C <7% without hypoglycemia, c) achieving A1C <7% at week Table. 24; and change in 24-hour glucose at week 2 assessed by continuous glucose monitoring. Mean±SD baseline data were age 55.5±9.6 years; A1C 9.1±1.0%; BMI 32.2±5.3 kg/m2, and were similar in both arms. DAPA + SAXA resulted in non-inferior reductions in A1C without weight gain, reductions in mean 24-hour glucose, and was associated with a lower prevalence of hypoglycemia and a greater proportion of patients achieving A1C <7% without hypoglycemia vs. INS (Table); there were no additional safety concerns. DAPA + SAXA therapy resulted in similar glycemic control, prevented weight gain and reduced risk of hypoglycemia compared with titrated INS in patients with T2D poorly controlled by MET ± SU. Table.

Supported By: AstraZeneca

GENETIC AND ETHNIC HETEROGENEITY AND THE ROAD TO PRECISION MEDICINE IN DIABETES

262‑OR Malate Dehydrogenase 2 (MDH2) as a New Diabetogene Causing Hyperglycemia in Families with Multigenerational Diabetes PRAPAPORN JUNGTRAKOON, SERENA PEZZILLI, ANTONELLA MARUCCI, LUCA PANNONE, ELISABETTA FLEX, TOMMASO BIAGINI, PATINUT BURANASUPKA- JORN, ORNELLA LUDOVICO, LUANA MERCURI, TIMOTHY HASTINGS, ROSA DI PAOLA, FEDERICA ALBERICO, CHRISTINE MENDONCA, JULIAN CERON, MONT- SERRAT PORTA DE LA RIVA, LORELLA MARSELLI, TOMMASO MAZZA, SIMONE MARTINELLI, VINCENZO TRISCHITTA, ALESSANDRO DORIA, SABRINA PRU-

ORALS DENTE, Bangkok, Thailand, Rome, Italy, San Giovanni Rotondo, Italy, Boston, MA, Barcelona, Spain, Pisa, Italy We are pursuing the identification of new diabetogenes by whole exome sequencing in families with multigenerational diabetes that do not carry MODY-gene mutations. Here we report two gain-of- function mutations (c.154 C>T p.Arg52Cys and c.478 G>A p.Val160Met) in the gene coding for Malate Dehydrogenase 2 (MDH2) that we have identified as segregating with hyperglycemia in two such unrelated families. The MDH2 enzyme is localized to the mitochondria where it catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the tri- Supported By: AstraZeneca carboxylic acid cycle. MDH2 also plays a pivotal role in the malate-aspartate shuttle that operates in the metabolic coordination between cytosol and 261‑OR mitochondria. By molecular dynamic simulation, both the Arg52Cys and Val- Dapagliflozin plus Saxagliptin Add-On vs. Glimepiride Add-On to 160Met substitutions were found to severely alter the atomic dynamics of Metformin in Patients with Poorly Controlled Type 2 Diabetes the MDH2 structure. MDH2 enzymatic activity was significantly increased JUAN P. FRIAS, GUILLERMO GONZALEZ-GALVEZ, EVA K. JOHNSSON, JILL (p<0.01) in HepG2 cells transfected with either MDH2_T154 or MDH2_A478 MAASKE, ANNE PETERS, Los Angeles, CA, Guadalajara, Mexico, Mölndal, Swe- as compared to WT_MDH2, despite no differences in MDH2 protein expres- den, Gaithersburg, MD sion levels. Increased MDH2 activity is known to decrease NAD/NADH Add-on therapy with a single oral antihyperglycemic agent is often insuf- ratio—a biochemical alteration reported to affect both insulin signaling and ficient to provide glycemic control in patients with type 2 diabetes (T2D) secretion. In genetically modified C. elegans strains obtained by means of uncontrolled on metformin (MET) monotherapy. This randomized, parallel- CRISPR/Cas9, the Arg52Cys mutant exhibited a significantly (p<0.05) pro- group, double-blind, 52-week trial (NCT02419612) evaluated the efficacy and longed lifespan and increased Paraquat resistance and lipid storage when safety of dual add-on therapy with dapagliflozin (DAPA) 10 mg/d + saxagliptin compared to WT strains. These changes resemble those reported for C.

ADA-Supported Research

A70 GENETIC AND ETHNIC HETEROGENEITY AND THE ROAD TO PRECISION MEDICINE IN DIABETES elegans with defective insulin signaling—a condition which in humans pre- ongoing. Our results show that the T1D-GRS is a useful discriminatory tool disposes to both insulin resistance and inadequate insulin secretion. between diabetes types and a strong predictor of whether a monogenic In conclusion, our data point to MDH2 as a new diabetogene, heterozy- cause is likely. More importantly, patients with a low T1D-GRS and negative gous mutations of which cause hyperglycemia in families presenting with for all known monogenic subtypes should be prioritized for exome or whole multigenerational diabetes. genome sequencing to maximize the chance of novel gene discoveries. Supported By: National Institutes of Health (R01DK55523); Italian Society of Supported By: National Institutes of Health (R01DK104942) Diabetology 265‑OR 263‑OR HLA-C Position 275, a Novel HLA Locus Identified in Autoantibody- Genotypic Associations with C-Peptide Persistence in People with Positive Type 1 Diabetes of Chinese Han Population Based on a Type 1 Diabetes Genome-Wide Association Study PAUL MCKEIGUE, ATHINA SPILIOPOULOU, STUART MCGURNAGHAN, MARCO KUANFENG XU, YANG CHEN, YONG GU, MENG ZHU, MEI ZHANG, MIN SUN, COLOMBO, TIMOTHY J. MCDONALD, HELEN COLHOUN, ON BEHALF OF THE XINYU XU, HSIANG-TING HSU, HENG CHEN, YUN SHI, YUN CAI, HAO DAI, SHUAI SDRNT1BIO INVESTIGATORS, Edinburgh, United Kingdom, Exeter, United Kingdom ZHENG, XUQIN ZHENG, HONGWEN ZHOU, LIPING YU, ZHIBIN HU, ZHIGUANG There is substantial variation in C-peptide levels in those with clinically ZHOU, JIANPING WENG, HONGBING SHEN, TAO YANG, Nanjing, China, Aurora, diagnosed type 1 diabetes (T1DM). We examined the association of C-pep- CO, Changsha, China, Guangzhou, China, Jiangsu, China tide levels with genotypic risk scores (GRS) for T1DM and T2DM in 5856 Type 1 diabetes (T1D) is a highly heritable autoimmune disease. Although genotyped people diagnosed with T1DM and requiring insulin within 1 year more than 50 non-HLA risk loci have been identified, the strongest associa- of diagnosis (the SDRNT1BIO). Region-specific GRS and residual genome tions were observed in HLA regions in caucasian populations. The aim of this wide scores for T1DM and T2DM were computed based on publicly avail- study is to identify T1D risk HLA loci in Chinese Han population and explore able summary GWAS data. Total genome wide scores were the sum of the the risk prediction models based on these loci. A two-stage genome-wide residual and region specific scores. C-peptide was measured using a Roche association study (GWAS) of T1D (at least one autoantibody positive) was ultrasensitive immunoassay on untimed serum. Overall 39% had detect- performed in Chinese Han population, the GWAS scan was conducted by the able C-peptide (≥5 pmol/L) with prevalence being highest with older age of Illumina Human OmniZhongHua platform and included 1,045 T1D cases and onset (p=6.9x10-05) and shorter duration (p=1.8x10-10). Heritability was 0.31 1,308 controls, and the replication included 1,378 cases and 3,774 controls. for age at onset and 0.26 for C-peptide adjusted for sex and age at onset. Promising associations were further validated by combining with the data Lower T1DM genome-wide score and higher T2DM score were positively from the Wellcome Trust Case Control Consortium (WTCCC). Association associated with C-peptide level, associations that were stronger with older analyses for T1D used logistic regression models assuming additive effects. age at onset (p=4.9x10-06 for T1DM score, p=0.009 for T2DM score inter- Results from different stages were combined with fixed effects model in actions with age at onset). The T1DM and T2DM GRS were uncorrelated meta-analysis. In the GWAS scan stage, we identified 18 distinct genomic except at the HLA region. Most T1DM region specific scores were lower with regions with P-values <1.00×10-5. As expected, the strongest associations increasing age at onset in particular at the HLA (p=3.2x10-34), INS (1.9x10-05) with T1D were located in the HLA region, and rs1770 at MHC (OR=4.54, CTSH (9.1x10 -06) and IKZF1 (p=0.0001) regions. Adjusted for age at onset, P<1.00×10-324), a previously identified caucasian T1D risk locus, showed higher C-peptide level was also associated with lower T1DM GRS at the the strongest association after combining the results from different stages. HLA (p=9.8x10-17) and INS (p=2.7x10-06) regions. Most T2DM region specific Further fine mapping in the HLA region identified five independent risk loci, scores were higher with older age at onset and adjusted C-peptide except a a novel Chinese specific locus HLA-C position 275 (P=9.78×10−12) was dis- T2DM associated HLA region score which was lower at higher age of onset covered except for rs1770, HLA-DRB1 position 11, HLA-DRB1 position74, and (p=1.7x10-17) and higher C-peptide (p=1.1x10-09). These results show that in HLA-A position 9. Risk prediction model based on these five independent people diagnosed with T1DM, especially at older ages, the variation in per- loci of HLA can achieve an AUC of 0.85 (0.84-0.87). A s the primary T1D GWA sistence of C-peptide secretion in part reflects a continuous spectrum of study in Asian population to date, the results identified a Chinese specific genetic risk for T1DM and T2DM. The lack of correlation in T1DM and T2DM HLA locus and would be the basis of precision medicine of Chinese T1D by GRS is in keeping with this continuum rather than “misdiagnosis” of T1DM risk prediction based on the identified HLA loci. as T2DM. Supported By: National Key Project of Research and Development Program of Supported By: JDRF; Diabetes UK China (2016YFC1000204, 2016YFC1305302, 2016YFC1305000, 2016YFC1305001); National Key Technology R&D Program of China (2015BAI12B13); Innovation-Driven 264‑OR Program of Central South University (2015CX009); Cheung Kong Scholars Program; Clinical Utility of the T1D Genetic Risk Score—Examples from the National Natural Science Foundation of China (81530026, 81530025, 81390543, U.S. Monogenic Diabetes Registry 81270897, 81670715, 81670756, 81370939, 81300668, 81400808, 81400813); MAY SANYOURA, LISA R. LETOURNEAU, ROCHELLE N. NAYLOR, LOUIS H. PHIL- Jiangsu Specially-Appointed Professor Project; Science and Technology Innova- IPSON, MICHAEL N. WEEDON, RICHARD A. ORAM, SIRI ATMA W. GREELEY, Chi- tion Team of Jiangsu Province, China; Top-Notch Academic Programs Project of cago, IL, Exeter, United Kingdom Jiangsu Higher Education Institutions (PPZY2015A067); Jiangsu Province Key Sci- ence and Technology Development Project (BE2017753); Jiangsu Province Science

Monogenic forms of diabetes are often difficult to distinguish from type 1 ORALS (T1D) and type 2 diabetes due to overlapping clinical features. As a result, a Foundation for Youth (BK20171082); Sun Yat-sen University (82000-31133402) growing majority of patients remain misdiagnosed and often inappropriately treated. We used a T1D genetic risk score (T1D-GRS), generated from 10 266‑OR T1D-associated common genetic variants, to help identify patients with a A Nonsynonymous Variant in GLP-1R Is Associated with Decreased low T1D-GRS, and thus a potential monogenic etiology. We genotyped 798 Risk of Type 2 Diabetes in Koreans probands from the U.S. Monogenic Diabetes Registry: 37% (n=293) had a SOO HEON KWAK, JEESOO CHAE, JIWON YOON, SUNGHEE CHOI, SOO LIM, MIN known monogenic cause and 63% (n=505) had an unknown cause or were KYONG MOON, YOUNG MIN CHO, HAK CHUL JANG, YOON SHIN CHO, HYUN not exhaustively sequenced. To improve the discriminatory power of the MIN KANG, JONG-IL KIM, KYONG SOO PARK, Seoul, Republic of Korea, Seong- T1D-GRS, participants were categorized using a T1D-GRS equivalent to the nam, Republic of Korea, Chuncheon, Republic of Korea, Ann Arbor, MI 5th, 25th, 50th, 75th, and 95th centile of the UK T1D cohort. 45% (132) of Type 2 diabetes (T2D) is a common complex disorder with strong genetic the registry participants with a confirmed monogenic cause had a T1D-GRS predisposition. Although different ethnic groups share most of the common below the 5th percentile, 35% (103) had a T1D-GRS between the 5-25th variants of T2D, there might be nonsynonymous variants that are specific percentile, 12% (35) between the 25-50th percentile, 5% (15) between the to an ethnic group. In this study, we used whole exome sequencing (N=917) 50-75th percentile, and 2% (7) between the 75-95th percentile. Of the par- and exome genotyping array (N=3,026) to identify nonsynonymous variants ticipants with an unknown cause, 24% (n=108) had a T1D-GRS below the in Korean T2D cases and controls (stage 1) and validated the findings in an 5th percentile, 30% (n=137) had a T1D-GRS between the 5-25th percentile, independent set of 15,095 T2D cases and controls (stage 2). Whole exome 16% (72) between the 25-50th percentile, 15% (67) between the 50-75th capture was prepared using Agilent SureSelect version 4+UTR and sequenc- percentile, and 15% (69) between the 75-95th percentile. We sequenced ing was performed by Illumina HiSeq 2000. Nonsynonymous variants were a total of 124 participants with a T1D-GRS below the 25th percentile using incorporated into customized Axiom Biobank Plus Genotyping array. Geno- a targeted next-generation sequencing library that includes more than type imputation was performed with 1,000 Genomes Project phase 3 refer- 140 genes known to be associated with diabetes. 21% (n=24) were found ence. In silico data look-up was done for stage 2 analyses. EPACTS software to have a pathogenic variant in a known diabetes gene. Further tests are was used for association testing and METAL was used for meta-analysis of

ADA-Supported Research

A71 ADIPOSE BROWNING AND ENERGY EXPENDITURE

stage 1 and 2 results. We identified 728,838 variants using whole exome (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, sequencing, which include 258,463 novel variants. A nonsynonymous variant KCNJ11, ABCC8, and APPL1) we considered patients to have MODY if they in GLP-1R (rs3765467, R131Q) was associated with decreased risk of T2D in carried one or more variants classified as likely pathogenic (LP) or pathogenic genome-wide significance (OR = 0.85, P = 3.01x10-8). This variant was associ- (P) according to current guidelines. We found 77 of 3650 patients carried an ated with lower fasting glucose concentration (P = 0.00020) and lower HbA1c LP/P variant in HNF4A (14 individuals), GCK (22), HNF1A (32), PDX1 (4), INS (3), level (P = 0.0085) in nondiabetic controls. The minor allele frequency of this and PAX4 (2). Compared to those with no LP/P variants, youth with MODY variant was 21.1% in Koreans but it was monomorphic in Europeans. We also had a younger age at diagnosis (12.9 ± 0.3 vs. 13.6 ± 0.1, P=0.01) and lower validated the previous association between PAX4 nonsynonymous variant fasting C-peptide levels (3.0 ± 0.2 vs. 4.5 ± 0.1, P=0.0001), although for both (rs2233580, R192H) and increased risk of T2D (OR = 1.52, P = 6.41x10-15). measures, the range of values overlapped between the two groups. Youth This variant was associated with lower age at diagnosis and decreased with MODY were less likely to have hypertension (6.9% vs. 18.2%, P=0.03) C-peptide level in T2D cases (P < 0.05). Gene-wise analysis revealed that and less likely to be on insulin (32.0% vs. 51.1%, P=0.001). Parental history of SLC30A8 was most significantly associated with decreased risk of T2D diabetes was not significantly different. MODY was found in all race/ethnic (P = 0.00010) by Madsen and Browning method. groups. In summary, we have identified East Asian specific nonsynonymous vari- Using a comprehensive set of MODY genes, we identified MODY in 2% of ants in GLP-1R and PAX4 to be associated with T2D in genome-wide signifi- youth with clinically diagnosed type 2 diabetes; in 88% (n=68) the specific cance. diagnosis would direct management. While these individuals differed signif- Supported By: Korean Ministry of Health and Welfare (HI15C1595, HI14C0060, icantly on several clinical characteristics, no criterion reliably separated the HI15C3131) two groups. Further study is needed to find ideal criteria to select individuals for genetic testing. 267‑OR Identifying Pathogenic Variants of Monogenic Diabetes Using Tar‑ 269‑OR geted Exome Sequencing Digenic Mutations in the FGF-21 Signaling Pathway Found in an SEUNG SHIN PARK, SE SONG JANG, YOUNG AH LEE, YOUNG MIN CHO, HAK Adolescent with Extreme Insulin Resistance CHUL JANG, KYONG SOO PARK, SOO HEON KWAK, Seoul, Republic of Korea, STEPHEN I. STONE, ELIZABETH A. ADESANYA, DANIEL J. WEGNER, JENNIFER Seongnam, Republic of Korea A. WAMBACH, F. SESSIONS COLE, DAVID M. ORNITZ, FUMIHIKO URANO, St. Background: Targeted exome sequencing is widely used for genetic diag- Louis, MO nosis of rare disease and it is replacing Sanger sequencing. In this study we Fibroblast Growth fFactor-21 (FGF-21) is a secretory molecule that has investigated pathogenic variants of monogenic diabetes genes in Korean multiple metabolic functions. However, its role in human insulin resistance early onset diabetes patients using targeted exome sequencing. remains unclear. Here we report a 12 year old female presenting to pedi- Method: The main eligible criteria for this study was: non-type 1 diabetes atric diabetes clinic with acanthosis nigricans, hirsutism, and acromegalic patients with age of onset < 30 years old and body mass index (BMI) < 30 kg/ features. The subject was noted to have extremely elevated insulin (2446 m2. Among the 2353 patients screened from the diabetes registry of Seoul uIU/mL) and elevated free testosterone (16 pg/mL). Due to her unique pre- National University Hospital Diabetes Clinic, a total of 93 participants were sentation, we hypothesized that she may have a genetic insulin resistance eligible. We analyzed 30 monogenic diabetes genes using targeted exome syndrome and whole exome sequencing was performed. 2 variants were sequencing. Identified genetic variants were evaluated by ACMG-AMP identified in genes critical for FGF-21 signaling, Fibroblast Growth Factor guideline for their pathogenicity. Receptor 1 (FGFR1) and beta Klotho (KLB). These mutations were inherited Result: Among the 93 index patients, a total of 83 rare (minor allele fre- in trans from each parent. FGFR1 and KLB are transmembrane co-factors quency < 0.5%), non-silent variants were found and evaluated for its patho- that bind FGF-21, leading to activation of intracellular tyrosine kinases, and genicity according to the ACMG-AMP guideline. These variants were classi- subsequent intracellular signaling. Fasting serum FGF-21 was elevated in fied as likely benign (N=8), uncertain significance (N=59), likely pathogenic the subject (391.3 pg/mL) compared to her father (104.3 pg/mL) and mother (N=13), and pathogenic (N=5). Three causative mutations were identified (225.2 pg/mL). A bicistronic expression plasmid was created recapitulating in HNF4A (p.R136W, p.M1I, p.R89Q), four mutations in five patients were the above mutations found in this family. These plasmids were transiently identified in HNF1A (c.G-124C, p.Y166N, p.L26Q, p.R278Q), four mutations transfected into L6 myoblasts, a cell line known to have low levels of FGF were found in GCK (p.G410S, p.N231S, p.L164F, p.H156fs) and one mutation receptor expression. ERK phosphorylation (pERK), the major downstream was identified in HNF1B (p.L168P). Other causative mutations were identified signal of FGF-21, was measured after the cells were treated with FGF-21. in WFS1 (p.R629W), INS (c.C-60G), ABCC8 (p.R933Q) and FoxP3 (p.Q200R). pERK was strongly upregulated in the cells transfected with wild type FGFR1 Mitochondria 3243 A>G variant was identified in two patients. Patients with and KLB (43,463 AU). However, mutations in either FGFR1 or KLB greatly pathogenic variants had higher MODY probability score and lower BMI than attenuated pERK (13,601.5 and 15,526 AU respectively). The combination of those without causative gene variants (p=0.015, p=0.039, respectively). mutations in both FGFR1 and KLB led to the lowest level of pERK (10,148.3 Conclusions: In this study, a total of 19 (20.4%) participants were identi- AU). We hypothesize that these digenic mutations in both FGFR1 and KLB fied to have monogenic cause of diabetes among 93 early onset diabetes are acting in a synergistic fashion leading to FGF-21 resistance. This helps patients. Further research is required to validate the clinical utility of tar- explain the severe insulin resistance found in this subject and may represent ORALS geted exome sequencing in early onset diabetes patients. a novel category of insulin resistance syndromes related to FGF-21. Supported By: National Institutes of Health (HL120002); Institute of Clinical and 268‑OR Translation Sciences/Children’s Discovery Institute (UL1TR000448) Monogenic Diabetes in the Progress for Diabetes Genetics in Youth (ProDiGY) Collaboration JENNIFER TODD, JEFFREY W. KLEINBERGER, SHYLAJA SRINIVASAN, SHERIDA ADIPOSE BROWNING AND ENERGY EXPENDITURE E. TOLLEFSEN, LYNNE L. LEVITSKY, LORRAINE E. KATZ, JEANIE B. TRYGGESTAD, FIDA BACHA, GIUSEPPINA IMPERATORE, JEAN M. LAWRENCE, CATHERINE 270‑OR PIHOKER, JASMIN DIVERS, JASON FLANNICK, DANA DABELEA, JOSE C. FLO- NFIA Colocalizes with PPARgamma and Activates the Cell-Type- REZ, TONI I. POLLIN, Boston, MA, Baltimore, MD, San Francisco, CA, St. Louis, Specific Enhancers to Control the Brown Fat Gene Program MO, Philadelphia, PA, Oklahoma City, OK, Houston, TX, Atlanta, GA, Pasadena, CA, YUTA HIRAIKE, HIRONORI WAKI, SHUICHI TSUTSUMI, HIROYUKI ABURATANI, Seattle, WA, Winston-Salem, NC, Cambridge, MA, Aurora, CO TOSHIMASA YAMAUCHI, TAKASHI KADOWAKI, Tokyo, Japan Monogenic diabetes, including maturity-onset diabetes of the young Brown fat dissipates energy as heat by means of the uncoupling protein-1 (MODY), is frequently misdiagnosed as type 1 or type 2 diabetes. Correct (UCP1) on the mitochondrial inner membrane. Brown fat activity is inversely diagnosis can result in a change in clinical treatment and impacts prediction correlated with body mass index in humans, and pilot studies have shown of complications and familial risk. Clinical algorithms can select individuals that interventions such as chronic cold exposure successfully recruit human for genetic testing; however, up to half of youth with MODY do not meet brown fat and increase systemic energy expenditure. Therefore, stimulating clinical criteria. Other barriers also limit access to genetic testing. development and/or function of brown fat would be a novel strategy for the To assess the prevalence of MODY in youth with a clinical diagnosis of treatment of obesity and its complications. Here, we identified nuclear fac- type 2 diabetes, we evaluated whole-exome sequence data from ProDiGY, a tor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide collaboration of the TODAY, SEARCH for Diabetes in Youth, and T2D-GENES open chromatin analysis of murine brown and white fat followed by motif studies. Focusing on 14 genes previously identified as causal for MODY analysis of the brown-fat-specific open chromatin regions. Introduction of

ADA-Supported Research

A72 ADIPOSE BROWNING AND ENERGY EXPENDITURE

NFIA into myoblasts results in brown adipocyte differentiation. Conversely, lead to development of targeted approaches to improve adipocyte and meta- the brown fat of NFIA knockout mice displays impaired expression of the bolic health and prevent type 2 diabetes in human obesity. brown-fat-specific genes and reciprocal elevation of muscle genes. In human Supported By: National Institutes of Health (R01DK080448, P30DK46200 to brown fat, expression of NFIA and the brown-fat-specific genes including S.K.F.), (R01DK101711 to V.P.); Joslin Diabetes Center (P30DK036836); Icahn School UCP1 is positively correlated. Mechanistically, NFIA co-localizes with the of Medicine at Mount Sinai (to M-J.L.); Osteopathic Heritage Foundations (to V.P.) master transcriptional regulator of adipogenesis, PPARgamma, selectively at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes 273‑OR and facilitates the binding of PPARgamma, leading to increased chromatin CRISPR-Engineered Human Brown-Like Adipocytes Promotes Met‑ accessibility and active transcription. Most recently, we performed mass abolic Homeostasis through Activation of Endogenous Brown Fat spectrometry analysis of NFIA protein complex to identify and characterize CHIH-HAO WANG, MORTEN LUNDH, ROKUS KRISZT, TIAN LIAN HUANG, LUIZ the crucial partner of NFIA-dependent brown-fat-specific chromatin regula- O. LEIRIA, FARNAZ SHAMSI, MATT LYNES, JUSTIN DARCY, BRICE EMANUELLI, tion. Collectively, these results indicate that NFIA is a key transcription fac- MICHAEL KIEBISH, YU-HUA TSENG, Boston, MA, Copenhagen, Denmark, Singa- tor that activates the cell-type-specific enhancers and facilitates the binding pore, Singapore, Framingham, MA of PPARgamma for controlling the brown fat gene program. Brown and brown-like beige adipocytes dissipate energy for thermogenesis and have been proposed to combat obesity and metabolic disorders. 271‑OR Uncoupling protein 1 (UCP1) is uniquely expressed in brown and beige adipo- STAT5 Deficiency in Mature Adipocytes Results in Metabolically cytes, and facilitates fuel utilization and energy expenditure. Ectopic UCP1 Healthy Adiposity, Reduced Growth Hormone Signaling in Adipose overexpression improves obesity and insulin resistance in mice. Recently, Tissue, and Decreased Energy Expenditure we utilized the CRISPR/Cas9 synergistic activation mediator and specific ALLISON J. RICHARD, HARDY HANG, TIMOTHY D. ALLERTON, CARRIE M. ELKS, gRNAs to boost endogenous UCP1 expression in human white adipocytes. JACQUELINE M. STEPHENS, Baton Rouge, LA The CRISPR/Cas9 engineered human brown fat-like (referred to as HUMBLE, STAT5 proteins promote adipocyte development and regulate adipose hereafter) cells had acquired human brown fat features when comparing tissue function in vitro and in vivo. To further elucidate the role of STAT5 bona fide human brown adipocytes derived from the same individual. Obese proteins in adipocytes, we used adiponectin-Cre and floxed STAT5 A/B mice mice receiving a transplantation of HUMBLE cells showed improved glucose to generate mice that lack both STAT5A and STAT5B in mature fat cells tolerance and insulin sensitivity, as well as increased energy expenditure. (STAT5AKO). Studies on several cohorts of both sexes of mice have revealed This was mediated, at least in part, via activation of endogenous brown fat, that STAT5AKO mice have increased adiposity. Notably, the adiposity phe- which showed an increase in heat production and glucose uptake in mice notype disappears upon high fat feeding when growth hormone (GH) levels receiving HUMBLE cells. Co-culture with HUMBLE cells or treatment of con- and GH signaling are diminished. Another striking observation is that obese ditional medium from HUMBLE cells promoted glucose uptake and thermo- STAT5AKO female mice are highly resistant to weight loss induced by low genesis in brown adipocytes. Metabolomic analysis identified an increased fat feeding when compared to floxed control mice that exhibit significantly ratio of arginine and asymmetrical dimethylarginine (ADMA) in the sera of enhanced weight loss and reduced adiposity. The adiposity of STAT5AKO mice receiving HUMBLE cell transplantation. Arginine is known to promote mice is not a result of alterations in food intake or lean body mass. Further- nitric oxide biosynthesis, which can activate thermogenic function of brown more, the increased adiposity in STAT5AKO mice is uncoupled from insulin fat. Consistently, the level of nitric oxide was increased in the endogenous resistance and metabolic dysfunction. It is well known that chronic eleva- brown fat of mice receiving HUMBLE cells. Inhibition of nitric oxide synthase tion of GH is associated with insulin resistance. However, in most mammals abolished the HUMBLE cells-mediated activation of brown adipocytes in a disruption of the IGF-1 axis accompanies altered GH signaling. Notably, vitro and in vivo. STAT5AKO mice do not have alterations in circulating IGF-1 levels. In addition, In summary, CRISPR/Cas9-mediated activation of endogenous UCP1 the adiposity phenotype of STAT5AKO mice does not appear to be associated expression in human white adipocytes creates brown fat-like features, with alterations in basal or stimulated lipolysis. However, STAT5AKO female and activates endogenous brown fat, potentially through arginine-related mice have a significant reduction in energy expenditure and fat oxidation. metabolites to improve the systemic glucose utilization. In summary, our extensive phenotypic characterization of STAT5AKO mice Supported By: National Institutes of Health; Ministry of Science and Technology has shifted our understanding of the metabolic actions of growth hormone of Taiwan and functions of STAT5 proteins. Supported By: National Institutes of Health (R01DK052968) 274‑OR Sirt5 Plays a Critical Role in Mitochondrial Protein Acylation and 272‑OR Mitochondrial Metabolic Homeostasis in Brown Fat Reprogramming of Human Adipocytes to a Briter Phenotype— GUOXIAO WANG, JESSE G. MEYER, WEIKANG CAI, MENGYAO E. LI, SAMIR Enhanced Fatty Acid Oxidation and Lipid Droplet Remodeling SOFTIC, C. RONALD KAHN, Boston, MA, Novato, CA MI-JEONG LEE, SUKANTA JASH, VISHWAJEET PURI, SUSAN K. FRIED, New York, Brown adipose tissue (BAT) is rich in mitochondria and plays important NY, Athens, OH roles in energy expenditure, thermogenesis and glucose homeostasis.

Converting energy storing white adipocytes to energy wasting ‘brite’ Recent studies have shown that mitochondrial activity can be regulated ORALS ones is now recognized to be feasible and may confer protection against by protein succinylation and malonylation, processes that are in part con- obesity and its related cardiometabolic diseases. Fragments of omental and trolled by Sirt5, the major mitochondrial desuccinylase and demalonylase. subcutaneous human adipose tissues were cultured for 7 days with insulin Compared to other tissues, brown fat has high levels of protein malonylation and dexamethasone with or without rosiglitazone (rosi) to induce britening. and succinylation, which were further increased in mice lacking Sirt5. BAT Rosi caused a metabolic reprograming of the adipocytes including increased specific Sirt5 KO mice (Sirt5-BKO) are intolerant to acute cold. Furthermore, rates of basal FA oxidation and the expression of mRNAs and proteins in when stressed with high fat diet or aging, Sirt5-BKO mice develop impaired multiple pathways that modulate FA metabolism, triacylglycerol synthesis, glucose homeostasis. Mass Spec analysis reveals that this correlates with cellular FA trafficking, and mitochondrial oxidative capacity (PGC1a, UCP1, an increase in succinylation of a variety of mitochondria enzymes and pro- CIDEA, PLIN5, FABP3). Furthermore, these changes were associated with a teins, including succinate dehydrogenase (SDH), glutamate dehydrogenase dramatic remodeling of the surface of the adipocyte lipid droplet (LD) such (GDH), and UCP1. Lysine to glutamine mutations of UCP1 at the two target that clusters of small LDs decorated with mitochondria were formed. Forma- sites, which mimic acylation, resulted in significantly decreased UCP1 activ- tion of these small LDs was blocked by inhibition of lipolysis and required FA ity. Likewise, hyperacylation of SDH and GDH secondary to Sirt5 deficiency activation and reesterification. Surprisingly, both omental and subcutaneous also impaired their activities. The reduced function of these and other pro- adipocytes were similarly ‘britened’. A similar remodeling was induced in teins in Sirt5KO BAT resulted in decreased mitochondrial membrane poten- primary cultures of newly-differentiated human adipocytes and these cells tial and increased accumulation of PINK1, a mitochondrial serine/threonine showed higher rates of basal and maximally-stimulated oxygen consumption. kinase that protect cells from stress-induced mitochondrial dysfunction. Consistent with transcriptome changes in both systems, lipidomics showed Depolarized mitochondria are often smaller in size. Consistent with this, increased levels of unsaturated FAs, phospholipid species, and cardiolipins mitochondria from BAT of Sirt5KO mice had higher Fis1 expression during in brite cells. Taken together, these results identify a coordinated metabolic fasting, leading to increased mitochondrial fission and smaller mitochondria. and structural reprogramming of mature human adipocytes toward a more Thus, succinylation and malonylation of key metabolic enzymes in mitochon- oxidative phenotype. Although rosi has unacceptable clinical side effects, dria is regulated by Sirt5 and plays an important role in BAT activity. Over- knowledge of the downstream effectors of this metabolic improvement may

ADA-Supported Research

A73 NOVEL MEDIATORS AND MECHANISMS OF OBESITY PATHOGENESIS IN ANIMALS

acylation impairs mitochondrial protein function and results in depolarized 277‑OR mitochondria and disturbed systemic metabolic homeostasis. Fibroblast Growth Factor-9 Regulates Adaptive Brown Adipose Tis‑ Supported By: American Diabetes Association (9-17-CMF-016 to G.W.) sue Mass Expansion FARNAZ SHAMSI, TIAN LIAN HUANG, YU-HUA TSENG, Boston, MA 275‑OR Brown adipose tissue (BAT) plays an essential role in adaptive thermo- BAF60a Orchestrates Thermogenic Chromatin Accessibility in genesis in response to environmental changes, such as cold and diet. Tar- Brown Fat geting BAT and modulating its mass and/or energy-dissipating capacity hold SIMING LI, TONGYU LIU, LIN MI, ZHUOXIAN MENG, JIANDIE LIN, Ann Arbor, MI, great promise for treatment of metabolic disorders. Expansion of BAT mass Hangzhou, China through increased cellular proliferation is a fundamental mechanism of cold Brown fat thermogenesis is important for cold defense and systemic adaptation. We have recently discovered Fibroblast Growth Factor 9 (FGF- energy balance. The activation of the thermogenic gene program during 9) as a cold-induced adipokine produced by BAT that regulates BAT mass. development and in response to cold requires global reconfiguration of Adeno-associated virus (AAV)-mediated overexpression of FGF-9 in vivo chromatin structure. Here we demonstrate that BAF60a, a component of increased BAT mass and improved glucose metabolism in obese mice. Addi- SWI/SNF chromatin-remodeling complex, is indispensable for cold-induced tionally, using double thymidine analogue labeling we demonstrated that thermogenesis. Mice with adipose tissue-specific inactivation of BAF60a FGF-9 overexpression in BAT increased cellular proliferation and enhanced exhibited impaired thermogenic gene expression and were exquisitely cold- cold-induced BAT expansion. Using an adipose tissue-specific Cas9 knock-in sensitive. Assay for Transposase-Accessible Chromatin using sequencing mouse model coupled with the AAV-mediated delivery of gRNAs targeting (ATAC) revealed that chromatin accessibility of many brown fat-enriched FGF-9, we specifically deleted FGF-9 in BAT and demonstrated that loss of genes was markedly reduced by BAF60a deficiency. Motif enrichment analy- FGF-9 resulted in reduction of cold-induced proliferation of brown adipocyte sis indicated that these BAF60a-dependent chromatin regions are highly progenitors, and led to impaired thermogenic capacity. Therefore, FGF-9 is enriched for binding sites for PPARγ, EBF2, and C/EBPβ, key transcriptional essential for cold-induced formation of new brown adipocytes and long term regulators of thermogenic gene expression, suggesting that BAF60a likely cold adaptation. Combining the results from these gain- and loss-of-function serves an important role in orchestrating the thermogenic chromatin state. studies, we conclude that FGF-9 acts as a growth factor for brown adipo- Adipose tissue inactivation of BAF60a augmented browning of inguinal cyte progenitors to promote BAT expansion in response to cold. These find- adipose tissue following cold acclimation. Mechanistically, this browning ings provide a novel regulatory mechanism modulating BAT mass, and may response was linked to compensatory activation of the ACTH/Melanocortin ultimately contribute to the development of novel therapeutic strategies to 2 Receptor signaling. Our studies outline a mechanism of global control of combat obesity and its comorbidities. chromatin accessibility during thermogenesis and reveal differential role of Supported By: American Diabetes Association (1-18-PDF-169 to F.S.); National BAF60a in brown and beige fat biogenesis. Institutes of Health Supported By: American Diabetes Association (1-15-BS-118 to J.L.); National Institutes of Health 278‑OR Endoplasmic Reticulum Membrane Protein Complex 10 (EMC10), a Novel Circulating Factor for Systemic Energy and Metabolic NOVEL MEDIATORS AND MECHANISMS OF OBESITY Homeostasis PATHOGENESIS IN ANIMALS XUANCHUN WANG, GUIFEN QIANG, YANLIANG LI, XINYI CAO, VICTORIA GIL, SHENGXIAN LI, KUANGYANG CHEN, XINRU WANG, MATTHIAS BLUHER, CHONG 276‑OR WEE LIEW, Shanghai, China, Beijing, China, Chicago, IL, Leipzig, Germany Targeting the Amyloid Precursor Protein (APP) to Mitochondria of Proteins secreted from metabolic organs play critical roles in fine-tuning White Adipose Tissues Triggers Mitochondrial Dysfunction and and maintenance of systemic metabolic and energy homeostasis. In this Obesity study, we identified a novel obesity-regulated circulating protein, EMC10, YU A. AN, PHILIPP E. SCHERER, Dallas, TX that regulates systemic energy homeostasis. We initially observed that The role of mitochondrial dysfunction in white adipose tissues (WAT) EMC10 is modulated in adipose tissues in diet-induced obesity (DIO) models under different metabolic conditions is not well understood. Here, we and subsequently in obese patients. Using an in-house-developed chemilu- report that under high fat diet (HFD) conditions, endogenous production of minescent immunoassay, we found that circulating EMC10 is significantly APP is significantly induced and, instead of being secreted, the protein is upregulated in overweight and obese patients. To determine the role of highly enriched in WAT mitochondria. To better understand the role of APP, EMC10, we subjected EMC10 KO mice to dietary treatment. We observed we established an inducible model by directing the full-length APP with a that ablation of EMC10 protects mice from DIO and associated metabolic mitochondrial targeting presequence quantitatively to mitochondria, selec- dysregulation including glucose intolerance, insulin resistance, hyperinsu- tively in adipocytes. We validated that APP mRNA and protein levels were linemia, hyperleptinemia, and hyperlipidemia. Conversely, enhanced circu- significantly elevated in subcutaneous WAT, and the protein was markedly lating EMC10 by expression of secretable AAV-hEMC10 in the liver promotes accumulated in isolated mitochondria from sWAT. EM imaging suggested obesity and associated metabolic dysfunctions in mice fed with either chow or high fat diet. Subsequent metabolic analysis revealed that EMC10 KO ORALS widespread loss of mitochondrial cristae. Respiratory measurements on a Seahorse instrument revealed a rapid impairment in mitochondrial functions mice on HFD have elevated whole body energy expenditure compared to in sWAT, concomitant with an increase in adipocyte size within a few days. WT controls via an increase in oxygen consumption in both the brown and APP transgenic mice displayed significantly enhanced fat mass, along with inguinal subcutaneous fat. We showed that ablation of EMC10 not only impaired glucose tolerance and reduced insulin sensitivity under a long term promotes basal UCP1 and PGC1a expression but also sensitized adipocytes HFD challenge. By H and E staining, we observed widespread inflammatory to beta-adrenergic stimulated thermogenic markers activation. Finally, our cell infiltration in WAT, and consistent with the adipose tissue dysfunction, preliminary data showed that activation of the transcription factor CREB and we also noticed massive hepatic steatosis. Adiponectin levels in plasma and p38 MAPK could potentially mediate EMC10 regulation of thermogenesis. sWAT were significantly reduced. We specifically eliminated endogenous Our findings implicate EMC10 as a novel therapeutic target for counteract- APP in WAT through a conditioned knock-out mouse model (Appflox/flox) to ing the development of obesity and obesity-induced metabolic dysfunction. demonstrate that adipocyte-specific deletion of APP can ameliorate high fat diet-induced mitochondrial dysfunction. 279‑OR In summary, accumulated APP in WAT mitochondria significantly induces HOXC10 Maintains White Adipose Tissue Identity and Its Loss mitochondrial dysfunction in adipocytes, leading to unhealthy adipose tis- Leads to Enhanced Browning of White Adipose Tissues sue expansion and eventually resulting in obesity and systemic insulin resis- ANGELINE H.Y. TAN, SINYEE GUN, WEIPING HAN, Singapore, Singapore, tance. Coppell, TX Supported By: National Institutes of Health (P01DK088761, R01DK55758, Increased thermogenesis via increased beige fat development enhances R01DK099110 to P.E.S.) energy expenditure and improves glucose homeostasis. However, thermogenic mechanisms regulating beige adipogenesis within white adipose tissue (WAT) are less understood. The homeobox-containing transcription factor, HOXC10, is traditionally thought to regulate musculoskeletal and patterning of limb development in mammals. We found that HOXC10 is expressed in subcutaneous WAT. Ectopic expression of HOXC10 in adi-

ADA-Supported Research

A74 NOVEL MEDIATORS AND MECHANISMS OF OBESITY PATHOGENESIS IN ANIMALS pocytes suppressed brown markers genes, while knockdown of HOXC10 along with the iWAT, actually exhibit a reduction in inflammatory marker upregulates brown genes expression. To investigate the functional role of expression. This ability to reduce adipose inflammation while enhancing lipid HOXC10 in adipose tissue, we generated adipose-specific HOXC10 knock- storage capacity allows the CREB3L3 KO mice to gain more weight on HFD, out (AHKO) mice using adiponectin-Cre mice. We show that development yet maintain a healthy metabolic profile. of functional beige adipocytes within subcutaneous WAT is enhanced in Supported By: National Institutes of Health (R01DK109015-01); American Heart AHKO mice, contributing to decreased white fat in the lean AHKO mice kept Association (16PRE30190011) at room temperature. Furthermore, AHKO mice show reduced weight gain, lower fat mass and improved glucose tolerance compared to wild type (WT) 282‑OR mice when fed on high fat diet. Interestingly, AHKO mice exhibit enhanced Androgens Activate Myeloid Cells during High-Fat Diet Exposure thermogenic response to cold exposure. These mice also show increased Promoting Sex Differences in Metabolic Disease energy expenditure, alongside increased in brown fat markers in subcutane- CAMERON A. GRIFFIN, SIMIN ABRISHAMI, MITA VARGHESE, KANAKADURGA ous WAT, with no effect on classical BAT. These data indicate that HOXC10 SINGER, Ann Arbor, MI plays a critical role in maintaining white adipose tissue identity, and loss of Men have a higher risk of metabolic and cardiovascular disease than HOXC10 promotes browning in WAT. pre-menopausal women, but the mechanisms of these differences are elu- Supported By: A*STAR Biomedical Research Council sive. Chronic myeloid inflammation during obesity contributes to metabolic disease risk and is significantly more robust in males. Given the paucity of 280‑OR data on male sex hormones contributing to macrophage responses in obe- A UCP1 Near-Infrared Reporter System for Monitoring Adipose sity, our objective was to understand the role of androgens in promoting “Browning” In Vivo Using Photoacoustic Imaging obesity-induced myeloid inflammation. Male and female C57Bl/6J mice DERRYN XIN HUI CHAN, SHIGEKI SUGII, WEIPING HAN, Singapore, Singapore, were fed a 60% high fat diet for 24 weeks. Glucose tolerance and insulin Coppell, TX sensitivity were measured. Monocyte transfer experiments assessed sex Obesity has reached pandemic proportions in the developed world. The differences in bone marrow myeloid responses to obesity independent of discovery that white adipocytes have the capacity to undergo browning to host sex. Gonadectomy, mice deficient of androgen receptor signaling (ARtfm) become metabolically-active beige cells has generated significant interest in and monocyte specific androgen receptor (AR) knockout mice (LysMAR-/y) the field of metabolic medicine. However, the study of adipose tissue brown- were used to model androgen deficiency. Compared to males, females ing has been hampered by a lack of imaging modalities that permit longitu- had dampened inflammatory responses with reduced CD11c+ ATMs and dinal and non-invasive monitoring of this process. To this end, we designed cytokines, even with increased adiposity. RNASeq demonstrated sex dif- near infra-red (NIR) fluorescence protein iRFP720-UCP1 reporter construct, ferences in gene pathways of both metabolic and inflammatory activation in which the expression of iRFP720 is driven by a mini UCP1 promoter. Non- in ATMs. Male monocytes transferred into females remained primed for invasive assessment of adipose beiging during adrenergic stimulation was pro-inflammatory response and induced an increase in adiposity. Castrated performed by viral transduction of mouse white adipocytes, followed by males exposed to high fat diet had impaired glucose tolerance and increased multi-spectral optoacoustic imaging technology with ultrasound tomogra- insulin sensitivity with increased adiposity and lower numbers of CD11c+ phy (MSOT-US). We observed increased iRFP720 fluorescence coupled with ATMs. These same findings were seen in ARtfm mice. Testosterone enhanced attenuated lipid signals upon stimulation. As proof of concept, we validated palmitate-stimulated myeloid colony production, and this response was our approach against the hybrid positron emission tomography combined dampened in castrated, ARtfm, and LysMAR-/y mice. These studies demon- with magnetic resonance (PET/MR) imaging modality, and quantified the strate that androgens play a critical role in enhanced metabolic dysfunc- extent of adipose browning by MRI-guided segmentation of PET signals. tion and enhanced myeloid inflammation in response to obesogenic cues. Notably, the browning extent detected by the iRFP720 reporter system with Androgen driven myeloid inflammation leads to metabolic dysfunction in a MSOT-US and the 18-FDG uptake in PET/MR are well correlated with UCP1 cell-autonomous manner driving sex-differences in obesity induced meta- induction. Together, these systems provide platforms for preclinical screen- inflammation. ing of compounds aimed to promote adipose browning and facilitate the Supported By: National Institutes of Health/National Institute of Diabetes and translation of these discoveries into pharmacological treatments for obesity. Digestive and Kidney Diseases (K08DK101755); University of Michigan Nutrition Supported By: A*STAR Biomedical Research Council Obesity Research Center

281‑OR 283‑OR Ablation of Adipose CREB3L3 Protects Mice from Obesity-Induced Targeting Intestinal Macrophages as a Potential Therapeutic Metabolic Dysfunction Option in Obesity MAXIMILIAN A. MCCANN, GUIFEN QIANG, VICTORIA GIL, HYERIM WHANG THERESA V. ROHM, SHEFAA ALASFOOR, ANGELA J. BOSCH, CLAUDIA CAVELTI- KONG, KEZHONG ZHANG, CHONG WEE LIEW, Chicago, IL, Beijing, China, WEDER, Basel, Switzerland Detroit, MI Background: Chronic inflammation such as systemic or tissue inflamma- Metabolically healthy obesity (MHO) is a subtype of obesity where tion has been well established in metabolic disease. Although macrophages

individuals lack the metabolic symptoms associated with obesity, despite thereby play a key role, not much is known about the initiation of inflamma- ORALS being obese. Our lab has found that the fat-specific ablation of cyclic-AMP tion. As the gut makes up the largest macrophage reservoir of the body and Responsive Element Binding Protein 3-like-3 (CREB3L3) creates a phenotype gastrointestinal changes occur in metabolic disease (altered gut microbiota, mimicking MHO in mice fed a high-fat diet (HFD). CREB3L3 is an ER-bound increased endotoxin and cytokines), the aim of our study was to assess the transcription factor that we have discovered to not only be expressed in role of intestinal macrophages (iMφs) in obesity. adipose tissue, but selectively downregulated in obese subcutaneous fat Research Design and Method: IMφs were isolated from the colon of from both mice and human patients. Subcutaneous fat is considered the C57BL/6, germ-free or CCR2-/- mice fed a high fat diet (HFD) or control diet more “metabolically-protective” subtype of white adipose tissue in obesity, up to three months and characterized by flow cytometry as CCR2+ (“inflam- so we hypothesized that this downregulation of CREB3L3 could contribute to matory”) or CCR2- (resident, anti-inflammatory) subpopulations. For dose- the healthier phenotype of obese subcutaneous fat, and the ablation of this dependent iMφ depletion, HFD-fed mice were orally treated with 50, 100 or protein specifically in adipose tissue could be metabolically beneficial. We 200 µg/g CSF-1R inhibitor (BLZ945) or its vehicle for up to ten weeks. found that the fat-specific knockout (KO) of CREB3L3 significantly increased Results: We found that within one week and up to three months of HFD, body weight and the mass of both the subcutaneous inguinal (iWAT) and inflammatory iMφs increase, preceding adipose tissue inflammation. Two visceral epididymal (eWAT) fat depots on HFD, as compared to wild type mouse models protected from metabolic disease - germ-free and CCR2-/- controls. However, the KO mice do not exhibit the insulin resistance or glu- mice - exhibited ≥10-fold lower inflammatory iMφ numbers when compared cose intolerance expected with their more obese phenotype, likely due to to WT mice, suggesting that a threshold of iMφs is required to elicit meta- enhanced lipid storage in adipose tissue, which sequesters lipids away from bolic disease. Based on this finding, we depleted macrophages by increas- the circulation and liver. Indeed, the KO mice do not exhibit dyslipidemia ing doses of CSF-1R inhibitor and observed a gradual iMφ-reduction, which and have reduced hepatic triglyceride content. The enhanced adipose lipid correlated with a dose-dependent improvement in fasting glucose, glucose storage is achieved via the enlargement of the iWAT and eWAT in the KO tolerance and insulin levels. mice. Morphometric analysis shows that both of these tissues have more Conclusion: HFD-induced obesity is associated with an increase in inflam- adipocytes, and the eWAT has more large adipocytes. While adipocyte matory iMφs, while mouse models protected from metabolic disease have hypertrophy typically contributes to metabolic dysfunction, the KO eWAT, lower numbers. Accordingly, dose-dependent depletion of iMφs is accompa-

ADA-Supported Research

A75 EVOLVING MECHANISMS IN CARDIOVASCULAR COMPLICATIONS

nied by gradual improvements in glucose metabolism. This suggests that the p=0.013). We conclude that high serum levels of C1QTNF1 significantly pre- number of iMφs is involved in initiation of metabolic disease and thus could dict MACE, in particular in patients with T2DM. serve as a potential therapeutic target in obesity. Supported By: University of Basel 286‑OR Endothelial/Nitric Oxide Regulation of Brown Adipose Tissue Acti‑ vating Lipokine, 12, 13-diHOME and Its Antiatherogenic Actions EVOLVING MECHANISMS IN CARDIOVASCULAR KYOUNGMIN PARK, QIAN LI, MATT LYNES, HISASHI YOKOMIZO, TAKANORI COMPLICATIONS SHINJO, RONALD ST-LOUIS, JIALIN FU, CHRISTIAN RASK-MADSEN, YU-HUA TSENG, GEORGE L. KING, Boston, MA 284‑OR Activation of brown adipose tissue (BAT) has been shown to decrease Insulin Resistance and Type 2 Diabetes Mellitus Are Associated atherosclerosis due to its systemic actions to improve insulin sensitivity with Impaired Mitochondrial Function in Human Ventricular Myo‑ and facilitate weight loss. However, the direct effect of BAT cytokines on cardium vascular arterial function and atherosclerosis has not been studied. We ELRIC ZWECK, DANIEL SCHEIBER, TOMAS JELENIK, PATRICK HORN, SOPHIE have reported that enhancing insulin’s action on the endothelial cells (EC) by -/- ALBERMANN, UDO BOEKEN, DIYAR SAEED, MALTE KELM, MICHAEL RODEN, overexpressing IRS1 to the EC on ApoE background (ECIRS1/ApoE-/- mice) RALF WESTENFELD, JULIA SZENDROEDI, Düsseldorf, Germany greatly increased insulin induced NO production and decreased atheroscle- Type 2 diabetes mellitus (T2DM) is related to increased cardiac morbid- rosis. ECIRS1 mice exhibited significant increases in BAT mass and activa- ity and mortality. Underlying mechanisms are yet unclear. Recent stud- tion when exposed to cold temperature (5°C), in parallel with elevation of ies detected mitochondrial abnormalities in atrial tissue acquired during BAT lipokine, 12,13-diHOME levels at basal, regular chow (RD) or high fat open-heart surgery of T2DM patients suffering from ischemic or valvular diet (HFD) compared to wild type mice (WT) by 188±33% and 187±39%, heart disease. We hypothesized that mitochondrial capacity and coupling respectively, (P<0.01). Recently, 12,13 diHOME has been reported to acti- efficiency are reduced in ventricular tissue of humans with T2DM and nor- vate BAT (Nat. Med, 2017. Elevation of 12, 13 diHOME induced by cold tem- mal heart function. High resolution respirometry was performed in trans- perature was inhibited by L-NAME, an inhibitor of NO. In EC, the addition of catheter ventricle biopsies of 29 healthy heart transplant recipients with 12,13-diHOME increase pAkt from 1-100nM, but without affecting peNOS normal left ventricular ejection fraction (64±8%) and no allograft rejection. and pErk. However, oxLDL-induced pErk activity and VCAM-1 expression We assessed oral glucose insulin sensitivity (OGIS) and redox potential were reduced in 12,13-diHOME (1nM) treated EC. Further, 12,13-diHOME (ORP) reflecting systemic oxidative stress. Glucose tolerant humans (CON; also increased intracellular calcium concentration with parallel elevation in n=16) and T2DM (n=13) had comparable age (54±14 vs. 58±12 years; p=0.45), NO production in EC. Functionally, 12,13-diHOME(1nM-10nM) increased vas- sex (81% vs. 85% male) and time since transplantation (26±24 vs. 25±26 cular relaxation in myography experiments significantly by 7.5%-24%. Using months; p=0.93), while body mass index was higher in T2DM (25.0±2.9 vs. antibody arrays for assessing the effects of 12,13-diHOME on NO production 28.1±4.9 kg/m2; p<0.05). State 3 respiration on octanoyl-carnitine was 20% in EC, signaling studies showed 12,13-diHOME activated mTOR, CREB and lower in T2DM (101±25 vs. 81±20 pmol/(s*mg); p<0.05). Lipid-linked respira- HSP27 (by 50, 2.5, and 4 fold, respectively) and down-regulated tyrosine- tion related negatively to HbA1C (r=‑0.45; p<0.05) and fasting blood glucose protein kinase (HCK) and GSK3α/β (by 30% and 26%, p<0.05) in EC. These levels (r=-0.41; p<0.05), but positively to OGIS (r=0.56; p<0.05). Respiratory studies for the first time showed that insulin’s action in EC can regulate BAT control ratios (RCR) on lipids were 21% lower in T2DM compared to CON lipokine 12,13-diHOME expression in a positive feedback loop to increase (1.6±0.4 vs. 1.2±0.3; p<0.05) and correlated negatively with HbA1C (r=-0.44; NO and increase vasodilation with associated decrease in atherosclerosis. p<0.05). Substrates of mitochondrial complex I and II induced 22% lower Supported By: National Institute of Diabetes and Digestive and Kidney Diseases RCR in T2DM (2.7±0.8 vs. 2.1±0.5; p<0.05). ORP was 24% higher in T2DM (P30DK036836, R01DK053105, DP3DK112192) (127±23 vs. 157±25 mV; p<0.01). Ventricular myocardium of T2DM and insulin resistant humans shows diminished mitochondrial oxidative capacity and 287‑OR coupling efficiency for lipid and glycolytic substrates which is related to Modified Mesenchymal Stromal Cells Promote Multiple Metabolic hyperglycemia and oxidative stress. This might indicate targets for treat- Benefits in Obese Diabetic Mice ment and prevention of T2DM-related heart failure. CLEYTON C. DOMINGUES, NABANITA KUNDU, YANA KROPOTOVA, NEEKI Supported By: Universitätsklinikum Düsseldorf AHMADI, SABYASACHI SEN, Washington, DC Background: Mesenchymal stromal cells (MSCs) are multipotent cells that 285‑OR can home-in to the sites of inflammation. Therefore, antioxidant-upregulated The Novel Adipokine C1QTNF1 Significantly Predicts the Incidence MSCs delivered intra-peritoneally can home-in to local inflamed fat pock- of Future Major Cardiovascular Events in Patients with Type 2 Dia‑ ets which may reduce inflammation and improve glucose tolerance in diet- betes induced obese and diabetic mouse model. AXEL MUENDLEIN, ANDREAS LEIHERER, CHRISTOPH H. SAELY, KATHRIN GEI- Methods: GFP-containing adenoviral constructs were used to upregulate GER, JANINE EBNER, EVA-MARIA BRANDTNER, BARBARA LARCHER, ARTHUR antioxidants Sod2 (mitochondrial) and Catalase (cytosolic) in human adi-

ORALS MADER, PETER FRAUNBERGER, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liech- pose-derived MSCs. Modified MSCs were delivered (IP) into mice subjected tenstein, Berne, Switzerland, Philadelphia, PA to 45% and 60% high-fat diet for 8-16 weeks. Increased serum levels of the novel adipokine C1q and tumor necrosis Results: Glucose tolerance was improved at week 4 in the antioxidant factor related protein 1 (C1QTNF1) have been linked with type 2 diabetes upregulated MSC-receiving groups with concomitant reduction in hyper- (T2DM) and ischemic heart disease. The impact of circulating C1QTNF1 on plasia in omental fat. A reduction in plasma levels of TNFa, an well-known the incidence of future major cardiovascular events (MACE) is unclear and is inflammatory marker, was found for all treated animal groups in compari- addressed in the present study. We measured C1QTNF1 serum levels in 542 son to control (null-MSCs). RT-PCR analysis of omental and pericardial fat patients undergoing coronary angiography for the evaluation of established showed significant up-regulation in mRNA expression of brown fat marker, or suspected coronary artery disease (CAD) using an enzyme-linked immu- Ucp1 (~1000-fold and 10-100-fold, respectively) which was confirmed by nosorbent assay. Prospectively, MACE were recorded over a mean follow- Ucp1-staining and concomitant increases in down-stream genes such as up period of 6.3 years. C1QTNF1 serum levels at baseline were significantly Pgc1a and Prdm16 mRNA expression. Remarkably, the treatment showed a increased in patients with T2DM (n=160) compared to those without diabe- significant reduction in liver fat content (by histology) and triglyceride con- tes (521.4 ± 224.8 vs. 429.5 ± 130.3 ng/ml; p<0.001). Prospectively, the inci- tent measurement. dence of MACE increased significantly through tertiles of C1QTNF1 (17.8%, Conclusion: Delivery of Sod2 and Catalase upregulated MSCs improved st nd rd glycemic control by reducing systemic inflammation, promotes browning of 24.7%, and 29.7% in the 1 , 2 and 3 tertiles, respectively; ptrend=0.010). Also after adjustment for age, sex, and T2DM as well as after additional white adipose tissue and reverses hepatic lipid accumulation. These results adjustment for body mass index, hypertension, LDL cholesterol, HDL choles- indicate that antioxidant upregulated MSCs can help to improve glucose terol, triglycerides, and angiographically determined baseline CAD, C1QTNF1 homeostasis, adipocyte energetics and hepatic lipid metabolism. Modified significantly predicted MACE, with adjusted HRs of 1.30 [1.04-1.61]; p=0.019 MSC therapy can be a promising therapy for type 2 diabetes, obesity and and 1.36 [1.09-1.70]; p=0.007, respectively. Patients with T2DM were at a fatty liver disease. significantly higher risk of MACE than those who did not have diabetes (48% Supported By: George Washington University vs. 26%; p=0.003). C1QTNF1 in subgroup analyses also in T2DM patients proved to be a strong predictor of MACE (adjusted HR 1.57 [1.10-2.24];

ADA-Supported Research

A76 NUTRITION 2018—NEW DATA, NEW PERSPECTIVES

288‑OR NUTRITION 2018—NEW DATA, NEW PERSPECTIVES Loss of Insulin Actions on Arterial Smooth Muscle Cells May Enhance Unstable Atherosclerotic Plaque Formation 290‑OR QIAN LI, JIALIN FU, KYOUNGMIN PARK, CHRISTIAN RASK-MADSEN, GEORGE Relationship between Amount and Type of Dietary Fat, Postprandial L. KING, Boston, MA Glycemia, and Insulin Requirements in Type 1 Diabetes Formation of unstable atherosclerotic plaque, a major cause of cardio- KIRSTINE BELL, SALLY DUKE, KYLIE M. ALEXANDER, MARGARET MCGILL, JEN- vascular mortality, is accelerated in insulin resistance and diabetes. Pre- CIA WONG, GREGORY FULCHER, STEPHEN M. TWIGG, JENNIE BRAND-MILLER, vious studies have suggested that endothelial dysfunction and systemic GARRY M. STEIL, Sydney, Australia, Boston, MA hyperlipidemia due to loss of insulin actions can accelerated its formation. ADA recommends individuals with type 1 diabetes (T1D) are taught to However, the mechanism for insulin resistance to accelerate the various adjust insulin for dietary fat however optimal adjustments are not yet clear. pathologies of unstable plaque, such as decreases in VSMC and extra cel- This study aimed to determine 1.) the relationship between the ‘amount’ and lular matrix (ECM) and increased inflammatory cells, have not been defined. ‘type’ of dietary fat and glycemia and 2.) the optimal insulin adjustments Since insulin actions on the vascular wall are diminished in insulin resistant for dietary fat. Six adults with T1D using insulin pump therapy attended the states, we assessed the loss of insulin action in VSMC and its effect on research clinic on 9 to 12 occasions. On the first 6 visits, participants con- the formation of atherosclerotic plaque. Insulin receptor (IR) was specifi- sumed meals containing 45g CHO with either 0g, 20g, 40g, or 60g fat and cally deleted from VSMC by breeding SM22 a promoter Cre and IR Flox mice either saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA) -/- to produce SMIRKO mice which were mated with ApoE mice to generate fat. Insulin was dosed using individual insulin: carbohydrate ratio as a dual- -/- -/- SMIRKO/ ApoE mice. Cultured VSMC from SMIRKO/ApoE mice showed wave 50/50% split over 2h. On subsequent visits, participants repeated the decreased insulin signaling and stimulated proliferation by >50%, but not to 20 to 60g fat meals with the insulin dose estimated using a model predictive PDGF. Weight, blood pressure, glucose, and systemic insulin tolerance were bolus, with up to 2 repeats/meal until glycemic control achieved. With the -/- -/- comparable between ApoE and SMIRKO/ApoE mice; which were fed same insulin dose, mean 5h incremental area under the curve (iAUC) was with normal chow to mimic pathophysiological hyperlipidemia encountered increased by 46%, 23% and 139% for the 3 fat loads relative to the 0g fat -/- clinically. Surprisingly, SMIRKO/ ApoE mice developed significantly more meal (0g: 509 ± 153; 20g: 742 ± 742; 40g: 626 ± 74; 60g: 1216 ± 434mmol/L. atherosclerotic aortic plaques (Sudan IV staining for lipid content) compared min; ns). The type of fat made small but non-significant differences to the -/- to ApoE mice with parallel increases of macrophage content and inflam- 5h iAUC. To achieve glycemic control, on average, participants required matory cytokines such as IL18 and ICAM1. In contrast, SMC as measured by 20-60% more insulin, delivered as a dual-wave over 1.25-2h. This study pro- SM22α staining was decreased with elevation of necrotic plaques (p<0.05). vides foundation for mealtime insulin dosing recommendations for dietary Further, proliferative index of VSMC as measured by Ki67 staining and fat in T1D. expression of hyaluronan synthase-2 were decreased. Thus, surprisingly, the loss of insulin’s specific actions on VSMC to increase proliferation increased Figure. inflammatory cell influx as observed in insulin resistance, may accelerated formation of unstable plaques in diabetes. Supported By: National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases

289‑OR Liraglutide Suppresses Atherosclerosis via AMP-Activated Protein Kinase Dependent and Independent Mechanisms in Diabetic Apoli‑ poprotein Enull Mice MASAKAZU KOSHIBU, YUSAKU MORI, HIDEKI KUSHIMA, MUNENORI HIRO- MURA, KYOKO KOHASHI, MICHISHIGE TERASAKI, TSUTOMU HIRANO, Tokyo, Japan Background: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have anti-atherosclerotic properties, possibly through activation of AMP-activated protein kinase (AMPK) in the vasculature. However, their relationship Supported By: Australian Diabetes Educators Association remains largely unknown. Methods: Streptozotocin-induced diabetic apolipoproteinE-null mice (male, 20 w) were randomly assigned to six treatment groups: saline (control, C), 291‑OR liraglutide at low or high doses (L- and H-Lira at 17 and 107 nmol/kg/d, Baseline Predictors and Influence of Early Weight Loss during an respectively), AMPK inhibitor dorsomorphin (AMPKI, 25 mg/kg/d), AMPKI+L- Intensive Weight Management Programme on Remission of Type 2 Lira, or AMPKI+H-Lira. Diabetes after 12 Months—Post-Hoc Analysis of the Diabetes Results: First, we confirmed that AMPKI inhibited H-Lira-induced AMPK Remission Clinical Trial (DiRECT) phosphorylation in the aorta (1.5-fold). After 4 w, biological parameters were MICHAEL E.J. LEAN, WILMA S. LESLIE, NAVEED SATTAR, GEORGE THOM, ELIZA- ORALS similar, except for HbA1c levels (C 9.8, L-Lira 8.6, H-Lira 8.4%, p<0.05). Both BETH L. MCCOMBIE, NAOMI T. BROSNAHAN, ALISON C. BARNES, ROY TAYLOR, L- and H-Lira suppressed intraplaque macrophage accumulation at the aortic ALEX MCCONNACHIE, FOR THE DIRECT STUDY TEAM, Glasgow, United Kingdom, sinus by 60%. H-Lira also suppressed aortic surface plaque area by 40%, Newcastle upon Tyne, United Kingdom which was not significant in L-Lira (25%). In AMPKI-treated mice, biologi- Introduction: An intensive weight management programme (Counter- cal parameters were similar. AMPKI completely eliminated L-Lira-induced weight-Plus), including low-energy formula diet for up to 16 weeks, then a anti-atherogenic effects but did not attenuate H-Lira-induced suppression of structured approach for weight-loss maintenance, delivered within routine atherosclerotic area (38%) and intraplaque macrophage accumulation (63%), primary care, has shown striking remissions of type 2 diabetes (T2DM diag- suggesting that AMPK mechanisms are L-Lira-dependent and H-Lira-inde- nosis <6 years). Remissions at 12 months were achieved by 46% overall, by pendent. In cultured human vascular endothelial cells, Lira (100 nM) reduced 73% with weight loss >10kg. TNF-induced inflammatory cytokine and adhesion molecule expression by 60 Methods: Baseline factors, and weight losses of 2-10kg from baseline, to 80%, which was retained at more than 50% after siRNA-knockdown of at 4, 6, and 8 weeks, were examined as potential predictors of remission AMPK (p<0.05). In cultured human monocytic cells (U937), Lira suppressed of T2DM at 12 months (nondiabetic HbA1c >48mmol/mol, on no antidiabe- LPS-induced inflammatory cytokine expression by 30% (p<0.05), which was tes medications) in the Intervention group (n=149, mean age 54 years, BMI 2 completely reversed by AMPKI. 34.5kg/m ). Conclusions: Lira suppressed atherosclerosis via AMPK-dependent and Results: Significant baseline predictors of diabetes remission at 12 -independent mechanisms; AMPK dependency is altered by GLP-1RA dose months, all modest, included older age, lower HbA1c, fewer antidiabetic and and cell type. more antihypertensive drugs (both stopped at baseline) and higher blood pressure. Weight losses at 4, 6, and 8 weeks were significantly associated with remission of diabetes at 12 months. Sixteen patients (11%) failed to achieve 2kg weight loss at 6 weeks, none of whom achieved remissions of diabetes. However, 14 of these patients failed to start, or withdrew from the intervention, within 6 weeks. At 8 weeks, 31 patients (21%) had failed

ADA-Supported Research

A77 NUTRITION 2018—NEW DATA, NEW PERSPECTIVES

to achieve 6kg weight loss, of whom only 5 achieved remission (95% sensi- participants (56.3%, 54/96) maintained mean blood beta-hydroxybutyrate tivity, 32% specificity). However, 15 of these patients had withdrawn from concentrations ≥0.5 mmol/L, indicating moderate adherence to nutritional treatment. Excluding earlier withdrawals from treatment, achieving <6kg advice. loss had only 17% specificity for identifying failure to achieve remission. These results demonstrate that patients with preT2D can be supported Conclusions: Early weight loss predicts treatment success. However, remotely with this continuous care model to improve risk factors associated many failing patients withdraw spontaneously and early ‘stopping rules’ with progression of preT2D to T2D. Ongoing research will evaluate the con- would deny treatment to a significant minority who gain remissions from tinued sustainability of this intervention and prevention of T2D. continued support. Supported By: Virta Health Corp Supported By: Diabetes UK 294‑OR 292‑OR A Plant-Based Diet Improves Beta-Cell Function and Insulin Resis‑ Acute Insulin Secretory Effects of a Classic Ketogenic Meal in tance in Overweight Adults—A 16-Week Randomized Clinical Trial Healthy Subjects HANA KAHLEOVA, ANDREA TURA, MARTIN HILL, RICHARD HOLUBKOV, NEAL SIMONA BERTOLI, ANGELA SPADAFRANCA, RAMONA DE AMICIS, ALESSAN- BARNARD, Washington, DC, Padua, Italy, Prague, Czech Republic, Salt Lake City, UT DRO LEONE, ANDREA MARI, ALBERTO BATTEZZATI, Piacenza, Italy, Milan, Italy, Background: The aim of this study was to test the effect of a 16-week Padova, Italy plant-based dietary intervention on beta-cell function and insulin resistance Classic Ketogenic Diet (KD) is a high-fat, low-carbohydrate diet that in overweight adults with no history of diabetes. induces ketone body production through fat metabolism with the goal of Methods: Participants (n=75) were randomized to follow a low-fat vegan mimicking a starvation state without depriving the body of necessary calo- diet (n=38) or to make no diet changes (n=37) for 16 weeks. At baseline and ries. KD is an established treatment of glucose transporter type 1 deficiency 16 weeks, beta-cell function was assessed during standard meal tests. Insu- syndrome and drug-resistant epilepsy, and it is currently evaluated in the lin secretory rate was calculated by C-peptide deconvolution, and beta-cell management of insulin resistant states as metabolic syndrome, obesity and function was quantified with a mathematical model. HOMA-IR (The Homeo- type 2 diabetes. Insulin is required for effective storage of meal substrates, stasis Model Assessment) index was used to assess insulin resistance while but insulin secretory rate (ISR) response after a classic ketogenic meal fasting. Repeated measure ANOVA was used for statistical analysis. (cKDm) has never been investigated. Results: A marked increase in glucose-stimulated insulin secretion was We measured ISR after a cross-over administrations of a cKDm and of a observed in the intervention group compared with controls (interaction Mediterranean meal (Mm) providing 2% vs. 60% carbohydrates, 88% vs. between group and time, Gxt, p<0.001). HOMA-IR index fell significantly 25% fat and 10% vs. 15% protein, respectively of 40% of individual’s total (p<0.001) in the intervention group (treatment effect -1.0 [95% CI, -1.2 to energy requirement in 12 healthy subjects (6F/6M, 19-31 year. Venous blood -0.8]; Gxt, p=0.004). Changes in HOMA-IR correlated positively with changes was sampled at 0, 10, 20, 30, 45, 60, 90, 120 and 180min to measure glucose, in body mass index (BMI) and visceral fat volume (r=0.34; p=0.009 and r=0.42; insulin and C-peptide concentrations. ISR was calculated from c-peptide p=0.001, respectively). The latter remained significant after adjustment for deconvolution and normalized to estimated body surface area. changes in BMI (r=0.41; p=0.002). Changes in glucose-induced insulin secre- Glucose and insulin concentrations and ISR responses were markedly tion correlated negatively with BMI changes (r=-0.25; p=0.04), but not with reduced (p<0,001 al parameters) after cKDm with respect to Mm (Figure). changes in visceral fat. For the first time we have shown that cKDm is disposed with only a mini- Conclusions: A 16-week low-fat vegan dietary intervention improved mal ISR compared to a Mm. This finding may be of interest for patients with beta-cell function and insulin resistance in overweight adults. The improve- insulin resistance and or insulin secretory defects. ment in insulin resistance was related to loss of visceral fat, independent of Figure. changes in BMI. Supported By: Physicians Committee for Responsible Medicine

295‑OR The Common Food Additive Carrageenan Increases Intestinal Permeability without Affecting Whole-Body Insulin Sensitivity in Humans—Results from a Randomized, Double-Blind Crossover Study ROBERT WAGNER, JANINE BÜTTNER, MARTIN HENI, LOUISE FRITSCHE, STEPHANIE KULLMANN, MORITZ WAGMÜLLER, ANDREAS PETER, HUBERT 293‑OR PREISSL, JUERGEN MACHANN, ULRICH-FRANK PAPE, GERRIT VAN HALL, PETER Continuous Remote Care Model Utilizing Nutritional Ketosis PLOMGAARD, REINHILD KLEIN, ANDREAS FRITSCHE, HANS-ULRICH HAERING, Improves Type 2 Diabetes Risk Factors in Patients with Prediabetes NORBERT STEFAN, Tübingen, Germany, Berlin, Germany, Copenhagen, Denmark AMY MCKENZIE, SARAH HALLBERG, NASIR H. BHANPURI, SHAMINIE J. ATH- It is not known whether food additives associated with western lifestyle,

ORALS INARAYANAN, JACKSON MCCUE, JAMES P. MCCARTER, JEFF VOLEK, STEPHEN such as the widely used carrageenan, also play a role in the pathogenesis PHINNEY, San Francisco, CA, Columbus, OH of diabetes. Animal data suggest that increased carrageenan consumption Prediabetes (preT2D) is a major U.S. public health concern, as over 84 mil- causes insulin resistance and diabetes, mainly by interfering with hepatic lion adults are at greater risk of cardiovascular disease and progression to insulin signaling. This is the first trial in humans that tested whether car- type 2 diabetes (T2D). Abnormal glucose, BMI, blood pressure, and blood rageenan added to the diet affects key metabolic traits. lipids are risk factors for progression to T2D. The purpose of this single-arm We conducted a randomized, double-blind, placebo-controlled, cross-over prospective longitudinal investigation was to assess change in risk factors trial (registered as NCT02629705). Healthy males (N=20) were randomly after one year of treatment with a continuous remote care model utilizing allocated to 14 days of carrageenan (250 mg twice daily) or matching pla- nutritional ketosis, behavior change, and support from a health coach, medi- cebo. After a washout-period of 30±7 days, they received the other com- cal provider, and peers provided via an online clinic. Patients with preT2D pound. At the end of each treatment phase, participants underwent an oral enrolled (n=116); at 1 year, 95 participants (82%) remained active. glucose tolerance test (OGTT), a hyperinsulinemic-euglycemic clamp with 1 Results are reported for participants with measurements completed labeled glucose, whole-body MR-tomography and H-MR-spectroscopy, at 1 year and baseline; n varied across measures (range: 77-95). Changes blood immune cell phenotyping and a lactulose-mannitol test for investigat- from baseline to 1 year included: weight -13.1±9.5 kg (from 110.6±23.5 kg, ing intestinal permeability. p<0.0001), BMI -5.0±3.5 kg/m2 (from 38.9 kg/m2, p<0.0001), HbA1c -0.3±0.3% Participants had a mean (±SD) age of 27.6±4.8 years and a BMI of 24.4±2.6 2 (from 5.9±0.2%, p<0.0001), fasting glucose -9±14 mg/dL (from 110±15 mg/dL, kg/m . Intestinal permeability significantly increased after carrageenan p<0.0001), systolic BP -6±14 mmHg (from 130±13 mmHg, p=0.0003), diastolic vs. placebo exposure (lactulose-mannitol-ratio 0.0196 vs. 0.015, p=0.03). BP -3±9 mmHg (from 83±8 mmHg, p=0.0032), triglycerides -43±55 mg/dL Whole-body insulin sensitivity index (ISI) did not differ between placebo (from 148±67 mg/dL, p<0.0001), and HDL-C +7±9 mg/dL (from 52±14 mg/dL, and carrageenan exposure (p=0.5 for both ISIclamp and ISIOGTT). No changes p<0.0001). Seventy percent (54/77) of participants lost >7% body weight. At of hepatic fat content, body fat mass, distribution, liver transaminases and 1 year, 51.1% (45/88) participants achieved fasting glucose <100 mg/dL (up inflammatory cytokines were observed (all p>0.4). from 24/94, 25.5% at baseline). No completer progressed to T2D, and 60.7% In young healthy males, short-term carrageenan intake resulted in (54/89) achieved HbA1c <5.7% (up from 14/94, 14.9% at baseline). Most increased gut permeability. This was not accompanied by changes of whole-

ADA-Supported Research

A78 PREVENTING AND TREATING HYPOGLYCEMIA body insulin sensitivity, body fat mass, distribution and liver fat content. PREVENTING AND TREATING HYPOGLYCEMIA Further investigations of hepatic and brain insulin sensitivity, immune phenotyping will reveal whether carrageenan intake affected these variables. 298‑OR Supported By: German Federal Ministry of Education and Research (01GI0925) Predictors of Hypoglycemia Avoidance in a Randomized Controlled rtCGM Trial (HypoDE) 296‑OR NORBERT HERMANNS, LUTZ HEINEMANN, GUIDO FRECKMANN, DELIA Dietary Rapeseed Oil Supplementation Reduces Hepatic Steatosis WALDENMAIER, DOMINIC EHRMANN, Bad Mergentheim, Germany, Düsseldorf, in Obese Men Germany, Ulm, Germany MICHAEL KRUSE, MARGRIT KEMPER, SOFIYA GANCHEVA, DIRK DANNEN- The HypoDE study, a randomized multi-center trial, showed that rtCGM BERGER, DANIEL F. MARKGRAF, MICHAEL RODEN, ANDREAS F. PFEIFFER, Nut- use reduces the number of low glucose events (<55 mg/dl for at least 20 hetal, Germany, Düsseldorf, Germany, Dummerstorf, Germany, Berlin, Germany minutes) per 28 days from 10.4 to 3.4 events compared to SMBG (13.5 to 13.2 Obesity is associated with nonalcoholic fatty liver disease (NAFLD) lead- events) in MDI-treated type 1 diabetic patients with hypoglycemia prob- ing to increased hepatic glucose production. NAFLD is one of the most lems. In this post-hoc analysis, we analyzed which baseline variables predict frequent liver diseases however, effective treatment is limited. Animal successful reduction of low glucose events. The criterion was a reduction studies showed a reduction in hepatic steatosis with dietary alpha linoleic to 0 events or by 50% from baseline. In a block-wise, multivariate logistic acid, which is highly enriched in rapeseed oil (RA). In this study we used a regression, demographic and medical variables, patient-reported-outcomes, nutritional therapeutic approach and investigated the effect of an isocaloric biochemical hypoglycemia at baseline (based on 4-week masked rtCGM diet supplemented with RA or olive oil (OL) on liver fat content, endogenous use), hours per day in time in range and hyperglycemia, and use of rtCGM glucose production, total body insulin sensitivity and serum lipids in obese were used as predictors. Model fit of the block-wise regression suggests humans suffering from NAFLD. 27 obese men (BMI 30-35, age 18-65) con- that none of the baseline characteristics did predict hypoglycemia avoid- sumed an isocaloric diet including 50 g of either RA (n=12) or OL (n=15) daily ance. However, entering use of rtCGM led to a rise of Nagelkerke´s R² from for eight weeks. Hepatic MRI, hyperinsulinemic-euglycemic clamp studies 0.061 to 0.331. This indicates that rtCGM use was the most important pre- with isotope labelled glucose and blood tests were performed prior to and at dictor for hypoglycemia avoidance. In the CGM Group, 76.1% of the patients the end of the study to assess liver fat content, endogenous glucose produc- compared to 26.3% in the Control group could avoid hypoglycemic events tion and cholesterol and free fatty acid levels, respectively. Body fat con- (Odds ratio 9.8, 95% CI 4.2 to 23.0). This suggests that rtCGM use is effec- tent and BMI did not change over the time of intervention for RA and OL. At tive in patients with type 1 diabetes and hypoglycemia problems regardless the end of the study a significant reduction in hepatic fat content (P=0.038) of diabetes duration, glycemic control, severity of hypoglycemia problems, could be observed for RA (13.09±1.61 before vs. 11.14±1.58% after interven- and exposure to biochemical hypoglycemia at baseline. tion) vs. OL (13.29±2.52 before vs. 15.73±2.74% after intervention). For RA, Table. Predictors of Hypoglycemia Avoidance. a 21% reduction (P<0.02) in serum free fatty acids was observed after eight weeks of oil consumption whereas no significant changes were seen for OL. Characteristics Nagelkerke’s Delta - Consuming RA resulted in a slight reduction in basal endogenous glucose R² Nagelkerke’s R² production (1.71±0.15 before vs. 1.49±0.08 mg/kg/min after intervention, Block 1 Demographic variables 0.005 P=n.s.) compared to OL (1.66±0.05 before vs. 1.65±0.08 mg/kg/min after (age, diabetes duration) intervention, P=n.s.). There were no significant changes in total body insulin Block 2 Block 1 + medical variables 0.022 0.017 sensitivity and serum cholesterol levels in RA or OL. Our results demonstrate (HbA1c, severe hypoglycemia a beneficial effect of the fatty acid composition of RA on hepatic lipid pro- in the past 12 month, duction as shown by reduced hepatic fat content and free fatty acid levels. hypoglycemia unawareness Supported By: German Union for the Promotion of Oil and Protein Plants Block 3 Block 2 + patient reported 0.029 0.027 outcomes (fear of hypoglycemia, diabetes distress, satisfaction 297‑OR with glucose monitoring, self- Gut Microbiota Metabolites, Amino Acid Metabolites, and Improve‑ reported health status) ments in Diabetes-Related Traits—The POUNDS Lost Trial Block 4 Block 3 + biochemical 0.058 0.009 YORIKO HEIANZA, DIANJIANYI SUN, GEORGE BRAY, LU QI, New Orleans, LA, hypoglycemia at baseline Baton Rouge, LA (time spent ≤70 and ≤55 mg/dl Gut microbiota alterations may be related to insulin resistance, diabetes, per day, baseline low and the impaired amino acid metabolism. We investigated whether changes glucose Events) in gut microbiota metabolite of trimethylamine N-oxide (TMAO) and its pre- Block51 Block 4 + time in range and time 0.061 0.003 cursors (choline and L-carnitine) were associated with improvements of dia- in hyperglycemia (>180 mg/dl) betes-related traits and diabetes-risk-related amino acids in a weight-loss Block 6 Block 5 + treatment 0.331 0.270 diet intervention. This study included 504 obese adults who were randomly (rtCGM vs. SMBG)

assigned to 1 of 4 energy-reduced diets varying in the macronutrient com- ORALS position. We calculated changes (Δ) in TMAO, choline, and L-carnitine from Supported By: Dexcom, Inc. baseline to 6 months after the diet intervention. Greater decreases in choline and L-carnitine were significantly (p <0.05) associated with greater improve- 299‑OR ments in fasting insulin levels and the Homeostatic Model Assessment of Differential Effects of the Insulin-Only and Bihormonal Configura‑ Insulin Resistance of insulin resistance (HOMA-IR) at 6 months. Particularly, tions of the Bionic Pancreas on Mean Glucose and Hypoglycemia the reduction of choline was predictive of long-term (2-year) improvements during the Daytime and Nighttime in fasting glucose (p=0.007), insulin (p=0.002), and HOMA-IR (p <0.001). We JORDAN SHERWOOD, COURTNEY A. BALLIRO, RABAB Z. JAFRI, FIRAS EL- found significant interactions between dietary fat intake andΔ TMAO for KHATIB, MICHELE MAHENO, MALLORY A. HILLARD, ALEXANDER J. O’DONOVAN, changes in fasting glucose, insulin, and HOMA-IR (Pinteraction <0.05); among RAJENDRANATH SELAGAMSETTY, HUI ZHENG, EDWARD DAMIANO, STEVEN J. participants who consumed a high-fat diet, greater increase in TMAO was RUSSELL, Boston, MA related to less improvements of the outcomes. In addition, ΔL-carnitine and The bihormonal bionic pancreas (BP) reduced both mean CGM glucose Δcholine were also significantly related to changes in amino acids (includ- (CGMG) and time in the hypoglycemic range relative to usual care in out- ing branched-chain and aromatic amino acids). However, the associations patient studies. We recently performed an outpatient study in which we of ΔTMAO, Δcholine, and ΔL-carnitine with the diabetes-related traits were compared insulin-only (IOBP) and bihormonal (BHBP) configurations of the independent of the changes in amino acids. BP with usual care (UC; conventional or sensor-augmented insulin pump In conclusion, weight-loss-diet-induced changes in TMAO, choline, and therapy) both with and without remote monitoring. We previously reported L-carnitine were significantly associated with the improvements in glyce- that monitoring did not affect the mean CGMG or hypoglycemia with either mia and insulin sensitivity, regardless concurrent changes in diabetes-risk configuration of the BP. The current analysis focuses on the data collected related amino acids. Dietary fat intake may modify the associations of from arms without remote monitoring (how the BP is eventually meant to be changes in TMAO with the improvements of diabetes-related traits. used) to determine how the effects of the BP differ between the day and night. As shown in the Table, both the IOBP (with glucose target of 110 mg/ dl) and BHBP (with glucose target of 100 mg/dl) significantly reduced mean

ADA-Supported Research

A79 PREVENTING AND TREATING HYPOGLYCEMIA

CGMG vs. UC during both the daytime and nighttime. In contrast, only the BHBP significantly reduced the percent of time spent with CGMG <60 mg/dl vs. UC. The BHBP similarly reduced percent of time <60 mg/dl vs. IOBP. The significant reductions in hypogycemia associated with the BHBP were driven primarily by reductions in hypoglycemia at night. Table.

Supported By: The Leona M. and Harry B. Helmsley Charitable Trust

300‑OR Relationship between A1C and Hypoglycemia Risk in Individual Patients Comparing Insulin Degludec with Insulin Glargine U100 Supported By: Novo Nordisk A/S ATHENA PHILIS-TSIMIKAS, WENDY LANE, ULRIK PEDERSEN-BJERGAARD, CAROL H. WYSHAM, LARS BARDTRUM, SIGNE H. ØSTOFT, SIMON HELLER, San Diego, CA, Asheville, NC, Hillerød, Denmark, Spokane, WA, Søborg, Denmark, Shef- 301‑OR field, United Kingdom Similar Glycemic Control and Less or Comparable Hypoglycemia Targeting a lower A1C may increase the hypoglycemia risk in patients with Insulin Glargine 300 U/mL (Gla-300) vs. Degludec 100 U/mL with diabetes. To investigate the relationship between A1C and hypogly- (IDeg-100) in Insulin-Naïve T2DM on Antihyperglycemic Drugs ± cemia risk on an individual level, this post-hoc analysis used data from two GLP-1 RAs—The BRIGHT Randomized Study double-blind, randomized, treat-to-target, two-period (32 weeks each) cross- ALICE Y.Y. CHENG, JULIO ROSENSTOCK, ROBERT RITZEL, ZSOLT BOSNYAK, over trials of insulin degludec (degludec) vs. insulin glargine 100 units/mL CHRISTINE DEVISME, PETER STELLA, ANNA M. CALI, XIANGLING WANG, JUAN (glargine U100) in patients with type 1 (T1D; SWITCH 1, n=501) or type 2 P. FRIAS, RONAN ROUSSEL, GEREMIA B. BOLLI, Mississauga, ON, Canada, Dal- diabetes (T2D; SWITCH 2, n=721). For each patient at each visit, A1C was las, TX, Munich, Germany, Paris, France, Boulogne-Billancourt, France, Tokyo, Japan, linked with the number of hypoglycemic events (blood glucose-confirmed Beijing, China, Los Angeles, CA, Perugia, Italy [<56 mg/dL] with symptoms or severe [third-party assistance]) since last BRIGHT is the first head-to-head clinical trial investigating the efficacy and visit. A 1% (10.9 mmol/mol) A1C reduction led to an 18% (degludec) and 34% safety of Gla-300 vs. IDeg-100. In this 24-week, multinational, open-label, (glargine U100) increased risk of hypoglycemia in T1D, and 45% (degludec) parallel-group, treat-to-target trial (NCT02738151), 929 insulin-naïve T2DM 2 and 67% (glargine U100) increased risk in T2D (Fig). Assuming an 11% (T1D) adults (mean HbA1c, 8.6%; DM duration, 10.6 years; BMI, 31.5 kg/m ), inad- and 30% (T2D) reduction in hypoglycemia risk, as seen in the SWITCH trials, equately controlled with oral antihyperglycemic drugs ± GLP-1 RAs, were this can be translated into a 0.61% (T1D) and 0.67% (T2D) A1C reduction with randomized 1:1 to once-daily Gla-300 or IDeg-100. Primary endpoint: HbA1c degludec with no increase in hypoglycemia risk vs. glargine U100. change from baseline to week 24. Secondary endpoints included hypoglyce- In conclusion, lowering A1C led to a higher hypoglycemia risk; however, mia, blood glucose levels, and adverse events. Non-inferiority of Gla-300 vs. the lower incremental hypoglycemia risk with degludec vs. glargine U100 IDeg-100 was demonstrated for the primary endpoint (Table). Gla-300 had may allow for a lower A1C target in both T1D and T2D with degludec than similar fasting SMPG reduction to IDeg-100 (Table), with final daily insulin with glargine U100 in clinical practice, when hypoglycemia is a limiting fac- doses of 0.54 and 0.43 U/kg from starting evening doses, per label, of 0.2 U/ tor for glycemic control. kg and 10 U/day (0.12 U/kg), respectively. Over the 24-week period, incidence of confirmed ≤( 70 mg/dL) or severe hypoglycemia was comparable, but event rates were lower with Gla-300 vs. IDeg-100, by 14% at any time of day (24h) and 19% at night (00:00-05:59 h) (Table). In summary, BRIGHT showed that Gla-300 provides similar glycemic control to IDeg-100, with less or comparable hypoglycemia, in previously inadequately controlled, insulin-naïve adults with T2DM. ORALS

ADA-Supported Research

A80 PREVENTING AND TREATING HYPOGLYCEMIA

and p=0.05). Incidences of hypoglycemia at any time of day (24 hours) appeared slightly higher in the ≥65 years, physician-led group. There was no evidence of heterogeneity of effect of self- vs. physician-led titration on hypoglycemia (p>0.05). Adverse events were similar in each group. Self-titration of Gla-300 resulted in similar glycemic target achievement to physician-led titration, without an increased risk of hypoglycemia, irrespective of age. There was a trend for more people to reach fasting SMPG targets without hypoglycemia with self- vs. physician-led titration, including those ≥65 years.

Supported By: Sanofi (NCT02738151)

302‑OR Efficacy and Safety of Insulin Degludec and Insulin Glargine U-100 in Hospitalized Patients with Type 2 Diabetes—An Open-Label, Randomized Controlled Study JUN SUZUKI, TADASHI YAMAKAWA, MINORI SHINODA, RIKA SAKAMOTO, KENICHIRO TAKAHASHI, YASUO TERAUCHI, Yokohama, Japan Aims/Introduction: The short-term efficacy and safety of insulin degludec (IDeg) in patients with type 2 diabetes have not previously been reported. We evaluated target glycemic control and glucose variability in hospitalized patients with type 2 diabetes. Material and Methods: In an open-label, multicenter, randomized controlled trial, 74 patients were randomly assigned to an insulin glargine U100 (IGla) group (38 patients) or insulin degludec group (36 patients) and were Supported By: Sanofi (EudraCT 2015-001626-42) administered Basal-bolus therapy during hospitalization. Continuous glucose monitoring was performed on Day 11 after the insulin treatment com- 304‑OR menced, to verify that day’s glucose variability. A Phase 3 Comparison of a Novel Liquid Glucagon Autoinjector Results: The proportion of patients achieving a fasting blood glucose level to Glucagon Emergency Kit for the Symptomatic Relief of Severe of 110 mg/dl and 2-hour postprandial blood glucose level of 180 mg/dl through- Hypoglycemia out at least a day during the observation period was 30.6% [95% confidence MARK P. CHRISTIANSEN, MARTIN J. CUMMINS, STEVEN J. PRESTRELSKI, POUL interval 18.2-45.5%, one-sided p-value = 0.057] (11/36) in the IGla group, STRANGE, Walnut Creek, CA, Chicago, IL, Princeton Junction, NJ and 31.3% [95% confidence interval 18.0-47.2%, one sided p-value = 0.056] Objective: A novel ready-to-use stable liquid glucagon autoinjector (GAI; (10/32) in the IDeg group. Although no significant difference was noted on Xeris Pharmaceuticals), was evaluated for relief of symptoms during rescue the 6-point SMBG profiles in mean blood glucose levels between the groups, treatment of severe hypoglycemia. the IDeg group showed a significant improvement in the percentage change Research Design and Methods: A randomized, controlled, double-blind, from baseline of mean blood glucose before lunch on Day 7 (p = 0.021) and crossover clinical trial was conducted in 80 adults with T1D (mean age 43.6 after dinner on Day 12 (Day 7 before lunch: IGla group -29.5 ± 26.3%, IDeg years) to compare subcutaneous 1 mg doses of GAI vs. Glucagon Emergency group -44.5 ± 22.1%, p = 0.021; Day 12 after dinner: IGla group -34.2 ± 22.5%, Kit (GEK; Eli Lilly) for the treatment of insulin-induced severe hypoglycemia. IDeg group -51.1 ± 21.7%, p = 0.021). No significant differences were noted Serial assessments of 4 autonomic and 4 neuroglycopenic symptoms and between the groups in the incidence of hypoglycemia or the glucose variability sensation of hypoglycemia were performed at each treatment visit. parameter data on continuous glucose monitoring. Results: The mean time to symptom relief was comparable between GAI Conclusion: This study suggests that IDeg is comparable with IGla in and GEK for both autonomic symptoms (15.4+12.1 min and 14.2+9.7 min, short-term efficacy and safety for patients with type 2 diabetes in the initial p=NS), and neuroglycopenic symptoms (16.3+10.9 min and 13.9+9.4 min, phase of basal-bolus therapy. p=NS). Mean time to resolution of the global feeling of hypoglycemia was ORALS also similar between groups. The incidence of all AEs was low in both groups; 303‑OR the most commonly reported AE was nausea (GAI 20.5%, GEK 12.7%, p=NS). Self- vs. Physician-Led Titration of Insulin Glargine 300 U/mL Conclusions: The prompt relief of neurologic symptoms is critical in the (Gla-300)—Improved or Comparable Efficacy at Week 24 without rescue of severe hypoglycemic emergencies. GAI achieved both autonomic Increased Risk of Hypoglycemia, Irrespective of Age (<65 or ≥65 and neuroglycopenic symptom relief during induced severe hypoglycemia, Years)—TAKE CONTROL and was safe and well tolerated. These results demonstrate that ready-to- KRZYSZTOF STROJEK, GREGORY BIGOT, MIREILLE BONNEMAIRE, ELIAS use GAI is a viable alternative to GEK. DELGADO, VIERA DONICOVA, MILAN KVAPIL, NIKOLAOS PAPANAS, LUIZA Figure. POPESCU, ROBERT RITZEL, BERND SCHULTES, LEA DUVNJAK, Zabrze, Poland, Levallois-Perret, France, Paris, France, Oviedo, Spain, Kosice, Slovakia, Prague, Czech Republic, Alexandroupolis, Greece, Bucharest, Romania, Munich, Germany, St. Gal- len, Switzerland, Zagreb, Croatia TAKE CONTROL, a 24-week, multicenter, randomized, open-label, parallel- group study, evaluated self- vs. physician-led titration of Gla-300 in people with T2DM. Efficacy and safety outcomes in people <65 years vs.≥ 65 years were assessed. Baseline HbA1c and fasting SMPG were comparable; in the older and younger groups, HbA1c (p=0.46 and p=0.29) and fasting SMPG (p=0.56 and p=0.15) reductions were similar between self- vs. physician-led titration. Attain- ment of the fasting SMPG target (80-130 mg/dL) without confirmed (<54 mg/dL) or severe hypoglycemia was slightly higher in older people; differences in self- vs. physician-led titration were not significant in older or younger groups (p=0.19

ADA-Supported Research

A81 RACE AND SOCIETAL INFLUENCES ON DIABETES

305‑OR variables, physical activity, caloric intake, depression, smoking, and alcohol BioChaperone Glucagon (BCG), a Stable Ready-to-Use Liquid Glu‑ intake, women in the assimilation class continued to have significantly lower cagon Formulation, Is Well Tolerated and Quickly Restores Eugly‑ TG and higher HDL levels compared to women in the separation class [β cemia after Insulin-Induced Hypoglycemia (95% CI): -23.7 (-41.9, -5.5) and 7.5 (2.9, 12.1); respectively]. There was no STANISLAV GLEZER, ULRIKE HOVELMANN, SOPHIE TENG, DANIELA LAMERS, significant association between acculturation and CMR factors in men. MARION ODOUL, JOSÉ CORREIA, ERIC ZIJLSTRA, MARTIN GAUDIER, OLIVIER Conclusion: SA women who employed an assimilation strategy had a more SOULA, DAVID DURACHER, Skillman, NJ, Neuss, Germany, Lyon, France favorable CMR profile compared to women using a separation strategy. BCG is a stable, ready-to-inject, aqueous formulation of human glucagon Future research should investigate the behavioral pathways linking accul- for hypoglycemia rescue therapy. In this randomized, double-blind, crossover turation strategies with CMR and potential incorporation into prevention trial we investigated the safety and efficacy of two BCG formulations vs. a interventions. commercially available glucagon (GlucaGen® HypoKit, GEN) in 27 patients with type 1 diabetes who received single subcutaneous doses of 1 mg of 307‑OR BCG1, BCG2 or GEN under insulin-induced hypoglycemic conditions (plasma Mediators of the Racial Disparity in Diabetes Incidence—The Rea‑ glucose (PG)<60 mg/dl). Both BCG formulations were safe and well tolerated sons for Geographic and Racial Differences in Stroke (REGARDS) with the most frequent adverse event being mild nausea with both BCGs and Study GEN. Both BCGs quickly restored PG of ≥ 70 mg/dl after hypoglycemia (BCG1 APRIL P. CARSON, D. LEANN LONG, ANDREA CHERRINGTON, GARETH R. DUT- 11.5±5.0 min; BCG2 10.0±3.5 min; GEN 7.3±1.8 min, p<0.001 vs. BCG1 and TON, VIRGINIA J. HOWARD, TODD BROWN, CLAUDIA MOY, MARY CUSHMAN, BCG2). PG≥70 mg/dl was reached within 30 minutes by all but one patient MONIKA SAFFORD, GEORGE HOWARD, Birmingham, AL, Bethesda, MD, Burling- with both BCGs and the mean PG increase at 15 minutes was 29±17 mg/dl ton, VT, New York, NY with BCG1, 36±16 mg/dl with BCG2 and 47±11 mg/dl with GEN (p<0.001 vs. Objective: To identify the contribution of individual and neighborhood fac- BCG1 and BCG2). tors to the racial disparity in diabetes incidence among older adults. In conclusion, the BioChaperone technology allows the formulation of Methods: This study included 7,304 black and white adults, aged 45-91 stable ready-to-use liquid formulations of human glucagon suited for rescue years at baseline (2003-07), without prevalent diabetes. Incident diabetes therapy of severe hypoglycemia with only slightly slower effects than GEN. was assessed at the 2nd in-home exam (2014-16) using plasma glucose or use Figure. of diabetes medications. Modified Poisson regression, stratified by sex, was used to obtain risk ratios. Mediation analyses using the change in beta coefficient method and bootstrapping were done to evaluate the contribution of socioeconomic, anthropometric, lifestyle, clinical and neighborhood factors, grouped separately and all factors combined. Results: The risk of diabetes was 75% higher for black women compared with white women and 35% higher for black men compared with white men (Table). Among men, only lifestyle factors were significant mediators of the racial disparity. In contrast, among women, all grouped factors, except clinical, were significant mediators. When taking into account all factors combined, only 4.2% and 28.1% of the racial disparity was explained for men and women, respectively. Conclusion: Although the factors underlying the racial disparity varied by sex, the excess diabetes risk for black vs. white adults remained after accounting for individual and neighborhood factors.

Supported By: Adocia

RACE AND SOCIETAL INFLUENCES ON DIABETES

306‑OR The Relationship of Acculturation to Cardiometabolic Risk Factors among U.S. South Asians—Findings from the MASALA Study

ORALS MOHAMMED AL-SOFIANI, SUSAN J. LANGAN, ALKA M. KANAYA, NAM- RATHA R. KANDULA, BELINDA NEEDHAM, CATHERINE KIM, DIANE M. BECKER, DHANANJAY VAIDYA, SHERITA GOLDEN, CLARE LEE, RITA R. KALYANI, Balti- more, MD, San Francisco, CA, Chicago, IL, Ann Arbor, MI Background: Immigration and the process of acculturation can lead to behavioral changes that exacerbate or reduce diabetes development. We investigated the association between acculturation strategies and cardiometabolic risk (CMR) factors among South Asian (SA) immigrants living in the U.S. Methods: Participants (n=849) in the Mediators of Atherosclerosis in SA Supported By: National Institute of Neurologic Disorders and Stroke Living in America (MASALA) study were categorized into 3 acculturation (U01NS041588) strategy classes based on self-reported questionnaire responses: separation (preference for SA culture), assimilation (preference for U.S. culture), and integration (similar preference for SA and U.S. cultures). Adjusted linear regression models examined associations of acculturation strategies with fasting and 2 hour glucose, lipids, blood pressure, waist circumference (WC), and body composition. Results: Overall, 22.4, 19.6, and 58% of men and 23.7, 26.2, and 50.1% of women were in separation, assimilation, and integration classes; respectively. Participants in the assimilation class had significantly lower levels of obesity, 2 hour glucose, insulin, HOMA-IR, triglycerides (TG), visceral fat, and higher HDL than those in the separation class (all p<0.05). In sex-stratified analyses, after adjusting for education, income, other socioeconomic

ADA-Supported Research

A82 RACE AND SOCIETAL INFLUENCES ON DIABETES

308‑OR 310‑OR Socioeconomic Profiles as Predictors of Glycemic Control in Youth Prevalence of Diagnosed Diabetes and Total Diabetes among Asian with Type 1 Diabetes Americans, 2011-2014 MELANIE W. SUTHERLAND, XIAONAN MA, JASON A. MENDOZA, BETHANY YILING J. CHENG, ALKA M. KANAYA, SHARON SAYDAH, MARIA ROSARIO ARA- A. BELL, BETH A. REBOUSSIN, ANNA R. KAHKOSKA, KATHERINE A. SAUDER, NETA, HENRY S. KAHN, GIUSEPPINA IMPERATORE, Atlanta, GA, San Francisco, CATHERINE PIHOKER, ANGELA D. LIESE, Columbia, SC, Seattle, WA, Winston- CA, Hyattsville, MD, La Jolla, CA Salem, NC, Chapel Hill, NC, Aurora, CO Asian Americans (AAs) comprise ~6% of the U.S. population. However, Various measures of socioeconomic status (SES) predict glycemic control there is little national information on the prevalence of diabetes (DM) among in youth and young adults (YYA) with type 1 diabetes; however, rarely have AAs. In addition, there are no national estimates of DM prevalence accord- multiple SES factors been considered simultaneously, including parental ing to AA ancestral subgroups. Among sampled AA adults (age ≥20 years) education and income, food security, health insurance status, and food we estimated the population prevalence of DM for all AAs and major geo- assistance. The purpose of this study was to identify multidimensional SES graphic AA ancestral subgroups using data from the U.S. National Health profiles in YYA with type 1 diabetes and to assess the association between and Nutrition Examination Survey 2011-2014. DM status was categorized these profiles and glycemic control. Data from 257 YYA with type 1 dia- as diagnosed (DiagDM), undiagnosed (UndiagDM; no DiagDM but HbA1c betes enrolled in the SEARCH Food Insecurity Ancillary Study in South ≥6.5% or fasting plasma glucose ≥126mg/dl or 2-hour plasma glucose ≥200 Carolina and Washington between the years 2013 and 2015 were included. mg/dl), or nondiabetic. We defined 4 AA ancestral subgroups as East AA We identified SES profiles utilizing latent class analysis and used multi- (Chinese, Japanese, and Korean), South AA (Indian, Pakistani, Sri Lankan, variable logistic regression to compare the profiles’ glycemic control, with Bangladeshi, Nepali, and Bhutanese), Southeast AA (Filipino, Vietnamese, HbA1c > 9% defined as poor glycemic control. Two profiles were identified: Cambodian, Laotian, Thai, Indonesian, Malaysian, Singaporean, and Hmong), Profile 1 (74%) included YYA that were of higher parental income and educa- and other AA. Age-sex adjusted prevalences and prevalence ratios were tion, and less likely to have government-funded health insurance, to receive computed for the 4 AA subgroups, as well as for non-Hispanic whites, non- food assistance, and to be food insecure. Profile 2 (26%) included YYA who Hispanic blacks, and Mexican Americans (Table). Compared to non-Hispanic were categorized opposite to that of profile 1. In profile 2, 60% of YYA had whites, East AAs had a similar prevalence of DiagDM but higher UndiagDM. poor glycemic control compared to 44% of YYA in profile 1. After adjustment The elevated prevalence of UndiagDM among AAs suggests the relatively for age, sex, race/ethnicity, site, diabetes duration, and diabetes medication, low awareness of their condition and reinforces the importance of diabetes YYA in profile 2 were significantly more likely to have poor glycemic control screening in these populations. relative to YYA in profile 1 (OR = 1.94, 95% CI = 1.01-3.71). Membership to a Table. Age-Sex Adjusted Prevalence of Diabetes and Prevalence Ratio, lower SES profile is associated with poor glycemic control among YYA with NHANES 2011-2014. T1D. Strategies to improve glycemic control, particularly in YYA who belong Race/ethnicity n Prevalence (%, 95% CI) Prevalence Ratio (95% CI) to lower SES profiles, are needed. (weighted N, Supported By: University of South Carolina million) DiagDM UndiagDM TotalDM DiagDM UndiagDM TotalDM 309‑OR Non-Hispanic AA 1517 (12.5) 11.2 (9.0, 13.5) 9.3 (6.9, 11.8) 20.6 (17.4, 23.7) 1.4 (1.1, 3.9) 2.6 (1.8, 9.0) 1.8 (1.5, 2.2) Neighborhood Walkability and Diabetes-Related Complications REEMA SHAH, JIN LUO, HERTZEL C. GERSTEIN, GILLIAN BOOTH, Hamilton, ON, East AA 594 (4.8) 7.2 (4.0, 10.4) 8.8 (5.1, 12.6) 16.0 (10.7, 21.3) 0.9 (0.6, 1.4) 2.5 (1.5, 4.1) 1.4 (1.0, 2.0) Canada, Toronto, ON, Canada South AA 345 (2.9) 15.4 (9.6, 21.2) 9.4 (4.9, 13.9) 24.8 (17.5, 32.2) 2.0 (1.4, 2.8) 2.7 (1.5, 4.5) 2.2 (1.6, 6.7) Neighborhoods that are more conducive to walking are associated with Southeast AA 414 (3.5) 14.1 (11.0, 17.3) 9.2 (5.5, 12.9) 23.3 (18.4, 28.2) 1.8 (1.4, 2.3) 2.6 (1.7, 3.9) 2.0 (1.6, 2.6) increased levels of physical activity and lower levels of obesity and dia- Other AA 164 (1.3) 9.8 (4.6, 15.0) 8.7 (1.9, 15.5) 18.5 (11.8, 25.2) 1.2 (0.7, 2.1) 2.4 (1.1, 5.5) 1.6 (1.1, 2.4) betes, but the effect of neighborhood walkability on the clinical course of patients with diabetes is not known. We conducted a retrospective cohort Non-Hispanic W 4472 (148.4) 7.9 (6.9, 8.8) 3.6 (2.6, 4.5) 11.5 (10.1, 12.8) 1.0 (Ref.) 1.0 (Ref.) 1.0 (Ref.) study in 439,392 adults using population-based administrative health data- Non-Hispanic B 2594 (25.5) 14.6 (13.3, 16.0) 6.2 (4.4, 7.9) 20.8 (18.4, 23.2) 1.9 (1.6, 2.2) 1.7 (1.2, 2.6) 1.8 (1.5, 2.2) bases to evaluate the effect of neighborhood walkability on the incidence Mexican Am 1296 (18.9) 14.8 (12.4, 17.2) 8.3 (6.4, 10.2) 23.1 (19.8, 26.5) 1.9 (1.6, 2.3) 2.3 (1.7, 3.2) 2.0 (1.7, 2.4) of diabetes related complications. Adults ( 30 years) with diabetes living in ≥ AA: Asian American; Non-Hispanic W: Non-Hispanic white; Non-Hispanic B: Southern Ontario cities were followed from April 1 2007 to March 31 2017. Non-Hispanic black; Mexican Am: Mexican American. Neighborhood walkability was derived from a validated index and classified into quintiles from lowest (Q1) to highest (Q5). Cox proportional hazards models were used to estimate the hazard of neighborhood walkability on 311‑OR retinopathy (laser photocoagulation/vitrectomy/VEGF therapy), nephropathy Food Insecurity in Older Adults with Diabetes (dialysis or kidney transplantation), foot complications (infection/amputa- EMILY B. SCHROEDER, CHAN ZENG, ANDREW STERRETT, TINA K. KIMPO, tion), and avoidable hospitalizations (hyper/hypoglycemia), adjusting for age, ANDREA R. PAOLINO, JOHN STEINER, Denver, CO sex, ethnicity, income, and comorbid conditions, as well as significant inter- Background: Food insecurity (FI) among individuals with diabetes has been actions between these and walkability. Overall, baseline characteristics associated with both hypoglycemia and worse glycemic control, but studies were similar across neighborhoods, however, low walkability areas tended of elderly insured populations are lacking. ORALS to be wealthier than high walkability areas. The cohort sustained 30457 reti- Methods: As part of its Medicare Annual Wellness Visit, Kaiser Per- nopathy events, 4883 nephropathy events, 40989 foot complications, and manente Colorado (KPCO) initiated a member survey in January 2012 that 24272 avoidable hospitalizations. There was an increased risk of retinopathy included a question about FI (“Do you always have enough money to buy the (Q1:Q5 adjusted HR 1.13, 95% CI 1.07-1.19, p<0.001), nephropathy (Q1:Q5 aHR food you need?”). We combined this survey with electronic health record 1.33, 95% CI 1.16-1.52, p<0.001), foot complications (Q1:Q5 aHR 1.27, 95% data and other data to conduct a nested case-control study of FI among CI 1.18-1.37, p<0.001), and avoidable hospitalizations (Q1:Q5 aHR 1.49, 95% KPCO members ≥ 65 years with diabetes. Of 32,666 members with diabe- CI 1.36-1.64, p<0.001) in lower walkability compared to higher walkability tes, 10,052 (31%) completed a survey. Of those, the 742 (7.4%) who had FI neighborhoods. Neighborhood design may have beneficial effects on diabe- were matched 1:3 based on date of the survey (± 7 days) to 2,226 individuals tes complications. This has important implications for policies to promote without FI. healthy environments to better manage the burden of chronic metabolic Results: Individuals with FI were slightly younger (mean age ± SD of 73.2 ± diseases. 6.1 vs. 73.9 ± 6.8), and were more likely to be female (60.6% vs. 46.8%), non- Supported By: Canadian Institutes of Health Research white (36.8% vs. 21.8%), living without a partner (49.6% vs. 33.0%), have less than high school education (21.2% vs. 9.0%), and have Medicaid (16.4% vs. 3.2%). Individuals with FI reported higher frequency of fair or poor general health (29.2% vs. 16.7%) and quality of life (23.6% vs. 10.5%), and were more likely to eat ≤ 2 meals a day (19.0% vs. 8.9%). Rates of hypertension and chronic kidney disease were similar, but individuals with FI were more likely to have depression on PHQ2 screening (24.8% vs. 9.1%). Individuals with FI were more likely to be taking any diabetes medication (70.5% vs. 63.6%) or insulin (25.4% vs. 19.4%), have an emergency department (ED) visit (19.0% vs. 13.9%) or hospitalization in the prior year (8.6% vs. 7.0%), and have an A1c > 9% (11.2% vs. 5.8%). Mean A1c for those with FI was 7.4 ± 1.4

ADA-Supported Research

A83 TRANSLATIONAL IMMUNOLOGY IN TYPE 1 DIABETES—A LOOK AT THE FUTURE

and those without FI was 7.1 ± 1.1. ED visits or hospitalizations for hypogly- Methods: To characterize the clinical and genetic features associated cemia or hyperglycemia were infrequent (≤10) in both groups. with the development of pediatric NAFLD, 503 obese adolescents (191 Conclusions: In this insured elderly population with diabetes, food insecu- caucasians, 134 African Americans, and 178 Hispanics) underwent a MRI rity was associated with higher health care utilization and A1c levels. Strate- to quantify hepatic fat content, an oral glucose tolerance test to assess gies to address FI in individuals with diabetes deserve further evaluation. glucose tolerance and insulin sensitivity, and the genotyping of three SNPs Supported By: Kaiser Permanente; National Institute of Diabetes and Digestive associated with NAFLD (PNPLA3 rs738409, GCKR rs1260326, and TM6SF2 and Kidney Diseases (K23DK099237) rs58542926). MRI and metabolic assessments were repeated in 133 patients after 2.27±1.44 years. 312‑OR Results: The prevalence of NAFLD was 42.9% in caucasians, 15.7% in Racial and Socioeconomic Disparities in Meeting Goals of Care in African Americans, and 59.6% in Hispanics (p<0.0001). Compared to cau- Adults with Type 1 Diabetes casians and Hispanics with NAFLD, African Americans adolescents with J. SONYA HAW, LIMIN PENG, FRANCISCO J. PASQUEL, PRIYATHAMA VELLANKI, NAFLD had higher fasting glucose (p=0.03), insulin (p=0.0004), C-peptide MAYA FAYFMAN, GEORGIA DAVIS, GUILLERMO E. UMPIERREZ, Atlanta, GA (p=0.04), 2-hour glucose (p=0.04), HbA1c (p<0.0001), and lower insulin sensi- Racial and socioeconomic (SES) disparities exist in youth with type 1 tivity (p=0.03), despite similar age (p=0.34) and gender distribution (p=0.37). diabetes (T1D). Black youth have worse glycemic control, higher frequency This translated in a higher prevalence of prediabetes and type 2 diabetes in of DKA, and less use of insulin pump therapy. However, data is sparse on African Americans (66.6%) compared to caucasians (24.4%) and Hispanics whether racial and SES disparities in meeting ABC (A1c, Blood pressure, (31.1%) with NAFLD (p=0.0003). Analysis of the patients without NAFLD at Cholesterol) goals of care exist in adulthood, when financial independence, baseline showed that ethnicity (p=0.02), high C-peptide levels (p=0.0006), access to healthcare, and cognitive maturity change. changes in z-score BMI over time (p=0.0006) and common gene variants We used T1D Exchange Network data, which enrolled youth and adults were strong predictors of NAFLD development at follow-up (AUROC curve 0.976). with T1D from 73 practices. We include adults (≥ 18 years) with available race/ethnicity data. Of 12,746 adults, 90% where white, 4.8% black and Conclusions: African American obese adolescents are relatively protected 5.7% Hispanic; annual household income in 13% was <$25K, 20% was $25- from NAFLD, but are more susceptible to the deleterious effects of NAFLD 50K, 34% was $50-100K, and 33% was >$100K. Logistic regression showed on glucose metabolism. The combination of ethnicity, markers of insulin increased probability of poor glycemic control (A1c >7%) with annual income resistance and genetic factors are strong predictors of pediatric NAFLD. <$25K (OR 2.29, 95% CI [1.92,1.74]), being black (OR 3.05, 95% CI [2.09,4.43]) Supported By: National Institutes of Health; American Heart Association or Hispanic (OR 1.42, 95% CI [1.10, 1.82]). Outcomes were similar in achieving SBP <140mmHg and LDL <100mg/dL. Data stratified by income showed significant disparities among races, with fewer black and Hispanic adults TRANSLATIONAL IMMUNOLOGY IN TYPE 1 achieving ABC goals compared to whites. There were no differences in mor- DIABETES—A LOOK AT THE FUTURE tality. Racial and SES disparities exist in adults with T1D. Racial disparities are 314‑OR evident after adjusting for income, suggesting socio-ecological factors play Selectively Agonizing Treg in Type 1 Diabetes with IL-2 Muteins a role in achieving optimal diabetes care in minority populations. LILIANE KHORYATI, SWARNIMA KUMARI, THI MINH NGUYET PHAM, MARIKA Table. BOGDANI, MARC GAVIN, Seattle, WA Regulatory T cells (Treg) play a pivotal role in maintaining self-tolerance. White, Black, Hispanic p-value non-Hispanic non-Hispanic For their development and function, Treg rely on interleukin-2 (IL-2) and efficiently compete with other immune cells for IL-2 by expressing the highest >$100K N=2924 N=45 N=77 levels of the IL-2 receptor alpha-chain (IL-2Ra or CD25). Treg deficiencies and A1c <7%, n (%) 920 (32) 4 (9.1) 14 (22) <0.001 alterations of the IL-2 pathway have been reported in several autoimmune SBP <140 mmHg, n (%) 2736 (95) 34 (79) 69 (92) <0.001 diseases including type 1 diabetes (T1D) where the loss of self-tolerance LDL <100 mg/dL, n (%) 1690 (69) 24 (57) 43 (67) 0.23 leads to the destruction of insulin-producing beta cells in the pancreas. Low- dose IL-2 therapy has been shown to be successful in several autoimmune No smoking, n (%) 2218 (95) 37 (95) 63 (90) 0.19 and inflammatory conditions; however, the potential for IL-2 to activate pro- $50K-100K N=2914 N=99 N=105 inflammatory immune cells raises concerns regarding its widespread utility, A1c <7%, n (%) 751 (26) 13 (14) 27 (26) 0.02 particularly in type 1 diabetes (T1D) where pathogenic autoreactive T cells SBP <140 mmHg, n (%) 2695 (93) 88 (89) 99 (95) 0.20 are activated by IL-2. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. We have generated a series LDL <100 mg/dL, n (%) 1607 (65) 53 (63) 48 (57) 0.27 of Fc-fused murine IL-2 muteins (Fc.IL-2 mutein) to optimize Treg-selectivity, No smoking, n (%) 2103 (90) 87 (97) 84 (97) 0.01 tissue distribution and PK. Twenty-eight Fc.IL-2 muteins were screened for $25K-50K N=1590 N=96 N=134 their signaling potency and surface retention on Treg. Eight of the mutein candidates, showing a reduced signaling in Treg but prolonged association ORALS A1c <7%, n (%) 333 (21) 8 (8.4) 16 (12) <0.001 with CD25, were further characterized in vitro. Two of the weakest muteins SBP <140 mmHg, n (%) 1439 (91) 84 (88) 124 (93) 0.37 were tested in vivo and showed a greater enrichment and Ki-67 induction LDL <100 mg/dL, n (%) 828 (65) 43 (62) 56 (59) 0.49 in Treg compared to wild type (WT) IL-2, and less effect on CD4+ T effec- No smoking, n (%) 1049 (83) 76 (87) 101 (85) 0.52 tor cells, CD8+ cells and NK cells. Finally, in the non-obese diabetic mouse <$25K N=964 N=125 N=149 model of T1D, we observed a significantly improved protection in mice treated infrequently with Fc.IL-2 mutein compared to WT IL-2, and, unex- A1c <7%, n (%) 159 (17) 6 (5.1) 19 (13) 0.001 pectedly, differences in efficacy between WT and mutein were more pro- SBP <140 mmHg, n (%) 890 (93) 97 (81) 141 (96) <0.001 nounced at a very high dose level. Altogether, our data suggest that Fc.IL-2 LDL <100 mg/dL, n (%) 460 (65) 55 (60) 61 (53) 0.04 mutein treatment represent a safer and more efficient alternative for the WT No smoking, n (%) 565 (75) 93 (79) 123 (89) 0.001 IL-2 therapy, allowing a selective Treg enrichment and a targeted control of the autoimmune response. Supported By: Type 1 Diabetes Exchange 315‑OR 313‑OR Hybrid Insulin Peptides Form in Islets of Nondiabetes Prone Mice Ethnicity and Common Genetic Variants Modulate Nonalcoholic TIMOTHY WILES, KATHRYN M. HASKINS, THOMAS DELONG, Aurora, CO, Fatty Liver Disease (NAFLD) Metabolic Phenotype in Obese Youth Denver, CO DOMENICO TRICO, SONIA CAPRIO, GIUSEPPINA R. UMANO, ALFONSO Using a proteomic strategy, we recently identified a new class of post- GALDERISI, MARIANA M. MATA, JESSICA NOUWS, BRIDGET PIERPONT, GRACE translational protein modification that occurs in pancreatic beta cells. This KIM, NICOLA SANTORO, New Haven, CT, Seattle, WA modification is a result of insulin fragments that become covalently linked to Background: Pediatric Nonalcoholic Fatty Liver Disease (NAFLD) repre- other protein fragments that form in beta cells. The resulting hybrid insulin sents a major risk factor for insulin resistance and type 2 diabetes in ado- peptides (HIPs) contain non-germline encoded amino acid sequences and lescents. provide a plausible explanation on how tolerance to self-antigens may be

ADA-Supported Research

A84 TRANSLATIONAL IMMUNOLOGY IN TYPE 1 DIABETES—A LOOK AT THE FUTURE lost leading to the development of type 1 diabetes (T1D). We demonstrated mice showed significant delay in T1D development, less islet-infiltrating that autoreactive CD4 T cells, isolated from the residual islets of T1D organ autoreactive CD8 T cells, reduced high-avidity IGRP autoreactive CD8 T cells donors, target various HIPs. Additionally, diabetes triggering CD4 T cells from in the spleen and pancreatic lymph node, and less inflamed islets. Interest- non-obese diabetic (NOD) mice, a major animal model for T1D, were shown ingly, we detect significant increase in serum levels of sCD137. Furthermore, to be HIP reactive. Using mass spectrometric analyses on cell extracts of we could not detect differences in CD137+ Treg populations between NOD beta cell tumors, isolated from NOD-RIPTAg mice, we confirmed the pres- and Tnfsf9-/- mice. In bone marrow transfer experiments, CD137L deficiency ence of HIPs in those cells. Key questions that arise from these observations in either hosts or donors was able to suppress T1D development. Tnfsf9-/- are whether HIP formation is a process that takes place in human beta cells, and wild type T cells showed similar capacity to induce T1D in NOD.Rag1-/- and whether individuals that do not develop diabetes also form HIPs. BALB/c recipients. We are working on identifying the cellular source of increased mice do not develop spontaneous diabetes and here we analyzed pancreatic sCD137 and the mechanism behind this elevation. We direct our efforts to islets from these mice by mass spectrometry. We verified the presence of understand how CD137-CD137L interaction can modulate T1D pathogenesis various HIPs in those islets. Data indicate that the quantity of HIPs formed and how to translate the data from NOD mouse to understand the pathogen- in NOD islets is equivalent to the quantity formed in BALB/c islets. However, esis of human diabetes. additional quantification steps will be required to validate these observa- Supported By: American Diabetes Association (1-16-IBS-049 to Y-G.C.); National tions. It is difficult to obtain sufficient amounts of residual islets from T1D Institutes of Health organ donors that allow a detailed proteomic analysis, but our findings now set the stage to look at more accessible islets of non-T1D organ donors to 318‑OR verify the formation of HIPs in humans. CD19+IgM+ Cells from NOD Mice Demonstrate Enhanced Therapeu‑ Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-17- tic Efficacy in Type 1 Diabetes Mellitus IBS-290 to T.D.) ANDREW D. VONBERG, MASSIMO PIETROPAOLO, Houston, TX We describe a protective effect on autoimmune diabetes and a reduced 316‑OR destructive insulitis in NOD.scid recipients following splenocyte injections Thymic Development of Beta-Cell Specific Regulatory T Cells in from diabetic NOD donors and sorted CD19+ cells compared to NOD.scid Neonatal NOD Mice recipients receiving splenocytes alone. This protective effect was age-spe- YONG LIN, THOMAS LEE, MARIA BETTINI, MATTHEW BETTINI, Houston, TX cific (only CD19+ cells from young NOD donors exerted this effect; p < 0.001). Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that Furthermore, we found that CD19+IgM+ cells is the primary cell subpopula- involves the destruction of insulin producing β cells found within the pan- tion of B cells that delayed adoptive transfer of diabetes mediated by diabe- creatic islets of Langerhans. Recent attention to neonatal thymocyte devel- togenic T cells from NOD mice (p = 0.002). In fact, removal of IgM+ cells from opment has revealed a window during which central tolerance is established the CD19+ pool did not result in diabetes protection. Blockade of IL-10 with in an Autoimmune Regulator (Aire) dependent manner. However, expression neutralizing antibodies at the time of CD19+ cell co-transfers also abrogated of Chromogranin A (ChgA) within the thymus has not been detected; there- the therapeutic effect, suggesting that IL-10 secretion was an important fore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) component of the protective effect. These results were strengthened by ex presenting ChgA to developing thymocytes. The relative contribution of vivo incubation of CD19+ cells with IL-5, which resulted in enhanced IL-10 thymically and peripherally derived antigens in the generation of neonatal production and significantly delayed diabetes progression compared to con- β cell antigen specific CD4+ regulatory T cells (Tregs) is not well understood. trols (p = 0.0005). To the best of our knowledge, this is the first study demon- For example, post-translational modification (PTM) of ChgA by fusion with an strating that CD19+IgM+ cells is the primary cell subpopulation of regulatory insulin peptide increases epitope immunogenicity by forming neo-antigens. B cells delaying diabetes mediated by diabetogenic T cells from NOD mice. Pathogenic T cells that are specific for neo-antigens uniquely expressed The potential to expand CD19+IgM+ cells, especially in response to increased in the pancreas may escape thymic selection. Therefore, we utilized two stimulation using IL-5 or by pharmacologic agents, may be a new therapeutic experimental designs to test the role of peripheral antigen exposure on neo- option for type 1 diabetes. natal development of β cell specific Tregs. Firstly, we adoptively transferred dendritic cells pulsed with ChgA peptides into BDC2.5 TCR Tg mice and WT 319‑OR NOD mice to study the role of migratory DCs on neonatal development of Contrast Enhanced Ultrasound with Submicron-Sized Contrast ChgA Tregs. Secondly, we targeted thymic Langerin+ dendritic cells with Agents Detects Diabetes Progression in Mouse Models of Type 1 anti-Langerin linked to ChgA peptides. In both experimental approaches, we Diabetes found a robust increase in the ratio and number of ChgA specific Tregs in the DAVID RAMIREZ, RICHARD K. BENNINGER, Denver, CO, Aurora, CO presence of the hybrid insulin/chromogranin A peptide (2.5HIP) while also In type 1 diabetes (T1D) the decline in beta-cell mass occurs many years observing an increase in negative selection. The expansion of thymic Tregs prior to clinical presentation. A non-invasive method to diagnose insulitis was observed in both adult and neonatal mice. Together our data suggests and beta-cell mass decline prior to diabetes does not exist. During T1D pro- neonatal exposure to PTM peptides enhances thymic development of β cell gression, the islet microvasculature increases permeability due to insulitis. specific Tregs and may alter T1D pathogenesis. Iron oxide nanoparticle MRI contrast accumulation has previously been Supported By: American Diabetes Association (1-17-JDF-013 to M.B.); Robert

suggested as a measure of ongoing insulitis. Contrast enhanced ultrasound ORALS and Janice McNair Foundation (CEUS) using gas-filled microbubbles (MBs) measures acoustic backscatter at sub-harmonic frequencies and is clinically approved. Sub-micron sized 317‑OR ‘nanobubbles’ (NBs) have previously been developed and show extravasa- Investigating the Role of CD137 Ligand in the Pathogenesis of tion in tumors. Here we tested whether these sub-micron sized NBs show Type 1 Diabetes disease-mediated extravasation in mouse models of T1D. We performed BARDEES FODA, MATTHEW H. FORSBERG, ASHLEY E. CIECKO, KEVIN W. MUEL- CEUS measurements following NB infusion in NOD mice and control LER, ARON GEURTS, YI-GUANG CHEN, Milwaukee, WI, Madison, WI (NOD;Rag1ko and C57Bl6) mice. We also examined fluorescent coverage Pathogenesis of type 1 diabetes (T1D) involves various interactions (FC) following rhodamine-labeled bubble delivery in these animals, through between genetic and environmental factors. The gene encoding the co-stim- isolating pancreata for histology and quantifying FC and insulitis scores. ulatory molecule CD137 is located within the Idd9.3 T1D susceptibility locus We observed progressive increased CEUS signal accumulation in pancreas and contributes to diabetes progression in NOD mice. We have previously of 4w and 10w NOD mice, which was absent in NOD kidneys (p < 0.001). shown that CD137 expression in T cells has dual functions: CD4+CD137+ Rag1ko controls showed minimal CEUS signal in pancreas and kidney. The T cells negatively regulate T1D development while CD8+CD137+ T cells FC was limited to the pancreata of NOD mice and was substantially higher showed potent diabetogenecity. The protective function of CD137 in CD4+ in endocrine compared to exocrine tissue (p = 0.002). NOD mouse islets also T cells is likely due to the significant amounts of soluble CD137 (CD137) had substantially more FC compared to Rag1ko (p < 0.0001), and showed a produced by Foxp3+ Tregs. The interaction between CD137 and its ligand correlation between NOD insulitis score and FC (p=0.043). Thus, CEUS with (CD137L) induces two signaling pathways, forward one driven by CD137 and NB contrast agents can detect disease progression prior to T1D onset, which the reverse signaling mediated by CD137L, both of which modulate T cell will be important for early T1D diagnosis and monitoring of disease preven- function. Here, we study the impact of CD137L deficiency on T1D to gain fur- tion or reversal. ther insight into disease pathogenesis. We successfully generated a mouse strain with the NOD background and knockout of the gene encoding CD137L (Tnfsf9) using CRISPR/Cas9 technology. Relative to wild type NOD, Tnfsf9-/-

ADA-Supported Research

A85 REGULATION OF ISLET FUNCTION IN HEALTH AND DISEASE

320‑OR We hypothesized that excessive GR activation is deleterious to beta-cells CD70 Modulates Thymocyte Development and Type 1 Diabetes in such that genetic GR depletion would preserve beta-cell mass and function NOD Mice in response to metabolic stress. To address this hypothesis, we generated CHENG YE, JOHN DRIVER, MICHAEL V. WILES, DAVE SERREZE, Gainesville, FL, beta-cell specific GR knockout (KO) mice, using an inducible Pdx1-CreERT2- Bar Harbor, ME loxP system. We then fed beta-cell GR-KO and WT littermates both regular CD70 is a tumor necrosis factor (TNF) superfamily member expressed by chow and high-fat diet for up to 30 weeks, followed by determination of activated T cells, B cells, dendritic cells, NK cells and mature macrophages. It beta-cell mass and function. We found that GR-deficient mice, as opposed interacts with CD27, a TNF receptor family member on T cells that regulates to WT littermates, developed high blood glucose levels and glucose intoler- T cell activation, proliferation and survival. Type 1 diabetes (T1D) is caused ance. This effect was not secondary to alterations in weight gain or insulin by T cell mediated beta cell destruction, however, T1D-prone NOD mice sensitivity. Instead, we detected a significant reduction in glucose-stimu- developed accelerated disease in the absence of CD70. This was associated lated insulin secretion, correlating with abnormal glucose metabolism in with altered proportions of double negative vs. double positive thymocytes GR-KO mice. We reproduced these findings in both male and female GR-KO in CD70 knockout mice, indicating that CD70 modulates global thymopoiesis. mice. Anti-insulin immunohistochemistry revealed that GR-KO mice had sig- Also affected, but in opposing directions, was the development of regula- nificantly reduced beta-cell mass, and decreased beta-cell replication, as tory T cells (Treg) and natural killer T (NKT) cells, two immunoregulatory visualized by in vivo BrdU-labeling assay. GR deficiency appears to impair lymphocyte subsets that are functionally and numerically deficient in NOD the ability of beta-cells to compensate for fat-elicited obesity and insulin mice. Tregs frequencies were reduced by 23% and 32% in CD70 heterozy- resistance, resulting in prediabetes in GR-KO mice. These results unveiled gous (CD70-/+) and homozygous (CD70-/-) knockout mice. In contrast, NKT cell an unexpected role of the glucocorticoid-GR signaling in safeguarding beta- frequencies were respectively increased by 48% and 61% in CD70-/+ and cell mass and function in response to metabolic stress. CD70-/- mice compared to standard NOD mice. The higher level of NKT cells Supported By: National Institutes of Health in CD70 knockout mice was unexpectedly associated with reduced levels of thymic CD1d, a molecule that is required for NKT cell development. Col- 323‑OR lectively, our results show that CD70 controls the development of several T Dapagliflozin Treatment Impactsβ , but Not α, Cell Function in Nor‑ cell populations that are known to modulate T1D development in NOD mice mal Human Islets and that, in the absence of CD70, T1D is exacerbated. These finding may CHUNHUA DAI, ALENA SHOSTAK, YASIR H. BOUCHI, NATHANIEL HART, GREG facilitate the development of clinical therapeutics aimed at enhancing the POFFENBERGER, DIANE C. SAUNDERS, RACHANA HALIYUR, DANIELLE DEAN, stimulatory function of CD70 as a way to suppress disease. RADHIKA ARAMANDLA, MARCELA BRISSOVA, MASAKAZU SHIOTA, DALE L. GREINER, LEONARD D. SHULTZ, RITA BOTTINO, ALVIN C. POWERS, Nashville, TN, 321‑OR Augusta, GA, Worcester, MA, Bar Harbor, ME, Pittsburgh, PA Stem Cell-Derived Tissue-Associated Regulatory T Cells Suppress Dapagliflozin (DAPA), a selective Sodium Glucose Cotransporter-2 (SGLT2) Autoimmune Diabetes inhibitor, is widely used for the treatment of type 2 diabetes (T2D). In T2D JIANXUN J. SONG, College Station, TX individuals, DAPA increases plasma glucagon, decreases insulin secretion, Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source and improves muscle insulin sensitivity. DAPA has been reported to increase of healthy regulatory T cells (Tregs) to treat autoimmune diabetes as they glucagon secretion in cultured normal human islets possibly because SGLT2 have the ability to create nearly entire cell types in the body, including Tregs. was more highly expressed in α cells. To investigate DAPA effect on human However, the right conditions for the development of auto antigen (Ag)-spe- islets in vivo, we transplanted normal human islets (n=3 donors) into immu- cific Tregs from PSCs (i.e., PSC-Tregs) has not been fully defined. Auto Ag- nodeficient mice and treated them with DAPA (2mg/kg/day) or NaCl by oral specific PSC-Tregs can be programmed to be tissue-associated and infiltrate gavage for 4 weeks. Basal blood glucose (NaCl vs. DAPA: 101±8 vs. 85±5 to local inflamed tissues (e.g., islets) to suppress autoimmune responses mg/dL, p=0.0858), glucagon (199.5±24.4 vs. 169.9±15.9 pg/mL, p=0.3107), after adoptive transfer, thus preventing possible overall immunosuppression and human insulin (1.15±0.17 vs. 0.99±0.14 ng/mL, p=0.4626) levels were from non-specific Tregs. In this study, a new approach was developed to gen- similar in both groups. However, 15 minutes after glucose challenge, DAPA- erate functional auto Ag-specific Tregs from induced PSC (iPSCs), i.e., iPSC- treated mice had lower blood glucose (375±21 vs. 287±16 mg/dL, p=0.003), Tregs, which have the ability to suppress autoimmunity in a murine model unchanged glucagon (86±14 vs. 75±8 pg/mL, p=0.4883), but lower human of autoimmune diabetes in auto Ag-associated fashion. Murine iPSCs were insulin levels (2.05±0.24 vs. 0.96±0.08 ng/mL, p<0.0001). The ratio of human retrovirally transduced with a DsRed reporter construct containing genes of insulin/blood glucose was lower in the DAPA group (0.52±0.07 vs. 0.36±0.04, ovalbumin (OVA)-specific T cell receptor (TCR) and the transcriptional fac- p=0.0322). Ultrastructure of transplanted human islets showed that the tor FoxP3. The DsRed+ iPSCs were in vitro differentiated into OVA-specific DAPA treatment decreased the % immature granules (52±2% vs. 43±2%, iPSC-Tregs with an OP9 stromal cell line in the presence of recombinant p=0.0109) but increased the % empty granules (5.7±0.6% vs. 13.7±0.7%, cytokines. A double transgenic (Tg) mouse model of autoimmune diabetes p<0.0001) in β cells. To investigate if these changes were a consequence of was established in F1 mice in which the first generation from RIP-OVA Tg improved insulin sensitivity or DAPA’s direct effect on β cells, we assessed mice that were crossed with OT-I T cell receptor (TCR) Tg mice was chal- insulin secretion by culturing human islets (n=3 donors) in 3.3 mM and 16.7 lenged with vaccinia viruses expressing OVA (VV-OVA). Adoptive transfer mM glucose with DAPA (0.5 µM) or NaCl for 1 hour. DAPA treatment had no ORALS of OVA-specific iPSC-Tregs dramatically suppressed autoimmunity in the effect on glucagon secretion at both glucose concentrations but reduced OVA-induced model of autoimmune diabetes, including the inflammation insulin secretion by 30% at 16.7 mM glucose (p=0.0259). By transcriptional and prevents the insulin-secreting pancreatic beta cells from destruction. Of profiling SGLT2 was expressed equally inα and β cells. These data sug- note, we demonstrated that the adoptive transfer significantly reduced the gest that DAPA does not directly affect glucagon secretion but has a direct higher ratio of CD8+ to CD4+ T cells in diabetic mice. These results indicate impact on human β cell function. that stem cells can be used to develop auto Ag-specific Tregs, which have a Supported By: National Institutes of Health; JDRF; U.S. Department of Veterans therapeutic potential for autoimmune diabetes. Affairs Supported By: National Institutes of Health (R01AI121180, R21AI128325) 324‑OR Foxo1-Expressing Cells in the Gut as a Source of Insulin for Diabe‑ REGULATION OF ISLET FUNCTION IN HEALTH AND tes Treatment DISEASE WENDY MCKIMPSON, TAIYI KUO, DOMENICO ACCILI, New York, NY The hallmark of type 1 diabetes is the irreversible destruction of insulin- 322‑OR secreting β-cells in the pancreas. Consequently, an attractive therapeutic Glucocorticoid Receptor Is Instrumental for the Mechanism of approach is to trigger the regeneration of functional β-cells elsewhere Beta-Cell Compensation for Insulin Resistance in Dietary Obesity within a diseased individual. We have previously demonstrated that removal JUN YAMAUCHI, SOJIN LEE, HENRY DONG, Pittsburgh, PA of Foxo1 in Neurogenin3 (Ngn3)-expressing cells creates functional insulin- The glucocorticoid receptor (GR) is known as a receptor of steroids, potent secreting cells within the intestine. The cells, however, only convert into anti-inflammatory ligands that are commonly used for the medication of insulin+ cells at a low frequency, limiting their therapeutic potential. Using allergy or asthma in children and adults. However, chronic use of steroids is a novel knock-in reporter mouse that encodes a Foxo1-Venus fusion protein associated with a significant risk of developing visceral adiposity and insu- (FoxV), we have identified a novel population of Foxo1-expressing cells in lin resistance, a medical condition that is known as the Cushing syndrome. the glandular epithelium of the stomach. These cells are distinct from other

ADA-Supported Research

A86 REGULATION OF ISLET FUNCTION IN HEALTH AND DISEASE gut FoxV+ cells in that, unlike Foxo1+ cells in the intestine, stomach FoxV+ forskolin, GLP-1, glucagon and GIP in mouse islets. The insulinotropic effect of cells rarely express serotonin. To identify the nature of Foxo1+ stomach cells, DHT is abolished using EPAC and PKA inhibitors as well as rapamycin indicat- we next isolated single cells from stomach and, using flow cytometry and ing that DHT amplifies the effect of GPCRs coupled to adenylate cyclase and qPCR, analyzed the gene expression of FoxV+ cells. Although Foxo1-positive stimulates GSIS via a cAMP/PKA/EPAC pathway and activation of mTOR. cells do not express markers of chief, pit, or neck cells, they have increased Supported By: National Institutes of Health (DK107444); U.S. Department of transcript levels of the parietal cell marker H+/K+ ATPase. We further Veterans Affairs (BX003725) validated these results by immunofluorescence and FACS. To determine if elimination of Foxo1 can convert stomach cells to insulin-producing cells, 327‑OR we next ablated Foxo1 in either Ngn3+ or parietal cells in mice using cre- Beta-Cell Glucotoxicity in KATP-Induced Diabetes Is Alleviated by mediated recombination. Strikingly, we detected cells positive for insulin Genetic Reduction of Glucose Metabolism and C-peptide in the stomach of mice harboring Foxo1 deletion. Similarly, MANUELA FORTUNATO, ZIHAN YAN, ZEENAT A. SHYR, HANNAH E. CONWAY, when we inactivated Foxo1 in primary stomach cultures, we detected mark- MARIA S. REMEDI, St. Louis, MO edly increased “β-like” gene expression and insulin+, C-peptide+ cells. Taken Glucotoxicity, reduced β-cell function and mass are key events in the together we have identified a new population of Foxo1-expressing cells in pathogenesis and progression of diabetes. We previously generated an the stomach and demonstrated that Foxo1 ablation is sufficient to convert insulin secretory-deficient mouse model of human K -induced diabetes. these cells to insulin-producing cells. ATP As predicted, KATP-GOF (gain-of-function) mice demonstrate severe diabetes Supported By: National Institutes of Health (T32HL007343-38) due to low circulating insulin levels, reiterating the human disease. However, as diabetes progresses, KATP-GOF mice show reduction of insulin content 325‑OR and β-cell mass, consequences of glucotoxicity. Islets from diabetic mice Maintenance of β-Cell Heterogeneity Is Required for Normal Islet also show increased glucose metabolism (NAD(P)H autofluorescence) and Function oxygen consumption rate (Seahorse analysis), and augmented mitochondrial DANIELA NASTESKA, GUY A. RUTTER, QIAO ZHOU, DAVID HODSON, Birming- membrane potential. Strikingly, they also demonstrate increased glucoki- ham, United Kingdom, London, United Kingdom, Cambridge, MA, Edgbaston, United nase (GK, first enzyme in glucose metabolism) and GLUT2 gene transcripts Kingdom and proteins. We hypothesized that augmented glucose metabolism in dia- Aim: Recent studies have shown the existence of discrete β-cell subpopu- betic mice will contribute to the glucotoxic reduction in in insulin content, lations with differing levels of maturity and function. We sought here to β-cell function and mass; and that these consequences will be alleviated understand how this diversity may influence islet function and insulin secre- by reducing glucose metabolism. To test this in vivo, we crossed KATP-GOF tion using a ‘loss of heterogeneity‘ model. mice with mildly glucose-intolerant heterozygous GK+/- knockout mice (with +/- Material and Methods: Overexpression was achieved using an adenoviral reduced glucose metabolism), to generate double-transgenic GK /KATP-GOF polycistronic construct for Pdx1, MafA, Ngn3 and mCherry (Ad3-NPM). Pdx1 mice. Progression of diabetes, assessed by blood glucose levels overtime, is +/- was silenced using short hairpin RNAs (shRNA). Gene and protein levels slower in GK /KATP-GOF mice, compared with KATP-GOF mice. Importantly 2+ +/- were detected by qPCR and immunohistochemistry (IHC), respectively. Ca also, glucose tolerance is enhanced in GK /KATP-GOF mice 12 days after dis- fluxes, ATP/ADP ratios and cAMP levels were analysed using dyes/biosen- ease onset, with respect to KATP-GOF mice, without significant changes in sors and high-speed spinning disk microscopy. Glucose- (GSIS) and incretin- insulin sensitivity. Insulin gene expression, insulin content and β-cell mass +/- stimulated insulin secretion (ISIS) were measured using HTRF assays. are partially preserved in GK /KATP-GOF mice. Together, these results dem- Results: Ad3-NPM increased Pdx1 (12-fold vs. CT; P<0.01) and MafA onstrate that hyperglycemia induce accelerated metabolism in KATP-GOF levels (2-fold vs. CT) without affecting Ngn3. IHC showed that Pdx1 over- mice damaging the islets, and that deceleration of metabolism by reducing low expression occurred predominantly in Pdx1 β-cells, forcing higher unifor- GK activity delays/prevent hypermetabolism induced-glucotoxicity in KATP- mity throughout the β-cell complement. The α/β-cell ratio did not change, GOF mice. indicating absence of transdifferentiation. Ca2+ responses to glucose were Supported By: National Institutes of Health (R01DK098584 to M.S.R.) markedly blunted (ΔF=0.81 vs. 0.44 AU, CT vs. Ad3-NPM; P<0.01), and this was accompanied by a decrease in β-cell-β-cell cell connectivity and hub 328‑OR number (12.6 vs. 5.6% hubs, CT vs. Ad3-NPM; P<0.05), the latter previously DAG Accumulation due to Type 1 DGK Inhibition Has Contradictory shown to be relatively immature cells with pacemaker features. ATP/ADP Dual Effect on Ca2+ Signaling in Pancreatic β Cells ratios and cAMP levels were unaffected. Glucose- and incretin-stimulated TOSHIAKI SAWATANI, YUKIKO K. KANEKO, TOMOHISA ISHIKAWA, Shizuoka, insulin secretion was impaired (% content secreted = 8.7 vs. 5.2 for GSIS Japan and 56.4 vs. 29.02 for ISIS, CT vs. Ad3-NPM; both P<0.01). Similar results for Pathophysiologically, excessive accumulation of diacylglycerol (DAG), a 2+ Ca dynamics, cellular connectivity and insulin secretion were obtained in lipid signal messenger, in pancreatic -cells has been shown to lead to the low β islets treated with Pdx1 shRNA to increase the size of the Pdx1 population. insufficiency of insulin secretion. Thus, the regulation of intracellular DAG Conclusion: Loss of β-cell heterogeneity, whether due to increased or levels is expected to be an important factor in maintaining β-cell function. decreased maturity, leads to islet failure and impaired insulin secretion. The intracellular DAG levels are strictly controlled by DAG kinases (DGKs). Thus, cellular diversity appears necessary for normal islet function.

We have previously shown that DGKα and γ, type 1 DGK isoforms, are highly ORALS Supported By: UK Medical Research Council; European Research Council expressed in mouse β-cells, and that DAG accumulation resulting from dysfunction of these DGKs reduces insulin secretion. In the present study, 326‑OR we investigated the effect of type 1 DGK dysfunction on the regulation of 2+ 2+ Molecular Mechanism of Androgen Receptor Stimulation of Insulin intracellular Ca concentration ([Ca ]i), which is correlated with insulin Secretion in Male β Cells secretion, in the β-cell line MIN6B. We also analyzed changes in expres- WEIWEI XU, FIONA B. ASHFORD, DAVID HODSON, FRANCK MAUVAIS-JARVIS, sion levels of DGKα and γ in β-cells of type 2 diabetes model NSY mice. 2+ New Orleans, LA, Birmingham, United Kingdom, Edgbaston, United Kingdom The amplitude of glucose-induced [Ca ]i oscillations was increased by 1 µM Androgen deprivation therapy increases the risk of type 2 diabetes mellitus R59949, a type 1 DGK inhibitor, and by 10 µM DiC8, a DAG analog. These (T2DM) in men. We previously showed that male pancreatic β-cell specific effects were abolished by Ro31-8220, a protein kinase C (PKC) inhibitor. In androgen receptor knockout (βARKOMIP) mice develop glucose intolerance contrast, 10 µM R59949 and 100 µM DiC8 suppressed the glucose-induced 2+ because AR potentiates glucose-stimulated insulin secretion (GSIS) through [Ca ]i elevation, which was insensitive to Ro31-8220, and furthermore increasing cyclic AMP (cAMP) production and amplifies the insulinotropic reduced voltage-dependent Ca2+ channel (VDCC) currents, which were mea- effect of glucagon-like peptide-1 (GLP-1). We show that male βARKOMIP mice sured by patch-clamp recording, implying that DAG accumulation due to 2+ exhibit impaired intraperitoneal (IP) glucose tolerance- because of impaired type 1 DGK dysfunction suppresses VDCC, thereby reducing [Ca ]i in β-cells. IP-GSIS- without alteration in oral glucose tolerance, suggesting that AR Real-time qPCR showed that the expression level of DGKα and γ was lower amplifies the islet-derived, but not the gut-derived GLP-1 to potentiate GSIS. in islets from NSY mice compared with control mice. Taken together, the Using male insulin-secreting β-cell line 832/3 transduced with exchange fac- results obtained here suggest that DAG accumulation due to type 1 DGK tor directly activated by a cAMP (EPAC)-based fluorescence resonance energy dysfunction has contradictory dual effect in β-cells depending on the degree transfer (FRET) sensor, we observe that AR agonist dihydrotestosterone (DHT) of accumulation; mild accumulation PKC-dependently induces a stimulatory allows GLP-1, glucagon and gastric inhibitory polypeptide (GIP) to increase effect on insulin secretion, whereas excessive accumulation suppresses it cAMP production above level of the individual hormones and to a similar level independently of PKC. DGK is likely a key molecule for the progression of to forskolin control. Accordingly, DHT increases the insulinotropic effect of type 2 diabetes.

ADA-Supported Research

A87 NEW INSIGHTS INTO LIPID AND LIPOPROTEIN ALTERATIONS IN PATIENTS WITH DIABETES

329‑OR Figure. Adiponectin Increases Glucose-Dependent Electrical Activity and Insulin Secretion in Mouse Pancreatic β Cells JOANA I. REAL, BELEN CHANCLON GARCIA, INGRID WERNSTEDT ASTERHOLM, PATRIK RORSMAN, Gothenburg, Sweden, Oxford, United Kingdom Decreased plasma adiponectin has been implicated as a cause of diabetes. However, the functional impact of adiponectin on glucose-dependent responses of β-cells remains poorly understood. Here we have investigated whether chronically and acutely increased adiponectin levels affect electrical activity (sharp electrodes) and insulin secretion in β-cells using respectively, adiponectin overexpressing (APNtg) mice and acute application of adiponectin. In the acute exposure experiments, adiponectin (20 µg/ml, 11 mM glucose) increased insulin secretion 42% and β-cell electrical activity (mea- Supported By: The Medicines Company sured as the fraction ‘active phase’) 32%. In islets from APNtg mice insulin secretion increased 44% and active phase increased 25%. The effects on 331‑OR electrical activity in APNtg β-cells correlated with membrane hyperpolariza- Angiopoietin-Like Protein 3 Directly Regulates HDL Metabolism tion and the resting membrane potential at 0 mM glucose averaged -60±2 in and Function control and -69±2 in APNtg β-cells. There was a moderate effect on action LONGYAN YANG, YAN WANG, YING FU, JIANAN LANG, DONG ZHAO, YINGMEI potential width (<20%) and a more pronounced impact on action potential FENG, Beijing, China frequency by exogenous adiponectin (+35%) and in APNtg β-cells (+50%). Background: Angiopoietin-like protein 3 (ANGPTL3) is produced by hepa- These results suggest that both acutely and chronically increased adiponec- tocytes. Once secreted, it inhibits endothelial lipase activity and regulates tin levels have a positive effect on electrical activity and insulin secretion angiogenesis. We previously reported the positive association between of pancreatic β-cells. serum ANGPTL3 and HDL function in nondiabetic participants. Hereby, we Supported By: Swedish Research Council; Wilhelm and Martina Lundgren investigated whether ANGPTL3 directly modulates HDL function. Foundation; Sigurd and Elsa Goljes Memory Foundation; Adlerbert Research Methods: HDLs were isolated from plasma. ANGPTL3 in HDLs was stud- Foundation ied by western blot and ELISA. Cholesterol efflux to HDLs was analyzed in macrophages preloaded with fluorescent cholesterol. Recombinant human ANGPTL3 was subjected to endothelial cells treated with TNF-α and ICAM-1 NEW INSIGHTS INTO LIPID AND LIPOPROTEIN expression was analyzed by FACS. Similar experiments were repeated in db ALTERATIONS IN PATIENTS WITH DIABETES mice injected with AAV encoding human ANGPTL3 cDNA or shRNA. Results: ANGPTL3 was present in human HDLs, which were lower in T2DM 330‑OR patients than controls (p=0.04). ANGPLT3 in HDLs was positively associ- Inclisiran Lowers LDL-C and PCSK9 Irrespective of Diabetes Status ated with cholesterol efflux in nondiabetic controls (r=0.33, p=0.044) but not without Worsening Glycemia in T2DM patients (r=0.05, p=0.77). Recombinant ANGPTL3 reduced ICAM-1 LAWRENCE LEITER, HWEE TEOH, DAVID KALLEND, R. SCOTT WRIGHT, ULF expression in endothelial cells exposed to TNF-α (p=0.0001). Similarly, ANG- LANDMESSER, PETER L. WIJNGAARD, JOHN J. KASTELEIN, KAUSIK K. RAY, PTL3 levels in HDLs were lower in db mice at 18 weeks old than 8 weeks old, Toronto, ON, Canada, Zürich, Switzerland, Rochester, MN, Berlin, Germany, Parsip- which was accompanied with reduced cholesterol reflux to HDLs and rendered pany, NJ, Amsterdam, Netherlands, London, United Kingdom inhibition of ICAM-1 expression in endothelial cells (p<0.05 for all). Follow- Inclisiran, an investigational PCSK9-specific RNA silencing molecule with ing AAV-mediated ANGPTL3 cDNA transfer, ANGTL3 levels were increased potential for a maintenance regimen of twice yearly dosing, significantly in HDLs of db mice, which increased cholesterol efflux and decreased ICAM-1 lowered LDL-C and PCSK9 in the dose-ranging ORION-1 trial. We report its expression compared with db mice with vector transfer. Vice versa, decreased efficacy and safety by diabetes status, and its impact on glycemia. ORION-1 ANGPTL3 levels in HDLs by AAV-mediated shRNA transfer abrogated HDL randomized 501 persons with atherosclerosis (ASCVD) or ASCVD-risk function mentioned above (p<0.05 for both). When placed on high fat diet for equivalents, and high LDL-C despite maximally tolerated LDL-C lowering 10 weeks, atherosclerotic plaque was larger in db mice with ANGPTL3 shRNA therapies, to inclisiran or placebo. Inclisiran significantly lowered LDL-C and transfer than other gene transferred groups (p<0.05). PCSK9 similarly in persons with and without diabetes at day 180 (Table). Conclusion: ANGPTL3 is a HDL component and direct regulates HDL func- Temporal A1C were similar in the placebo and incisiran arms (Figure). These tion. Decrease of ANGPTL3 expression may reduce HDL function and accel- data suggest that inclisiran is efficacious and does not worsen glycemia in erate atherosclerosis in T2DM. persons with ASCVD or ASCVD-risk equivalents, regardless of their diabetes Supported By: National Natural Science Foundation of China (81470566, status, over 180 days. 81670765); Capital Medical University Science Funding in China (PYZ2017049)

ORALS 332‑OR Prevalence and Predictors of Residual Hypertriglyceridemia According to Statin Use in U.S. Adults with Diabetes WENJUN FAN, SEPHY PHILIP, CRAIG B. GRANOWITZ, PETER P. TOTH, NATHAN D. WONG, Irvine, CA, Bedminster, NJ, Sterling, IL Background: Hypertriglyceridemia (HTG) is associated with increased cardiovascular risk and is common in diabetes (DM); however, statin therapy remains the primary treatment in most with moderate HTG. We examined the extent of residual HTG despite statin treatment and in those with well- controlled LDL-C in adults with DM. Methods: We studied 1,448 subjects with DM projected to 24.4 million (11.1% of U.S. adults) in the U.S. National Health and Nutrition Examination Surveys 2007-2014 who had available morning fasting triglyceride (TG) levels. We compared the distribution of TG levels by statin use. Results: The Table shows in DM overall and by statin use the proportions with TG <150 mg/dl, 150-199 mg/dl, 200-499 mg/dl, and >500 mg/ dl; nearly 40% despite statin use had TG >150 mg/dl. In addition, despite an LDL-C<100 mg/dl (<70 mg/dl in those with atherosclerotic cardiovascular disease), TG>150 mg/dl and TG>200 mg/dl were prevalent in 35.7% and 15.0% of statin users, respectively, and 36.2% and 17.4% of non-statin users, respectively. Conclusions: Residual HTG despite statin use remains common in more than a third (approximately 10 million) of U.S. adults with DM, even among statin

ADA-Supported Research

A88 NEW INSIGHTS INTO LIPID AND LIPOPROTEIN ALTERATIONS IN PATIENTS WITH DIABETES users with well-controlled LDL-C levels. Greater efforts are needed to promote terol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual adherence to lifestyle measures, as well as use of pharmacologic therapies immunohistochemistry in combination with morphometric analysis revealed where indicated, to address the risks associated with residual HTG. that ApoC3-transgenic and wild type littermates had similar beta-cell and Table. Estimated Proportions (%) of Adults within Different Levels of Fasting alpha-cell masses as well as islet size and architecture. These effects cor- Triglycerides among Those With Diabetes With and Without Statin Use, related with similar amplitudes of glucose-stimulated insulin secretion and NHANES 2007-2014 (Sample n and Weighted n Indicated in Parenthesis). similar degrees of postprandial glucose excursion in ApoC3-transgenic U.S. Adults with Diabetes vs. wild type littermates. ApoC3-transgenic mice did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and All On Statin Not on Statin cholesterol depositions in the pancreata. ApoC3-transgenic mice, despite Trigly<150mg/dL 867 (14.1M) 464 (7.5M, 60.5%) 403 (6.6M, 54.6%) persistent hypertriglyceridemia, maintained euglycemia under both fed and (n=7070, 163.0M) fasting conditions without manifestation of insulin resistance and fasting 150<=Trigly<200mg/dL 255 (4.9M) 131 (2.5M, 20.0%) 124 (2.4M, 20.1%) hyperinsulinemia. We concluded that hypertriglyceridemia per se is not an (n=1287, 29.5M) independent risk factor for beta-cell dysfunction. 200<=Trigly<500mg/dL 291 (4.8M) 124 (2.2M, 18.1%) 167 (2.6M, 21.7%) Supported By: National Institutes of Health (n=1141, 25.3M) Trigly>=500mg/dL 35 (0.6M) 11 (0.2M, 1.4%) 24 (0.4M, 3.6%) 335‑OR (n=95, 2.1M) Unaltered Fatty Acid Uptake, Utilization, and Mitochondrial Respi‑ Total 1448 (24.4M) 730 (12.4M) 718 (12.0M) ration in Right Atrial Appendage of Type 2 Diabetic Human Heart (n=9593, 219.9M) NANDINI RJ, RAJI SR, VIVEK V. PILLAI, JAYAKUMAR K., SRINIVAS GOPALA, p=0.0686 comparing distribution of TG levels in those on vs. not on statins. Thiruvananthapuram, India Previous studies on diabetic human heart have shown decreased uti- Supported By: Amarin Pharma Inc. lization of fatty acid (FA)/carbohydrate substrates. We hypothesized that increased mitochondrial dysfunction with concomitant decrease in fatty 333‑OR acid utilization would be found in diabetic hearts of patients from the Indian Circulating ANGPTL8 and ANGPTL4 Levels Are Increased in subcontinent that would explain their increased propensity of cardiovas- Patients with Hyperglycemia Combined Dyslipidemia cular complications. We have compared mitochondrial respiration in atrial XUEFENG YU, PUHAN LU, XI CHEN, Wuhan, China appendage tissue from nondiabetic and diabetic human subjects undergoing Context: Angiopoietin-like protein 8 (ANGPTL8) and Angiopoietin-like elective coronary artery bypass graft (CABG) surgery using high-resolution protein 4 (ANGPTL4) are regarded as protein regulating triglyceride (TG) respirometry with FA and carbohydrate substrate combinations. In the pres- metabolism in mice. However, whether ANGPTL8/4 are involved in glucose ence of FA, diabetic mitochondria showed unimpaired complex I and electron and/or lipid metabolism in human remain unclear. transferring flavoprotein (ETF) mediated respiration while significantly lower Objective: To examine circulating ANGPTL8/4 levels in subjects with oxygen consumption was noted when succinate was added, indicating the hyperglycemia and/or dyslipidemia and their correlation with glucose and possible derangement of complex II respiration. Unlike other human studies, lipid metabolism. Design, Setting and Participants: Serum ANGPTL8/4 levels we report no significant reduction in FA-mediated mitochondrial respira- were measured using ELISA in age-, sex-, and BMI- matched healthy control tion. However, the carbohydrate protocol indicated impairment of complex subjects (CON) (n=92), subjects with isolated hyperglycemia (IHG) (n=87), I mediated respiration. There was no significant change in the expression with isolated dyslipidemia (IDL)(n=86) and hyperglycemia combined dyslip- of OXPHOS proteins. Unchanged levels of FA uptake proteins like CD36 idemia (HCD)(n=99) from the Risk Evaluation of cAncers in Chinese diabeTic and FABP, FA oxidation enzyme, long chain acyl CoA deydrogenase (LCAD), Individuals: a lONgitudinal (REACTION) study. regulators of FA metabolism like sirtuins and nuclear receptors, PPARα Results: Serum ANGPTL8 and ANGPTL4 levels were both elevated in and transcription coactivators, PGC1α/β were observed in diabetic heart. patients with HCD compared to CON subjects. After controlling for age, sex The acetylation status of mitochondrial specific proteins involved in fatty and blood lipid, ANGPTL8 levels positively correlated with fasting plasma acid oxidation (acetylated LCAD and PGC1α/β) also showed no significant glucose (FPG) (partial r = 0.154), HbA1c (partial r = 0.110) and triglycerides and changes. Diabetic patients showed equal expression of the antioxidant glucose (TyG) index (age, sex adjusted)(partial r = 0.224). After controlling for enzymes and other markers of oxidative stress. The unimpaired FA uptake, age, sex and FPG, ANGPTL8 levels positively correlated with TG (r = 0.163), oxidation and mitochondrial respiration indicates absence of overt mito- TG/HDL (r = 0.147) and non-HDL/HDL (r = 0.134). ANGPTL4 were positively chondrial dysfunction in type 2 diabetic human heart. correlated with FPG, 2h-glucose, HbA1c and TyG index. Among lipid-related Supported By: Government of India Science and Engineering Research Board variables, ANGPTL4 were positively correlated with TG, TG/HDL, non-HDL/ (SB/SO/HS-051/2013 to S.G.); Government of India Council of Scientific and Indus- HDL and negatively correlated with HDL-cholesterol. After controlling for trial Research; Government of India Department of Science and Technology related variables, the correlations were the same. Conclusion: Circulating ANGPTL8 and ANGPTL4 levels were increased in 336‑OR patients with HCD and might play an important role in linking glucose and

Effect of a New Type of Nanomaterial on Glucose and Lipid Metabolism ORALS lipid metabolism. JIN WU, YINGBO CHEN, LIPING LIU, QING YAO, XIAOCEN LIU, YINGJIE WU, Supported By: National Natural Science Foundation of China (81570740); Minis- Dalian, China, Jinan, China try of Science and Technology of the People’s Republic of China (2016YFC0901200, Diabetes mellitus is a systemic metabolic disease characterized by dis- 2016YFC0901203); Tongji Medical College rupted glucose and lipid metabolism. With an alarming rise of incidence each year, there is therefore a pressing need to find more efficient medi- 334‑OR cines and methods for its treatment. Using the obese type 2 diabetes db/ Hypertriglyceridemia per se Is Not an Independent Risk Factor for db mouse model, in our pervious work we found that a new type of nano- Beta-Cell Dysfunction material could inhibit the formation of insulin resistance. The underlying JUN YAMAUCHI, TING ZHANG, SOJIN LEE, HENRY DONG, Pittsburgh, PA mechanisms of its therapeutic effect in the glucose and lipid metabolism Hypertriglyceridemia results from increased production and decreased are further investigated in the present study. Compared with the control clearance of triglyceride-rich very low-density lipoproteins, a pathological mice, the blood glucose of the mice decreased significantly after the nano- condition that accounts for heightened risk of ischemic vascular diseases material injection, accompanied by a dose-dependent hypoglycemic effect. in obesity and type 2 diabetes. Despite its intimate association with insulin The impaired glucose tolerance and insulin resistance were significantly resistance, whether hypertriglyceridemia constitutes an independent risk improved in mice treated with the material. We found that the expression of for beta-cell dysfunction in diabetes is unknown. Answering this fundamen- PCK1 and G6Pc were reduced at both RNA and protein levels, whilst p-AKT tal question is stymied by the fact that hypertriglyceridemia is intertwined was stimulated at the protein level after the nanomaterial injection, which with hyperglycemia and insulin resistance in obese and diabetic subjects. To indicated its positive impact on glucose metabolism. Reductions in total, circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)- adipose and liver weight were also present in db/db mice treated with the transgenic mice, a model with genetic predisposition to hypertriglyceride- material, compared with the control. In the aspect of lipid metabolism, H and mia without alterations in glucose metabolism. We showed that ApoC3- E and oil red O staining of liver tissue revealed an amelioration of fatty liver transgenic mice, as opposed to age/sex matched wild type littermates, symptoms in mice injected with the material. Consistent with the observed develop hypertriglyceridemia with concomitant elevations in plasma choles- phenotype, the mRNA expression of the FA synthesis enzymes ACC and FAS

ADA-Supported Research

A89 ADIPOSITY, INSULIN RESISTANCE, AND TYPE 2 DIABETES MELLITUS—WHAT IS NEW IN RISK FACTORS AND BIOMARKERS?

were decreased significantly after the treatment, as confirmed by q-PCR in epidemiological, observational and/or interventional studies investigating analysis. Meanwhile, an observably lower nucleus abundance of SREBP1c, changes in adipocyte insulin sensitivity in obese youth with IGT and T2D. the important transcriptional regulator of ACC and FAS, was detected fol- Table. lowing the treatment, using immunohistochemical approach. These results demonstrate the striking effects of the nanomaterial on the glucose and lipid Variables, mean ± SEM NW Obese-NGT Obese-IGT Obese-T2D P, metabolism. Taken together, our data implies the significance of this novel (n=49) (n=89) (n=38) (n=29) ANCOVA type of nanomaterial as the next-generation therapy for diabetes and fatty Age (years) 14.4 ± 0.3 14.6 ± 0.2 14.9 ± 0.4 15.2 ± 0.3 NS liver, yet the relevant mechanisms are to be further examined. BMI (kg/m2) 20.6 ± 0.3 33.9 ± 0.7 36.8 ± 1.0 36.8 ± 1.0 <0.0001 Supported By: National Natural Science Foundation of China (81471000, Visceral adipose tissue (cm2) 24.4 ± 2.2 60.8 ± 4.1 85.1 ± 5.1 83.6 ± 7.7 <0.0001 81600668) Fasting glucose (mg/dL) 95.1 ± 0.8 95.4 ± 0.6 96.8 ± 1.2 121.5 ± 4.8 <0.0001 337‑OR Fasting insulin (µU/mL) 16.2 ± 0.8 31.3 ± 1.5 50.4 ± 4.9 57.6 ± 9.6 <0.0001 Glycation and Deamidation Result in HDL Dysfunction in Patients Fasting FFA (mmol/L) 0.25 ± 0.02 0.28 ± 0.01 0.35 ± 0.02 0.32 ± 0.02 0.001 with Type 2 Diabetes AT-IR (µU/mL x mmol/L) 4.06 ± 0.38 8.98 ± 0.69 17.51 ± 1.99 18.59 ± 3.00 <0.0001 TAKHAR KASUMOV, MAKAN GOLIZEH, SANGEETA KASHYAP, Rootstown, OH, Montreal, QC, Canada, Cleveland, OH Supported By: Eunice Kennedy Shriver National Institute of Child Health and Diabetes is associated with HDL dysfunction and perturbed iron metabo- Human Development (K24HD01357, R01HD27503 to S.A.); National Center for lism. ApoAI, transferrin (Trf) and ceruloplasmin (Cp) are the key HDL proteins Advancing Translational Sciences (UL1TR000005); National Center for Research involved in cholesterol efflux and iron metabolism. We tested the hypoth- Resources (UL1RR024153) esis that hyperglycemia-induced glycation contributes to HDL dysfunction and altered iron metabolism in diet-controlled patients with T2D. HDL from 339‑OR patients with T2D and matched healthy controls (n=9/group) was isolated Serum Uric Acid (SUA), Urinary Albumin Excretion (UAE), and and it’s anti-oxidant and cholesterol efflux properties were quantified. HDL Hypertension (HTN) in Adolescents with Type 2 Diabetes (T2D) in proteome composition and post-translational modification of proteins were the TODAY Study 2 quantified by proteomics aproach. Metabolic H2O-labeling was applied to PETTER BJORNSTAD, LORI M. LAFFEL, JANE L. LYNCH, LAURE EL GHORMLI, quantify HDL proteome dynamics. Patients with T2D and controls had similar RUTH S. WEINSTOCK, SHERIDA E. TOLLEFSEN, KRISTEN J. NADEAU, Aurora, CO, lipid (triglycerides, total cholesterol, and HDL cholesterol) profile. HDL from Boston, MA, San Antonio, TX, Rockville, MD, Syracuse, NY, St. Louis, MO, Denver, CO T2D patients had reduced anti-oxidant (PON1 activity) and macrophage- Elevated SUA is increasingly recognized as a risk factor for kidney disease cholesterol efflux capacity (P<0.05) and was enriched with glycated apoAI in adults with diabetes, yet there are limited data in youth. We hypothesized and transferrin (Tf), and deamidated ceruloplasmin (Cp), the key HDL proteins that elevated SUA would predict development of elevated UAE and HTN over involved in cholesterol and iron transport. Both ApoAI and Trf glycation were time in teens with T2D. Serum creatinine, cystatin C, SUA, and urine albu- directly correlated with HbA1c and the extent of glycations were associated min to creatinine ratio (ACR) were assessed in 539 youth, ages 12-17 with with their increased degradation. ApoAI glycation was inversely correlated T2D duration <2 years at baseline in the TODAY study. Estimated GFR (eGFR) with cholesterol efflux activity of HDL. Trf glycations at Lys-206 and Lys- was calculated using creatinine and cystatin C. HTN was defined as systolic 534 sites involved in iron coordination, were 2-3 fold higher in T2D than in (SBP) or diastolic blood pressure (DBP) ≥130/80 mm Hg and elevated albumin the controls. The PTM search on Cp revealed five asparagine deamidation excretion (UAE) as ACR ≥30mg/g. Mean arterial pressure (MAP) was calcu- sites in T2D patients with N-943 being located on one of the oxidation-prone lated ([SBP + 2 (DBP)]/3). Generalized estimating equations and Cox propor- motifs and may result in the loss of Cp’s ferroxidase activity. In vivo HDL flux tional hazard models evaluated the relationship between SUA and outcome study demonstrated that glycated apoAI and Tf, and deamidated Cp spe- variables longitudinally over 7 years, adjusting for age, sex, race/ethnicity, cies were degraded 3, 10, and 2 fold faster than the respective non-modified BMI, A1c, eGFR, ACEi/ARB use, and TODAY treatment group assignment. native proteins. HDL dysfunction and oxidative stress in T2D is related to Hyperuricemia (≥6.8 mg/dL) was present in 25.6%, HTN in 18.7%, and glycation- and deamidation-induced instability of HDL proteins, including elevated UAE in 6.1% at baseline, and boys had higher baseline SUA than ApoAI, Tf and Cp. girls (6.7±1.4 vs. 5.4±1.2 mg/dl, p<0.0001). Over 7 years, 37.4% developed Supported By: American Diabetes Association (1-15-IN-31 to T.K.); American HTN and 18.0% elevated UAE. Baseline SUA correlated with increase in Heart Association SBP (β±SE: 0.66±0.16, p<.0001), DBP (0.37±0.15, p=0.01), MAP (0.47±0.14, p=0.001), and log UAE (0.05±0.02, p=0.01) over time in multivariable models. Higher baseline SUA increased risk of incident HTN (HR: 1.20, 95% CI 1.05- ADIPOSITY, INSULIN RESISTANCE, AND 1.36, p=0.007, per 1 mg/dL increase in SUA) and incident elevated UAE (HR: TYPE 2 DIABETES MELLITUS—WHAT IS NEW IN 1.23, 95% CI 1.03-1.47, p=0.02, per 1 mg/dL increase in SUA) in fully adjusted RISK FACTORS AND BIOMARKERS? models. Hyperuricemia was common in youth with T2D; higher baseline SUA independently increased risk for onset of HTN and elevated UAE over 7 years. Therapies lowering SUA may hold promise to impede development of ORALS 338‑OR Adipocyte Insulin Resistance Index in Youth along the Span of Gly‑ diabetic kidney disease and HTN in T2D youth. cemia from Normal Glucose Tolerance (NGT) to Impaired Glucose Supported By: National Institute of Diabetes and Digestive and Kidney Dis- Tolerance (IGT) to Type 2 Diabetes (T2D) eases (T32DK063687, U01DK61212, U01DK61230, U01DK61239, U01DK61242, JOON YOUNG KIM, FIDA BACHA, HALA TFAYLI, SARA MICHALISZYN, SILVA U01DK61254) ARSLANIAN, Pittsburgh, PA, Houston, TX, Beirut, Lebanon, Youngstown, OH One of the pathophysiological components of T2D is adipocyte insulin 340‑OR resistance. We demonstrated diminished insulin suppression of lipolysis, The TCF7L2 Variant rs7903146 Affects the Risk of Youth-Onset 2 using the hyperinsulinemic-euglycemic clamp combined with [ H5]glycerol Type 2 Diabetes by Reducing the Incretin Effect on Insulin Secretion tracer, in obese IGT vs. NGT youth (Diabetes 66: 2017). Herein we examined ALFONSO GALDERISI, BRIDGET PIERPONT, DAVID D’ALESSIO, NICOLA SAN- a surrogate index of adipose tissue insulin resistance (AT-IR= fasting insulin TORO, SONIA CAPRIO, New Haven, CT, Durham, NC x FFA) to test the hypothesis that AT-IR increases from normal weight (NW) Background: Common variants of the transcription factor-7-like-2 gene to obese NGT to IGT to T2D. A total of 205 youth (70 M/135 F; Tanner IV-V) (TCF7L2) have been implicated in the progression of prediabetes to T2D. We had body composition, visceral adipose tissue (VAT), fasting glucose, insulin explored the effect of the rs7903146 variant on the incretin effect in youths and free fatty acids (FFA), and AT-IR evaluated. Despite hyperinsulinemia in with dysglycemia. obese IGT youth, fasting FFA was higher compared with NW and obese NGT Methods: 5 carriers with the risk-genotype of TCF7L2 (TT at the peers (Table). AT-IR was 2.2 fold higher in obese NGT, 4.3 fold higher in IGT rs7903146, 14.4±2.3y, BMI 34.7±6.4kg/m2) and 5 with the wild type genotype and 4.6 fold higher in T2D compared with NW before and after adjusting for CC (16.2±1.9y, BMI 36.8±6.0kg/m2) underwent a 3-hour OGTT followed, a sex, Tanner stage, BMI and VAT. week later, by an isoglycemic intravenous glucose infusion (IVGTT), designed In conclusion, the surrogate index of AT-IR reflects pathophysiologi- to match the plasma glucose concentrations during the OGTT. The incre- cal alterations in adipose tissue insulin sensitivity showing progressive tin effect was measured as 100*(SROGTT-SRIVGTT)/SROGTT where SR was the increase in AT-IR in youth from normal weight to obese, and from NGT to secretion rate of c-peptide. IGT to T2D. This AT-IR, derived from fasting blood samples, could be used

ADA-Supported Research

A90 ADIPOSITY, INSULIN RESISTANCE, AND TYPE 2 DIABETES MELLITUS—WHAT IS NEW IN RISK FACTORS AND BIOMARKERS?

Results: As shown in the Figure, the plasma glucose profile in the OGTT Table. Association Between Change in Predictor Variables and Decline in ALT and IVGTT were well matched in both groups. The increase in SR during the from Baseline to 6 Months. OGTT compared to IVGTT was significantly lower in the carriers of the TT com- Change in predictor Estimate (SE) p-value Estimate (SE) p-value pared to CC genotype (ΔiAUCc-peptide 1281±688.6 vs. 5632±1604 pmol/L*min, variables over adjusted for age, adjusted for age, race, p < 0.025). Thus, relative to the control group, carriers of the TT-risk geno- 6 months race, sex sex, change in BMI type had a significant reduction of the incretin effect (TT 0% [-11.74, 16.84], Decline in BMI 0.64 (0.13) <0.01 CC: +35% [12.05, 55.71]; p <0.018). Conclusion: The TCF7L2 variant rs7903146 impairs the incretin effect in Decline in fasting glucose 0.40 (0.07) <0.01 0.28 (0.08) 0.01 obese youths carrying the risk-genotype (TT), suggesting a causative mech- Decline in HbA1C 4.59 (2.28) 0.04 1.57 (2.32) 0.50 anism for the association of this variant with progression to T2D. Decline in HOMA-IR 1.11 (0.22) <0.01 0.82 (0.27) <0.01 Figure. Decline in Triglycerides 0.17 (0.02) <0.01 0.15 (0.02) <0.01 Increase in HDL -0.75 (0.15) <0.01 -0.52 (0.16) <0.01 Decline in TG/HDL ratio 5.11 (0.56) <0.01 4.85 (0.66) <0.01

Supported By: National Institute of Diabetes and Digestive and Kidney Dis- eases; National Institutes of Health

342‑OR DNA Methylation at Birth Is Associated with Adiposity and Infant Growth during the First Six Months of Life in the Healthy Start Study SARAH J. BORENGASSER, ANNE P. STARLING, WEIMING ZHANG, JACOB FRIED- MAN, RICHARD F. HAMMAN, IVANA YANG, KATERINA KECHRIS, DANA DABE- LEA, Aurora, CO In utero exposures have been shown to impart increased risk for obesity in the offspring and are associated with epigenetic changes. DNA methylation was measured in 351 umbilical cord blood (UCB) samples within the Healthy Start Study, a well-characterized prebirth cohort that is following mother-infant pairs from pregnancy-early childhood. The primary objective was to test if DNA methylation at birth was associated with fat mass (FM) gains and infant growth (weight-for-age Z-score, WAZ) during the first 6 months (mos) of life to identify potential biomarkers of future obesity risk. DNA methylation was assessed in UCB via the Illumina 450K array. Adipos- ity was measured at birth and 4-6 mos of age via PeaPod. Infant weight was obtained from medical records. After adjusting for cellular heterogeneity, sex, and race we found 742 methylation sites (CpGs) through a univariable analysis that were associated with the rate of FM (g/day) in the first 6 mos of life (p < 0.001). Then, partial least squares (PLS) fitting using a multivariable Supported By: National Institute of Diabetes and Digestive and Kidney Diseases analysis selected a subset of 235 predictor CpGs in this list for KEGG path- (R01DK-111038 to S.C.); National Institutes of Health (R01HD-40787, R01HD-28016, way analysis. Similarly, 1314 CpGs were associated with WAZ (p < 0.001); K24HD-01464, (UL1RR-0249139 to S.C.); Robert E. Leet and Clara Guthrie Patterson PLS selected a subset of 198 CpGs for KEGG analysis. Using a FDR < 0.001 Trust (to A.G.) for KEGG analysis, 3 pathways were associated with FM gain (Metabolic pathways, Glyoxylate and dicarboxylate metabolism, Neurotrophin signal- 341‑OR ing) and 8 pathways for WAZ gain (Gastric acid secretion, Oocyte meiosis, Metabolic pathways, Endocytosis, Tight Junction, Oxytocin signaling, GnRH Nonalcoholic Fatty Liver Disease (NAFLD) Outcome Post-Bariatric signaling, and Inflammatory mediator regulation of TRP channels). The KEGG: Surgery in the Teen-Longitudinal Assessment of Bariatric Surgery Metabolic pathways was identified for both FM and WAZ gain, and 3 gene (Teen-LABS) Consortium targets enriched in this pathway, ATP5C1, CYP26A1, and IDH3A are known FIDA BACHA, RESMI GUPTA, TODD M. JENKINS, MARY BRANDT, THOMAS H. to be involved in fat metabolism. Our studies have identified potential can- INGE, DAVID KLEINER, STAVRA XANTHAKOS, TEEN-LABS CONSORTIUM, Hous- didate methylation biomarkers, present at birth, which can be used for early ton, TX, Cincinnati, OH, Aurora, CO, Bethesda, MD risk detection in order to mitigate development of childhood obesity. Teen-LABS is a multicenter, prospective observational study of ado- Supported By: National Institutes of Health (R01DK076648, R01ES022934,

lescents undergoing bariatric surgery. Predictors of resolution of NAFLD ORALS K01DK109077-02, K99ES025817) post-surgery are unclear. We evaluated changes in serum ALT levels, an acceptable surrogate when liver biopsy is not feasible, in 219 Teen-LABS subjects 3 years post gastric bypass or vertical sleeve gastrectomy. Three 343‑OR years post-surgery, median BMI declined to 38.4 from 52.6 kg/m2 at baseline Risk Factors of Youth Type 2 Diabetes (Y-T2DM) and Prevalence of (p<0.01). Median ALT (IU/L) decreased between baseline (36.5 ± 2.6 [95% Dysglycemia (DG) CI: 31.4, 41.7]) and 6 months after surgery (28.9 ± 2.5 [95% CI: 24.4, 33.9]), MOHAMED SALEH, JOON YOUNG KIM, CHRISTINE MARCH, SHAHWAR YOU- and remained stable at 1, 2 and 3 years, all p<0.01 vs. baseline. 58% had SUF, SILVA ARSLANIAN, Pittsburgh, PA abnormal ALT at baseline, which decreased to 10% at 3 years. After adjust- The incidence of Y-T2DM is increasing parallel with the childhood obesity ment by logistic regression, 6 month improvement in BMI, fasting glucose, epidemic. The ADA consensus recommends screening for Y-T2DM or predia- HOMA-IR, triglycerides, TG/HDL, and HDL were independently associated betes in asymptomatic overweight youth who have any two additional risk with reduced ALT. Improvement in metabolic indices remained significantly factors (RFs): family history of T2DM in 1st or 2nd degree relatives, minor- associated with decline in ALT, after adjusting for BMI (Table). ity race/ethnicity, insulin resistance [acanthosis nigricans, polycystic ovary In conclusion, while weight loss was associated with decreased ALT by syndrome, hypertension, dyslipidemia, and small for gestational age], and 6 months post-surgery, improved fasting glycemia, insulin resistance and maternal GDM during the child’s gestation. However, the evidence for these dyslipidemia indices were also independently associated with ALT decline. recommendations is minimal, if not nonexistent. In an established pediatric obesity registry, OGTT data of 618 youth >10 years old, BMI ≥ 85th%, were analyzed to evaluate the prevalence of DG based on the frequency of RFs. Participants were divided into 4 groups according to the number of RFs. Par- ticipants were 70% females, 70% caucasians, 31% with prediabetes, and 5.1% with Y-T2DM. With increasing RFs, the prevalence of normal glucose tolerance (NGT) declined, DG increased, fasting glucose concentration did not change, but fasting insulin concentration increased significantly (Table:

ADA-Supported Research

A91 INNOVATIONS IN INSULIN FORMULATION AND DELIVERY

mean ± SE). Participants with 1 RF had ~25% prevalence of DG. The odds for sion pattern 1 month after bariatric surgery in obese adolescents, in concur- DG with 2, 3 and ≥ 4RFs were 1.6, 1.7, and 2.5 respectively compared to 1 rent with a modest initial weight loss (Pre-op weight=138.6± 5.3kg, Post-op RF (P=0.01). weight=123.3± 5.0kg). In conclusion, the prevalence of prediabetes and T2DM in overweight/ Conclusion: Drastic changes in exosome profile were observed 1 month obese youth is high ~25% in the presence of only 1 RF, and escalates with after bariatric surgery in severely obese adolescents. It is interesting that increasing number of RFs. the most significant change happens in the first month after the surgery with Table. a modest initial weight reduction, indicating the presence of molecular processes responsible for the improvements in metabolic condition following Obesity + 1 RF Obesity + 2 RF Obesity + 3 RF Obesity + ≥4RF P-ANOVA bariatric surgery, independent of weight loss. These findings suggest that (n=45) (n=165) (n=223) (n=185) the exosomes play a key role in the profound metabolic improvements fol- Age (years) 13.7 ± 0.3 14.0 ± 0.2 14.3 ± 0.1 14.4 ± 0.2 NS lowing bariatric surgery, and serve as a novel biomarker or a therapeutic Glycemic status 34 (75.6%)/ 108 (65.5%)/ 143 (64.1%)/ 103 (55.7%)/ 0.009 target for metabolic diseases. (NGT/Dysglycemia), n (%) 11 (24.4%) 57 (34.5%) 80 (35.9%) 82 (44.3%) BMI percentile 97.6 ± 0.4 98.0 ± 0.3 98.4 ± 0.2 98.6 ± 0.2 NS Fasting glucose (mg/dl) 92.4 ± 1.2 93.9 ± 0.9 94.0 ± 0.9 94.9 ± 1.2 NS INNOVATIONS IN INSULIN FORMULATION AND DELIVERY Fasting insulin (μu/ml) 25.6 ± 2.3 31.0 ± 1.9 34.7 ± 1.6 42.5 ± 2.7 < 0.0001 OGTT 2-hour Glucose (mg/dl) 117.6 ± 4.7 125.9 ± 3.0 129.4 ± 2.7 138.4 ± 3.3 0.001 346‑OR OGTT 2-hour Insulin (μU/ml) 121.5 ± 17.6 160.7 ± 11.2 169.9 ± 10.8 217.0 ± 14.3 < 0.0001 Pharmacodynamic Effect of Novel Oral Insulin Tregopil in Relation to Meal Composition in Type 2 Diabetes Mellitus Patients ANAND P. KHEDKAR, VINU JOSE, HAROLD E. LEBOVITZ, ALAN D. CHER- 344‑OR RINGTON, GILBERT ALEXANDER FLEMING, SANDEEP N. ATHALYE, ASH- Maternal Obesity, Diabetes during Pregnancy, Gestational Weight WINI VISHWESWARAMURTHY, Bangalore, India, Brooklyn, NY, Nashville, Gain, Breastfeeding, and Child’s BMI Growth Trajectory from Ages TN, Harpers Ferry, WV 2 to 6 Years Our aim was to evaluate pharmacodynamics (PD) of insulin tregopil (IN- XINHUI WANG, MAYRA P. MARTINEZ, ANNY XIANG, Pasadena, CA 105) in relation to meal composition in type 2 diabetes mellitus patients in Limited studies have evaluated the interplay among maternal obesity, placebo-controlled, crossover study. Eighteen patients were randomised to maternal diabetes including preexisting type 1 (T1D), type 2 (T2D) or gesta- 6 sequences and administered insulin tregopil 30 mg or placebo, 20 minutes tional diabetes mellitus (GDM), excessive gestational weight gain (EGWG), prior to 2 meals (separated by 5 hours). Morning meal was ADA/high-fat/ and breast feeding (BF) in association with childhood growth trajectory. high-fibre meal while afternoon was ADA meal. PD sampling was done from Here we assessed their associations with child’s BMI trajectory from ages 2 dosing up to 180 minutes post dose. AUC0-180min (glucose exposure), Cmin to 6 years. Data included 71,892 singleton children born at 28-44 gestational (minimum glucose concentration observed) and tmin (time of peak PD effect) weeks in 2007-2011 at Kaiser Permanente Southern California hospitals who are presented in Table 1. Insulin tregopil administration following high-fat had at least 5 BMI measurements between ages 2 to 6. First, BMI trajec- and high-fibre meal in morning resulted in glucose exposure (GM ratio of tory patterns for these children were identified using group-based trajectory AUC0-180 min, baseline-corrected) of 92% and 98% compared to ADA meal, modeling method. Second, the relationships between maternal exposures respectively; and 99% and 94%, respectively, after afternoon meal, com- and growth trajectory groups were evaluated using multinomial logistic pared to ADA meal. Maximal reduction in plasma glucose for ADA-ADA, regression adjusting for maternal age at delivery, race/ethnicity, education, high fibre-ADA and high fat-ADA meal groups was 42.0mg/dL, 38mg/dL and and child’s sex. Three distinct BMI trajectory groups were identified: group 33.4mg/dL, respectively, in morning and 29.3mg/dL, 16.7mg/dL and 37.9mg/ 1 (59% of the cohort) had stable low BMI over time, group 2 (35% of the dL, respectively, in afternoon. This peak PD effect was observed 37 to 47 cohort) had stable median BMI over time, and group 3 (6% of the cohort) had minutes post dose, indicating ultra-short action profile. No clinically signifi- high and increasing BMI over time. Relative to group 1, the adjusted odds cant difference in PD response to insulin tregopil was observed irrespective ratio (OR[95% CI]) of being in group 3 associated with maternal exposures of meal composition, in the same or subsequent meal. were 8.0 [7.3-8.8] for pre-pregnancy obesity, 2.8 [2.6-3.1] for pre-pregnancy Table 1. Pharmacodynamic Parameters for Plasma Insulin Tregopil and overweight, 2.1 [1.2-3.8] for T1D, 1.7 [1.5-2.0] for T2D, 1.2 [1.1-1.4] for GDM, Placebo. 1.5 [1.4-1.6] for EGWG, and 1.3 [1.2-1.4] for BF<6 months after adjusting for each other. ORs of being in group 2 were much smaller and were insignificant Baseline-Corrected Plasma Glucose (Relative to time of Meal Administration) for T1D and GDM. Further adjustment for birth weight and gestational age at Type of meal during morning AUC0-180 min Cmin (mg/mL) Tmin (min) delivery had a small effect on the OR estimates but did not change the con- a b c clusion. Thus, among the four exposures, high and increasing BMI trajectory and afternoon Time of meal (min*mg/mL) ± SD ± SD ± SD in offspring from age 2 to 6 years was strongly associated with maternal Insulin tregopil administered before both the meals obesity and overweight; modestly with maternal T1D, T2D, and EGWG; and ADA - ADA Morning 190.68 ± 29.12 0.67 ± 0.16 40.58 ± 8.99 ORALS slightly with BF<6 months and GDM. Afternoon 216.13 ± 38.41 0.71± 0.14 38.23 ± 14.67 High-fat - ADA Morning 173.81 ± 30.82 0.69 ± 0.15 42.22 ± 11.66 345‑OR Afternoon 215.28 ± 56.74 0.82 ± 0.12 37.44 ± 25.29 The Role of Exosomes in Improvement of Insulin Sensitivity in Obese Adolescents following Bariatric Surgery High-fibre - ADA Morning 186.71 ± 30.23 0.73 ± 0.15 46.47 ± 37.23 AHLEE KIM, KAZUTOSHI MURAKAMI, VISHNUPRIYA J. BORRA, LAWRENCE M. Afternoon 205.46 ± 37.86 0.64 ± 0.13 41.17 ± 7.81 DOLAN, AMY SHAH, TAKAHISA NAKAMURA, Cincinnati, OH Insulin tregopil administered before morning meal and placebo administered Background: Exosomes, a type of extracellular vesicle, are a novel mode of before afternoon meal tissue-to-tissue communication. Recent work suggests a role of exosomes in ADA - ADA Morning 191.76 ± 27.20 0.70 ± 0.13 38.44 ± 13.74 the development of obesity-related metabolic disease, such as insulin resistance or type 2 diabetes. With bariatric surgery, individuals undergo significant Afternoon 256.99 ± 43.38 0.91 ± 0.06 22.77 ± 14.87 weight reduction and dramatic metabolic improvements. We hypothesize that High-fat - ADA Morning 169.65 ± 18.63 0.66 ± 0.16 48.82 ± 20.88 exosomes mediate the metabolic improvements following bariatric surgery. Afternoon 238.68 ± 31.76 0.92 ± 0.07 24.88 ± 14.09 Methods: We performed comprehensive exosome analyses, includ- High-fibre - ADA Morning 197.49 ± 35.23 0.75 ± 0.13 40.88 ± 37.67 ing measuring size and serum concentration of exosomes, and evaluating exosomal microRNAs expression pattern in 30 teenagers who underwent Afternoon 257.13 ± 35.19 0.93 ± 0.07 19.23 ± 13.27 sleeve gastrectomy (age=16.7± 0.4 years; gender=26 females, 4 males; race= a, b, c, d, e, f Baseline corrected arithmetic mean values are presented. ADA: 19 caucasians, 11 African Americans). Individuals who had ever been diag- American Diabetes Association; SD: Standard Deviation. nosed with diabetes were excluded from the study. Results: We observed a significant reduction in serum exosome concentration (pre-op= 4.47x108± 6.28X107particles/mL; post-op=2.56x108± 2.44X107 particles/mL, p=0.01), and a drastic shift of exosomal RNA expres-

ADA-Supported Research

A92 INNOVATIONS IN INSULIN FORMULATION AND DELIVERY

347‑OR Figures 1a-1b. Basal Oral Insulin Analog I338 Enhances Meal-Related Hepatic Glu‑ cose Disposal MARY C. MOORE, ERICA NISHIMURA, CHRISTIAN L. BRAND, THOMAS KJELD- SEN, PETER MADSEN, HANNE H. REFSGAARD, KARSTEN WASSERMANN, SANNE GRAM-NIELSEN, MARTA S. SMITH, L. MERKLE MOORE, BEN FARMER, JON R. HASTINGS, PHILLIP E. WILLIAMS, ALAN D. CHERRINGTON, Nashville, TN, Måløv, Denmark, Copenhagen, Denmark, Søborg, Denmark Oral insulin avoids the need for injections, and insulin enters via the hepatic portal vein (Po), the normal route of secretion. We examined the effect of I338, an acylated analog designed for oral use, dosed either Po to match the oral absorption profile or IV to match the steady state basal subcutaneous (SC) plasma profile, on a Po glucose challenge. To achieve steady- state concentrations in normal dogs, I338 was infused IV 45 min daily for 4 days. On day 5 a primed, continuous infusion of 3-3H glucose was given. After 90 min of equilibration and 30 min of basal sampling, a clamp was conducted (0-300 min), with somatostatin to inhibit pancreatic secretion and basal glucagon replacement. In the LOW dogs (n=5) I338 (pmol/kg/min) was Supported By: MannKind Corporation infused either Po at 40 (0-45 min to mimic oral absorption) or IV at 1 (0-300 min to mimic subcutaneous [SC] delivery), approximately an equivalent daily 349‑OR dose (1800 vs. 1440 pmol/kg, Po vs. IV). High dose dogs (HI; n=4) received BioChaperone Technology Enables the Development of Pramlint‑ I338 at 50 (Po) or 5 (IV) pmol/kg/min. A Po glucose infusion mimicking meal ide-Prandial Insulin Combinations absorption was given via computer algorithm from 30-252 min. All dogs were GRÉGORY MEIFFREN, ALEXANDRE GEISSLER, YVES MEYER, AYMERIC RANSON, studied twice, 2 weeks apart in random order, receiving both Po and IV I338. CHARLES FORTIER, OLIVIER SOULA, RÉMI SOULA, BERTRAND ALLUIS, RICHARD Po vs. IV, respectively, resulted in: 1.) lower glycemic levels (peak glucose CHARVET, Lyon, France LOW 6.2±0.3 and 10.3±0.6,* HI 4.5±0.3, and 7.8±0.8* mM); 2.) increased net Pramlintide (Symlin®) is currently used on top of mealtime insulin therapy hepatic glucose uptake (AUC30-240 min Low 218.4±151.8 and -852.0±371.4,* by T1D or T2D patients to achieve a better control of post-prandial glucose HI 732.6±338.5 and -58.5±578.4* µmol/kg); 3.) enhanced glucose clear- excursion. Indeed, pramlintide affects the rate of postprandial glucose ance (AUC30-240 min LOW 481.9±29.7 and 359.4±33.7,* HI 874.3±99.8 and appearance by slowing down gastric emptying, reducing postprandial glu- 495.3±25.9* mL/kg) and 4.) greater peak hepatic fractional glucose extrac- cagon secretion and modulating satiety, which affects caloric intake. Never- tion (LOW 0.05±0.02 and -0.01±0.01,* HI 0.10±0.05 and 0.04±0.02*)(*P<0.05 theless, the use of pramlintide is currently limited as it cannot be combined vs. corresponding Po treatment). In short, Po basal I338 delivery produced with prandial insulin due to formulation pH incompatibility and results in a superior glucose lowering and hepatic glucose disposal during a morning high burden of the number of injections. BioChaperone (BC) technology stabi- “meal” compared to SC-like delivery. Thus basal oral I338 can improve the lizes pramlintide in aqueous solution at neutral pH and enables a pramlintide, glycemic response to the first meal after dosing, potentially improving glu- prandial insulin co-formulation. BC pramlintide-human insulin (BC Pram Ins) cose disposal at subsequent meals via the 2nd meal effect. formulation is physically and chemically stable for at least 6 weeks at 30°C and 9 weeks at 25°C. Physical stability was evidenced by visual inspection 348‑OR and MFI analysis. Chemical stability (recovery measured by RP-HPLC and Improved Postprandial Blood Glucose (PPBG) Excursions with high molecular weight species measured by SE-HPLC) was similar to that of Technosphere Inhaled Insulin (TI) Compared with Aspart in T1D commercial Humulin® and Symlin®. Under simulated in-use pump conditions Patients—STAT Study at 37°C, BC Pram Ins formulation shows excellent physical and chemical sta- HALIS K. AKTURK, JANET K. SNELL-BERGEON, AMANDA REWERS, LESLIE J. bility for at least 1 week, with insulin and pramlintide recoveries higher than KLAFF, ANNE PETERS, BRUCE W. BODE, TIMOTHY S. BAILEY, SATISH K. GARG, 95% and a formulation essentially free of particles. The pharmacokinetics Aurora, CO, Denver, CO, Renton, WA, Los Angeles, CA, Atlanta, GA, Escondido, CA of pramlintide was evaluated following single subcutaneous administration Post-prandial hyperglycemia is difficult to control due to lack of an ideal (0.1875 U/kg insulin, 1.125 µg/kg pramlintide) of BC Pram Ins formulation and prandial insulin. TI (Afrezza®) has the most rapid onset of action, lasts for pramlintide co-injected with human insulin (separate injections) to fasted 2 hours. healthy pigs. BC Pram Ins shows a slower absorption of pramlintide (LSM Sixty patients with T1D on multiple daily injections (MDI) were ran- ratio [95% CI] ΔAUCPram0-30min: 0.45 [0.20; 1.05]) while the late exposure to domized in a multi-center study, stratified by baseline A1c values (<8.5% pramlintide is higher (ΔAUCPram60-180min: 2.65 [1.44; 4.90]) compared to the or ≥ 8.5%) to the control group using aspart (n=34) vs. TI group (n=26). The separate injections. TI arm was advised to take extra inhalations at 1 and 2 hours after meals In conclusion, the in-vitro and preclinical PD properties of BC Pram Ins based on PPBG. Baseline characteristics were similar (Figure 1a). Forced support its clinical development to afford a better mealtime treatment for Expiratory Volume did not differ at all times. We used intent-to-treat analy- T1D and T2D patients. ORALS sis, and examined outcomes over a 4-week period using linear regression with repeated measures. 350‑OR Mean CGM glucose, SD, time in range (70-180 mg/dL), % time in hyper- Fully Closed-Loop Glucose Control in Noncritical Care Settings—A (>180 mg/dL) or hypoglycemia (<70, <60, or <50 mg/dL) were similar in both Randomised, Controlled Two-Centre Study groups. PPBG at 1 hour was lower in the TI group (mean ± SE PPBG difference LIA BALLY, HOOD THABIT, SARA HARTNELL, EVELINE ANDEREGGEN, YUE RUAN, -31.7±6.6 mg/dL, p<0.0001). PPBG was numerically lower at 2 hours (mean ± MALGORZATA E. WILINSKA, MARK EVANS, MARIA M. WERTLI, ANTHONY P. SE PPBG -13.0±7.1 mg/dL, p=0.07) with no difference at 3 and 4 hours (Figure COLL, CHRISTOPH STETTLER, ROMAN HOVORKA, Berne, Switzerland, Manches- 1b). The TI group increased bolus insulin dose (mean ± SD of 47.8 ± 23.9 ter, United Kingdom, Cambridge, United Kingdom, Oxford, United Kingdom U/day) compared to the control group (23.0 ± 9.8 U/day; p<0.0001) in week 1. Automated fully closed-loop (CL) insulin delivery system without meal- Bolus insulin dose in the TI group was higher (28.2 U/day; p<0.0001), and did bolusing was evaluated in medical and surgical non-critical care wards of not differ by study week (p=0.25). two acute hospitals in Switzerland and United Kingdom. In a randomised We conclude that TI improves PPBG when treat-to-target algorithms are controlled parallel design study, 136 adults with inpatient hyperglycaemia used in patients with T1D on MDI. requiring subcutaneous (s/c) insulin were randomised to receive either CL- directed s/c insulin delivery (n=70) or conventional s/c insulin therapy as per local practice with masked continuous glucose monitoring (n=66) for up to 15-days. Participants consumed self-selected hospital meals and were matched for age (68±10 vs. 67±13 years; CL vs. control), HbA1c (7.8±2.5 vs. 8.0±1.9%) and BMI (32.7±8.2 vs. 32.3±8.1kg/m2). During CL, participant’s usual insulin and sulphonylurea therapy were withheld. In an intention to treat analysis, the proportion of time when sensor glucose was in target range from 5.6 to 10.0mmol/l was significantly higher during CL (p<0.001,

ADA-Supported Research

A93 INNOVATIONS IN INSULIN FORMULATION AND DELIVERY

Table). CL reduced time spent above target (p<0.001), and reduced mean and standard deviation of sensor glucose (p<0.001). Time spent hypoglycaemic was low and comparable. Total daily insulin delivery was not different. No severe hypoglycaemia or serious adverse events occurred in either group. In conclusion, fully closed-loop without meal-bolusing is safe, and may improve glucose control in a heterogeneous inpatient population. Table. Closed-loop Conventional P insulin delivery insulin therapy (n=70) (n=66) Time spent at sensor glucose levels (%) 5.6 to 10.0 mmol/l 65.7±16.8 41.5±16.9 <0.001 Supported By: Eli Lilly and Company >10.0 mmol/l 23.7±16.6 49.5±22.8 <0.001 <5.6 mmol/l 10.6±6.7 9.0±13.3 0.37 352‑OR <3.0 mmol/l 0.0 (0.0, 0.1) 0.0 (0.0, 0.0) 0.80 Improvements in A1c and Time-in-Range in DIY Closed-Loop Mean sensor glucose (mmol/l) 8.5±1.6 10.5±2.4 <0.001 (OpenAPS) Users DANA M. LEWIS, RICHARD S. SWAIN, THOMAS W. DONNER, Seattle, WA, Col- SD of sensor glucose (mmol/l) 2.6±1.0 3.2±1.1 <0.001 lege Park, MD, Baltimore, MD Total daily insulin (U) 44.0 (27.5, 73.3) 40.2 (26.5, 65.6) 0.36 Overview: We performed a retrospective study that analyzed outcomes Data are mean ± SD or median (IQR). from a subset (n=20) of the Do It Yourself (DIY) closed loop community. This already well-controlled, highly motivated T1D population realized further Supported By: Diabetes UK; European Foundation for the Study of Diabetes; UK improvements in A1c and glucose time in range (TIR), and a reduction in time National Institute for Health Research; Cambridge Biomedical Research Centre; spent high and low during all timeframes after beginning a DIY hybrid closed Bern University Hospital; Abbott Diabetes Care loop “artificial pancreas” system. Objective: To compare mean BG, TIR (70-180 mg/dl), and time above and 351‑OR below clinically meaningful thresholds before and after OpenAPS initiation. Human Regular U-500 Insulin via Continuous Subcutaneous Insu‑ Methods: We performed a retrospective cross-over analysis of continu- lin Infusion vs. Multiple Daily Injections in Adults with T2D—The ous BG readings recorded during 2-week segments 4-6 weeks before and VIVID Study—All Randomized Population after initiation of OpenAPS (Johns Hopkins IRB00121066). Mean BG and TIR GEORGE GRUNBERGER, ANUJ BHARGAVA, TRANG T. LY, HOWARD ZISSER, LIZA were analyzed overall, as well as by day (7am-11pm) and night (11pm-7am), ILAG, JAMES K. MALONE, SHUYU ZHANG, JENNAL JOHNSON, Bloomfield Hills, and statistical significance was assessed using paired t-tests. MI, West Des Moines, IA, Billerica, MA, Santa Barbara, CA, Indianapolis, IN Results: Mean BG and TIR improved in every time category. Overall, mean VIVID is the first prospective randomized controlled trial evaluating CSII BG (mg/dl) improved (135.7 to 128.3); as did mean estimated HbA1c (6.4 to (using an investigational Omnipod U-500™ Insulin Management System) vs. 6.1%). TIR increased from 75.8 to 82.2% overall. Overnight, BG time <70 was MDI (TID) with U-500R (Humulin® R U-500). This was a 26-week, open-label, reduced from 6.4 to 4.2%, and time <50 was reduced from 2.3 to 1.0%. Over- multicenter, parallel study in adults with T2D on high dose insulin (201-600 all, BG excursions >300 were reduced from 1.7 to 0.35%. units/day) with or without other antihyperglycemic agents (NCT02561078). Conclusion: Even with tight initial control, persons with T1D saw meaning- We present results for the all randomized population (N=420); results for ful improvements in estimated A1c, TIR, and a reduction in time spent high the primary population (excluding participants on concomitant GLP-1 RAs and low, during the day and at night, after initiating OpenAPS. or SGLT2 inhibitors) are reported separately. Baseline characteristics were Table. similar between arms. Without adjusting for multiplicity, CSII demonstrated significantly greater reduction in A1C and FPG at a lower total daily dose vs. MDI at 26 weeks. Also, a higher percentage of participants on CSII reached A1C targets <7.0%, <7.5%, and <8.0%. Severe hypoglycemia rates were low. Other hypoglycemia rates were similar (documented symptomatic) or higher (nocturnal) for CSII vs. MDI. Weight gain was similar (Table). In the all randomized population, U-500R via CSII demonstrated greater efficacy at a lower dose with a higher nocturnal hypoglycemia rate vs. MDI. CSII with U-500R may be a viable option for patients with T2D on high dose insulin. Individualized dose titration will be important for safely achieving glycemic targets. ORALS Table.

ADA-Supported Research

A94 NOVEL ASPECTS OF METABOLIC REGULATION

knockout of the miRNA-processing enzyme Dicer (ADicerKO) exhibit lipodystrophy and metabolic syndrome. This is due, in part, to a loss of multiple circulating exosomal microRNAs and their effects on gene expression in liver and other tissues. In this study, we explored the role of exosomal proteins in metabolism by comparing the serum exosomal proteome of wild type (WT) and AdicerKO mice. To this end, we isolated serum exosomes from WT and KO mice and performed quantitative proteomics by LC-MS. A total of 277 exosomal proteins were identified, of which 12 were significantly downregulated and 16 were upregulated by >1.5 in AdicerKO mice. Among these were two important adipokines. Thus, the insulin sensitizer adiponectin was downregulated in exosomes, while the insulin resistance-associated adipokine retinol-binding protein-4 (Rbp4) was upregulated. Transthyretin, which 353‑OR binds to and extends Rbp4 half-life, was also increased in serum exosomes Digitally Enhanced Insulin Therapy—A Multicenter Clinical Trial from AdicerKO mice. Additionally, serum amyloids A-1 and A-4, serum amy- ISRAEL HODISH, RICHARD M. BERGENSTAL, MARY L. JOHNSON, REBECCA A. loid component P, haptoglobin and ceruloplasmin, which are adipose and/ PASSI, ANUJ BHARGAVA, NATALIE YOUNG, DAVIDA F. KRUGER, ANGELA HAI- or liver-specific in origin and often altered in insulin resistant states, were LEY, EMILY UNGER, ERAN BASHAN, Ann Arbor, MI, Minneapolis, MN, West Des down-regulated in the exosomes of knockout mice. Moines, IA, Detroit, MI, Livonia, MI In summary, serum exosomes contain multiple proteins which can modu- A quarter of patients with type 2 diabetes (T2D) use insulin, yet most late insulin sensitivity or insulin resistance. Induction of lipodystrophy by KO remain under treated. The shortcoming of the therapy stems from its dynamic of Dicer in adipose markedly alters the circulating exosomal proteome due to nature which necessitate frequent dosage titration. In reality, current over- loss of fat or the secondary hepatosteatosis this produces. These exosomal burdened health-care systems fall short of time to delivering sufficient insu- proteins provide potential new signals in the crosstalk between fat, liver and lin titrations. The d-Nav® Insulin Guidance Service overcomes this barrier. other tissues in response to metabolic disease. d-Nav is a handheld device that automatically titrates insulin dosage at least weekly, based on glucose readings that the patient is already scheduled to 355‑OR take with the d-Nav. Additionally, the service includes dedicated support of The Role of GLP-1 Receptor Expressed in Pancreatic -Cells in care specialists. We enrolled 181 sub-optimally controlled T2D patients to α Regulating Glucagon Secretion a multicenter, 1:1 randomized control trial to assess the effectiveness and YANQING ZHANG, KESHAB R. PARAJULI, GENEVIEVE E. SMITH, RAJESH GUPTA, safety of the d-Nav service compared to close follow-up of diabetes special- WEIWEI XU, LAUREN U. NGUYEN, ANADIL F. ZAKARIA, FRANCK MAUVAIS-JAR- ists. Both groups received 7 interactions in the 6-month study. VIS, VIVIAN FONSECA, KYLE W. SLOOP, HONGJU WU, New Orleans, LA, Toledo, d-Nav group: automatic titrations occurred 1±0.2 times per week (Figure); OH, Kenner, LA, Indianapolis, IN A1c reduction 8.7±0.8% to 7.7±1.0%; 94.6% retention; high-level of satisfac- Glucagon-like peptide-1 (GLP-1) inhibits glucagon secretion from -cells tion and comfort; minor hypoglycemia (<55mg/dl) 0.3±0.6/month. α and stimulates insulin secretion from cells in a blood-glucose dependent Control: A1c reduction 8.5±0.8% to 8.2±0.9% (p<0.0001 between groups). β manner. Whereas the effects of GLP-1 on cells are mediated by GLP-1 Severe hypoglycemia: statistically similar and low. Expansion of super- β receptor (GLP-1R) has been described in detail. The action of GLP-1 on vised automated insulin titrations is feasible as long as it is simple to use -cells, however, is not clear. To determine whether the inhibition is due by the patient and it does not increase providers’ burden. It may lead to a α to a direct effect that occurs through GLP-1R of -cells, or indirect effect on sizable reduction in complications and costs. α α-cells through insulin stimulation from β-cells, we generated α-cell specific GLP-1R knockout (αGLP-1R KO) mice after GLP-1R expression was verified by glucagon-producing α-cells at protein avnd mRNA level in both mouse and human pancreas. Compared to control mice, the αGLP-1R KO female mice had impaired glucose tolerance following intraperitoneal glucose administration, and had more impaired glucose tolerance after 1m high fat diet-fed, but their insulin secretion and insulin sensitivity were maintained. Interestingly, non-fasting glucagon level of αGLP-1R KO mice was significantly higher than that of the control mice, whereas insulin and GLP-1 levels were similar between the two groups of mice. More importantly, we found glucagon secretion was stimulated by glucose 15 minutes following glucose administration in the αGLP-1R KO mice, but not in the control mice. Further- more, we isolated islets from αGLP-1R KO mice and directly examined the effects of αGLP-1R deletion on glucagon secretion. Glucose-stimulated insulin secretion assay showed the αGLP-1R KO islets had no significant differences from the control islets. However, arginine significantly stimulated ORALS glucagon secretion in the two group islets and the stimulation was significantly enhanced in αGLP-1R KO islets. Taken together, these data showed that αGLP-1R deletion disturbed glucose-dependent glucagon secretion, whereas insulin secretion was not affected, demonstrating αGLP-1R plays a direct role in regulating glucagon secretion. Supported By: National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (2R42DK085974-02A1) 356‑OR FoxK1/K2 Are New, Important Components of IR and IGF1R Signal‑ ing and Control of Cell Proliferation and Metabolism NOVEL ASPECTS OF METABOLIC REGULATION MASAJI SAKAGUCHI, WEIKANG CAI, CHIH-HAO WANG, THIAGO M. BATISTA, EIICHI ARAKI, C. RONALD KAHN, Kumamoto, Japan, Boston, MA Insulin and IGF-1 are regulators of cell growth and metabolism. Despite 354‑OR considerable progress in understanding the molecular mechanisms underly- Serum Exosomal Proteins—A New Component of Intercellular ing these effects, except for members of the FoxO family, many regulators of Communication in Metabolism the transcriptional effects of these hormones remain poorly understood. To BRUNA B. BRANDAO, EMRAH ALTINDIS, RUBEN GARCIA MARTIN, C. RONALD identify new components of IR/IGF1R signaling, we created brown preadipo- KAHN, Boston, MA cyte lines in which both endogenous IR and IGF1R genes had been deleted Adipose tissue is an important endocrine organ; both excess or deficiency and then reconstituted these cells with normal IR, IGF1R or chimeric recep- of adipose is associated with metabolic syndrome. MicroRNAs (miRNAs) tors containing the extracellular domain of IR fused to intracellular domain play a fundamental role in regulating adipocyte function and metabolism. of IGF1R and vice versa. Mass spectroscopic proteomic analysis revealed Recently, we showed that a large fraction of circulating miRNAs comes from a number of proteins co-precipitated with each receptor construct, in both exosomes released by adipose tissue and that mice with an adipose-specific

ADA-Supported Research

A95 NOVEL ASPECTS OF METABOLIC REGULATION

ligand stimulation-dependent and -independent manners. Among the pro- expression. Moreover, adenovirus-mediated overexpression of RARβ restored teins that associated with both receptors and chimeric receptors in a ligand- hepatic impairment of RARβ and systemic glucose intolerance and decreased dependent manner was FoxK1. In contrast to FoxO1, the transcription factor hepatic gluconeogenesis by obesity. Mechanistically, overexpression of RARβ FoxK1 was translocated from the cytoplasm to the nucleus after insulin stim- decreased obesity-induced expression of MPC1 and MPC2, key regulators of ulation, a pattern that is reciprocal to that of FoxO1 after insulin stimulation. gluconeogenesis from pyruvate. Increasing RARβ in ob/ob mice also inhibited We show that FoxK1 and FoxK2 are phosphorylated in two major domains the induction of alanine transaminase (ALT), a key enzyme that converts ala- and that translocation to the nucleus is dependent on the Akt-mTOR medi- nine into pyruvate for glucose production. Moreover, overexpression of RARβ ated phosphorylation, while their localization to the cytoplasm in the basal downregulated lipogenic gene expression, upregulated fatty acid oxidation, state is dependent on GSK3 phosphorylation. Knockdown of FoxK1 and and improved hepatic steatosis and hyperlipidemia in ob/ob mice. Hepatic FoxK2 in a mouse hepatocyte cell line causes marked alteration of the tran- and systemic insulin resistance were also reduced by overexpressing RARβ. scription of genes associated with apoptotic pathway and down-regulation In sum, hepatic defective RARβ may contribute to the upregulation of MPC/ of genes involved in control of cell cycle and metabolism. This resulted in ALT and gluconeogenesis in diabetes and RARβ can be a novel drug target to decreased cell proliferation and altered mitochondrial fatty acid metabolism normalize metabolic dysregulation in diabetes. in these cells. Thus, insulin stimulates the reciprocal translocation of FoxKs Supported By: American Diabetes Association (1-15-BS-216 to F.K.N.); National and FoxO1 between the cytoplasm and nucleus turning off FoxO1-regulated Institutes of Health genes and turning on FoxK-regulated genes resulting in an elaborate balance in insulin regulation of metabolism, growth and cell survival. 359‑OR Supported By: National Institutes of Health; Takeda Foundation; Merck Founda- Macrophages React to Postprandial Signals and Regulate the tion; Kanae Foundation for the Promotion of Medical Science Response to Feeding through Akt-mTOR-Dependent Production of IL-10 357‑OR GOTARO TODA, NOZOMU KAMEI, KOTARO SOEDA, YUKARI MASUDA, YOSHI- Loss of CEACAM1 in Endothelial Cells Contributes to the Develop‑ HIKO IZUMIDA, NAOKI KOBAYASHI, TAKAYOSI SASAKO, KAZUYUKI TOBE, ment of Cardiac Fibrosis MORRIS J. BIRNBAUM, TAKASHI KADOWAKI, KOHJIRO UEKI, Tokyo, Japan, Hiro- HARRISON T. MUTURI, HILDA E. GHADIEH, SAJA S. KHUDER, SUKANTA JASH, shima, Japan, Toyama, Japan, Philadelphia, PA RAGHD ABU HELAL, NIGEL A. DANIELS, JAMES LIU, VISHWAJEET PURI, GUILL- We explored the role of the serine/threonine protein kinase Akt in mac- ERMO VAZQUEZ, RAJESH GUPTA, SONIA M. NAJJAR, Athens, OH, Toledo, OH, rophages, using conditional knockout models. Myeloid specific Akt1 Akt2 Baltimore, MD double knockout mice (MAktDKO mice), fed a normal chow diet, showed Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) higher plasma glucose levels in the fed state. Among the various responses has been implicated in various physiological processes in the vascular sys- to feeding, the liver of MAktDKO mice did not show the repressed gluco- tems, including blood vessel formation, and endothelial barrier function. neogenic gene expression in response to feeding. As this was also mim- Consistent with a role for hepatic CEACAM1 in promoting insulin sensitivity icked in mice treated with multiple antibiotics to eradicate the intestinal by mediating insulin clearance and maintaining physiologic plasma insulin flora, lipopolysaccharide (LPS), which increases in the blood after feeding levels, we have recently identified a role for hepatic CEACAM1 in preventing may play a role in this context. Among genes Akt dependently induced cardiac hypertrophy and dysfunction. The aim of this study was to eluci- by LPS, IL-10 was induced in 72 hours when stimulated by LPS alone, but date the contribution of endothelial cell CEACAM1 in cardiac function and this induction was markedly shortened to 3 hours when also stimulated by fibrosis that has remained elusive. To this end, we generated an endothelial insulin. Elevations of plasma concentrations of IL-10 could be detected in cell-specific Ceacam1 knockout mouse line (VECadCc1-/-). Gomori Trichrome the portal vein after feeding, and such concentrations of IL-10 augmented Blue Staining revealed a significant perivascular and interstitial fibrosis in suppression of gluconeogenic gene expression in primary hepatocytes by the heart of VECadCc1-/- mice compared to their control littermates. Con- physiologically detected levels of insulin. Deficient suppression of gluconeo- sistently, the mRNA levels of markers of fibrosis [collagen 1A1,α -SMA and genic gene expression in these mice was rescued when mTOR signaling was fibroblast-specific protein1 (FSP1)] were elevated with a reciprocal decrease strengthened by deletion of TSC2, implicating an important role of Akt-mTOR of endothelial cell makers (CD31), indicating endothelial mesenchymal tran- signaling in the feeding response. Moreover, mice injected with adenovirus sition (EndMT) in these mice. Cardiac fibrosis was associated with increased expressing short hairpin RNA targeting the IL-10 receptor showed deficient activation of the TGF-β1-Smad2/3 signaling pathway. Immunofluorescence suppression of post prandial gluconeogenic gene expression, in support of staining co-localized CD31 with α-SMA in the myocardial perivascular sec- a model in which macrophages respond to postprandial signals, regulating tion of VECadCc1-/- hearts. Moreover, mRNA level of α-SMA, FSP1, SNAIL glucose homeostasis in the liver by expressing IL-10 in an Akt-mTOR depen- and SLUG were up-regulated while that of CD31 was down-regulated in dent process. mouse heart endothelial cells (MHEC) isolated from VECadCc1-/- mice. The Supported By: Japan Society for the Promotion of Science Akt/eNOS pathway was basally inactivated, leading to lower nitric oxide (NO) level in MHEC isolated from VECadCc1-/- mice. Moreover, loss of endo- 360‑OR thelial CEACAM1 caused reduced association with SHP-2 phosphatase and NAD-Mediated Metabolic Reprogramming Epigenetically Regu‑ consequently, reduction in insulin-stimulated IRS-1/Akt/eNOS activation.

ORALS lates Gene Expression to Promote Preadipocyte Differentiation These studies identified an important role for the loss of CEACAM1 in endo- KEISUKE OKABE, ISAO USUI, ALLAH NAWAZ, SHIHO FUJISAKA, TOMONOBU thelial cells as a mechanism underlying cardiac fibrosis. KADO, YOSHIKO IGARASHI, KUNIMASA YAGI, KAZUYUKI TOBE, TAKASHI NAK- AGAWA, Toyama, Japan, Shimotsuga, Japan 358‑OR During cell differentiation, intracellular energetic pathways are drasti- Hepatic Overexpression of Retinoic Acid Receptor β Downregu‑ cally reorganized. Although this alteration had been considered as a pas- lates Mitochondrial Pyruvate Carrier 1 and Protects from Metabolic sive process to adapt to environmental change during differentiation, recent Dysregulation in ob/ob Mice studies suggested that the metabolic alteration rather actively regulates FARNAZ KEYHANI NEJAD, DONGHWAN LEE, HANQING CHEN, ZERONG LIANG, the differentiation process. It is known that the dynamic metabolic repro- NICOLAS MUSI, MENGWEI ZANG, San Antonio, TX gramming occurs during differentiation of preadipocytes, however its Our recent studies identify that retinoic acid receptor β (RARβ), a vitamin implication in adipogenesis is unknown. In this study, we employed mass A-related nuclear receptor, regulates mitochondrial fatty acid oxidation in spectrometry-based metabolomics and investigated the metabolic change hepatocytes and in vivo in a fasting-inducible manner, which contributes to during differentiation of 3T3-L1 preadipocytes. We found that the level of the metabolic adaptation to fasting. Mitochondrial pyruvate carrier (MPC) has nicotinamide adenine dinucleotide (NAD), a cofactor mediating redox reac- been recently identified to control gluconeogenesis in hepatocytes, which con- tion and protein modification, was significantly increased during the dif- tributes to the pathogenesis of diabetes. The present study aims to determine ferentiation. Consistently Nampt, a rate-limiting enzyme of NAD synthesis, whether RARβ is linked to the control of glucose metabolism. We observed was also upregulated in the early phase of adipogenesis. Further, we found hepatic downregulation of RARβ occurred in high fat, high sucrose (HFHS) diet- that pharmacological or genetic inhibition of Nampt reduced NAD synthesis fed mice and in genetically obese ob/ob mice. Treatment with all-trans-retinoic and repressed the metabolic reprogramming during adipogenesis. Notably, acid (RA), a natural ligand of RARβ and an approved drug for anticancer treat- inhibition of Nampt blocked the gene expression of Pparg and impeded the ment, at doses of 2.5- 5.0 mg/kg/day for 4 weeks lowered fasting glucose preadipocyte differentiation. The effect of Nampt inhibition was cancelled levels and ameliorated glucose intolerance and insulin resistance in HFHS- by the supplementation of nicotinamide mononucleotide, a precursor of fed mice, which was associated with increased hepatic gluconeogenic gene NAD. Our metabolomic analysis also revealed that TCA cycle intermedi-

ADA-Supported Research

A96 HUMAN OBESITY PATHOGENESIS—FROM BRAIN TO PERIPHERAL ORGANS ate, α-ketoglutarate (αKG) was upregulated during the differentiation and 0.11±0.14 (p<0.01). Cognitive performance improved in all subjects (MMSE: Nampt inhibition disturbed the rise of αKG. Interestingly, αKG is known as 30, range 28-30; MoCA: 28.5, range 24-30; both p=0.03 or less vs. baseline). a cofactor of the demethylation of histones. The ChIP assay revealed that There was no correlation between MoCA and metabolic changes though it αKG mediated the demethylation of H3K9me3 on Pparg promoter region and was positively correlated with NP post-RYGB (p=0.04). promote the gene expression of Pparg during adipogenesis. This process Conclusion: Morbid obese subjects have subclinical cognitive impair- was inhibited by Nampt inhibitor treatment, and was reversed by the supple- ments and brain plasticity alterations that significantly improve after RYGB. mentation of αKG. Altogether, our data indicated that Nampt-mediated NAD While no correlation was found between metabolic and hormonal changes, synthesis is necessary for differentiation of preadipocyte. NP was positively associated with MoCA score.

361‑OR 363‑OR CMHX008, a Novel Thiazolidinedione-Like PPARγ Partial Agonist, Effects of High BMI on Synaptic Function and Metabolic Connectiv‑ Enhances Insulin Sensitivity with Minor Influences on Bone Loss ity in the Brain—Evidence of Gender Difference XIAOQIU XIAO, YI HOU, XUEMEI CAO, JIAYU LI, Chongqing, China ARIANNA SALA, MAURA MALPETTI, ANNA FERRULLI, LUIGI GIANOLLI, LIVIO Objective: Traditional thiazolidinediones (TZDs), rosiglitazone for instance, LUZI, DANIELA PERANI, Milan, Italy, San Donato Milanese, Italy are peroxisome proliferator-activated receptor γ (PPARγ) potent agonists Several studies reported a significant association with obesity and cogni- and can treat type 2 diabetes but carry unwanted effects including increased tive deficits in aging, especially for executive, attention and memory domains. risk for fracture. Here we compared insulin-sensitizing efficacies and bone- The neural effects of obesity and the correlated cognitive dysfunction have loss side effects of CMHX008, a novel TZDs-like PPARγ partial agonist, with yet to be elucidated. We retrospectively collected a large cohort of healthy rosiglitazone. elderlies (N=222; age=74.03±5.88y; M/F=112/105; BMI range=19.21-38.79), Methods: TR-FRET PPARγ competitive binding assay was used to compare from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. All the binding affinity between CMHX008 and rosiglitazone. High fat diet fed subjects underwent in vivo positron emission tomography with 18F-Fluoro- and ob/ob mice were administered with vehicle, CMHX008 or rosiglitazone. deoxyglucose (FDG), thus we were able to evaluate the correlation between Bone marrow mesenchymal stem cells (BMMSCs) were used to examine dif- BMI, synaptic function and metabolic connectivity in the whole brain and ferences in differentiation into osteoblasts after treated with CMHX008 and within the resting-state neural networks, crucially considering the effect rosiglitazone. of gender. We found a significant positive correlation between BMI levels Results: TR-FRET showed lower affinity to PPARγ by CMHX008 compared and brain metabolism in female group only (R=.435; P<0.001), with high BMI with rosiglitazone. Mice treated with CMHX008 for 16 weeks had the com- associated with increased glucose consumption in the orbitofrontal cortex. parable effect of insulin sensitization as rosiglitazone, which was related Whole brain connectivity analysis revealed an altered pattern in frontal to significant inhibition on PPARγ Ser 273 phosphorylation and improved regions in females with high BMI, namely a decreased connectivity between insulin sensitivity by facilitating phosphorylation insulin receptor and AKT orbitofrontal regions and high-order prefrontal cortex, and an abnormally in adipose tissues. Micro-CT and histomorphometric analyses demonstrated elevated connectivity with nucleus accumbens. The connectivity analysis of that the degree of trabecular bone loss after treated with CMHX008 was the resting-state brain networks confirmed that in elderly females with high weaker than rosiglitazone as evidence by consistent changes in BV/TV, Tb.N, BMI, the core of vulnerability is within the frontal regions, with impaired Tb.Th, Tb.Sp, and mineral apposition rate. BMMSCs treated with CMHX008 connectivity in executive and salience networks. In contrast, elderly males showed higher ALP activity and mRNA levels of bone formation markers with high BMI showed loss of long-distance connections between frontal than those treated by rosiglitazone in osteoblast differentiation test. and parietal cortices, invariantly across attentional, executive and poste- Conclusion: PPARγ sparing eliminates the skeletal side effects of TZDs while rior default mode networks. Our findings support a strong detrimental effect maintaining their insulin-sensitizing properties. CMHX008 shared the compa- of high BMI levels on brain metabolism and neural connectivity in healthy rable insulin-sensitizing effects as rosiglitazone with lower risk of bone loss. aging, with gender differences. Notably, high BMI in females affects the Supported By: National Natural Science Foundation of China; Fundamental executive and reward systems. This evidence brings to considerations for Science and Advanced Technology Research of Chongqing medical practice and health policy.

364‑OR HUMAN OBESITY PATHOGENESIS—FROM BRAIN TO Brain Insulin Sensitivity Predicts Adiposity—Nine-Year Results of PERIPHERAL ORGANS the Tulip Study VERA SCHMID, OTTO TSCHRITTER, JÜRGEN MACHANN, ROBERT WAGNER, 362‑OR NORBERT STEFAN, HUBERT PREISSL, HANS-ULRICH HAERING, ANDREAS FRIT- Cognitive Function and Brain Plasticity in Obese Patients—The SCHE, MARTIN HENI, Tübingen, Germany, Stuttgart, Germany Impact of Bariatric Surgery Lifestyle intervention reduces body weight and prevents type 2 diabetes. ANGELA DARDANO, GIUSEPPE DANIELE, CLAUDIA LUNGHI, ANNAMARIA One crucial modulator of food intake and whole body energy metabolism CICCARONE, FERRUCCIO SANTINI, GIOVANNI CECCARINI, CARLO MORETTO, is insulin action in the brain. We now test whether brain insulin sensitiv-

GIUSEPPE PENNO, ROBERTO MICCOLI, MARIA CONCETTA MORRONE, STEFANO ity is a long-term predictor of weight and body fat distribution during and ORALS DEL PRATO, Pisa, Italy after a lifestyle intervention program. Before, after 9 months, at the end of Background and Aim: Obesity adversely affects brain function, but the lifestyle intervention at 24 months, as well as after 8.7 ±1.6 years follow- effect of bariatric surgery on cognitive function is still poorly evaluated. We up, participants of the Tuebingen Lifestyle Intervention Program underwent assessed metabolic parameters and cognitive function in obese patients a 75g OGTT, and whole body MRI. Brain insulin sensitivity was measured before and after bariatric surgery (RYGB). at baseline by changes in neuronal signals (theta frequency band) with Methods: A 75g OGTT was performed in 10 nondiabetic obese patients magnetoencephalography during a hyperinsulinemic-euglycemic clamp in a (BMI 46.0±5.4 kg/m2; age 43.6±10.5 years; HbA1c 43.3±4.6 mmol/mol) before subgroup of 28 participants of whom long-term follow-up was available in and 6-months after RYGB for measurements of metabolic parameters. Mini 15 persons. Higher brain insulin sensitivity before lifestyle intervention was Mental State Examination (MMSE) and Montreal Cognitive Assessment associated with a more pronounced reduction in total and visceral fat dur- (MoCA) were administered; neuronal plasticity (NP) of the visual cortex was ing the lifestyle intervention (all p<0.01). High brain insulin sensitivity was measured as change in ocular dominance after 120 min monocular depriva- also associated with less regain of fat mass during the long-term follow-up. tion. No subject with drugs or alcohol abuse, psychiatric illness, head injury, In contrast to visceral fat, changes in subcutaneous adipose tissue were brain tumor, dementia, epilepsy, learning disorder, developmental disability, independent of the brain insulin responsiveness. Our results extend previous and impaired sensory function were included. findings on the role of brain insulin for the success of lifestyle intervention Results: At baseline median MMSE score was 28.5 (range 25-30) and to a long-term perspective. Insulin action in the human brain appears to be median MoCA score 26.5 (range 21-30), indicating early cognitive abnor- not only important for weight loss, but seems to determine regain of body malities (MoCA<26 points). Six months after RYGB, BMI decreased to weight and especially total and visceral fat mass thereafter. Experimental 35.2±6.1 kg/m2 (p=0.004) and glucose tolerance did not change. HOMA-IR findings demonstrated that insulin administration to the brain modulates (from 5.1±2.3 to 1.6±1.1) and disposition index (from 3.3±4.6 to 35.0±41.0) peripheral energy fluxes in humans. In line, our results indicate fat depots all improved (p<0.05); post-OGTT GLP-1 increased from 5336.6±2262.6 to specific effects of brain insulin with particular benefits in the visceral com- 11132.3±3412.2 pmol/lx120min (p<0.05) and fasting plasma leptin decreased partment. These findings support the role of brain insulin in the regulation of from 80.0±46.7 to 13.1±6.6 pmol/l (p<0.008). NP increased from 0.03±0.1 to body fat distribution and body weight.

ADA-Supported Research

A97 HUMAN OBESITY PATHOGENESIS—FROM BRAIN TO PERIPHERAL ORGANS

365‑OR nomenon is important for T2D remission in human patients after RYGB, RL Hyperinsulinemia Invokes Greater Brain Perfusion in Reward and biopsies were obtained at the time of surgery and at 1 and 6 months post- Motivation Regions in Obesity operatively. Differentially expressed genes (DEG) were determined using DEREK GROSKREUTZ, RAJITA SINHA, CHERYL LACADIE, WAI LAM, MUHAM- Affymetrix HTA 2.0 microarrays; morphology was examined via electron MAD HAMZA, CHRISTIAN P. SCHMIDT, DONGJU SEO, JANICE HWANG, RENATA microscopy, revealing a significant increase in microvillus length over time BELFORT-DEAGUIAR, JESSICA LEVENTHAL, SAMUEL ROSENBERG, LISA PARIKH, (P<0.01). Enrichment analysis showed robust changes cellular proliferation, TODD CONSTABLE, ROBERT SHERWIN, ANIA M. JASTREBOFF, New Haven, CT cell cycle regulation, and cytoskeletal remodeling pathways at both time- Elevated insulin levels are often observed in obesity, but the effects of points. DEG suggest a metabolic shift away from fatty acid oxidation, toward hyperinsulinemia on cerebral blood flow (CBF) are not known. While insulin utilization of glucose and amino acids. Common DEG (FDR<0.05) at 1 and 6 acts as a satiety factor in lean humans, it is unclear if this CNS effect is months were strongly correlated (R2=0.899, P<0.0001), suggesting that RL maintained in the setting of hyperinsulinemia in obesity. This study aimed signatures are established early and persist. Interestingly, 91% of the top to isolate the effects of elevated insulin levels during euglycemia in lean 100 DEG at 6 months were downregulated; GSEA revealed significant enrich- (LN: n=6, BMI 21.1 kg/m2, age 32 years) and obese (OB: n=8, BMI 32, age 30) ment of genes regulating epigenetic reprogramming (P<0.01), e.g., DNMT1 humans. Participants underwent a 2 hour oral glucose tolerance test [mean and DNMT3B at 1 and 6 months. Remarkably, change in DNMT1 expression 120-min insulin LN 41 mU/ml vs. OB 113 mU/mL (p=.49) and glucose LN 101 at 1 month, controlling for baseline body weight, predicted 6-month HbA1c mg/dL vs. OB 112 mg/dL (p=.72)] and on 2 separate days received either a change (R2=0.744, P=0.002). This suggests a mechanistic role for methyla- saline infusion (control) or underwent a euglycemic-hyperinsulinemic clamp tion to underlie glycemic improvement, and points to a potential predictive (insulin 2 mU/kg/min, glucose 90 mg/dL) during functional magnetic reso- role for early RL DEG. We hypothesize that post-RYGB T2D improvement may nance imaging (fMRI). Whole-brain analyses comparing neural responses on relate to RL remodeling including increased energy utilization, and that the hyperinsulinemic vs. saline control days demonstrated increased CBF in the stimulus may be epigenetic reprogramming as a result of altered intestinal putamen, insula, anterior cingulate cortex, and middle temporal gyrus in the nutrient flow. OB group, but not in the LN group (p=.01, whole-brain corrected; Figure). We Supported By: Endocrine Fellows Foundation (to M.A.S.); Pediatric Endocrine conclude that obesity invokes different CBF responses to hyperinsulinemia, Society (to M.A.S.); National Institutes of Health specifically in regions implicated in impulsivity, reward processing, and interoceptive signaling. Thus, while in lean humans increased insulin levels 367‑OR signal satiety and reduce food-seeking behavior, this may not be the case in Glucagon-Like Peptide-1 (GLP-1) Increases Skeletal and Cardiac obese humans. Muscle, Microvascular Perfusion, and Improves Metabolic Insulin Figure. Action in Obese Humans NASUI WANG, ALVIN WAI KIT TAN, LINDA JAHN, LEE HARTLINE, KEVIN W. AYLOR, EUGENE BARRETT, ZHENQI LIU, Charlottesville, VA, Singapore, Singapore Obesity is associated with metabolic and microvascular insulin resistance. The latter is characterized by impaired insulin-mediated microvascular recruitment. GLP-1 engenders vasodilation in addition to its glycemic actions. At physiological concentrations, GLP-1 acutely recruits skeletal and cardiac muscle microvasculature in healthy humans and this action is preserved in the insulin resistant rodents. To examine whether GLP-1 recruits microvasculature and improves insulin’s action in obese humans, 15 obese adults (age 27 ± 2.2, BMI 33.7 ± 1.2) were studied thrice at random order after an overnight fast. Subjects received an intravenous infusion of GLP-1 (1.2 pmol/kg/min) for 150 mins or an intravenous infusion of either normal saline or GLP-1 (1.2 pmol/kg/min) for 150 mins with a euglycemic insulin clamp (1 mU/kg/min) superimposed over the last 120 mins. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity (MFV) and blood flow (MBF) were determined at 0, 30 and 150 min. Steady-state glucose infusion rate (GIR) was calculated. Insulin infusion alone did not change MBV or MBF in either skeletal or cardiac muscle, confirming the presence of microvascular insulin resistance. GLP-1 infusion alone increased MBV by ~30% and ~40% in skeletal and cardiac muscle respectively (p<0.05) without affecting MFV, leading to a significant increase in MBF in both skeletal and cardiac muscle. Superimposition of insulin infusion to GLP-1 infusion did not further increase MBV or MBF in either skeletal or cardiac muscle, but raised the steady-state GIR from 4.1± ORALS 0.4 to 5.0 ± 0.5 mg/kg/min (p<0.05). We conclude that in obese humans with microvascular insulin resistance GLP-1’s vasodilatory actions are preserved in both skeletal and cardiac muscle microvasculature. This may contribute to improving metabolic responses to insulin and decreasing cardiovascular morbidity and mortality associated with obesity and diabetes. Supported By: American Diabetes Association (1-17-ICTS-059 to Z.L.); National Supported By: National Institutes of Health Institutes of Health (R01HL094722, R01DK102359); National Natural Science Foun- dation of China (81300657) 366‑OR Intestinal Epigenetic Reprogramming Relates to Glycemic Improve‑ 368‑OR ment after Roux-en-Y Gastric Bypass Surgery Regulation of Lipocalin 2 by Estrogen in Human Adipose Tissue—A MARGARET A. STEFATER, COURTNEY PANCIOTTI, HENRY A. FELDMAN, WIL- Link between ERβ Pathway and Insulin Resistance LIAM F. GOURASH, ELEANOR SHIRLEY, ANITA COURCOULAS, NICHOLAS STY- PRASAD G. KAMBLE, MARIA J. PEREIRA, GRETHA J. BOERSMA, KRISTINA E. LOPOULOS, Boston, MA, Pittsburgh, PA ALMBY, JAN W. ERIKSSON, Uppsala, Sweden Despite the remarkable, early remission of T2D after RYGB, the surgery’s We recently showed that the adipokine lipocalin 2 (LCN2) causes insulin key mechanisms have yet to be defined in a way that can be translated to resistance in human adipose tissue (AT). Its expression is induced by dexa- less invasive therapies. Recently, we demonstrated that improved glucose methasone (Dex) in AT from pre-menopausal women, but not from post- homeostasis after RYGB in rodents is associated with intestinal metabolic menopausal women or men. We also found that males had higher levels reprogramming: the Roux Limb (RL) exhibits dramatic morphologic and of LCN2 mRNA than females. Together, this proposes a regulation of LCN2 metabolic remodeling, leading to augmented utilization of metabolic fuels by glucocorticoids and sex steroids. Subcutaneous (SC) AT from post-meno- to support the increased bioenergetic demand. To address whether this phe- pausal women (n=29, 67±7 Y, BMI 28±5 kg/m2) was incubated ± 17 β-hydroxy

ADA-Supported Research

A98 ALPHABET SOUP—GABA, FGF, ERA—RECIPE FOR CENTRAL NERVOUS SYSTEM CONTROL OF METABOLIC HOMEOSTASIS AND DIABETES REMISSION estradiol (E2, 0.01-100 nM) and ± Dex (0.3 µM) for 24 hours and LCN2 mRNA non-sense suppression-based, inducible genetic system is implemented to levels were measured. AT was co-treated with E2 (1 nM) and 100 nM and rapidly delete a specific group of LepR signaling from adult mice within 4 either estrogen receptor (ER) α or β antagonist, MPP or PHTPP, respectively, days. Acute deletion of LepR from AgRP neurons show a drastic increase of or with an ERβ agonist (DPN, 100 nM) for 24 hours. E2 (1nM) increased food intake, massive weight gain, and severe glucose intolerance, develop- LCN2 mRNA (p<0.01) and protein (p<0.05) levels by~3 fold in AT, which was ing in a rate with tight manifestation of the global leptin deficiency. Fur- reversed by ERβ antagonist. Notably, E2 in presence of MPP induced LCN2 thermore, a neural circuit comprising of LepR-expressing AgRP neurons and mRNA by 6 fold (p<0.05) compared to control. Likewise, ERβ agonist also downstream GABAergic neurons in the DMH is mapped by genetic and func- induced LCN2 mRNA by 3.5 fold (p<0.05). Next, E2 induced ERβ mRNA by 1.8 tional tracing approaches. Optogenetic manipulation of AgRPLepR-DMHGABA fold (p<0.05), but not ERα mRNA or protein levels. Dex alone did not change neural circuit or the post-synaptic DMHMC4R neurons strongly altered food LCN2 mRNA levels in AT from post-menopausal females. Dex reduced ERα intake and glucose tolerance in a bidirectional controlled manner. Among mRNA and protein levels by 20% (p<0.001) and 35% (p<0.01), respectively, all GABAA subunits examined, Gabra3 (encoding GABAA α3 subunits) dis- but increased ERβ mRNA by 220% (p<0.001). Dex-induced reduction in ERα played the most robust changes in response to genetic inactivation of Lep- mRNA levels was associated with BMI (r=0.569, p<0.01), waist circum- RAgRP signaling. Using CRISPR/Cas9 gene editing technique, loss of Gabra3 ference (r=0.565, p<0.01), fasting insulin (r=0.531, p<0.05) and c-peptide in DMHMC4R neurons reduced food intake and enhanced glucose intolerance. (r=0.560, p<0.01). In multivariate analysis BMI (std β coefficient = 0.626, Surprisingly, chronic infusion of bicuculline into the DMH fully reversed the p < 0.01; model: r 2 = 0.392) remained as significantly associated variable. obesity and glucose intolerance as a result of acute deletion of LepRAgRP In summary, E2 induced LCN2 expression in SCAT of postmenopausal signaling. Furthermore, genetic ablation of GABADMH neurons blunted leptin- women and this may be mediated via ERβ. Dex stimulation and inhibition of mediated control of glucose metabolism and food intake. ERα and ERβ respectively implies a selective glucocorticoid mechanism to In conclusion, we unveil a novel AgRPLepR-DMHGABA neural circuit and promote ERβ mediated transcriptional pathways. Overall, our data suggest associated GABAA receptor signaling system in fundamental control of that ERβ pathway may promote LCN2 expression and hence insulin resis- leptin-associated obesity and diabetes. tance in AT. Supported By: American Diabetes Association (7-13-JF-61 to Q.W.); National Institutes of Health (1R01DK109194, 1R56DK109194) 369‑OR Functional and Developmental Heterogeneity in Human Adipose 371‑OR Tissue Depots Microglia Activation Regulates Systemic Glucose Tolerance JAMES W. JOHNSON, CHERYL CERO, ALANA O’MARA, JOYCE D. LINDERMAN, JOHN DOUGLASS, MARTIN VALDEARCOS, SR., SUNEIL K. KOLIWAD, JOSH ALISON S. BASKIN, AARON CYPESS, Bethesda, MD THALER, Seattle, WA, San Francisco, CA Obesity results from the accumulation of excess white adipose tissue, Obesity and high fat diet (HFD) consumption in rodents is associated and its increasing rates worldwide pose a significant risk to overall health with hypothalamic inflammation and activation of microglia, the resident due to its multiple comorbidities, like type 2 diabetes. A novel treatment CNS immune cells. Recently, we provided evidence that microglia are criti- strategy is to increase energy expenditure through activation of brown and cal intermediary cells that transmit the inflammatory signal induced by HFD beige adipocytes, which can use uncoupling protein 1 (UCP1) for thermo- exposure. Mice with a conditional knockout of IKKb specifically in microg- genesis. In addition to having different developmental lineages, brown and lia (CX3CR1CreERT2 x IKKbeta fl/fl, MGKO) have reduced susceptibility to beige adipocytes differ functionally regarding the additional mechanisms by HFD-associated hyperphagia and DIO compared with IKKb fl/fl littermate which they generate heat. Most human fat depots are not easily accessible, controls (Ctl). Surprisingly, despite their reduced fat mass, HFD-fed MGKO so little is known about their developmental and functional characteris- mice have equal glucose intolerance to Ctls as well as no improvement in tics. In this study, we collected adipose tissue from 11 patients (age 16-84, white adipose tissue inflammation or hepatic triglyceride content. Together 4 female/7 male) who had undergone autopsy. We sampled from 7 anatomi- these data suggest dissociation between microglial regulation of energy and cally distinct fat depots: subcutaneous (sc), omental (om), retroperitoneal glucose homeostasis. To address this possibility, we pair-fed MGKO mice to (rp), pericardial (pc), periadrenal (pa), paraspinal (ps), and supraclavicular (sv). Ctl mice, resulting in matched body weights during HFD feeding. Pair-fed We measured mRNA levels of lineage and functional genes associated with MGKO mice showed a greater degree of impairment in glucose tolerance brown, beige, and white adipocytes. Energy storing white adipocytes were and insulin sensitivity with preserved insulin secretion compared to con- defined by high leptin expression, while energy expending brown and beige trols. Similarly, chemical inactivation of microglia using centrally-adminis- cells were defined by high UCP1; these were further distinguished as having tered minocycline worsens insulin sensitivity in HFD-fed rats. Finally, using a brown lineage using Zic1 and beige via Tbx1. Principal component analysis several distinct genetic models, we show that microglial activation acutely showed three different clusters of genes consistent with the brown, beige, improves glucose tolerance and insulin sensitivity. Together, these data and white lineages. Functionally, based on expression of leptin and UCP1 for reveal a novel aspect of CNS regulation of metabolism in which microglial white and brown fat, respectively, the sc and om were white (P<0.05), while inflammatory activation promotes HFD overconsumption and weight gain rp, pc, pa, ps, and sv were mostly brown (P<0.05). Regarding lineage of the but offsets obesity-associated dysregulation of glucose homeostasis. These UCP1+ depots, ps had both beige and brown cells, while rp, pa, ps, and sv results indicate the need for pathway-specific targeting to develop glial- were predominantly brown. based therapeutics for obesity and diabetes. In summary, human adipose tissue is not merely white or brown but instead Supported By: American Diabetes Association/Pathway to Stop Diabetes (1-14- ORALS is composed of many depots with distinct developmental lineages and func- ACE-51 to J.T.); Novo Nordisk tional capacities. These differences will be important when trying to use brown and beige adipocyte thermogenesis to treat obesity and diabetes. 372‑OR Supported By: National Institutes of Health An Action in the Hypothalamic Arcuate Nucleus Is Sufficient to Explain the Sustained Remission of Diabetes Induced by Central Administration of Fibroblast Growth Factor-1 (FGF-1) ALPHABET SOUP—GABA, FGF, ERA— JENNY M. BROWN, JARRAD SCARLETT, MILES E. MATSEN, ANNA SECHER, RECIPE FOR CENTRAL NERVOUS SYSTEM CONTROL RASMUS JORGENSEN, GREGORY J. MORTON, MICHAEL W. SCHWARTZ, Seat- OF METABOLIC HOMEOSTASIS AND DIABETES tle, WA, Måløv, Denmark REMISSION Our recent finding that a single intracerebroventricular (icv) injection of FGF-1 elicits sustained diabetes remission in rodent models of type 2 diabe- 370‑OR tes supports a growing consensus that the brain is a key target for diabetes drug development. The current work was undertaken to pinpoint the brain Deciphering a GABAergic Neural Circuit in Critical Control of area responsible for this action of FGF-1. To this end, we considered the Leptin-Mediated Feeding, Body Weight, and Glucose Homeostasis following preliminary findings: 1.) both FGF Receptor 1 and integrin recep- QI WU, YONG HAN, Houston, TX Leptin plays a major role in control of glucose metabolism and feeding tor αvβ3 (known to be involved in sustained FGF-1 signaling) are concen- behavior by acting upon the LepR neurons that widely distribute through the trated in the hypothalamic arcuate nucleus (ARC), 2) neuronal activation and brain. Although several LepR-expressing neuronal groups were identified sustained induction of the MAP Kinase/ERK pathway occurs in this brain in distinct nuclei, a dominant neural circuit that mediates the physiological area following icv FGF-1 injection, and 3) labeled FGF-1 is concentrated in roles of leptin remains elusive, largely due to the inadequate phenotypes as this area following icv injection. We therefore hypothesized that in rodent a result of developmental and neural compensations. In this report, a novel, models of T2D, an action in the ARC (a brain area known to participate in

ADA-Supported Research

A99 ALPHABET SOUP—GABA, FGF, ERA—RECIPE FOR CENTRAL NERVOUS SYSTEM CONTROL OF METABOLIC HOMEOSTASIS AND DIABETES REMISSION

glucose homeostasis) mediates the sustained diabetes remission elicited by accounts for the reduced glucose-sensing capability of these ERαvlVMH neu- icv FGF-1 injection. To test this hypothesis, we investigated whether a single rons; enhanced AMP kinase activity selectively in ERαvlVMH restores the microinjection of a low dose of FGF-1 into the ARC (0.3 µg bilaterally, for a glucose response in female mice depleted of estrogen. Thus, our results total of 0.6 µg) can mimic the response elicited by icv injection of a higher indicate that estrogen-ERα-AMP kinase signals in ERαvlVMH neurons are dose (3.0 µg). We report that in Zucker Diabetic Fatty rats, a single micro- required for these neurons to detect hypoglycemia and therefore are indis- injection of FGF-1 bilaterally into the ARC (via an indwelling dual guide can- pensable for defending against severe hypoglycemia in female mice. nula) induced remission of hyperglycemia lasting >3 week, whereas intra- Supported By: American Diabetes Association (1-17-PDF-138 to Y.H.) arcuate microinjection of saline vehicle was without effect (P<0.05 vs. Veh). Since icv injection of the same FGF-1 dose (0.6 µg) had no significant effect 375‑OR on glycemia, sustained diabetes remission induced by intra-ARC microinjec- A Single-Cell Transcriptomics Roadmap to Investigate Diabetes tion of FGF-1 cannot be explained by leakage into ventricular cerebrospinal Remission Induced by the Central Action of Fibroblast Growth fluid. We conclude that an action in the ARC is sufficient to explain the effect Factor-1 (FGF-1) of FGF-1 to induce sustained diabetes remission. MARIE A. BENTSEN, DYLAN RAUSCH, JARRAD SCARLETT, KIMBERLY M. Supported By: National Institutes of Health (DK101997, DK083042); Novo Nor- ALONGE, PASCAL N. TIMSHEL, ZAMAN MIRZADEH, ANNA SECHER, RASMUS disk A/S JORGENSEN, TUNE PERS, MICHAEL W. SCHWARTZ, Seattle, WA, Copenhagen, Denmark, Phoenix, AZ, Måløv, Denmark, Gentofte, Denmark 373‑OR Recently, lasting remission of hyperglycemia was achieved in rodent mod- Sustained Diabetes Remission Induced by the Central Action of els of type 2 diabetes (T2D) by a single intracerebroventricular (icv) injec- Fibroblast Growth Factor-1 (FGF-1) Requires Activation of Integrin tion of FGF-1. While the mechanism underlying this effect is unknown, the Receptor ανβ3 lack of association with changes of body fat mass or risk of hypoglycaemia JARRAD SCARLETT, JENNY M. BROWN, BAO ANH N. PHAN, EDDIE L. KHAV, raises the possibility that icv FGF-1 normalizes the defended level of glyce- MILES E. MATSEN, NIKHIL K. ACHARYA, HONG T. NGUYEN, ANNA SECHER, mia, rather than simply lowering blood glucose levels. Recent work from RASMUS JORGENSEN, GREGORY J. MORTON, MICHAEL W. SCHWARTZ, Seat- our lab has identified gluco-regulatory neurocircuits in the mediobasal hypo- tle, WA, Glendale, AZ, Måløv, Denmark thalamus (MBH) as targets for this FGF-1 effect. To investigate the mecha- The brain is implicated in the antidiabetic effects of members of the nism underlying FGF-1 action in this brain area, we used large-scale single fibroblast growth factor (FGF) peptide family. Previously, we reported that cell RNA-sequencing to identify and characterize FGF-1-responsive cells in a single intracerebroventricular (icv) injection of FGF-1 induces remission mouse MBH. Based on >70,000 single cell MBH transcriptomes from dia- of hyperglycemia that is sustained for weeks in rodent models of type 2 betic ob/ob mice harvested 5d after a single icv injection of either FGF-1 or diabetes (T2D). The integrin receptor ανβ3 plays a key role in FGF-1 sig- vehicle, we identified >20 cell clusters and >900 cell type-specific differen- naling, and crosstalk between ανβ3 and the FGF-1-FGF receptor (FGF-R) tially expressed genes (p<0.01). Among these potential targets of the MBH complex is necessary for increased DNA synthesis and proliferation in response to FGF-1 in diabetic mice, we found that FGF-1 treatment robustly cultured cells. These cellular responses downstream of FGF-1-FGFR-ανβ3 increased the proportion of newly formed oligodendrocytes, a response result from sustained (e.g., lasting for 5 hours) activation of the extracel- that can be predicted to yield new mature oligodendrocytes and associated lular signal-regulated kinase (ERK) signal transduction pathway. In cultured myelination of axons. These preliminary results demonstrate that transcrip- fibroblasts, prolonged ERK activation induced by FGF-1 is not replicated tional identification and characterisation of icv FGF-1-responsive cell clus- with a mutant FGF-1 peptide (R50E) that activates FGFR normally, but does ters may both advance our understanding of brain control of glucose homeo- not activate ανβ3 signaling. Similarly, we report that whereas icv FGF-1 stasis and identify novel targets for diabetes prevention and treatment. (3 µg) induces phosphorylation of ERK1/2 at both early (20 minute) and late Supported By: National Institutes of Health; Novo Nordisk A/S; Lundbeck Foun- (5 hr) time points in hypothalamic punches obtained from wild type mice, icv dation; Novo Nordisk Foundation injection of the same dose of R50E elicited this effect only at the early time point. Thus, R50E fails to activate hypothalamic FGF-1-FGFR-ανβ3 signaling in vivo. To investigate whether activation of ανβ3 is required for the prolonged antidiabetic effect of icv FGF-1, diabetic ob/ob mice received a single injection of either vehicle (Veh), recombinant FGF-1 (3 µg), or R50E (3 µg). Whereas R50E mimicked the effect of native FGF-1 to reduce food intake, body weight and blood glucose for the first week after icv injection (P<0.001 vs. Veh), the mutant peptide failed to elicit the sustained diabetes remission observed in animals receiving icv FGF-1. We conclude that signaling by the full FGF-1-FGFR-ανβ3 complex is necessary for the effect of FGF-1 signaling in the brain to elicit sustained remission of diabetic hyperglycemia in rodent models of T2D. Supported By: National Institutes of Health (DK114474, DK101997, DK083042, ORALS DK089056, DK035816)

374‑OR Estrogen-Responsive Neurons in the Ventrolateral VMH Regulate Glucose Balance YANLIN HE, PINGWEN XU, YAN XIA, YONGJIE YANG, XING CAI, ILIRJANA HYSENI, YONG XU, Houston, TX Brain glucose-sensing neurons can detect glucose fluctuations and prevent severe hypoglycemia. We observed that estrogen receptor-α (ERα)- expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamic nucleus (vlVMH) are all glucose-sensing neurons, being either glucose-inhibited neurons (GI-ERαvlVMH) or glucose-excited neurons (GE-ERαvlVMH). In particular, hypoglycemia inhibits GE-ERαvlVMH neurons vlVMH through opening the KATP channel, and activates GI-ERα neurons via enhanced t-type voltage-gated calcium currents. GI-ERαvlVMH neurons preferentially project to the medioposterior arcuate nucleus of the hypothalamus (mpARH) and GE-ERαvlVMH neurons preferentially project to the dorsal Raphe nuclei (DRN); selective activation of the ERαvlVMH◊mpARH circuit and inhibition of the ERαvlVMH◊DRN circuit both increase blood glucose. Importantly, loss of estrogen-ERα signals impairs glucose-sensing properties of ERαvlVMH neurons and attenuates hyperglycemic response to the central glucopenia in female mice. Depletion of estrogen impairs the AMP kinase pathway which

ADA-Supported Research

A100