COMPLICATIONS—HYPOGLYCEMIA

COMPLICATIONS—HYPOGLYCEMIA tolerance during 1g/kg glucose intraperitoneal glucose tolerance tests (p<0.05). During 160 minute hyperinsulinemic hypoglycemic clamp studies, HET and Guided Audio Tour: Hypoglycemia—From Basic Science to Private Practice HOM-KO mice needed signifi cantly less dextrose than controls (table 1). It is not (Posters: 388-P to 395-P), see page 19. clear yet whether this was due to reduced hepatic insulin sensitivity, increased CRR or both with plasma epinephrine responses being non-signifi cantly greater

& 388-P in HOM-KO than controls(1130 ± 364 vs 642 ± 137 pg/ml). Our data suggest that neuronal GK, presumably through its role in glucose- Achieving FPG Target Without Nocturnal Hypoglycemia: A Pooled POSTERS sensing, plays a central role in maintaining blood glucose. Complications Analysis of Studies in T2D Comparing Insulin Degludec vs. Insulin Acute and Chronic Glargine Table 1 BERNARD ZINMAN, JITEN VORA, MARCUS NIEMEYER, MARI-ANNE GALL, *=p<0.001 Genotype n Dextrose Infusion Plasma glucose CHANTAL MATHIEU, Toronto, ON, Canada, Liverpool, United Kingdom, Søborg, Rate (mg/kg/min) - at end of clamp Denmark, Leuven, Belgium last 20 minutes (mM) Insulin degludec (IDeg), a new basal insulin that forms soluble multi- of clamp hexamers after sc injection, has an ultra-long and stable glucose-lowering Clamp Study 1 HET-KO 8 7.2 ± 1.3* 2.57 ± 0.14 effect. These properties may lead to less nocturnal hypoglycemia compared to other basal insulin analogues, and should allow more patients to reach Controls 10 12.0 ± 2.7 2.69 ± 0.13 fasting plasma glucose (FPG) target safely. In this pooled analysis we investigated the proportion of patients with T2D Clamp Study 2 HOM-KO 7 4.9 ± 1.6 *** 2.87 ± 0.06 achieving target FPG (<90 mg/dL [5 mmol/L]) without nocturnal confi rmed hypoglycemia in 4 open-label, randomized, treat-to-target trials where Controls 12 13.3 ± 1.9 2.99 ± 0.07 patients (N=2380) received either IDeg or insulin glargine (IGlar), both once- daily in combination with OADs, for 26 or 52 weeks. Confi rmed hypoglycemia Supported by: Yousef Jameel MRC Wellcome Trust was defi ned as PG < 56 mg/dL (3.1 mmol/L) or severe episodes requiring assistance, and nocturnal confi rmed hypoglycemia defi ned as episodes from 00:01 to 05:59. & 390-P A greater proportion of patients achieved the FPG target, and fewer Simplifi cation of Insulin Regimens in Older Adults With Diabetes patients experienced nocturnal confi rmed hypoglycemia with IDeg than Decreases Hypoglycemia, while Maintaining Glycemic Control with IGlar (table). The chance of achieving FPG target without confi rmed MEDHA N. MUNSHI, ALISSA R. SEGAL, COURTNEY RYAN, CHRISTINE SLYNE, nocturnal hypoglycemia was 82% higher with IDeg: estimated odds ratio NORA SAUL, Boston, MA IDeg/IGlar = 1.82 [1.49; 2.22]95%CI. Older adults with diabetes on complex insulin regimens are known to have In conclusion, T2D patients are more likely to reach FPG target without high risk of hypoglycemia at all levels of glycemic control. We evaluated the nocturnal confi rmed hypoglycemia with IDeg than with IGlar. These fi ndings effect of simplifi cation of insulin regimen on frequency (number of excursions may have important implications for achieving target levels of glycemic <70) and duration (total time spent <70) of hypoglycemia measured by control in clinical practice. continuous glucose monitoring (CGM), and glycemic control (A1C) in older adults with type-2 diabetes. Thirty patients (mean age 77±6 years, duration Trial ID; Number (%) Number (%) Number (%) meeting Statistical diabetes 20±10 years and A1C 7.6±1%) taking ≥2 insulin injections and with N FAS IDeg/ meeting FPG target without nocturnal FPG target without Analysis ≥1 episode of hypoglycemia over 5-day CGM are enrolled. At present, 17 IGlar (<90 mg/dL) confi rmed nocturnal confi rmed Est. odds ratio patients have completed a simplifi cation protocol which entails switching hypoglycemia hypoglycemia to once daily glargine + other non-insulin agents. At study entry, patients IDeg IGlar IDeg IGlar IDeg IGlar IDeg/IGlar were taking average 3.4±1.3 insulin injections per day. Simplifi cation was 3579; 773/257 302 (39.1) 62 (24.1) 667 (86.3) 218 (84.8) 254 (32.9) 50 (19.5) 1.82 achieved in all patients; all receiving only one injection of glargine with 8 patients requiring one and 9 requiring 2 additional non-insulin agents. The 3672; 228/229 86 (37.7) 66 (28.8) 214 (93.9) 209 (91.3) 79 (34.6) 56 (24.5) [1.49; 2.22]95%CI number of hypoglycemic episodes declined in 15 (88%), with no episodes 3586; 289/146 124 (42.9) 53 (36.3) 231 (79.9) 111 (76.0) 100 (34.6) 40 (27.4) detected in 7 patients during CGM. The overall frequency of hypoglycemia 3668; 228/230 109 (47.8) 72 (31.3) 204 (89.5) 181 (78.7) 97 (42.5) 59 (25.7) decreased from 5.2±3 to 1.6±2 (p<0.001), and the overall duration of Total; 1518/862 621 (40.9) 253 (29.4) 1316 (86.7) 719 (83.4) 530 (34.9) 205 (23.8) hypoglycemia decreased from 339±314 to 106±188 (p<0.02) minutes after The endpoint was analyzed using a logistic regression model with logit link. The model included treatment, simplifi cation. Simplifi cation did not adversely affect A1C which was 7.4±1% sex, trial, antidiabetic treatment at screening, and region as fi xed factors, and age and baseline FPG as covari- at baseline and 7.3±1% after simplifi cation (p=NS). In 11 of 17 (65%) patients ates. Subjects were insulin-naïve in trials 3579, 3672, 3586. Trial 3668 included both insulin-naïve and non- A1C remained stable or improved, with mean lowering of 0.32%. In the 6 naïve subjects. In trial 3668, subjects in the IDeg fl exible-dosing group were excluded. N FAS = number of sub- patients whose A1C worsened after simplifi cation, 5 patients showed jects in the full analysis set. In all trials, subjects were treated with basal insulin once daily + OADs. improvement in the duration of hypoglycemia and the increase in A1C was ≤0.5% in 4 patients. Diabetes related distress measured by Problem Areas Supported by: Novo Nordisk, Inc. in Diabetes (PAID) improved in 13 of 17 patients and remained same in 1 patient after simplifi cation. In conclusion, simplifi cation of insulin regimens & 389-P in older adults with type-2 diabetes decreases hypoglycemia and diabetes- Brain Glucokinase Plays a Central Role in Blood Glucose Homeo- related stress, while maintaining glycemic control. stasis Supported by: Sanofi EMMANUEL O. OGUNNOWO-BADA, CHRISTINE RICHES, WILLIAM J. MARSH, JING XIA, JEFFREY W. DALLEY, MARK L. EVANS, Cambridge, United Kingdom Glucose sensing in specialised brain areas helps maintain blood glucose & 391-P by triggering counterregulatory responses (CRR) to hypoglycemia and/ Improved Hypoglycemic Accuracy, Alerts and Detection With the or controlling hepatic glucose fl ux. We investigated the role of brain G4 PLATINUM CGM System glucokinase (GK) in control of blood glucose by crossing mice expressing Cre THOMAS PEYSER, LUCAS BOHNETT, KATHERINE NAKAMURA, San Diego, CA recombinase under nestin promotor with GK-fl oxed mice to create neuronal Use of continuous glucose monitoring (CGM) devices improves glycemic GK knockout (KO) mice. We anticipated greater CRR during hypoglycemia control. Previous generations of CGM devices have reported acceptable and/or impaired glucose tolerance. performance, but accuracy in the hypoglycemic range has been a Mice were born at Mendelian ratios and studied 5-8 weeks old. Data shortcoming of earlier systems. We report here the accuracy of the Dexcom below are for heterozygote (HET-KO; nes-cre+/- GKlox/WT ) or homozygote KO G4 PLATINUM (DG4P) CGM in the hypoglycemic range and compare the (HOM-KO; nes-cre+/- GKlox/lox) compared with littermate controls (nestin- results to the previous generation. cre-/- or GKWT/WT). The performance of the DG4P was evaluated in a multicenter study with Confi rming a brain specifi c GK KO, ex vivo hypothalamic but not liver GK 72 diabetic subjects. CGM values were compared with Yellow Springs activity was reduced in HET-KO compared with controls (n=6-9, Vmax 10 vs Instrument (YSI) blood glucose measurements every 15 minutes. There were 13 mU/mg, p<0.01). As predicted, HET-KO mice showed impaired glucose 1,373 paired points with YSI values below 80 mg/dL. There were 83% of CGM

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A99 COMPLICATIONS—HYPOGLYCEMIA

values within 20 mg/dL of the YSI values, as shown in a density intensifi ed Bland-Altman Bias plot, compared to a 73% in the previous generation. & 393-P The true hypoglycemia alert rate is the percentage of time the CGM Hypoglycemia Increases Platelet Reactivity in Patients With Type alarmed when the YSI was at or below the alert setting within a 15 minute 2 Diabetes window before or after the event. The true alert rate for the DG4P at an 80 ELAINE Y.K. CHOW, AHMED IQBAL, EMMA WALKINSHAW, ALEXANDRA LUBI- mg/dL alert setting was 87%, a 16% increase to the previous generation. NA SOLOMON, REBECCA DALY, HEATHER M. JUDGE, ROBERT F. STOREY, SIMON Similarly, when the YSI indicated severe hypoglycemia (55 mg/dL or less), R. HELLER, Sheffi eld, United Kingdom the DG4P indicated biochemical hypoglycemia (70 mg/dL or less) 88% of Increased cardiovascular (CV) mortality has been reported in trials of POSTERS

Complications the time, a 15% increase to the previous generation. Improved accuracy intensive glycemic control in patients with Type 2 diabetes. There is strong

Acute and Chronic in the hypoglycemic range addresses a fundamental historic problem with evidence that hypoglycemia predicts subsequent mortality. We reasoned CGM and may lead to improved patient confi dence and glycemic control in hypoglycaemia might increase platelet reactivity, contributing to CV events patients using this technology. following an episode. Nine patients with type 2 diabetes completed paired hyperinsulinemic clamp studies separated by at least 4 weeks. Patients were on no antiplatelet drugs except aspirin in two. Glucose was maintained at hypoglycemia (2.5mmol/L) or euglycemia (6mmol/L) for two 60 minute periods. Platelet aggregation was measured at baseline, end of clamp, recovery,1 and 7 days later using whole blood impedance aggregometry (Multiplate) and whole blood single platelet counting (WBSPC). Collagen- and ADP-induced platelet aggregation (% change area under curve.minute) by impedance aggregometry increased at recovery after hypoglycemia (both p <0.01 vs euglycemia) and up to day 1 (Fig. 1). % Platelet aggregation assessed by WBSPC (3µM ADP) was also elevated at the end of hypoglycemia (51±35% vs 14±40%, p=0.04).These changes did not persist at day 7. We have shown for the fi rst time that moderate hypoglycemia, frequently observed clinically, can increase platelet reactivity substantially in individuals with Type 2 diabetes acutely and after the event. This could contribute to CV mortality during intensive glycemic therapy by triggering coronary thrombosis.

& 392-P The Adrenomedullary Epinephrine Response to Declining Plasma Glucose Concentrations Is a Signaling Event that Is Not Caused by a Decrease in the Cerebral Metabolic Rate of Glucose JO ANN V. ANTENOR-DORSEY, NADIA KHOURY, YI SU, ANGELA M. SHACKL- EFORD, KRISHAN G. JETHI, WILLIAM J. POWERS, PHILIP E. CRYER, ANA MARIA ARBELAEZ, St. Louis, MO, Chapel Hill, NC Declining glucose concentrations signal an array of physiological responses. The glycemic threshold for epinephrine secretion is ~3.6-3.9 mmol/L, while that for symptoms is ~2.8-3.0 mmol/L. The extent to which these responses are due to decreased brain glucose metabolism is unclear since the level of low circulating glucose that becomes limiting to brain glucose metabolism is not known. We hypothesized that declining glucose Supported by: NIHR concentrations signal an array of responses that are dependent on changes in brain glucose metabolism. We measured the global cerebral metabolic rate of glucose (CMRGlc) with [1-11C]glucose positron emission tomography in 394-P healthy young adults during two sets of 2-hour euglycemic-hyperinsulinemic WITHDRAWN glucose clamps: 1) ~5 and ~3.3 mmol/L (n=10) and 2) ~4.2 and ~2.5 mmol/L (n=7). Mean (+SE) fi ndings are described in table below. These data indicate that the glycemic threshold for a decrease in global CMRGlc is lower than that for an increase in adrenomedullary epinephrine secretion and approximates that for symptoms. Therefore, the epinephrine secretory response to declining plasma glucose concentrations is a signaling event that is not caused by a decrease in global cerebral metabolic rate of glucose.

Plasma glucose CMRGlc Epinephrine Symptom (mmol/L) (umol·100g-1·min.-1) (pmol/L) Score 5.3+0.1 25.9+0.7 431+142 2.9+0.5 4.2+0.05 23.5+0.7 320+138 3.1+1.3 3.5+0.05 25.7+0.7 1100+270b 5.8+1.4d 2.6+0.05 20.5+1.1a 3360+715c 11.2+3.3e As compared to ~5 mmol/L: ap<0.001; bp<0.05; cp<0.005; dp<0.04; ep<0.05 Supported by: Harold Amos Medical Faculty Development Program; Child Health Research Center

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A100 COMPLICATIONS—HYPOGLYCEMIA

& 395-P Interactions between Glucocorticoid Signaling and Insulin Mediate & 397-P the Impact of Recurrent Hypoglycemia on Hippocampal Function Incidence and Frequency of Patient-Reported Hypoglycaemic DANIELLE M. OSBORNE, EWAN C. MCNAY, Albany, NY Events in Spain Recurrent hypoglycemia (RH) initiates multiple compensatory mechanisms, DOMINGO OROZCO-BELTRAN, PEDRO MEZQUITA RAYA, ANTONIO RAMIREZ including several within the brain (e.g., alterations in hypothalamic glucose- DE ARELLANO, MANUEL GALAN, San Juan de Alicante, Spain, Almería, Spain, sensing). We have focused on the sequelae of RH in the hippocampus, which Madrid, Spain Hypoglycaemia is a common side effect of insulin therapy and can act as a is critically regulated by glucose availability and where we have previously POSTERS shown RH to alter function at cognitive, metabolic, and synaptic levels: barrier to optimal diabetes management with a negative impact on diabetes Complications these alterations include markedly impaired cognitive ability during further patients’ quality of life. However, few data on hypoglycaemia are available Acute and Chronic hypoglycemic episodes, with attendant increase in risk. Currently we are outside of a clinical trial setting. investigating the mechanisms of physiological changes that occur following We investigated patient-reported rates of non severe hypoglycaemic RH in the hippocampus. Given the high levels of glucocorticoid (GR) and events (NSHE), hypoglycaemia awareness and reporting hypoglycaemia to mineralocorticoid receptors (MR) in the hippocampus and corticosterone’s general practitioners (GPs) or specialists in Spain. Type 1 (T1) and insulin- (CORT) role in mediating the stress response, which is vital to controlling treated Type 2 (T2) diabetes patients (basal only, T2BOT; basal-bolus, T2BB; the body’s glycemic levels, we have focused on CORT and its receptors and other regimen, T2O) >15 yrs old were recruited via existing panels to as a possible mechanism in RH. Using hippocampal neuron cultures we complete four questionnaires at 7-day intervals. NSHE data were reported have found that CORT, both independently and synergistically with insulin, for a 7-day recall period; SHE were reported as events in the last year. NSHE signifi cantly increases pAMPKalpha, pERK1/2, pCREB, pAMPA receptor was an event with symptoms, with or without blood glucose measurement GluR1 subunit, and serum and glucocorticoid inducible kinase-1 (SGK1). (BGM), or low BGM without symptoms, which the patient could manage These fi ndings are supported by in vivo results. RH caused increases in without assistance. SHE was low BGM and help from a third party to translocation of hippocampal plasma membrane GR, insulin-responsive manage. glucose transporter-4, NMDAR2B, and AMPA receptor GluR1, without In total 630 patients (47% T1 and 53% T2) completed 2238 patient weeks altering total protein levels. There was also a signifi cant increase, following (table). RH, in total levels of SGK1 and a trend for increased MR in the hippocampus. Mean NSHE per patient week were 1.7 (T1), 0.4 (T2BOT), 0.8 (T2BB) and 0.6 Following these initial molecular-level fi ndings, we also report the role of (T2O) corresponding to 88, 18, 42 and 30 annual events respectively. Many GR and MR in mediating the cognitive impact of RH, using an experimental patients (33-56%) reported impaired, or unawareness of, hypoglycaemia and approach of antagonizing GRs and MRs in the hippocampus prior to RH 18-32% reported that they rarely or never communicate their hypoglycaemia in order to determine their role in mediating RH-induced changes in both to a GP/specialist. NSHE are a common occurrence amongst T1 and insulin- protein expression and spatial working memory. treated T2 patients in Spain and the real burden may be underestimated.

Guided Audio Tour: Hypoglycemia—The Barrier (Posters: 396-P to 403-P), see page 19.

& 396-P Cardiovascular Autonomic Neuropathy Predicts Severe Hypoglyce- mia in Patients With Type 2 Diabetes Mellitus: A Ten-Year Follow Up Study YU-BAE AHN, JAE-SEUNG YUN, HYUK-SANG KWON, JUNG-MIN LEE, SUNG- RAE KIM, JAE-HYOUNG CHO, YONG-MOON PARK, SEUNG-HYUN KO, Suwon, Gyeonggi-Do, Republic of Korea, Seoul, Republic of Korea We investigated whether cardiovascular autonomic dysfunction might infl uence the development of severe hypoglycemia (SH) in patients with Type 2 diabetes. From 2000 to 2002, cardiovascular autonomic function testing (AFT) was performed on 955 patients with type 2 diabetes, and these patients were followed-up in 2011 and 2012. Tests for AFT measured heart rate variability parameters (expiration-to-inspiration [E/I] ratio, responses to the Valsalva maneuver, and standing). AFT scores were determined from the results of the each test (0: normal, 1: abnormal). We used Cox proportional hazard regression analysis to test associations between the SH episodes and potential explanatory variables. The median follow up time was 9.5 years. At baseline, the mean age and diabetic duration of the total study population were 56.0 ± 10.0 years Supported by: Novo Nordisk, Inc. and 9.1 ± 6.4 years, respectively. The incidence of SH was 1.54 per 100 patient-years. The patients with SH had more ratio of female, were older, had a longer duration of diabetes and received more insulin and ACE & 398-P inhibitor treatment. Poor glycemic control, renal impairment, and diabetic HbA1c and Risk of Severe Hypoglycemia in Type 2 Diabetes: The microvascular complications also were more presented in the group with SH Diabetes & Aging Study at baseline. Cox hazard regression analysis revealed that the development KASIA J. LIPSKA, E. MARGARET WARTON, ELBERT S. HUANG, HOWARD H. of SH was associated with abnormal AFT score (Normal vs. mild autonomic MOFFET, SILVIO E. INZUCCHI, HARLAN M. KRUMHOLZ, ANDREW J. KARTER, dysfunction, HR 2.41, P = 0.005; normal vs. moderate to severe autonomic New Haven, CT, Oakland, CA, Chicago, IL dysfunction, HR 4.33, P < 0.001) in univariate analysis. After adjustment Clinicians may assume that risk of hypoglycemia is highest among type 2 for sex, age, diabetes duration, estimated glomerular fi ltration rate, HbA1c, diabetes (T2DM) patients whose HbA1c levels are lowest. We examined the treatment of insulin and ACE inhibitor/ARB, severe autonomic dysfunction association between HbA1c level and hypoglycemia in T2DM. predicted the development of SH (Normal vs. moderate to severe autonomic T2DM patients aged 30-75 years on anti-hyperglycemic therapy were dysfunction, HR 2.43, P = 0.004) and higher AFT score tended to have higher asked to report severe hypoglycemia requiring assistance within the risk of the development of SH (P for trend = 0.015). previous year in the Diabetes Study of Northern California (DISTANCE) The development of SH was independently associated with cardiovascular survey conducted 2005-2006. Poisson regression models were specifi ed to autonomic dysfunction in patient with type 2 diabetes. examine relative risk (RR) of hypoglycemia across HbA1c levels, and were adjusted for age, sex, race, comorbidities, renal function, DM duration, history of prior hypoglycemia, polypharmacy, and DM medication category. Interactions between HbA1c level and potential effect modifi ers (age, DM medication category, DM duration) were tested.

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A101 COMPLICATIONS—HYPOGLYCEMIA

Among 9,094 eligible survey respondents (mean age 59.5 ± 9.8 years, HbA1c 7.5 ± 1.5%, DM duration 10.6 ± 8.4 years, 22% on insulin), 985 (10.8%) & 400-P reported severe hypoglycemia in the past year. The RR of hypoglycemia Severe Hypoglycemia and Adverse Events in Older People With or across HbA1c categories in the fully adjusted model is shown in the Figure. Without Diabetes: Population-Based Cohort Study The HbA1c-hypoglycemia relationship was not signifi cantly modifi ed by age, SUMIT R. MAJUMDAR, BRENDA R. HEMMELGARN, MENG LIN, KERRY baseline DM medication or DM duration. MCBRIEN, BRADEN J. MANNS, MARCELLO TONELLI, Edmonton, AB, Canada, Self-reported severe hypoglycemia was common among T2DM patients Calgary, AB, Canada in a large integrated healthcare system. Hypoglycemia frequently occurred Little is known about the prognostic impact of hypoglycemia that is POSTERS

Complications across all levels of glycemic control, but the risk tended to be highest in suffi ciently severe to require hospitalization. We hypothesized that such

Acute and Chronic patients with near-normal glycemia and very poor control. severe hypoglycemia would be associated with increased long-term morbidity and mortality, irrespective of diabetes status. Therefore, we undertook a cohort study using linked administrative healthcare and laboratory databases in Alberta, Canada. From January 1, 2004 to March 31, 2009 we included all outpatients 66-years and older who had at least one serum creatinine and one A1c measured. To examine the independent association between hospitalization for severe hypoglycemia and all-cause mortality we used time-varying Cox proportional hazards (adjusted hazard ratio [aHR]) and for all-cause hospitalizations we used Poisson regression (adjusted incidence rate ratio [aIRR]). Results were based on a fi nal cohort that included 85,810 patients: mean age 75-years, 51% female, and 50% had diabetes defi ned by administrative data. Overall, 440 patients (0.5%) had severe hypoglycemia and most (93%) had diabetes. During 4-years follow-up, 16,320 (19%) patients died. Severe hypoglycemia was independently associated with increased mortality (60% vs 19% mortality for no hypoglycemia, aHR 2.55, 95%CI 2.25-2.88), and this increased in a dose-dependent manner (aHR for no hypoglycemia=1.0 vs 1 episode=2.49 vs 1 or more=3.78, p-trend<0.001). Severe hypoglycemia was also independently associated with subsequent hospitalizations (aIRR for no hypoglycemia=1.0 vs 1 episode=1.90 vs 1 or more=2.61, p-trend<0.001) and recurrent hypoglycemia (aHR for no hypoglycemia=1.0 vs 1 episode=2.45 vs 1 or more=9.66, p-trend<0.001). In conclusion, we found that older people who have an episode of severe hypoglycemia are easily identifi ed and at substantially increased risk of morbidity and mortality, and that this risk is Supported by: NIA (T32AG1934) independent of diabetes or its treatments.

& 399-P & 401-P Hypoglycemia, Not Glycemic Variability, Predicts Adverse Hospital Negative Binomial Regression Analysis of Hypoglycemia Data from Outcomes Diabetes Clinical Trials: The Effect of Adjusting for Baseline Hypo- YOOJIN KIM, KUMAR B. RAJAN, SHANNON A. SIMS, KRISTEN E. WROBLEWS- glycemia Rates KI, SIRIMON REUTRAKUL, Chicago, IL JUNXIANG LUO, SCOTT J. JACOBER, MELVIN J. PRINCE, MICHELLE A. CAREY, Increased glycemic variability (GV) is associated with adverse hospital YONGMING QU, Indianapolis, IN, Blue Bell, PA outcomes in critically ill patients, but data in non-critically ill patients Baseline hypoglycemia rates are generally not collected or included as a is lacking. We conducted a retrospective review of inpatient medical covariate in statistical models used for analyzing hypoglycemia data. The admissions to explore the association between GV, measured as % objective was to examine the effect of adjusting for baseline hypoglycemia coeffi cient of variation (%CV), and hospital outcomes. Outcomes included ICU on estimation effi ciency in a negative binomial regression (NBR) model. Data transfer, hospital acquired infections (HAI), acute renal failure (ARF), death, were collected from 15 randomized, controlled insulin trials (≥8 weeks) with composite outcomes (≥ 1 of outcomes), and length of stay (LOS). We also available baseline hypoglycemia data (2796 subjects, 52% male, mean age examined if GV correlation with outcomes is independent of hypoglycemia. 57 years, mean BMI 29.9 kg/m², mean A1C 8.9%) in patients with type 1 We analyzed 1,276 admissions in which insulin was given, with ≥ 6 point diabetes mellitus (T1DM, n=210), type 2 diabetes mellitus (T2DM) and insulin- of care glucose (POC) values and LOS between 2-30 days (mean 5.4± 3.8 treated prior to randomization (T2DM-insulin treated, n=1511) or T2DM and days). A total of 30,652 POC glucoses were obtained: mean 186.3± 48.6 mg/ insulin-naïve (n=1075). There was a moderate correlation between baseline dL and %CV 34.2± 11.1. One or more hypoglycemic events (POC glucose <50 and postbaseline hypoglycemia rates (nocturnal and total) in patients with mg/dl) occurred in 12.5% patients. ICU transfer occurred in 3.3%, HAI 4.8%, T1DM or T2DM-insulin treated (Table 1). Adjusting for baseline hypoglycemia ARF 8.3%, death 0.9% and composite outcomes 13.8%. Analyses adjusting reduced the standard error (SE) of NBR for patients with T1DM or T2DM- for age, sex, race and Charlson score found that every 10 unit increase in insulin treated (Table 1). Simulations confi rmed that adjusting for baseline %CV was associated with an increase in LOS of 0.27 days (p<0.01) and reduced the SE for NBR and increased testing power in hypoglycemia ARF [OR 1.19 (95% CI 1.00-1.42), p=0.06]. The association with ARF became analysis. These fi ndings suggest that 1) baseline hypoglycemia rate is non-signifi cant after adjustment for hypoglycemia. For LOS, there was signifi cantly correlated with postbaseline hypoglycemia rate for patients a signifi cant interaction between %CV and presence of hypoglycemia. with T1DM or T2DM-insulin treated, and 2) adjustment for baseline Although there was no correlation between %CV and LOS in patients hypoglycemia may improve the estimation effi ciency for hypoglycemia data without hypoglycemia, %CV and LOS were inversely correlated in patients analyses in clinical trials. with hypoglycemia (p<0.05). The probability of hypoglycemia increased with increasing %CV. When considered simultaneously with %CV, hypoglycemia was signifi cantly associated with adverse outcomes, including increased LOS of 3 days for those with average %CV (p<0.001), HAI [OR 2.99 (1.39- 6.43), p<0.01], death [OR 5.46 (1.02-29.2), p<0.05] and composite outcomes [OR 1.75 (1.06-2.90), p<0.05]. Our results show that hypoglycemia, but not GV, is predictive of adverse outcomes in hospitalized, non-critically ill patients.

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A102 COMPLICATIONS—HYPOGLYCEMIA

Hospital. A retrospective review was performed of patients receiving Table 1 alerts from 7/9-12/9/2012. Alerts were answered Monday through Friday, Population n Hypoglycemia n Hypoglycemia Correla- % 0700-1700. Preventable SH events (events that occurred after the alert (baseline) ratea (postbaseline) ratea tionb Change was generated) occurred in 2.6% (21/818) intervention patients versus (baseline) (postbaseline) of SEc the expected rate of (82/818) (p<.001). This refl ects a 74% reduction in SH Nocturnal T1DM 210 0.44±1.02 210 0.84±1.23 0.46* -6.8 attributable to the nurse-driven process. The total rate for SH was 5.6% T2DM insu- 1477 0.31±1.24 1477 0.26±0.68 0.37* -6.0 (46/818) alerted patients versus the expected rate of 82/818 (p<.001). This refl ects a 44% reduction in overall SH, acknowledging that alerts were not

lin-treated POSTERS T2DM insu- 932 0.01±0.15 936 0.09±0.63 0.00 1.1 activated from 1701-0659. This alert process contributed to the overall Complications lin-naïve reduction in hospital SH rates. The 2011 12-month rate was 1.31 per 1000 Acute and Chronic patient days (42 events per month) compared to 2012 with 1.08 per 1000 Total T1DM 210 3.74±4.14 210 6.74±6.86 0.63* -11.8 patient days (26 per month). T2DM insu- 1493 0.79±2.00 1511 1.70±2.83 0.42* -2.2 Conclusion: A real-time informatics alert plus trained nurse responders lin-treated signifi cantly decreased SH events. The ability to respond in real-time to the T2DM insu- 1075 0.09±0.59 1075 0.56±1.62 0.10† 0.6 event is critical to the success of this intervention. lin-naïve Supported by: Barnes-Jewish Foundation aPer patient per 30 days (mean±SD) bCorrelation between hypoglycemia rate at baseline and hypoglycemia rate 404-P during treatment. cThe study sample size weighted average change in standard error after A Novel Glucagon Analogue, ZP-GA-1, Displays Increased Chemical adjustment for baseline hypoglycemia rate. and Physical Stability in Liquid Formulation *p<0.0001. DITTE RIBER, FRANCESCA MACCHI, LISE GIEHM, METTE S. ANDERSEN, TOR- †p=0.0013. BEN ØSTERLUND, PIA NØRREGAARD, ANDERS VALEUR, TRINE S.R. NEERUP, Glostrup, Denmark Supported by: Eli Lilly and Company Glucagon is a peptide hormone produced in response to low blood sugar levels. Pharmaceutically, glucagon is used for emergency treatment of & 402-P severe hypoglycemia in diabetic subjects taking insulin. Glucagon possesses Comparative Preclinical Safety-Pharmacology Study of Soluble poor solubility in aqueous buffers at or near physiological pH, and is also Non-Aqueous Glucagon (G-Pen™) vs. Lilly Glucagon™, for Treatment notorious for having poor chemical and physical stability. of Severe Hypoglycemia We here present data for ZP-GA-1, a novel glucagon analogue, which BRETT J. NEWSWANGER, JACQUES PAPPO, STEVEN J. PRESTRELSKI, Austin, displays increased solubility and stability, while retaining high potency at TX the glucagon receptor. Severe hypoglycemia remains a signifi cant unmet medical need. Currently The physical stability of ZP-GA-1 in solution has been tested over a period approved rescue products (Lilly, Glucagon™ for Injection; Novo Glucagen®) are of 14 days at 40°C (pH 7.5, with agitation) at concentrations of 1 and 5 mg/ based on lyophilized formulations which require reconstitution, complicating ml. No aggregation was detected as measured by thiofl avin T and tryptophan ease of administration in emergency situations. Xeris has developed a fl uorescence and turbidity. ZP-GA-1 also exhibits improved chemical stability simple, ready-to-use, soluble glucagon formulation based on biocompatible, over native glucagon in a simple liquid formulation. non-aqueous solvents that effectively suppress the fi brillation of glucagon The effect of ZP-GA-1 on acute blood glucose release in rats was observed in aqueous solutions. The G-Pen™ formulation has demonstrated investigated and compared to native glucagon. ZP-GA-1 (60 nmol/kg) induced superior chemical stability after 6 months of storage at room temperature. an increase in blood glucose reaching a maximal response at t = 30 minutes Comparative pharmacology studies in a rodent model (n = 10/sex/group) before returning to baseline levels 90 minutes after s.c. injection. demonstrated that at half the dose, subcutaneously (SC) injected G-Pen™ ZP-GA-1 thus demonstrates superior physicochemical properties over glucagon resulted in similar (p > 0.05) pharmacokinetic endpoints (AUC, native glucagon along with a comparable pharmacodynamic profi le. These Cmax, Tmax, and T1/2) to 5 µg SC doses of the Lilly Glucagon™ reference studies suggest ZP-GA-1 as a potential candidate for the treatment and/or drug. Injection of 2.5 µg G-Pen™ glucagon showed rapid absorption (Tmax ~ prevention of severe hypoglycemia in the form of a ready-to-use rescue kit 5 min), and marked elevation of blood glucose levels in male rats (TBGmax or in an artifi cial pancreas. ~ 11.5 min). BGmax was normally distributed (p=0.2007) between groups. There was a proportional relationship between the G-Pen™ glucagon injection dose (2.5 µg, 5 µg, or 50 µg) and the measured plasma glucagon concentrations. In a concurrent 14-day GLP safety/toxicology study, there were no signifi cant histopathological fi ndings indicative of toxicity. Overall these data support the development of G-Pen™ as a ready-to-use rescue drug for severe hypoglycemia, as well as a glucagon formulation suitable for a bi-hormonal (insulin-glucagon) infusion pump. Supported by: NIH/NIDDK (2R44DK085809-02)

& 403-P Implementation of a Real-Time Informatics Alert With Clinical De- cision Support for Prevention of Severe Hypoglycemia ELIZABETH S. PRATT, MICHAEL ELLIOT, MARY CLARE BLACKBURN, STEPHEN J. SCHAFERS, KEVIN HEARD, JOHN LYNCH, COREEN VLODARCHYK, JANET MCGILL, RACHEL KILPATRICK, GARRY S. TOBIN, St. Louis, MO Inpatient severe hypoglycemia (SH), blood glucose (BG) <40 mgs/dl, is 405-P associated with morbidity. We previously developed and tested a predictive Duration and Impact of Hypoglycemic Events With Insulin Degludec model in research to identify and intervene in the inpatients at risk for and Insulin Glargine—A Meta-Analysis SH with a nurse-driven process. The alert was triggered when a patient STEWART B. HARRIS, JITEN VORA, TORSTEN E. CHRISTENSEN, RAHUL KAPUR, exceeded a specifi ed informatics generated risk score and experienced a BG MERYL BROD, London, ON, Canada, Liverpool, United Kingdom, Søborg, Denmark, <90 mg/dl in the setting of active diabetes therapy. The nurse and provider Mill Valley, CA collaborated, utilizing an algorithm, to identify a safe diabetes regimen in Insulin degludec (IDeg) is a new-generation, ultra-long-acting basal order to prevent SH. This alert process, tested on 12 acute care units in 2011, insulin, with a distinct mechanism of protraction resulting in a fl at and has a positive predictive value of 10% for the development of SH. Initial stable pharmacokinetic profi le and a low rate of hypoglycemia. However, results yielded a 68% reduction in SH on the intervention units. little has been published regarding the duration and consequences of these This successful model was implemented hospital-wide over a fi ve month hypoglycemic events. period. The research team trained 58 nurse experts in the alert process This meta-analysis comprised 3 treat-to-target, open-label, non-inferiority covering 26 acute care and fi ve progressive care units at Barnes- Jewish trials (26-52 wks duration) comparing IDeg and insulin glargine (IGlar) in

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A103 COMPLICATIONS—HYPOGLYCEMIA

1,925 insulin-naïve patients with type 2 diabetes on a basal insulin-only age was 58.8 (10.9) yr; duration of diabetes 9.6 (7.0) yr; BMI 31.9 (6.4) kg/ regimen (where there was no infl uence from bolus insulin). Across these m2; 40% were female; 23% insulin users. At baseline, 43.9% had never had trials, rates of overall confi rmed hypoglycemia (plasma glucose <56mg/dL a hypo;27.5% had a hypo in the past 4 weeks (50.1% & 22.8% of 1028 non- or severe requiring assistance) were signifi cantly lower for IDeg vs IGlar insulin users, and 22.9% & 43.5% of 301 insulin users respectively); 85.2% (rate ratio 0.83, 95%CI: [0.70; 0.98]). Patients were interviewed to discuss of those with hypo self-managed their most recent,4.7% required healthcare their hypoglycemic events at each visit; if more than one event was reported professional help. Among those with baseline HbA1c values (n=393), those only the last event was analysed to minimize recall bias. The interview without hypo in the 4 weeks prior to baseline reported lower HbA1c than captured: time to recognize event, duration of event, time to recover, any those with ≥1hypo (7.11% (1.39) vs. 7.51% (1.60), p <0.01). Patients with hypo POSTERS

Complications contact to healthcare professionals (HCP) and impact on usual activities. in the 4 weeks prior to baseline also reported more worry and more hypo-

Acute and Chronic 6,065 events were recorded and analysed using an ANOVA model controlling avoidance behaviors on the HFS, as well as higher total hypo fear at baseline for covariates. (see Table). There were no statistical differences in the characteristics of a In conclusion, subjects in this study who reported experiencing hypo also hypoglycemic event between IDeg and IGlar for any of the parameters reported higher HbA1c values and higher hypoglycemia fear (worry and tested. Time to recognize: 7.2 min for both IDeg and IGlar. Duration: 26.4 vs behavior). 28.2 min for IDeg vs IGlar. Time to recover: 34.2 vs 32.4 min for IDeg vs IGlar. 35.4% of patients reported that the event had an impact on daily activities with IDeg vs 33.0% for IGlar. After an event 9.2% (IDeg) vs 10.2% (IGlar) contacted a HCP. In conclusion, rates of confi rmed hypoglycemia were 17% lower with IDeg vs IGlar and there were no differences in terms of recovery time, impact on daily activities or use of healthcare resources. Thus, the ultra-long-acting profi le of IDeg did not affect the duration or impact of a hypoglycemic event vs IGlar. Supported by: Novo Nordisk, Inc.

406-P Can Diabetes Alert Dogs Truly Detect Hypoglycemia? 408-P DANA S. HARDIN, JENNIFER CATTET, WESLEY ANDERSON, ZACHARY The Effect of Recurrent Hypoglycemia on Cerebral Electrical SKRIVANEK, Indianapolis, IN Activity in Patients With Type 1 Diabetes and Hypoglycemia Un- Our group previously documented the positive impact of a diabetes alert awareness dog (DAD) on a diabetes patient’s quality of life. To our knowledge, no ANNE-SOPHIE SEJLING, TROELS W. KJÆR, ULRIK PEDERSEN-BJERGAARD, LINE randomized controlled studies were published regarding the ability of DADs S. REMVIG, ANINE LARSEN, MARTIN N. NIELSEN, LISE TARNOW, BIRGER THOR- to detect hypoglycemia. Our current work aimed to test DADs in 2 controlled STEINSSON, CLAUS B. JUHL, Odense, Denmark, Copenhagen, Denmark, Hillerød, trials. Denmark, Lyngby, Denmark, Gentofte, Denmark, Esbjerg, Denmark Four service dogs from the Indiana Canine Assistance Network previously Episodes with hypoglycemia induce changes in the spontaneous trained in both basic and advanced obedience and mobility assistance were electrical activity of the brain as measured by electroencephalogram (EEG). placed in a hypoglycemia alert training program. The training introduced the While repeated exposure to hypoglycemia attenuates counterregulatory dogs to perspiration and breath samples from patients with type 1 diabetes, and symptomatic responses, the effect on the EEG is not known. This is followed by positive reinforcement for successful recognition (alert) of particularly relevant in patients with type 1 diabetes and hypoglycemia hypoglycemic (low) samples. For the purposes of the current 2 studies, unawareness who are exposed to frequent episodes of hypoglycemia. samples were placed in separate cups on a randomization device (Lazy Suzan Thirteen patients with type 1 diabetes (7 men, age 58±9 years (mean±SD), wheel). One cup contained the low sample, 3 contained normoglycemic duration of diabetes 32±10 years, HbA1c 7.9±1.0 %) and hypoglycemia samples from the same patient, and 3 contained gauze without samples. unawareness (assessed by a validated method) were recruited. The patients In both studies, 4 separate wheels, containing samples from 4 separate were studied by hyperinsulinemic hypoglycemic clamp on two consecutive patients were used and dogs (study 1 n=2, study 2 n=4) replicated the search days. EEG was recorded at euglycemia (blood glucose 5.3±0.7 mmol/l on 2 times/wheel. The placement of the samples was based on a pre-specifi ed day 1 and 5.6±0.6 mmol/l on day 2) and at hypoglycemia (2.3±0.2 mmol/l randomization scheme. on day 1 and 2). Sensitivity (proportion of correct alerts) and specifi city (proportion of sniffs EEG was recorded by a 10-20 cap system. Data from C3 and P3 were without alert on normoglycemic samples) were calculated after pooling data analyzed during standardized rest periods by quantitative EEG analysis with across all trials in both studies (2 dogs participated in both studies). Overall, respect to absolute amplitude of the theta band, centroid frequency of the the best dog performed at 71% (percent) sensitivity / 90% specifi city. The alpha band and the peak frequency of the unifi ed theta-alpha band. dog with the poorest performance was 22% sensitivity / 75% specifi city. The amplitude of the theta band increased during hypoglycemia when This variability in dog performance refl ects differences in training level and compared to euglycemia on both days (p<0.01 and p<0.001). Hypoglycemia other individual animal characteristics. associated changes in theta band amplitude, alpha centroid frequency and Our results demonstrate that DADs are able to identify chemical com- theta-alpha peak frequency did not differ between day 1 and 2. pounds specifi c to hypoglycemia and therefore be trained to alert to its Hypoglycemia-induced EEG changes were not affected by antecedent presence. We are continuing studies to elucidate best practices for training hypoglycemia in patients with impaired hypoglycemia awareness. This DADs and ultimately to identify the chemical signature that the dogs are is in contrast to the well-known adaptation of counterregulatory and detecting. symptomatic responses. Supported by: Eli Lilly and Company 409-P 407-P Does CSII Early Morning Insulin Programming Increase Hypoglyce- Hypoglycemic Events and Glycemic Control among Adults With mia? Type 2 Diabetes in the United Kingdom (UK) MATTHEW BOUCHONVILLE, ELIZABETH DURAN-VALDEZ, DAVID S. SCHADE, BETH MITCHELL, JEFFREY VIETRI, ANTHONY ZAGAR, MATTHEW REANEY, Albuquerque, NM BRADLEY CURTIS, Indianapolis, IN, Milan, Italy Programming insulin pumps to increase early morning insulin delivery to The aim of this research was to assess if hypoglycemic episodes (hypo) minimize the Dawn Phenomenon is a common practice in T1DM. However, are associated with poorer control of blood glucose as measured by self- if the Dawn Phenomenon is minimal or absent, dangerous early morning reported HbA1c levels, in adults with type 2 diabetes. Members of an hypoglycemia may result. Our study compared the frequency of early internet survey panel in the UK identifi ed as having diabetes and currently morning hypoglycemia in adult well-controlled T1DM subjects on continuous taking prescription anti-hyperglycemic medication participated in a series subcutaneous insulin infusion (CSII) who programmed an increase in insulin of monthly surveys. Information on hypo in the prior 4 weeks, HbA1c level, delivery between 3:00 am and 7:00 am with similar T1DM subjects who the Hypoglycemia Fear Survey (HFS), and other measures, were collected. did not program an increase in insulin delivery to counteract the Dawn Baseline was completed by 1329 subjects (response rate 19%). Mean (SD) Phenomenon.

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A104 COMPLICATIONS—HYPOGLYCEMIA

Twenty eight T1DM from the community were studied on multiple the POCT machines (to track those that did or did not recheck within the occasions (n=323) over an eight month period with overnight continuous allotted time), email feedback from the lab to those who failed to recheck glucose monitoring to determine the incidence of hypoglycemia and the within the allotted time, and a weekly dashboard listing compliance rates Dawn Phenomenon. Twenty of these subjects normally programmed an (by unit) and non-compliant users (by name). These interventions resulted in increase in early morning insulin delivery to combat the Dawn Phenomenon signifi cant improvements in the rates of blood glucose rechecks within 30 and eight did not. Hypoglycemia was defi ned as a continuous glucose and 60 minutes (Table), such as decreasing the time for recheck POCT BG<70 monitoring glucose level less than 70 mg/dl between 3 a.m. and 8 a.m. mg/dL. The established policies/protocols may be used by other hospitals to and the Dawn Phenomenon as an increase from nadir to pre-breakfast in improve their process of blood glucose re-testing. POSTERS continuous glucose monitoring glucose greater than 10 mg/dl. Table. POCT for BG<70mg/dL and BG<40mg/dL, Pre and Post JC-ADA Certifi - Complications We observed 101 hypoglycemic events and 112 Dawn Phenomenon cation Acute and Chronic events out of a total of 323 overnight observations. T1DM subjects who Dates March-April May-June Pre Visit March-April May-June Post Visit P-Value programmed an early morning increase in insulin delivery had a signifi cant 2011 2011 JC-ADA 2012 2012 JC-ADA reduction in the incidence of the Dawn Phenomenon (37%) compared to non- (March-June (March-June programmers (48%) (p<0.01) but also had an increase in the incidence of 2011) 2012) hypoglycemia (37%) compared to non-programmers (15%) (p<0.01). Glucose #Total POCT BG 80,106 78,574 158,680 72,042 71,779 143,821 levels less than 50 mg/dl (severe hypoglycemia) were observed in 44% of all hypoglycemic episodes. #BG<70mg/ 1,744 1,830 3,574 1,532 1,573 3,105 0.081 Programming an increase in early morning insulin delivery with CSII dL(%) (2.18%) (2.33%) (2.25%) (2.13%) (2.19%) (2.16%) results in a reduction in the incidence of the Dawn Phenomenon but also #BG<40mg/ 195 175 370 163 189 352 0.515 increases the incidence of hypoglycemia. This hypoglycemia may be more dL(%) (0.24%) (0.22%) (0.23%) (0.23%) (0.26%) (0.24%) hazardous to the T1DM patient than the hyperglycemia induced by the Dawn #BG Retests in 644 612 1,256 1,266 1,290 2,556 <0.001 Phenomenon. 30 minutes(%) (36.93%) (33.44%) (35.14%) (82.64%) (82.01%) (82.32%)

410-P #BG Retests in 1,190 1,229 2,419 1,441 1,479 2,920 <0.001 Does Severe Hypoglycemia Predict Total and/or Cause-Specifi c 60 minutes(%) (68.23%) (67.16%) (67.68%) (94.06%) (94.02%) (94.04%) Mortality in Long Duration Type 1 Diabetes (T1D)? The Pearson chi-square test was used and statistical analyses were done GEORGIA PAMBIANCO, TREVOR J. ORCHARD, Pittsburgh, PA with SAS 9.3. A past history of severe hypoglycemia has recently been shown to be associated with increased mortality in patients with type 2 diabetes. 412-P However, little is known about how a history, and/or the frequency of severe Association between Hypoglycemia and Inpatient Mortality, hypoglycemia events, may affect overall mortality in type 1 diabetes. Length of Hospital Stay and Readmissions in Diabetic Hospitalized Data from the Pittsburgh Epidemiology of Diabetes Complications Patients (EDC) study of childhood onset T1D (mean age 28 and duration 19 years, ANGEL ASENJO, BARBA RAQUEL, NOEMI GONZALEZ PEREZ DE VILLAR, RICAR- n=658) were thus examined to determine if self reported history of severe DO GOMEZ-HUELGAS, JESUS CANORA, SONIA GONZALO, JUAN HINOJOSA, hypoglycemia at baseline (1986-1988, n=626) or subsequent frequency of JUSTO RUIZ, ANTONIO ZAPATERO, Madrid, Spain, Málaga, Spain severe hypoglycemic events, is associated with total and/or cause-specifi c Objective: To assess the impact of hypoglycemia on clinical outcomes (CAD, Renal Disease) mortality assessed up to 2011. The frequency of severe among internal medicine hospitalized diabetic patients. hypoglycemia events was restricted to subjects attending the third exam Methods: In a retrospective study, hospitalizations between 2005-2010 in cycle (1990-1992, n=443) at which point the question was fi rst introduced Internal Medicine wards in Spain were identifi ed from an inpatient electronic into the study. Severe hypoglycemia was defi ned as unconsciousness: database. All diabetic admissions were included, except for those with a requiring external assistance and/or having a low blood sugar reaction (<50 length of stay <24 hours. In an analysis, associations between hypoglycemic mg/dl) without recognizing it. episodes and inpatient mortality, readmissions (<30 days), and length of Death was confi rmed by death certifi cate and classifi ed according to stay were evaluated. Those diabetics (ICD-9-MC: 249.00-251.99) who had Diabetes Epidemiology Research International (DERI protocol) by a physician a secondary diagnosis of hypoglycemia were analyzed (ICD-9-MC codes committee. Multivariable analyses were adjusted for baseline diabetes 251.2, 250.30, 250.31, 250.80, 250.81, 249.30, 249.31, 249.80, 249.81). duration, gender and the number of study visits completed. A history of Results: Among 921,306 diabetic admissions, hypoglycemia occurred in ever experiencing severe hypoglycemia was not independently related to 46408 (5%). Inpatient mortality occurred in 10.2% of hospitalizations with a total or cause-specifi c mortality, with >70% of decedent and non-decedent hypoglycemic event, and in 9.5% in patients without event (OR 1.08 CI95% participants reporting a past history of the event. 1.07-1.11). However, the frequency of biennial severe hypoglycemia events during In multivariate logistic regression analyses adjusting for age, gender, and follow up, was an independent predictor of both total (HR=1.25, 1.1-1.4) and selected comorbidities, hypoglycemia was associated with a signifi cant renal-related (HR=1.46, 1.1-1.8), but not CAD-related, mortality (HR=1.15, increase in inpatient mortality risk (adjusted odds ratio (OR)=1.04 [95% .95-1.4), adjusting for diabetes duration and number of study visits. Why CI: 1.02, 1.07]). Length of stay was increased in hospitalizations in which frequency of severe hypoglycemia relates to total and renal-related, but not hypoglycemia was reported (mean 10.8 days vs. 9.9 days; p<0.0001). CAD-related, mortality in type 1 diabetes warrants further investigation. Readmission rate was also higher in those patients (16.3% vs 15.4%, OR 1.06 CI95% 1.03-1.09). 411-P Limitations: Due to the nature of the data source, some data were not Improvements in Blood Glucose Re-Testing after Hypoglycemia in a available including type and dose of antidiabetic treatment, outpatient Medical University Hospital Setting medical histories (including diabetes history and comorbidities), KATHIE L. HERMAYER, DANIELLE B. SCHEURER, PAMELA C. ARNOLD, MARY- cardiovascular risk, pre-hospitalization medications, and cause of death ELIESE MERRILL, YUSHENG ZHU, JUANITA A. EPPS, ANGELA B. STRICKLAND- Conclusions: Hypoglycemia was common among diabetic hospitalized HEDGPETH, AMY HUTTO, AMY R. WILSON, DAWN ROBERTSON, PATRICK J. patients and, while a direct causal relationship cannot be assumed, it was CAWLEY, Charleston, SC associated with an increased risk of inpatient mortality, increased number A signifi cant patient safety concern worldwide is inpatient hypoglycemia, of readmissions and increased length of hospital stay. and its subsequent treatment and re-evaluation. The Medical University of South Carolina (MUSC) instituted a multi-pronged process improvement 413-P plan for improving the lag time between Point of Care Testing (POCT) blood The Effect of RAS Blockade on Cognitive Function, Hormonal Coun- glucose (BG)<70 mg/dL, and recheck within 30 minutes, for patients with ter-Regulation and Symptom Responses during Hypoglycemia in and without diabetes, as part of The Joint Commission American Diabetes Patients With Type 1 Diabetes Association Certifi cation (JC-ADA) for Advanced Inpatient Diabetes Care. LOUISE H. FÆRCH, BIRGER THORSTEINSSON, LISE TARNOW, JENS J. HOLST, MUSC is a 700+ hospital bed facility. The process improvement plan ULRIK PEDERSEN-BJERGAARD, Hillerød, Denmark, Gentofte, Denmark, Copenha- included changes to policies/protocols to refl ect the need for 30 minute gen, Denmark rechecks, nursing/ancillary education on how and when to recheck blood High activity of the renin-angiotensin system (RAS) is associated with glucose, timers on all nursing units, badge cards for all personnel using increased risk of severe hypoglycemia in type 1 diabetes. High spontaneous

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A105 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

RAS activity as opposed to low RAS activity is suggested to result in more pronounced cognitive impairment during mild hypoglycemia which in turn & 415-P may confer an increased risk of severe hypoglycemia in type 1 diabetes. Omega-3 Docosahexaenoic Acid (DHA) Not Only Inhibits LPS- We studied if RAS blockade with an angiotensin 2 receptor (AT2R) inhibitor Induced IL-6 Expression, but Also Attenuates Synergistic Stimula- in subjects with type 1 diabetes and high RAS activity improves cognitive tion of IL-6 by LPS and Palmitic Acid in Macrophages function during mild insulin-induced hypoglycemia. JUNFEI JIN, XIAOMING ZHANG, ZHONGYANG LU, YANCHUN LI, MARIA F. LOPES- Nine patients with type 1 diabetes and high RAS activity were included VIRELLA, YUSUF A. HANNUN, YAN HUANG, Charleston, SC, New York, NY in a double-blind, randomized, balanced, crossover study of the effect of Increasing studies have reported that omega-3 polyunsaturated POSTERS

Complications 32 mg candesartan or placebo for one week on cognitive function, counter- fatty acids (PUFAs) have benefi cial effects on cardiovascular disease in

Acute and Chronic regulatory hormonal response and symptoms during two hyperinsulinemic diabetic patients. However, the underlying mechanisms have not been hypoglycemic clamps separated by at least 4 weeks. We measured cognitive well elucidated. Since it is known that infl ammation plays an important function in terms of reaction time (CALCAP), selective attention and cognitive role in cardiovascular disease and that patients with type 2 diabetes have fl exibility (Stroop’s test) and information processing (Trailmaking test). increased plasma lipopolysaccharide (LPS) activity and saturated fatty Compared to placebo, candesartan did not change performance of acids (SFAs), we postulated that SFA augments infl ammatory response CALCAP, Stroop’s or trailmaking tests at a plasma glucose concentration of of macrophages to LPS, but omega-3 PUFAs antagonize LPS/SFA-induced 2.6 (±0.16 and ±0.19) mmol/l, respectively. We did not see any difference infl ammatory response. In the present study, we found that 100 microM of in counter-regulatory hormones (adrenaline, cortisol, growth hormone and palmitic acid (PA), the most abundant SFA in diabetic patients, augmented glucagon (all p>0.1)) or substrates (ketones, glycerol, alanine, FFA or lactate LPS (1 ng/ml)-induced interleukin (IL)-6 secretion (258 ± 7 vs. 139 ± 14 pg/ (all p>0.1) on the two experimental days. We did neither fi nd any changes in ml) from RAW264.7 macrophages. Similar observation was also made autonomous (p=0.068) nor in neuroglycopenic symptomscores (p=0.57). in human monocyte-derived macrophages. In contrast, omega-3 PUFA We conclude that candesartan had no effect on consequences of or docosahexaenoic acid (DHA) not only inhibited LPS-induced IL-6 secretion responses to experimental mild hypoglycemia in subjects with type 1 by 53 ± 6% (mean ± SD of 3 experiments), but also attenuated the diabetes and high RAS activity. synergistic effect of PA and LPS on IL-6 secretion by 72 ± 9% (mean ± SD of 3 experiments). Our further studies showed that LPS and PA had a synergistic effect on ceramide production (Control: 6.7, LPS: 6.9, PA: 21.6; LPS + PA: COMPLICATIONS—MACROVASCULAR— 28.4 pmol/nmol phosphate), but DHA inhibited LPS/PA-increased ceramide ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND production by 46% (Control: 6.7; LPS + PA: 28.4; LPS + PA + DHA:18.4 pmol/ HUMAN DIABETES nmol phosphate). Finally, our studies showed that blockade of ceramide production by myriocin or fumonisin B1, inhibitors of ceramide synthesis, signifi cantly reduced the effect of PA and LPS on IL-6 expression, indicating Guided Audio Tour: Cardiovascular Disease Risk and Detection in Diabetes an important role of ceramide in IL-6 upregulation. Taken together, this (Posters: 414-P to 421-P), see page 15. study demonstrates that omega-3 DHA not only inhibits LPS-induced IL-6 expression, but also attenuates synergistic stimulation of IL-6 expression by & 414-P LPS and PA by reducing ceramide production in macrophages. Supported by: U.S. Dept. of Veterans Affairs; NIH (DE016353) Reduced Paraoxonase 1 Activity Contributed to Dysfunctional HDL in Patients With Type 2 Diabetes HIROSHI MURAKAMI, JUTARO TANABE, YUKI KIMURA, KOKI MATSUMURA, & 416-P MAKI YAMASHITA, KOTA MATSUKI, HIROSHI MURAKAMI, JUN MATSUI, NAOKI Previous Cardiovascular Event Predicts Severe Hypoglycemia in TAMASAWA, Hirosaki, Japan Patients With Type 2 Diabetes Mellitus We revealed the mechanism of dysfunctional HDL in patients with type SEUNG-HYUN KO, JAE-SEUNG YUN, YONG-MOON PARK, HYUK-SANG KWON, 2 diabetes, illustrating the relationship between the cholesterol effl ux JUNG-MIN LEE, SUNG-RAE KIM, SANG-AH CHANG, JAE-HYOUNG CHO, YU-BAE capacity and paraoxonase1 (PON1) activity. PON1 is seen mostly in the HDL AHN, Suwon, Gyeonggi-Do, Republic of Korea, Seoul, Republic of Korea particle and protect the HDL particle from oxidation. We investigated whether previous cardiovascular event, defi ned as (1) Subjects were patients with type 2 diabetes admitted to our hospital for a myocardial infarction, heart failure, resuscitation from ventricular diabetic control and education (n=36) and controls without diabetes (n=9). (2) tachycardia or fi brillation, angina, or need for coronary revascularization, Plasma HDL fraction was separated by PAGE and protein recovery equipment might infl uence the development of severe hypoglycemia (SH) in patients (Jokor). (3) We investigated HDL-mediated cholesterol effl ux considering with Type 2 diabetes. both the quality (Effl ux-hdl) and quantity (Effl ux-whole) of HDL particles. From 2000 to 2002, patients aged 25-75 years with type 2 diabetes Cholesterol effl ux capacity was determined as reported previously. Effl ux- without chronic kidney disease (estimated glomerular fi ltration rate (eGFR) whole was calculated using the ApoA-I concentration of HDL fraction, which ≥ 60 mL/min/1.73m2) were enrolled (n = 894) and these patients were evaluates total cholesterol effl ux in the native HDL fraction. (4) PON1 activity followed-up in 2011 and 2012. SH was defi ned as hypoglycemic episodes in both HDL fraction and plasma were determined using the paraoxonase requiring hospitalization or medical care in an emergency department. We measuring kit (JaICA). used the Cox proportional hazard regression analysis to test associations (1) Effl ux-hdl in the patients with diabetes was signifi cantly decreased between the SH episodes and potential explanatory variables. 7.3% compared with controls. 624 patients completed a follow-up evaluation, and the median follow-up This 7.3% decrease was equivalent of 26.4% decrease of Effl ux-whole. (2) period was 9.5 years. At baseline, the total study population consisted of PON1 activity in the HDL fraction in patients with diabetes was positively 372 (59.6%) women with mean age of 55.5 ± 9.9 years. Duration of diabetes correlated with Effl ux-hdl (p=0.019), and showed negative tendency with was 8.9 ± 6.3 years. The incidence of SH was 1.31 per 100 patient-years. The HbA1c (p=0.10). (3) Plasma PON1 activity was signifi cantly reduced in patients with SH were older, had a longer duration of diabetes and received patients with diabetic retinopathy (p=0.036) and negatively correlated more insulin, and had more diabetic microvascular complications at baseline. with urine albumin (p=0.073). Plasma PON1 activity also showed negative The Cox hazard regression analysis revealed that the development of SH was correlation with mean IMT (p=0.016) and PWV (p=0.006), respectively. associated with a history of cardiovascular events (HR 3.81; 95% CI 1.81 - In this study, we found HDL particle in patients with diabetes could lose 8.01, P < 0.001) and ischemic stroke (HR 2.47, 95% CI 1.20 - 5.09; P = 0.014) in effl ux capacity owing to reduced PON1 activity, which inversely correlates univariate analyses. After adjusting for sex, age, DM duration, eGFR, HbA1c, with chronic high blood glucose levels (HbA1c). Plasma PON1 activity were treatment of insulin or ACE inhibitor/ARB, previous cardiovascular events well correlated with diabetic angiopathy. These results mean reduced PON1 still remains the risk factor of SH (HR 2.29, 95% CI 1.06 - 4.98; P = 0.036) activity drives HDL remodeling, leading to dysfunctional HDL, and this may The development of SH was independently associated with previous explain the pathogenesis of diabetic angiopathy. cardiovascular events in patients with Type 2 diabetes. Therefore, more clinical attention and diabetic education are needed in patients with type 2 diabetes who have a history of cardiovascular events.

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& 417-P & 419-P Microalbuminuria, but Not Reduced GFR, Is Independently Associ- Presence of Albuminuria can Determine Eligible Patients With Type ated With Arterial Stiffness and Carotid Plaque in Patients With 2 Diabetes Who Need to be Undergone Coronary CT Angiography Type 2 Diabetes KWANG JOON KIM, SANGHOON SHIN, HYUK-JAE CHANG, BYOUNG-WOOK EUN SOOK KIM, EUN YOUNG MO, SUNG DAE MOON, JE HO HAN, Incheon, Re- CHOI, EUN SEOK KANG, BYUNG-WAN LEE, Seoul, Republic of Korea public of Korea Object Controversy still exists regarding the screening of subjects with Background: Both abluminuria and reduced estimated glomerular fi ltration diabetes mellitus (DM) for coronary artery disease (CAD). The hypothesis POSTERS

rate (eGFR) have been shown to increase the cardiovascular disease in of this study states that presence of albuminuria can determine eligible Complications addition to end stage renal disease. This study investigated the independent subjects for the screening of CAD, evidenced by coronary computed Acute and Chronic association between albuminuria and GFR with atherosclerotic vascular tomography angiography (cCTA), in patients with DM. changes in patients with type 2 diabetes. Methods This study included 3,971 asymptomatic subjects (DM: 302, Methods: A cross-sectional analysis was performed among 712 subjects 7.6%). An 1:3 matching between DM and non-DM subjects was performed with type 2 diabetes. GFR was estimated by the Modification of Diet in by a propensity score calculated using age, gender, BMI, past history. Renal Disease formula and albuminuria was assessed by urinary albumin- Albuminuria was defi ned as albumin-to-creatinine ratio greater than to-creatinine ratio (ACR). Arterial stiffness was assessed by brachial-ankle 30mg/g, and the risk of coronary atherosclerosis [coronary artery calcium pulse wave velocity (PWV) and carotid intima-media thickness (IMT) and score (CACS) >10, CACS >100, the presence of CAD and obstructive CAD plaque grade were assessed by B-mode ultrasonography. (greater than 50% stenosis)] was calculated in subgroup of non-DM, DM Results: Mean PWV and plaque grade linearly increased according without albuminuria, and DM with albuminuria. to albuminuria status whereas no differences were observed in IMT. In Results We selected 1510 subjects (1208 non-DM vs. 302 DM, propensity- contrast, reduced eGFR (< 60 mL/min/1.73 m2) was not associated with any score matched) and all the baseline characteristics were similar. DM markers including PWV, IMT, and plaque grade. Albuminuria was signifi cantly patients withour albuminuria had similar prevalence of CAD compared with associated with atherosclerotic vascular changes, with microalbuminuria non-diabetic patients. However, the prevalence of CAD was signifi cantly and macroalbuminuria increasing high PWV (odds ratio 1.17 [95% confi dence increased in DM patients with albuminuria. (OR: 4.33, 95% CI: 2.09-8.95) interval 0.68-2.00] and 3.14 [1.31-7.59], respectively; P for trend = 0.039) and Conclusions The presence of albuminuria remarkably contributes the plaque presence (1.90 [1.13-3.20] and 2.80 [1.19-6.61], respectively; P for risk of coronary atherosclerosis in asymptomatic DM. Screening for CAD trend =0.009) after adjustment for conventional cardiovascular risk factors. could be considered in patients with asymptomatic DM, who present with Further adjustment for eGFR did not change the signifi cance. In multivariate albuminuria. linear regression analysis, ACR was independently associated with PWV (β = 0.14, P < 0.001) and plaque grade (β = 0.08, P = 0.007). Conclusion: Microalbuminuria is associated with arterial stiffness and plaque grade independently of reduced eGFR in subjects with type 2 diabetes. Long term follow-up studies are needed to clarify if microabluminruia has a greater impact on the development and progression of cardiovascular disease over reduced eGFR.

& 418-P Fibrin Clot Structure in Type 1 Diabetes: No Gender Differences but Relations to Microangiopathy SARA TEHRANI, GUN JÖRNESKOG, ANNA ÅGREN, PER-ERIC LINS, HÅKAN & 420-P WALLÉN, ALEKSANDRA ANTOVIC, Stockholm, Sweden Serum Omentin Signifi cantly Predicts Cardiovascular Events Both Patients with type 1 diabetes have a tighter fi brin network formation, in Patients With the Metabolic Syndrome and in Subjects Who Do which may contribute to their increased risk of cardiovascular disease Not Have the Metabolic Syndrome (CVD). We investigated the fi brin clot structure in patients with type 1 CHRISTOPH H. SAELY, ANDREAS LEIHERER, AXEL MUENDLEIN, ALEXANDER diabetes in relation to gender as well as to risk factors of CVD. A total of VONBANK, DANIELA ZANOLIN, KATHRIN GEIGER, PHILIPP REIN, HEINZ DREXEL, 235 patients (129 males) with no history of CVD were included. Mean age Feldkirch, Austria, Triesen, Austria, Philadelphia, PA and diabetes duration were 45±13 (mean±SD) and 22±14 years, respectively. Some recent small cross-sectional studies have described associations Fibrin clot structure was assessed by determination of the permeability of the novel adipocytokine omentin with atherosclerosis. However, no coeffi cient (Ks). Turbidimetry clotting and lysis assays were performed to prospective data on the power of omentin to predict cardiovascular events further explore the fi brin polymerization rate, clot density and fi brinolysis. are available. Ks did not differ between men and women with type 1 diabetes (10.4±4.0 We therefore measured serum omentin in a series of 297 patients vs 10.8±3.9 cm2x10-9, p=0.5, respectively). Women (<50 years) had as tight undergoing coronary angiography for the evaluation of established or fi brin clot as age matched men (11.5±4.2 vs 11.3±4.2 cm2x10-9, p=0.8). No suspected stable CAD; the metabolic syndrome (MetS) was defi ned gender differences were found regarding P-fi brinogen levels or other fi brin according to national cholesterol education programme adult treatment clot characteristics. Women had worse glycemic control (HbA1C 7.2±1.5 vs panel III criteria; cardiovascular events were recorded over a mean follow- 6.8±1.2 % in men, p=0.02, Mono-S) despite lower BMI (24.3±3.9 vs 25.5±3.6 up period of 3.2 years. kg/m2 in men; p=0.02). Prevalences of retinopathy and microalbuminuria During the follow-up period, 18.4% of our patients suffered cardiovascular were 62 and 22% in men vs 64% and 29% in women (ns). Patients with events, corresponding to an annual event rate of 5.8%. In the total study retinopathy and microalbuminuria had lower Ks levels (10.1±3.7 and 9.7±4.0 population, serum omentin signifi cantly predicted cardiovascular events cm2x10-9) compared with those without retinopathy and microalbuminuria, both univariately (standardized adjusted HR 1.47 [1.21-1.78]; p <0.001) respectively (11.5±4.2 and 11.0±3.9 cm2x10-9, p<0.05 for both). Ks was related and after adjustment for age, gender, BMI, diabetes, hypertension, LDL to P-fi brinogen (r=-0.6, p<0.001) with similar correlations in men and women. cholesterol, HDL cholesterol and smoking (HR 1.49 [1.21-1.82]; p<0.001). No other relevant correlations were found between Ks and the investigated From our patients, 98 had the MetS and 199 did not have the MetS. In both biochemical variables. The present study shows that women with type 1 of these patient subgroups serum omentin strongly predicted cardiovascular diabetes have as tight fi brin clot as men at the same age. This fi nding may events both univariately (HRs 1.51 [1.15-2.00]; p=0.003 and 1.41 [1.08-1.84]; in part explain why the protective effect of female gender on cardiovascular p=0.011, respectively) and after adjustment for age, gender, BMI, diabetes, morbidity is abolished in patients with type 1 diabetes. Notably, a tighter hypertension, LDL cholesterol, HDL cholesterol and smoking (1.56 [1.09- fi brin network was found in patients with microangiopathy. 2.25]; p=0.016 and 1.48 [1.12-1.97]; p=0.006, respectively). From this fi rst prospective evaluation of the cardiovascular risk associated with serum omentin we conclude that elevated serum omentin is a strong predictor of cardiovascular events both among patients with the MetS and among subjects who do not have the MetS.

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A107 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

a similar incidence of hypopneas events was present in diabetic and non- & 421-P diabetic patients (18.3±4.2 vs. 16.7±5.3 e/h; p=0.951). By contrast, both the Novel Biomarkers Appear Consequence of Atherosclerosis in Type rate of apneas and CT90 were signifi cantly increased in diabetic patients 1 Diabetics (15.2±2.8 vs. 10.3±1.9 e/h; p=0.048, and 18.0±6.7 vs. 10.3±7.7 %; p=0.042, KELLY J. HUNT, NATHANIEL L. BAKER, RICHARD L. KLEIN, PATRICIA A. CLEARY, respectively). No differences between the rate of obstructive and central GABRIEL VIRELLA, MARIA F. LOPES-VIRELLA, THE DCCT/EDIC RESEARCH GROUP, apneas were observed. In addition, diabetic patients also showed higher Charleston, SC, Rockville, MD daily sleepiness (7.5±3.1 vs 6.0±4.7, p=0.012) than non-diabetic subjects. In We measured biomarkers in DCCT/EDIC participants with type 1 diabetes conclusion, T2DM patients have a higher rate of apneas than non-diabetic POSTERS

Complications at DCCT baseline and approximately 16 to 18 years later (EDIC years 10 subjects, leading to more severe nocturnal hypoxemia and daily sleepiness.

Acute and Chronic to 12) and studied their association with internal carotid intima-medial Supported by: CIBERDEM; CIBERES; FIS (12/00803); ISCIII; SEEN thickness (IMT), a subclinical marker of atherosclerosis. Three composite scores were created by combining Z-scores of individual biomarkers: acute & 423-P phase reactants (fi brinogen and CRP); cytokines/adipokines (TNFR 1 and 2, Mechanisms of Cognitive Impairment in Type 2 Diabetes Mellitus PAI-I active, PAI-1 total and IL-6); and thrombosis (fi brinogen, PAI-1 active CHRIS MORAN, THANH PHAN, RICHARD BEARE, LEIGH BLIZZARD, ALISON and PAI-1 total). Internal carotid IMT was measured at EDIC years 1, 6 and VENN, TIM GREENAWAY, GERALD MUNCH, JOSEPHINE FORBES, VELANDAI 12. Elevated internal carotid IMT at each time point and IMT progression SRIKANTH, Clayton, Australia, Hobart, Australia, Sydney, Australia from EDIC year 1 to 12 were defi ned as being in the upper quintile of their We aimed to compare cognitive function, global MRI measures of respective distributions. Using logistic regression models, all three composite cerebrovascular disease and brain atrophy and regional distribution biomarker scores measured 10 to 12 years into EDIC (around the time of fi nal of cerebral atrophy between a large sample of people with T2DM and a IMT measurement) were associated with elevated internal carotid IMT as population-based comparison sample without T2DM. In addition, we well as IMT progression (see Table); in contrast, composite biomarker scores aimed to study the infl uence of brain MRI measures on observed cognitive measured at DCCT baseline (~6 years prior to initial IMT measurement) differences between groups. were not signifi cantly associated with internal carotid artery IMT or IMT We prospectively studied participants from Southern Tasmania over 55 progression (results not shown). Regression models were adjusted for years of age. We used a neuropsychological battery to assess cognitive DCCT treatment group, retinopathy cohort, age, gender, diabetes duration, performance. We performed brain MRI on each participant which HbA1c, LDL, HDL, SBP, smoking status and IMT reader. These associations was subsequently graded for the presence and number of infarcts and suggest that novel biomarkers may be a consequence rather than a cause microbleeds. We measured total grey matter, white matter volume and of atherosclerosis. white matter lesion volume. Using Voxel-based Morphometry, we looked for regional differences in grey matter volume. We assessed the association between T2DM and cognitive performance using linear regression modelling and assessed for effect measure modifi cation by MRI structural brain measures. T2DM was present in 339 of 712 participants. The mean age was 70 years of age. Participants with T2DM performed poorer in spatial function tasks(p<0.001) after adjustment for potential confounders. There was no statistically signifi cant difference in executive function, speed or memory scores between the two groups. Participants with T2DM had smaller hippocampal and grey matter volume(p<0.001) and more cerebral infarcts (p<0.001). Hippocampal volume, grey matter volume and number of cerebral infarcts modifi ed the effect of T2DM on spatial factor performance after adjustment for potential confounders. The location of grey matter atrophy that modifi ed the relationship between T2DM and spatial factor performance was predominately in the precentral, parahippocampal and superior temporal gyri(p<0.001). The fi ndings suggest that hippocampal and grey matter volume lie on the causal pathway linking T2DM and spatial factor performance. Supported by: NHMRC (Australia) Supported by: NIDDK (R01-DK081352) & 424-P Guided Audio Tour: Interventions to Improve Cardiovascular Disease in HDL Composition Is Profoundly Altered in Patients With Type 2 Dia- Diabetes (Posters: 422-P to 429-P), see page 15. betes (T2D) and Atherosclerotic Vascular Disease CECILIA MORGANTINI, DAVID MERIWETHER, SIMONA BALDI, ELENA VENTURI, & SILVIA PINNOLA, ALAN M. FOGELMAN, ELE FERRANNINI, ANDREA NATALI, 422-P SRINIVASA T. REDDY, Pisa, Italy, Los Angeles, CA Characterization of Sleep Apnea Syndrome in Type 2 Diabetic Pa- We have shown that in T2D patients the anti-infl ammatory and anti- tients (Sweet Sleep Study) oxidant functions of HDL are impaired. Recent studies suggest that HDL ALBERT LECUBE, CRISTINA HERNÁNDEZ, GABRIEL SAMPOL, ODILE ROMERO, dysfunction correlates with the lipoprotein content of oxidized fatty acids JORDI MESA, RAFAEL SIMÓ, Barcelona, Spain derived from arachidonic (HETEs) and linoleic acid (HODEs). In this study, we There is growing evidence suggesting than sleep apnea is highly prevalent examined whether HDL from T2D patients contain elevated levels of oxidized in patients with type 2 diabetes mellitus (T2DM). However, whether diabetic fatty acids and correlate with macrovascular complications (MVC). patients share the same sleep breathing pattern than non-diabetic patients HDL contents of HETEs and HODEs were determined by LC-MS/MS in 40 remains to be elucidated. For this purpose we designed a case-control study nondiabetic controls (ND), 40 T2D without MVC (DMVC-) and 38 T2D with between 119 T2DM patients and 238 non-diabetic subjects closely matched known history of MVC (DMVC+). HDL oxidant index was evaluated by a by age, gender, BMI, waist and neck circumferences, and smoking status. The cell-free assay using dichlorofl uorescein as described previously. Twenty- exclusion criteria included chronic respiratory disease, neuromuscular and six randomly selected subjects from the three groups underwent coronary cerebrovascular disease, alcohol abuse, use of sedatives, and pregnancy. calcium score evaluation (CAC). DMVC+ patients showed signifi cantly lower Examination included a respiratory polysomnography, oxygen saturation total cholesterol, LDL-C and HDL-C, and signifi cantly higher levels of plasma measures as the cumulative percentage of time spent with oxygen triglycerides. Other major cardiovascular risk factors were similar among the saturations below 90% (CT90), and the degree of sleepiness using the groups. HETEs and HODEs contents, expressed as ng per mg of HDL-C, were Epworth Sleepiness Scale (ESS). Apnea was defi ned as cessation of airfl ow signifi cantly increased in DMVC+ as compared to DMVC and ND patients with duration of at least 10 seconds, and an accurate differentiation between (p<0.01): 5-HETE 68.4±75.3 vs. 42.2±34.8 and 26.1±16.5; 12-HETE 18.5±20.3 obstructive and central apneas was performed. The apnea-hypopnea index vs. 11.2±7.6 and 10.6±14.7; 15-HETE 9.4±1.2 vs. 5.2±4.7 and 2.7±1.6; 9-HODE (AHI) was defi ned as the sum of apneas plus hypopneas divided by time in 11.2±12.9 vs. 7.1±6.1 and 4.5±2.3; 13-HODE 14.2±13.4 vs. 9.0±6.3 and 5.1±3.1. bed. A higher AIH was observed in T2DM patients (34.5±9.3 vs. 26.2±8.1 HDL oxidant index was not different among the three groups; however, it events/hour; p=0.017). When apnea and hiponea were evaluated separately,

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A108 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES was signifi cantly higher in patients with CAC score >100 than in patients Plasma levels of protein-bound pentosidine, Nε-(carboxymethyl)lysine with CAC score <100 (1.7±0.4 vs. 1.2±0.4; p<0.008). (CML), Nε-(carboxyethyl)lysine (CEL) and 5-hydro-5-methylimidazolone (MG- In conclusion, patients with diabetes and diabetes+MVC are characterized H1) were measured with ultra- or high-performance liquid chromatography- by a severe, graded enrichment of oxidized fatty acids on HDL. A loss of HDL tandem mass spectrometry and adjusted for the level of protein (lysine) in function (as estimated by the HDL oxidant index) is observed only in patients the sample. Tetrahydropyrimidine (THP) was measured with ELISA. HsCRP, with more advanced atherosclerosis. and sVCAM-1 and vWF were measured with ELISA, as markers for LGI and ED, respectively. The association of plasma AGEs with T1DM and CAC was analyzed with ANCOVA and adjusted for age, sex, BMI, waist-to-hip ratio, & 425-P POSTERS Results of Phase 3 Studies of PA32540 in Diabetic Patients Requir- smoking, blood pressure, lipid profi le, eGFR and diabetes status. Complications ing Aspirin for Secondary Cardiovascular Disease Prevention People with T1DM had higher plasma levels of the AGEs pentosidine Acute and Chronic JOHN G. FORT, JAY L. GOLDSTEIN, ANGEL LANAS, DAVID WHELLAN, YING ((geometric) mean 0.69 (95%-CI) (0.65-0.73) vs. 0.51 (0.48-0.54); p<0.001) ZHANG, INGRID C. WITHERELL, Chapel Hill, NC, Evanston, IL, Zaragoza, Spain, nmol/mmol LYS, CML (105 (102-107) vs. 93 (90-95); p=0.001) nmol/mmol Philadelphia, PA, Winnetka, IL LYS, MG-H1 (177 (171-184) vs. 169 (163-175); p=0.06) nmol/mmol LYS and Introduction: Patients with diabetes and prior cardiovascular (CV) events THP (126 (118-134) vs. 113 (106-120); p=0.03) U/mL, compared with controls, may require daily life-long treatment with aspirin. Nearly 40% of patients respectively. Levels of pentosidine and THP were higher in subjects with a had a history of diabetes in Phase 3 studies for PA32540 (325 mg enteric- moderate to high compared to a low CAC score, 0.69 (0.60-0.79) vs. 0.58 coated (EC) aspirin/40 mg immediate-release omeprazole). Ulcer rates and (0.56-0.61) nmol/mmol LYS; p=0.02 and 136 (117-157) vs. 117 (112-123) U/mL; treatment continuation were evaluated in this subpopulation. p=0.08, respectively. These results were not attenuated by adjustment for Methods: Subjects prescribed 325 mg daily aspirin for ≥3 months for LGI and ED. secondary CV prevention were randomized to once-daily PA32540 or 325 The AGEs pentosidine, CML, MG-H1 and THP were higher in T1DM. Higher mg EC aspirin in two phase 3 double-blind, multicenter studies. Endoscopic levels of the AGEs pentosidine and THP were associated with higher CAC assessments for gastric or duodenal ulcers were performed (screening; 1, score, independent of LGI and ED. 3, and 6 months). An independent blinded endpoint committee adjudicated Supported by: Center for Translational Molecular Medicine major adverse cardiovascular events (MACE). Results: Table 1 shows the diabetic population. Over 6 months, the PA32540 & 427-P vs EC aspirin subjects had signifi cantly lower rates of gastroduodenal ulcers Glycemic Variability Is Related to Duration of Diabetes Rather than and discontinuations due to upper GI adverse event/symptoms, with similar Cardiovascular Risk Factors MACE incidence. SO-YEON YOO, DAE-HO LEE, SANG AH LEE, HYOUN-JUNG CHIN, GWANPYO Conclusion: In patients with diabetes and prior CV events, PA32540 was KOH, Jeju, Republic of Korea associated with a signifi cantly lower rate of endoscopic gastroduodenal The role of glycemic variability in the development of cardiovascular ulcers, a higher rate of treatment continuation, and similar CV event diseases remains controversial. We investigated the relationship between incidence, compared with aspirin alone. These fi ndings support the use of indices of glycemic variability and cardiovascular (CV) risk factors in diabetic a single integrated tablet of omeprazole and aspirin for improving overall patients. effi cacy of antiplatelet therapy for secondary CV prevention in diabetic Two hundred sixty three diabetic patients performed a seven-point self- patients. monitoring of blood glucose during each month for 3 consecutive months. Table 1. Baseline Characteristics and Results (Diabetes Subjects, Intent-to- From these records, glycemic variability indices (standard deviation (SD) and Treat Population) M-value) were calculated. HbA1c was measured on the last day of the third month. Body mass index (BMI), waist circumference (WC), blood pressures, BASELINE PA32540 ASA MACE AT STUDY PA32540 (N=213) ASA CHARACTERISTICS (N=213) (N=188) END (N=188) duration of diabetes, high-sensitivity C-reactive protein (hsCRP), fi brinogen, (6 months) alanine aminotransferase (ALT), γ-glutamyltransferase (GGT), creatinine, uric acid, total cholesterol, triglyceride (TG), HDL, LDL, Apo B and Apo AI Age, mean (SD), years 66.2 (7.0) 65.8 (6.8) Non-fatal myocar- 32 Male gender, n (%) 157(73.7%) 131(69.7%) dial infarction levels, urine albumin:creatinine ratio (UACR) and ankle-brachial pressure index (ABI) were assessed. Race (n (%) - -165(87.8%) Transient ischemic 10 The SD was signifi cantly correlated with duration of diabetes (r=0.281; White 188(88.3%) 22(11.7%) attack p<0.001) and UACR (r=0.131, p<0.05), but not with BMI, WC, blood pressures, Black 25(11.7%) hsCRP, fi brinogen, ALT, GGT, creatinine, uric acid, lipid profi le and ABI. The Lipid-lowering therapy, n (%) 182(85.4%) 160(85.1%) Coronary artery 10 M-value was correlated with duration of diabetes (r=0.288; p<0.001), UACR disease (r=0.198; p<0.01), TG (r=0.132; p<0.05) and ABI (r=-0.212; p<0.001), but not Insulin therapy, n (%) 62(29.1%) 56(29.8%) Acute coronary 02 with other parameters. Using multiple linear regression to adjust for HbA1c artery syndrome and other covariates, only diabetes duration (β=0.184; p<0.01 and β=0.134; RESULTS AT STUDY END PA32540 ASA P value Heart failure 0 1 p<0.01, respectively) remained independent correlate of the SD and M-value. (6 months) (N=213) (N=188) CV risk factors failed to maintain its independent association. Endoscopic gastroduodenal 5 (2.3%) 21 (11.2%) P < 0.001 Planned CABG 1 0 In this study, duration of diabetes rather than CV risk factors was an ulcers, n (%) (0.8% - 5.4%) (7.0% - 16.6 independent variable of indices of glycemic variability. These fi ndings suggest 95% CI %) that glycemic variability is largely determined by β-cell function which Discontinuations due to upper GI 3 (1.4%) 11 (5.9%) P = 0.018 TOTAL MACE 6 events in 6 5 events in 4 deteriorates with increasing duration of diabetes, but not cardiovascular event, n (%) subjects (2.8%); subjects complication. 4 treated with (2.1%); clopidogrel 1 treated with & 428-P clopidogrel Time Course to Myocardial Infarction in Relation to Diagnosis and Duration of Type 2 Diabetes NATALIE NANAYAKKARA, JEFFREY LEFKOVITS, ANDREW E. AJANI, PETER G. & 426-P COLMAN, SPIROS FOURLANOS, Melbourne, Australia Despite extensive literature documenting the 2-3 fold higher risk of Plasma Levels of Advanced Glycation Endproducts are Associated myocardial infarction (MI) in type 2 diabetes patients (T2D), there is less With Type 1 Diabetes and Coronary Artery Calcifi cation information on the age at T2D diagnosis and expected time course to MARCELLE G.A. VAN EUPEN, MIRANDA T. SCHRAM, HELEN M. COLHOUN, JEAN sustaining a MI1. This study examines the relationship of T2D across different L.J.M. SCHEIJEN, COEN D.A. STEHOUWER, CASPER G. SCHALKWIJK, Maas- age groups and diabetes duration prior to MI. tricht, Netherlands, Dundee, United Kingdom The Royal Melbourne Hospital coronary care database (n=12961, 2001- Advanced glycation endproducts (AGEs) may play a role in the development 2012) was used to identify 3317 patients presenting with their fi rst MI of of coronary artery calcifi cation (CAC) in type 1 diabetes (T1DM). We studied whom 975 (29%) had diabetes. Diabetes details (including type and duration) whether plasma AGE levels are: associated with 1) T1DM and 2) CAC; and 3) and cardiovascular risk profi les for 500 of these patients were obtained from whether the latter association could be explained by low-grade infl ammation their primary care physicians and hospital records. (LGI) and endothelial dysfunction (ED). We studied 178 people with and In this cohort of 500 patients (69% male), the mean age at diagnosis 174 people without T1DM, both with mean age of 38 years. Coronary of T2D was 55±14y and mean age at MI was 67±11y (mean 12±9y interval calcifi cation was quantifi ed in a CAC score based on computed tomography.

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A109 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

between the diagnosis of T2D and MI). Younger onset T2D had a greater response element (ARE)-luciferase activity as well as 4-HHE. DHA (25-100 duration to MI (Figure 1a) but developed MI at a younger age overall (Figure µM) or 4-HHE (5 µM) increased mRNA and protein expression of HO-1 and 1b). In T2D diagnosed before age 50y (n=171, age 40±7) versus after age 50y the siRNA of Nrf2 signifi cantly reduced those effects. Furthermore, DHA (n=329, age 63±9y) there was a signifi cantly greater time to MI (mean 18±10y prevented the oxidant-induced ROS production and cell death, and the vs 10±7y, p<0.0001). effects of DHA were disappeared by HO-1 inhibitor or the siRNA of Nrf2. In People diagnosed with diabetes at a younger age had a longer mean conclusion, we found the preconditioning effects of DHA against oxidative duration to MI, however the mean age at MI was still younger. These data stress through Nrf2 activation in vascular endothelial cells, which may may assist determining which diabetes patients, based on age at diagnosis explain the mechanism of the cardioprotective effects of DHA. POSTERS

Complications and duration, require more focussed counselling and screening for coronary Acute and Chronic artery disease. 431-P C ardiac O verexpres sion of Me t allo thionein Rescue A k t 2 De fi ciency- Induced Impairment of Cardiac Insulin Signaling and Cardiac Func- tion YI TAN, XIAOKUN LI, LU CAI, Louisville, KY, Wenzhou, China Akt1 is known to regulate the physiologic and pathological growth of cardiomyocytes while Akt2 predominantly regulates glucose metabolism in response to insulin stimulation. Mice lacking Akt2 display a profound diabetic phenotype, indicating that Akt2 plays a key role in insulin receptor downstream signal transduction (J Biol Chem 281:32841-51). Whether and how metallothionein (MT) can improve Akt2 defi ciency induced cardiac dysfunction remains unknown. We cross bred cardiac overexpression MT 1. S. Goya Wannamethee et al, Arch Intern Med. 2011; 171(5) 404-410 (MT-tg) mice with Akt2 defi ciency (Akt2-ko) mice to generate a double transgenic line (MT-tg/Akt2-ko). Akt2 defi ciency induced hyperglycemia, & 429-P hyperinsulimia, and impaired glucose tolerance and insulin sensitivity from 2 to 5 months old in Akt2-ko mice compared to wild type (WT) mice. NonAlcoholic Fatty Liver Disease and Cardiovascular Events in In- Cardiac overexpression of MT, though did not affect non-fasting blood dian Type 2 Diabetes Population glucose and plasma insulin levels, signifi cantly attenuated Akt2 defi ciency- MANOJ SALUJA, Kota, India induced impairment of glucose tolerance and insulin sensitivity in MT-tg/ Nonalcoholic fatty liver disease (NAFLD) is the most common liver Akt2-ko mice compared to Akt2-ko. Global Akt2 defi ciency also induced disease in type 2 diabetes patients. We tried to evaluate the future risk systemic abnormalities including hypertension and large organ hypotrophy of cardiovascular events among Indian type 2 diabetes population having at 5 months after birth. For the heart, Akt2 defi ciency signifi cantly reduced NAFLD, independent of metabolic syndrome features and other classical systolic function, decreased ejection fraction and increased left ventricle risk factors. We carried out a prospective case-control study in 1060 type hypotrophy in Akt2-ko mice at 5 months old compared to age-matched 2 diabetic patients with NAFLD (an ultrasound diagnosis) who were not WT mice; all these changes were not observed in MT-tg/Akt2-ko mice. having any diagnosed cardiovascular disease (using standard criteria)at Mechanistic study revealed that Akt2 defi ciency induced systemic glucose the time of recruitment. Two hundred and fi fty age and sex matched type metabolic disorder along with the increase of cardiac oxidative stress 2 diabetics without NAFLD served as controls. The study population was (indicated by increased lipid peroxidation) and impaired insulin stimulated followed up for fi ve years. During 5 years of follow-up, 213 cases with Akt signaling (defi ned by decreased phosphorylation of GSK-3 and GS), NAFLD developed cardiovascular events including coronary heart disease β which were prevented in MT-tg/Akt2-ko mice. These results suggest that (myocardial infarction and coronary revascularization procedures), ischemic MT rescue Akt2 defi ciency-induced impairment of cardiac function probably stroke, or cardiovascular death. After adjustment for age, sex, smoking through its antioxidant potency and compensation of Akt function. history, diabetes duration, HbA1c, LDL cholesterol, liver enzymes, and use Supported by: NSFC 30971209; 81200239; Y20100001; 81273509 of medications, the presence of NAFLD was signifi cantly associated with an increased CVD risk (odds ratio 1.78, 95% CI 1.4-2.1, P < 0.001). In conclusion, NAFLD is signifi cantly associated with signifi cantly increased risk of cardio- 432-P vascular morbidity and mortality among type 2 diabetic individuals. This Beta 2 Microglobulin and Subclinical Atherosclerosis in Normo- relationship was independent of classical risk factors and could only partly albuminuric Type 2 Diabetes Mellitus be explained by occurrence of metabolic syndrome. EUN-HEE JANG, MEE-KYOUNG KIM, KI-HYUN BAEK, KI-HO SONG, BONG-YUN CHA, HYUK-SANG KWON, Seoul, Republic of Korea 430-P Objective: Chronic kidney disease is associated with increased risk of cardiovascular disease. Serum 2-microglobulin ( 2M) levels are elevated in A Novel Mechanism of Preconditioning Effect by Docosahexaenoic β β renal failure and are suggested surrogate marker of cardiovascular mortality Acid on Endothelial Antioxidant Activity via Nrf2 Pathway for patients with chronic kidney disease. However, the relation between KATSUTARO MORINO, ATSUSHI ISHIKADO, YOSHIHIKO NISHIO, FUMIYUKI NA- serum 2M levels and subclinical atherosclerosis in the patients with KAGAWA, YOKO SONO, OSAMU SEKINE, TAKESHI YOSHIZAKI, SATOSHI UGI, β normoalbuminuric type2 diabetes is not yet clear. TAKETOSHI MAKINO, TOMIO OKAMURA, ATSUNORI KASHIWAGI, HIROSHI Methods: A total of 196 patients with type 2 diabetes mellitus who showed MAEGAWA, Otsu, Japan, Kagoshima, Japan, Osaka, Japan normoalbuminuria were enrolled consecutively in this study. Exclusion criteria Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 was serum creatinine over 2.0mg/dL. Serum 2M levels were measured using PUFAs) have direct antioxidant and anti-infl ammatory effects in vascular β solid-phase, two-site chemiluminescent immunometric assay. Brachial-ankle tissues, explaining their cardioprotective effects. We have tested the pulse wave velocity (PWV) and carotid artery intima-media thickness (C-IMT) hypothesis that n-3 PUFAs increase antioxidant activity through the were measured as indicators of subclinical atherosclerosis. activation of Nrf2, a master regulatory transcriptional factor, in vascular Results: Mean age and BMI of our study population were 58 ± 15 years tissues. First, C57BL6 and Nrf2 knockout mice (Nrf2-KO) were fed with (mean±SD) and 25.1± 3.9 kg/m², respectively. Their average HbA1c and the fi sh oil diet (FD) for 3 weeks. FD signifi cantly increased mRNA or protein duration of diabetes were 9.3 ± 2.1 years and 10.5 ± 8.6 years. Average expression of heme oxygenase-1 (HO-1), an Nrf2 target gene, by 2.1 or 1.5 serum 2M levels was 1.97± 0.69 mg/L. Patients with higher 2M levels fold in thoracic aorta, while the effect of FD was not observed in Nrf2- β β showed older age, a longer duration of diabetes, higher cystatin C, higher KO. FD also signifi cantly increased acetylcholine-dependent vasodilatory homocysteine, increased PWV and increased C-IMT compared with response in control mice but not in Nrf2-KO, and had no effects on sodium patients of lower 2M. Serum 2M levels was positively correlated with nitroprusside-dependent response in both, indicating that FD increases the β β PWV (r=0.406, p<0.0001) and C-IMT(r=0.334, p<0.0001). After adjusting for Nrf2-mediated vasodilatory response in an endothelium-dependent manner. age, duration of diabetes, hypertension, HbA1c and creatinine , the serum Second, human umbilical vein endothelial cells were stimulated with 2M levels and age remained as independent risk factors for PWV (B=99.6; docosahexaenoic (DHA) or eicosapentaenoic (EPA) acid. We found that DHA β R2=0.453; P = 0.038) and C-IMT(B=0.089; R2=0.207; P = 0.012). (75 µM) but not EPA signifi cantly increased intracellular 4-hydroxy hexenal Conclusion: Higher serum 2M levels in the patient s with nor moalbuminuric (4-HHE, 5.0 fold), an end-product of n-3 PUFAs peroxidation, accompanied β type2 diabetes was positively associated with subclinical atherosclerosis. with ROS production. DHA but not EPA increased the nuclear expression (8.4 fold) and DNA binding (4.5 fold) of Nrf2. DHA also increased antioxidant

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A110 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

433-P benefi cial effect of an antihypertensive class. The aim of this study was to A Meta-Analysis: Screening Test for Silent Myocardial Ischemia in analyze the effi cacy of different classes of antihypertensives in mortality of Patients With Asymptomatic Type 2 Diabetics hypertensive patients with type 2 DM. We reviewed randomized controlled LIHUA ZHANG, HONG LI, YU FENG LI, SIMIN ZHANG, XIUYING ZHANG, JIE LIN, trials from 1950 up to November 2012, comparing fi ve antihypertensives HONG YAN BI, LINONG JI, Beijing, China, Kunming, China classes used alone or in combination for treatment of hypertension in Coronary artery disease (CAD) is the major cause of mortality and morbidity patients with type 2 DM: thiazides, betablockers, calcium channel blockers in patients with type 2 diabetes mellitus (T2DM). But the utility of screening (CCBs), angiotensin converting inhibitors (ACEi) and angiotensin receptor blockers (ARBs). A network meta-analysis method was used to obtain pooled patients with T2DM for asymptomatic CAD is controversial. This study was POSTERS aimed to assess the validity of screening for silent myocardial ischemia estimates. A total of 27 studies were included with 49418 patients reporting Complications (SMI) in asymptomatic patients with T2DM as being at high cardiac risk 5647 deaths from which 1306 were due to cardiovascular (CV) events. ACEi Acute and Chronic and whether it affects patients’ cardiac outcomes (cardiac death, nonfatal and CCB combination showed a signifi cant reduction in CV mortality as myocardial infarction, heart failure, unstable angina, ventricular rhythm compared to placebo (median HR, 95% Credibility Intervals: 0.16, 0.01 to disorders and so on ). The authors searched several electronic databases, 0.82), betablockers (0.20, 0.02 to 0.98), CCBs (0.21, 0.02 to 0.97) and ARBs among which PUMED, The Cochrane Library, EMBASE, and Proquest (0.18, 0.02 to 0.91). ACEi and CCB combination showed a borderline benefi t Dissertations & Theses database (all up to Oct 2012) were combined with in total mortality compared to placebo (0.34, 0.08 - 1.03). Total mortality was hand searches.Four random controlled trials (RCTs) and 2 cohort studies with not signifi cantly different for any other pairwise comparisons between the follow up ranging from 22 to 72 months were identifi ed. A meta analysis classes. In these analyzed studies, end of trial systolic and diastolic blood was performed pooling data and we applied a random-effects model or pressure levels were also signifi cantly lower with ACEi and CCB combination fi xed-effects model to combine odds ratio and 95% confi dence intervals. as compared to placebo (-5.24, -8.91 to - 1.81 mmHg) and to CCBs (-2.87, -5.82 For a total of 1275 asymptomatic patients with T2DM the cumulative to -0.03 mmHg) although it was not different from ARBs and betablockers. prevalence rate of SMI was 21.0%. Silent ischemia screening test (Four Reduction of CV mortality was observed in hypertensive patients with type trial ,SPECT) were available to diagnose occult coronary disease, yet after 2 DM treated with ACEi and CCB combination. This may be probably related follow up the endpoint events in screened group had no difference with not- to higher blood pressure reduction effect rather to a particular benefi cial screened group (Cardiac death: odd ratio[OR]=0.92, 95% confi dence interval effect of this combination of antihypertensive agents. [CI]:0.51-1.64; P=0.37. Nonfatal cardiac event: OR=0.91, 95%CI: 0.65-1.26; Supported by: CNPq (307015/2010-6) P=0.13). Same result was got from the subgroup analysis of patients with SMI group and patients without SMI group. No signifi cant difference was 436-P observed about heart death (OR= 0.84, 95%CI: 0.14-5.28, P=0.86) and other Cystatin C: A Strong Marker to Prompt the Lower Limb Ischemia in cardiac events (OR= 1.63, 95%CI: 0.97-2.16, P=0.07). These results suggest Chinese Type 2 Diabetic Patients? that the systematic and routine detection of silent ischemia in high-risk JING SHEN, FANG LIU, HUI ZENG, LIANXI LI, WEIPING JIA, Shanghai, China asymptomatic patients with T2DM is unlikely to provide major benefi t on Objective: Cystatin C is growing to be an ideal indicator for renal function hard cardiac outcomes in patients. and cardiovascular events. The aim of this study was to investigate the relationship between serum cystatin C levels and lower extremity artery 434-P disease and to verify its predictive value for lower limb ischemia in diabetic Increased Expression of FC-Gamma Receptors in Nascent Meta- population. bolic Syndrome Methods: A total of 1609 T2DM patients were included in this cross- ISHWARLAL JIALAL, SRIDEVI DEVARAJ, Sacramento, CA, Houston, TX sectional study. They were divided to two groups: with lower limb ischemia Metabolic Syndrome (MetS) affects 1 in 3 US adults and confers increased and without. Lower limb ischemia was defi ned by ABI < 0.9 and ultrasound propensity to diabetes and cardiovascular disease (CVD). CRP has been shown examination. Their clinical and biochemical characteristics were detected to predict CV Events in patients with MetS and the proatherogenic effects and compared. appear to be mediated by the Fc gamma receptors (FcgR), CD32 and CD64. Results: There were signifi cant differences between patients with and We have previously shown that subjects with nascent MetS exhibit a pro- without lower limb ischemia in age, duration of diabetes, systolic blood infl ammatory state characterized by high levels of high sensitive C-reactive pressure, GA, CRP, RBC, Hb, neutrophils, TP, albumin, BUN, Cr, cystatin C, protein (hsCRP) and increased cellular infl ammation following adjustment for GFR, AKP, carotid and femoral IMT, prevalence of hypertension, coronary adiposity. FcgR are well-characterized cell surface immune receptors that artery disease and cerebral infarction (all P<0.01) and HbA1c (P<0.05). mediate phagocytosis, release of infl ammatory mediators and stimulation of Prevalence of lower limb ischemia rose with decreasing GFR and increasing the immune response. The main FcgRs on monocytes include FcgRI (CD64), cystatin C (both P<0.01). Binary logistic regression analysis found the FcgRIII (CD16), and FcgRII (CD32). FcgR/apo E Double knockout mice have independent risk factors were age (P=0.000, OR=1.131), diabetes duration reduced atherosclerosis. However, there is no data on FcgR expression in (P=0.033, OR=1.044), the presence of hypertension (P=0.004, OR=2.627), MetS compared to controls and this was the aim of the study since these GA (P=0.014, OR=1.053), cystatin C (P=0.002, OR=2.783) and neutrophils patients had BMI adjusted elevated CRP level (p=0.002). Monocytes were (P=0.041, OR=1.040). Further ROC curve analysis showed the cut point of isolated by negative magnetic separation from subjects with nascent MetS cystatin C for lower limb ischemia was 1.2 mg/L. Cystatin C levels also without diabetes or CVD and age, gender matched controls (n=42/group). elevated with carotid IMT. Age (β=0.545, P=0.000), cystain C (β=0.188, Surface expression of CD32 and CD64 on monocytes was examined by P=0.000), the prevalence of coronary artery disease (β=0.112, P=0.000), fl ow cytometry. Surface expression of CD32 and CD64 were signifi cantly hypertension (β=0.097, P=0.002) and cerebral infarction (β=0.097, P=0.028) increased in MetS patients compared to controls even following adjustment were independent risk factors for lower extremity lesion. for adiposity (CD32: C: 37 +/- 19, MetS, 54 +/- 21 mfi /million cells, p=0.0009, Conclusions: There was a strong and independent association between CD64: C: 44 +/-17 vs MetS: 65 +/- 23 mfi /million cells, p<0.0001). Expression cystatin C and peripheral arterial disease, and cystatin C higher than 1.2 of both these FcgR correlated signifi cantly with the number of features of mg/L may predict high risk of lower limb ischemia. MetS (p for trend < 0.001). Furthermore, there was a signifi cant correlation Supported by: NSFC (81270397), (81070650) of CD32 and CD64 receptors and levels of hsCRP (r=0.23 and r=0.31, p<0.05). These novel data point to a potential role of CD64 and CD32 in mediating increased systemic and cellular infl ammation in MetS and thus predisposing 437-P to increased risk for CVD. Percutaneous Recanalization in Type 2 Diabetic Patients With Criti- cal Limb Ischemia (CLI): Comparison of Angiographic and Clinical 435-P Results According to Different Classifi cation Systems Classes of Antihypertensive Agents and Mortality in Hypertensive ELISABETTA IACOPI, LOREDANA RIZZO, ALBERTO COPPELLI, CHIARA MATTALIA- Patients With Type 2 Diabetes—Network Meta-Analysis NO, IRENE BARGELLINI, ROBERTO CIONI, ALBERTO PIAGGESI, Pisa, Italy LUCIANA R. REMONTI, SOFIA DIAS, CRISTIANE B. LEITÃO, CAROLINE K. KRAM- To evaluate three different angiographic classifi cations in describing the ER, LUCAS P. KLASSMANN, NICKY WELTON, A.E. ADES, JORGE L. GROSS, Porto actual pattern of diabetic macro-angiopathy (DMA) and in predicting the Alegre, Brazil, Bristol, United Kingdom clinical outcomes of critically ischemic diabetic foot (DF), we traced back all Diabetes (DM) and hypertension are commonly associated. Reduction DF patients submitted to percutaneous transluminal angioplasty (PTA) in our of blood pressure is effective in reducing diabetes complications. There Department in the years 2009 - 2010, with a follow up of 13.4±9.7 months. is controversy if this effect is due to blood pressure reduction only or to a DMA severity was assessed according to three different classifi cation

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A111 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

systems based on angiographic pattern: the Trans Atlantic Society Consensus The overall median level of osteocalcin was 20.51 (16.71-24.98) ng/mL and II (TASC II), the Joint Vascular Society Council (JVSC), and the morphological of C-IMT was 0.60 (0.55-0.65) mm. Four-hundred-and-sixteen (31.5%) of the classifi cation proposed by Graziani et al. We eventually correlated the clinical participants had increased C-IMT. Participants with increased C-IMT had results (healing rate, healing time, rate and level of amputations), with the signifi cantly lower serum osteocalcin levels than those with C-IMT <0.65 severity of vascular involvement before and after the revascularization mm (19.77 [16.17-24.52] ng/mL vs. 20.84 [16.92-25.39] ng/mL, P =0.01). The procedures as expressed by the three different classifi cation systems. serum osteocalcin level was significantly and negatively correlated with We evaluated 202 consecutive PTA performed in 166 diabetic patients C-IMT (P <0.01) and was identifi ed as the independent factor signifi cantly (age 72.8±9.8 yrs, duration of diabetes 20.5±12.1 yrs, HbA1c 7.8±1.8%). TASC infl uencing C-IMT (standardized β = -0.117, P <0.01). POSTERS

Complications II was not applicable in 55.4% of patients pre- and in no patient post-PTA Serum osteocalcin level was negatively associated with subclinical

Acute and Chronic because of the absence of sovra-popliteal arterial stenosis. JVSC scored athero sclerosis in Chinese postmenopausal women. 7.8±1.7 pre- and 4.8±2.3 (p<0.01) post-PTA while Graziani classifi cation scored 4.8±0.9 pre- and 1.4±0.5 post-PTA (p<0.01). 66.8% of patients healed 440-P in 28.4±23.7 weeks, while 3.9% underwent to major amputations. Among Contribution of Asymmetric Dimethylarginine to the Suppression of the three angiographic scores only JVSC signifi cantly related with the clinical Mitochondrial Biosynthesis in Myocardium of Type 2 Diabetic Rats outcome (p=0.04).In conclusion TASC II is inadequate to describe vascular YAN XIONG, CHUN-XIA HAI, WEI-JIN FANG, NI QIU, ZHI-MIN HE, Guangzhou, involvement in diabetic patients with CLI. While both the other classifi cations China are effective in describing both basal and post-PTA conditions of DMA, but Mitochondrial dysfunction is related to diabetic cardiomyopathy. only JVSC is a reliable predictor of outcome in DF patients with CLI. Mitochondrial biogenesis (MB) regulated by nitric oxide (NO) is suppressed in diabetes and contributed to mitochondrial dysfunction. The endogenous 438-P NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) plays an FoxO1 Acetylation Is Correlated to Expression of Infl ammatory Fac- important role in the development of diabetic cardiovascular complications. tors in Atherosclerotic Plaques from Subjects With and Without However, it is unclear whether elevated endogenous ADMA is implicated Diabetes in suppressed MB of diabetic myocardium. This study was to determine MASSIMO FEDERICI, ROSSELLA MENGHINI, MARINA CARDELLINI, EUGENIO the relationship between them in myocardium of type 2 diabetic rats and MARTELLI, VIVIANA CASAGRANDE, FRANCESCA DAVATO, ALESSIA VALENTINI, then to investigate the direct effect of ADMA on MB and its mechanisms ANTONIO TAMMONE, MARTA FABRIZI, ROBERT STOEHR, OTTAVIA PORZIO, STE- in cardiomyocytes. The ratio of the copy numbers of mitochondrial gene to FANO RIZZA, ARNALDO IPPOLITI, RENATO LAURO, Rome, Italy nuclear gene was measured to represent MB. Contents of ADMA & NO, The aim of this study was to investigate the activation of FoxO1 in human activity or expression of NOS and dimethylarginine dimethylaminohydrolase atherosclerosis. In human atherosclerotic plaques biobank (n=100) we (DDAH), the major metabolic enzyme of ADMA were analyzed to show the have determined mRNA expression of FoxO1, VCAM-1, TLR4, MMP9, TNF- changes in DDAH/ADMA/NOS/NO pathway. The transcription of peroxisome alpha and CCL2 by quantitative real time PCR (qPCR) normalizing to 18S proliferator-activated receptor-γ coactivator (PGC-1α) & uncoupling protein2 and analysing with the deltadeltaCT method. In protein extracts we have (UCP2) and oxidative stress were detected to explore the potential mechanisms. analyzed the levels of VCAM-1, FoxO1, FoxO1 Lysine acetylation (FoxO1Ac) Results showed that ADMA content was elevated in myocardium of diabetic and FoxO1 acetylation mediated by SirT1 on Lysine 241 (FoxO1-Sirt1Ac), rats compared to control. This elevation was associated with inhibitions of MB normalizing to tubulin and FoxO1 levels. mRNA expression levels were and ATP production. These inhibitions were paralleled with downregulated normalized using logarithmic transformation. PGC-1α transcription, upregulated UCP2 transcription, decreased NO In atherosclerotic plaques we found that FoxO1Ac was positively correlated production and increased oxidative stress. Incubation of cardiomyocytes with with the levels of several infl ammatory markers, such as VCAM-1 (R=0.53 ADMA not only reduced MB, ATP & NO contents and PGC-1α transcription, but p<0.0001), TLR4 (R=0.44 p=0.002) and MMP9 (R=0.44 p<0.0001) mRNA also increased oxidative stress and UCP2 transcription. These results indicate expression, whereas FoxO1-Sirt1Ac was mildly correlated only with VCAM- that the elevated endogenous ADMA contributes to suppressed MB and 1 mRNA (R=0.25 p=0.005) and VCAM-1 protein levels (R=0.22 p=0.008). ATP production in myocardium of diabetic rats, and the mechanisms may be Moreover, FoxO1 mRNA expression resulted in correlation with VCAM-1 related to the decreases of NO content & PGC-1α transcription and increases (R=0.73 p<0.001), TLR4 (R=0.78 p<0.001), TNF-alpha (R=0.75 p=0.002), of oxidative stress & UCP2 transcription. CCL2 (R=0.7 p<0.001) mRNA expression and with the FoxO1Ac levels Supported by: NSFC (30873062), (81170778) (R=0.34, p=0.005). Metabolic analysis indicate that FoxO1Ac is correlated with Fasting Plasma Glucose (R=0.23 p=0.039), therefore we divided our samples in 4 groups according to the metabolic status (Normal Glucose 441-P Tolerance n=35, Impaired glucose Tolerance n=31, Type 2 Diabetes Mellitus Comparison of Atherosclerotic Disease Assessed by Carotid Inti- with new diagnosis and no treatment n=18, Type 2 Diabetes Mellitus under ma-Medium Thickness, Endothelial Function and C-Reactive Pro- treatment=16); by ANOVA statistical analysis, we found that the degree of tein in Male Patients With Type 2 Diabetes With Normal and Low metabolic status is positively correlated with FoxO1Ac (p=0,002) whereas Testosterone Levels the FoxO1 mRNA expression is similar in the different groups. JAVIER M. FARIAS, MARINA KHOURY, GUILLERMO E. UMPIERREZ, MATIAS TI- In conclusion, our study suggests that FoxO1 acetylation status is physio- NETTI, Buenos Aires, Argentina, Atlanta, GA logically relevant and may contribute to the progression of athero sclerosis Type 2 Diabetes (T2D) and low serum testosterone are associated with in human subjects. increased risk of atherosclerotic complications. The magnitude of such association; however, in middle-aged patients with T2D has not been 439-P determined. Accordingly, we evaluated atherosclerotic markers in patients Relationship Between Serum Osteocalcin Levels and Carotid Inti- with T2D with normal and low total testosterone in 115 male patients < 70 ma-Media Thickness in Chinese Postmenopausal Women years without previous cardiovascular events with normal (≥ 3,5 ng⁄ml, RONG YANG, XIAOJING MA, JIANXIN DOU, FEIFEI WANG, YUQI LUO, DI- n=79) and low (<3,5 ng⁄ml, n=36) total testosterone (TT). We measured ANCHENG LI, JIAAN ZHU, YUQIAN BAO, WEIPING JIA, Shanghai, China serum C reactive protein (CRP), carotid artery intima-media thickness (IMT), Serum osteocalcin is closely related to metabolic risk factors. Therefore, the presence carotid atherosclerotic plaque (Plaque) by high-resolution B-mode aim of our study was to evaluate the association between serum osteocalcin ultrasound, and endothelial dysfunction (ED) by brachial artery fl ow mediated levels and subclinical atherosclerosis in Chinese postmenopausal women. dilación. Results: There were no difference in age 56.6±7, DM duration: A total of 1319 postmenopausal women (age range: 41-78 years) without 6.6±3 yrs, BMI 30.1±3 kg⁄m2, LDL 119.3±23 mg⁄dl, HDL 39.9±7 mg⁄dl, HbA1c a history of cardiovascular disease (CVD) or carotid plaque were analyzed. 6.9±0.6%, between normal and low TT groups, p= NS. Frequencies of ED Electrochemiluminescence immunoassay was used to measure total serum 53.91% (n=62) and plaque, 52.17% (n=60). Mean IMT (0,100±0,014 mm) was osteocalcin levels. B-mode ultrasound measurement of carotid intima- signifi cantly correlated with TT, r: -0.39 (p <0.0001) Compared to patients media thickness (C-IMT) was used to evaluate subclinical atherosclerosis. with normal T, those with low T have higher IMT ≥0.100 (80% vs 39%, odds Participants with the upper quartile of C-IMT measurements (≥0.65 mm) were ratio (OR) 6.41(CI95%: 2.5-16.4), p<0,001 and atherosclerotic plaques 68.5% classifi ed as having increased C-IMT in the present study. C-IMT association vs. 45%, OR 2.60 (1.12-6.03), p<0.0001; ED (80.5 vs 42.5%, OR 5.77 (2.77- with metabolic parameters was assessed by Spearman correlation analysis, 14.77), p <0001, and higher CRP (2.74±582 vs 0.89±0.88 mg⁄L, p<0.0001). while that with serum osteocalcin was assessed by multiple stepwise Using multiple logistic regression analyses adjusted for age, diabetes regression adjusted for potential confounders. duration, HbA1c, lipids, treatment effect, and BMI, we found that TT levels

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A112 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES were associated with greater IMT [OR: 8.43 (2.5-25.8)] and ED [OR: 4.98 444-P (1.72-14.37)], but not with the presence of atherosclerotic plaques (p=NS). Effect of a Single Dose of Canakinumab on hsCRP Levels in T2DM In summary, male T2D patients with low total testosterone have greater Patients IMT, ED and CRP compared to diabetic patients with normal TT. Low CAMPBELL HOWARD, ADELE NOE, ANN TAYLOR, TOM THUREN, East Hanover, testosterone in middle age T2D is associated with more advanced athero- NJ, Basel, Switzerland, Cambridge, MA sclerotic markers. IL-1β plays a key role in atherothrombosis and worsening of islet β-cell function. Canakinumab (CKM), a human monoclonal antibody, targets IL-

442-P 1β-dependent infl ammation and reduces hsCRP and other biomarkers. POSTERS Control of Cardiovascular Risk Factors in Asymptomatic Type 2 Dia- This multicenter, randomized, double-blind, placebo (PL)-controlled, dose- Complications betic Patients: Consequences on Myocardial Ischemia and Silent escalation study aimed to report pharmacokinetics (PK) and hsCRP levels in Acute and Chronic Coronary Stenoses 231 T2DM patients on stable metformin dose. EMMANUEL COSSON, ANTOINE AVIGNON, ARIANE SULTAN, MINH TUAN PK profi le was assessed at 0 h, 2 h, and Days 1, 13, 27, 55, 83, 167 and NGUYEN, PAUL VALENSI, Bondy, France, Montpellier, France changes in hsCRP levels were assessed at Weeks 4, 12, 18 and 24 after a Silent myocardial ischemia (SMI) is a common complication of diabetes. single IV administration of 5 doses of CKM. SMI may be due to functional coronary disorders and/or coronary stenoses Systemic clearances across dose groups were comparable, indicating that (CS). We hypothesized that control of cardiovascular risk factors (CVRF) at the PK of CKM was dose-related (Fig 1). Signifi cant dose-related reduction in time of investigation would be associated with a lower prevalence of SMI. hsCRP was observed at Week 4, with greater reductions vs PL for higher A total of 1627 asymptomatic type 2 diabetic patients with at least one doses (Fig 2). Reduction in hsCRP was maintained at Week 12 for CKM 1.5 additional CVRF were screened for SMI with stress and/or dipyridamole and 10 mg/kg. myocardial scintigraphy. SMI was detected in 412 patients (25.3%); a A single dose of CKM provides dose dependent reductions of hsCRP coronary angiography was performed in 298 of those with SMI and found sustained over 12 weeks in T2DM patients. CS in 131 patients (44%). The patients were classifi ed according to control of blood pressure (≤140/90 mmHg), HbA1c (≤7.5%), LDL cholesterol (≤2.6 mM) and triglycerides (≤2.3 mM). SMI prevalence was inversely associated with the number of controlled CVRF (39.6%, 35.7% 24.7%, 19.8%, 17.7% in patients with none, 1, 2, 3 or 4 controlled CVRF, respectively; p<0.001). In multivariate analysis taking into account the number of controlled CVRF (3 or 4 vs 0-2), age, diabetes duration, gender, retinopathy, nephropathy, smoking, HDL cholesterol, peripheral vascular disease, SMI was associated with a lower number of controlled CVRF (odds ratio 0.53[95CI 0.39-0.74]), male gender (2.4 [1.8-3.3]) and peripheral vascular disease (1.6[1.02-2.5]). Among the patients with SMI, the prevalence of CS was lower in the patients with 3-4 controlled CVRF than in those with 0-2 controlled CVRF (34.5 vs 47.7%, p<0.05), even after multivariate analysis. To conclude, in patients with type 2 diabetes, SMI is markedly lower and is less concordant with CS when the number of controlled CVRF is higher, even after adjustment on confounders. This strongly suggests the preventive effi cacy of CVRF control on silent coronary artery disease.

443-P Sirt1 Signaling and Cardiovascular Risk after Roux-en-Y Gastric Bypass Surgery YANHUA PENG, MICHEL MURR, STEVEN RAKITA, JAMES LEE, Tampa, FL Obesity and diabetes increase cardiovascular disease risk. It has been demonstrated that Roux-en-Y gastric bypass surgery (RYGB) cures obesity- related diabetes and attenuates the risk of cardiovascular diseases. Our previous data have demonstrated that, within the liver, AMPK and Sirt1 are up-regulated while infl ammatory/oxidative stress and insulin sensitivity are improved after RYGB. The molecular mechanisms for these are not well understood. We have hypothesized that downstream signaling of Sirt1 plays an important role in the improvements in cardiovascular risk following RYGB. Fasting glucose, OGTT, serum markers of infl ammatory and oxidative stress, C-reactive protein (CRP), TNF-α, IL-6, the ratios of adiponectin/ leptin, high molecular weight (HMW) adiponectin/total adiponectin, and malondiadehyde (MDA) were measured. Rats were split into RYGB and sham Supported by: Novartis Pharma AG with weight matched control. Sirt1, PGC1α, Nrf2, iNOS, FOXO1 and p53 were assessed in the blood vessel. Glucose was used for mimicking obesity-induced glucotoxicity in human Endothelial cell EAhy926, and SiRNA techniques for 445-P depletion Sirt1. All proteins above were measured in the cell study. Glucagon-Like Peptide-1 (GLP-1) Activates AMPK and Diminishes RYGB greatly lowered fasting plasma glucose, CRP, TNF-α, IL-6 and MDA; Infl ammation in Human Aortic Endothelial Cells improved insulin sensitivity (p<0.001); the nuclear protein fractions of Sirt1, NADIA M. KRASNER, JOSE M. CACICEDO, YASOU IDO, NEIL B. RUDERMAN, PGC1α and Nrf2, and the protein level of PGC1α were signifi cantly increased, Boston, MA while the protein levels of iNOS and FOXO1, and the nuclear fraction of p53 GLP-1 based therapies are prescribed for type 2 diabetes due to their were signifi cantly decreased (p<0.001) in the blood vessels of RYGB rats. In effects on insulin sensitivity and secretion, glucagon secretion, and food EAhy926 cell, Sirt1 depletion consequently leads to Sirt1 75% reduction, and intake, resulting in improved glycemic control. Recent epidemiological Sirt1 downstream signaling separately and signifi cantly decreased (PGC1a, studies also suggest that GLP-1 may have cardiovascular benefi ts; however, Nrf2) or increased (iNOS, FOXO1, p53) (all p<0.001). the mechanism responsible is unknown. AMP- activated protein kinase Our results suggest that the improvement in cardiovascular risk by RYGB (AMPK) has been implicated as a therapeutic target for the prevention is mediated by preventing endothelial cells from undergoing apoptosis, of cardiovascular disease by virtue of its effects on lipid metabolism, reducing infl ammatory/oxidative stress through Sirt1 and Sirt1-regulated infl ammation, and other atherogenic events in vascular cells. In the present downstream signaling. study, we examined whether and how GLP-1 activates AMPK, and its Supported by: University of South Florida affects on infl ammation in cultured human aortic endothelial cells (HAECs). We found that GLP-1 treatment resulted in increased intracellular Ca2+

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and transient activation of AMPK in HAECs, with phosphorylation of its to 18.1, eGFR 81 ± 18 ml/min/1.73m2, ranging from 60 to 155, and median substrate acetyl-CoA carboxylase (ACC), after 5-15min. pCREB was also urine albumin-creatinine ratio (ACR) 9.8 (5.2 - 19.7) (interquartile range) mg/ activated after 20min, suggesting involvement of additional mechanisms. gCre. FMD was negatively correlated with age (r = -0.213, p = 0.026), systolic GLP-1 also signifi cantly decreased the infl ammatory response (indicated by blood pressure (SBP) (r = -0.190, p = 0.047), and positively with eGFR (r = increased expression of adhesion factors; VCAM-1, ICAM and TNFa) caused 0.284, p = 0.003), but not signifi cantly with log ACR (r = -0.058, p = 0.550) by hyperglycemia (HG, 25mM), lipopolysaccharide (LPS, 1ug/mL) or TNFα in simple regression analyses. In multiple regression analyses including (10ug/mL). Treatment of HAECs with LPS or TNFα also increased monocyte age, sex, duration of diabetes, body mass index, SBP, smoking, glycated adhesion, this too was diminished by GLP-1. RT-PCR revealed that HAECs hemoglobin, fasting insulin, total cholesterol, use of statins, use of ACE POSTERS

Complications also express dipeptidyl peptidase-4 (DPP4), the primary enzyme responsible inhibitors/ARBs, and eGFR or log ACR, only eGFR (β = 0.334, p = 0.006), but Acute and Chronic for GLP-1 degradation. Incubation with HG increased HAEC DPP4 activity, as not log ACR, was an independent contributor to FMD (R2 = 0.219, p = 0.018). well as GLP-1 degradation, potentially explaining the reduced level of GLP-1 In conclusion, eGFR, but not albuminuria, is independently associated with in diabetic patients. The results suggest that the benefi cial effects of GLP-1 vascular endothelial function in type 2 diabetic patients. on atherosclerotic risk could be mediated by AMPK activation, resulting from calcium infl ux, and the resulting decrease in infl ammation. Further studies 448-P are needed to determine if the effects of GLP-1 are attributable to AMPK, as Coronary Artery Calcifi cation, but Not its Progression, Predicts well as additional mechanisms potentially working in conjunction. CAD Incidence in Type 1 Diabetes TREVOR J. ORCHARD, RACHEL G. MILLER, Pittsburgh, PA 446-P Though coronary artery calcifi cation (CAC) has been to be shown to High Molecular Weight Adiponectin (HMW-A) and Cardiovascular strongly correlate with the prevalence or history of coronary artery disease (CV-) Death in Patients With Type 2 Diabetes (T2D) events (CAD) in type 1 diabetes (T1D), how well it, or its progression, CLAUDIA MENZAGHI, LUCIA SALVEMINI, CONCETTA DE BONIS, SIMONETTA predicts future events is unclear. We thus examined 289 participants in the BACCI, VINCENZO TRISCHITTA, San Giovanni Rotondo, Italy Pittsburgh Epidemiology of Diabetes Complications (EDC) study of childhood Cardiovascular disease is the fi rst-cause of death in T2D. Novel markers onset T1D. When CAC (C-150 scanner, GE Imatron, South San Francisco, CA) are needed in order to predict and prevent such event. Recently, high was fi rst determined, mean age and duration of T1D were 38 and 31 years adiponectin has been surprisingly associated with increased mortality in respectively. During a mean of 12 years follow up, 49 incident events (CAD elderly people with T2D; whether a similar counter-intuitive association death, myocardial infarction, coronary revascularization, EDC physician is observed also with CV-death in T2D is not known. Our aims were to diagnosed angina or ischemic ECG on Minnesota Coding)) were observed. Log investigate the association between serum HMW-A and CV-death in T2D CAC was the strongest predictor (Hazard ratio 1.35 95% CI 1.2-1.5) along with and whether such association is a causal one. T1D duration and total cholesterol in a forward stepwise Cox proportional We studied 359 patients with T2D and coronary artery disease from the hazard model that also allowed for the entire lipid profi le, HbA1c, smoking, Gargano Heart Study and performed a “mendelian randomization analysis” hypertension, albumin excretion, estimated GFR, BMI, pulse rate and white testing the association between rs822354 (a SNP in the adiponectin locus blood cell count all previously shown to predict CAD in this study. However, strongly associated with HMW-A in GWAS) and CV-death in the same in the subgroup (187) with repeated CAC measures approximately 5 years cohort. later who did not have an incident event before repeat CAC, progression During a 7 years follow-up, 58 CV-death/1,934 person-year occurred. of CAC (determined by the Hokanson method) did not add to the prediction HMW-A predicted CV-death in a model comprising age, sex, smoking habit, of the 14 subsequent incident events for which baseline CAC was the BMI, HbA1c, insulin therapy, hypertension, total cholesterol, HDL cholesterol only signifi cant predictor (HR 2.13 (0.6-6.90) and 1.4 (1.1-1.8) respectively). and triglycerides levels: HR per SD increment of 1.38 (1.10-1.74). This Similarly, for 87 individuals with prevalent CAD at fi rst CAC measure, only association was strongly signifi cant among males (n=242, HR=1.58, 95%CI baseline CAC, and not its progression, predicted the 37 recurrent events. 1.22-2.05), but not females (n=117, HR=0.91, 95%CI 0.55-1.47), thus pointing All analyses of CAC progression were adjusted for years between the 2 CAC to a sex specifi c effect of HMW-A on CV-death (p value for HMW-A-by-sex measures. We thus conclude that while CAC is a powerful predictor of CAD interaction=0.045). events in T1D, determining its rate of progression may not substantially add Notably, rs822354 (similarly associated with HMW-A levels in both males to its prediction of CAD events. and females: per allele adjusted β±SE=0.22±0.09 µg/ml, p=0.01 and 0.26±0.15 µg/ml, p=0.08, respectively), was associated with CV-death among males 449-P (HR=1.80, 95%CI 1.16-2.80), but not females (HR=0.85, 95%CI 0.42-1.71; p for High Glucose Induced ANRIL Expression in Human Macrophage via SNP-by-sex interaction=0.050), thus mirroring the sex specifi c association Transcription Factor NF-kB and AP-1 observed between HMW-A levels and CV-death and strongly supporting a JIANJIN GUO, WEI REN, JIE LIU, WEIPING JIA, Nanjing, China, Shanghai, China, cause-effect relationship underlying the association in males. Taiyuan, China In conclusion, in patients with T2D high serum HMW-A is an independent ANRIL, a large noncoding RNA has been identifi ed associated with risk factor for CV-death among males, but not females. Further studies are coronary disease, intracranial aneurysm and also type 2 diabetes in recently needed to confi rm this fi nding. genomewide association studies (GWAS). Furthermore, the expression level of ANRIL was signifi cantly increased in patients with high atherosclerotic 447-P plaque. In this study, we examined the regulation of human monocyte- Vascular Endothelial Function Is Not Associated With Albuminuria derived macrophage (MDM) ANRIL expression by high concentration But With eGFR in Type 2 Diabetic Patients glucose. Treatment of human MDMs with glucose (5.6 to 30 mmol/L) for SATOSHI IMAMURA, TOMOAKI MORIOKA, YUKO YAMAZAKI, NAOYA KAWANO, 24 to 72 hours or 48 hours, ANRIL mRNA levels was increasing in a dose- RYUTARO NUMAGUCHI, HIROMI URATA, KOKA MOTOYAMA, KATSUHITO MORI, and time-dependent manner. Several NF-kB and activated protein-1 (AP-1) SHINYA FUKUMOTO, TETSUO SHOJI, MASANORI EMOTO, MASAAKI INABA, binding sites were identifi ed in the promoter of ANRIL gene. Exposure of Osaka, Japan MDMs to a high glucose environment increased the promoter activities and Chronic kidney disease (CKD) is recognized as an independent predictor of enhanced the binding of NF-kB and AP-1 to promoter. In addition, induction cardiovascular mortality. Vascular endothelial dysfunction contributes to the of ANRIL expression by high glucose was abolished by BAY 11-7085 (NF-kB initiation/progression of atherosclerosis and a predictive surrogate marker inhibitor) and curcumin (AP-1 inhibitors). Overall, these results indicate that of cardiovascular events. However, clinical association of renal function high glucose concentrations enhance ANRIL expression in human MDMs and/or albuminuria with vascular endothelial dysfunction has not been fully and that is regulated by transcription factor NF-kB and AP-1. elucidated in type 2 diabetes. In this study, we investigated the association between vascular endothelial function and renal function or albuminuria in 450-P patients with type 2 diabetes. One hundred and nine patients with type 2 Saxagliptin Treatment Enhances eNOS Function and Expression diabetes whose estimated glomerular fi ltration ratio (eGFR) greater than 60 While Reducing Nitroxidative Stress in Aortic and Glomerular 2 ml/min/1.73m , were included in this study (mean age 60 years, duration Endo thelia of Obese Zucker Rats of diabetes 8.1 years, 88 patients with normo-, 16 with micro-, and 5 with R. PRESTON MASON, ROBERT F. JACOB, J. JOSE CORBALAN, LU-LIN JIANG, macro-albuminuria). Flow-mediated dilatation (FMD) of the brachial artery TADEUSZ MALINSKI, Beverly, MA, Athens, OH as an endothelial function was assessed by ultrasound (UNEXEF 18G, Unex, Endothelial cell (EC) dysfunction associated with hyperglycemia is Japan). The subjects exhibited mean FMD 7.0 ± 3.9 (SD) %, ranging from 0.7 characterized by reduced nitric oxide (NO) bioavailability linked to endothelial

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NO synthase (eNOS) uncoupling and increased peroxynitrite (ONOO-) HbA1c values of <5.7% (normal according to ADA criteria), 5.7-6.4% (at production. In this study, we hypothesized that EC dysfunction is reversed risk of diabetes according to ADA criteria), and ≥6.5% (diabetes according to with saxagliptin, a DPP4 inhibitor, through enhanced eNOS expression ADA criteria) were found in 36.4%, 56.2%, and 7.4% of women and in 44.2%, and activity. To test this hypothesis, obese Zucker rats were fed a high-fat 46.6%, and 9.1% of men, respectively. The prevalence of angiographically diet and treated with saxagliptin at 10 mg/kg/day for 6 weeks. Aortic and diagnosed coronary atherosclerosis in these HbA1c categories was 31.2%, glomerular ECs were then assayed ex vivo for NO and ONOO- release using 38.2%, and 47.2% among women (ptrend=0.041) and 63.2%, 65.3% and amperometric nanosensors. Saxagliptin treatment was compared to acute 64.8% among men (ptrend=0.589). In logistic regression models, HbA1c as administration of S17834, an NAD(P)H oxidase inhibitor (tested at 40 µM), or a continuous variable was a strong predictor of coronary atherosclerosis POSTERS superoxide dismutase (SOD) at 100 units/mL. Changes in EC function were among women (adjusted OR for a 1% increase in HbA1c 1.61 [95% CI 1.07- Complications correlated with fasting glucose (FG) levels and eNOS expression measured 2.43]; p=0.024) but not among men (OR 0.92 [0.74-1.13]; p = 0.416). The Acute and Chronic by qPCR. The results showed that saxagliptin treatment increased aortic and interaction HbA1c by gender was signifi cant (p=0.022), indicating that HbA1c glomerular NO release by 23% (p<0.05) and 18% (p<0.02), respectively, with was a signifi cantly stronger predictor of coronary atherosclerosis among comparable reductions in ONOO- levels; the NO/ONOO- ratio, an indicator of women than among men. NO synthase coupling, increased by 53% in aortic ECs (p<0.01). S17834 and We conclude that gender has a signifi cant impact on the association of SOD further enhanced NO bioavailability, increasing aortic NO release by HbA1c with angiographically diagnosed coronary atherosclerosis among 48% (p<0.01) and 196% (p<0.001), respectively, over levels observed with subjects without previously known diabetes. saxagliptin alone. Saxagliptin treatment also increased eNOS expression in ECs by 31% (p<0.01). Reversal of EC dysfunction was observed prior to 453-P any reductions in FG levels. These data suggest that saxagliptin reverses EC Diabetic Retinopathy and CVD: A Continuum of Micro- and Macro- dysfunction in obese rats by enhancing eNOS function and expression while angiopahty Is Suggested by NT-ProBNP reducing nitroxidative stress. KUMIKO HAMANO, IKUE NAKADAIRA, JUN SUZUKI, MEGUMI GONAI, Kawasaki, Japan 451-P Diabetic retinopathy (DMR) has been associated with increased all-cause Extracellular Superoxide Dismutase (SOD3) Variants and Risk of and CVD mortality risk. Identifi cation of DMR could possibly add to the diabetic Cardiovascular Events and All-Cause Mortality in Type 1 Diabetic patient’s CVD risk stratifi cation. However,the underlying mechanism linking Subjects different organs are not well elucidated. N-terminal proBNP (NT-proBNP) KAMEL MOHAMMEDI, SAMY HADJADJ, RONAN ROUSSEL, FRÉDÉRIC FUM- is postulated as a prognostic biomarker of heart failure and cardiovascular ERON, MICHEL MARRE, GILBERTO VELHO, Paris, France, Poitiers, France mortality in type 2 diabetes. We published that NT-proBNP could be a marker Extracellular superoxide dismutase (SOD3) is a major extracellular of silent myocardial ischemia and have reported that NT-proBNP could antioxidant enzyme. It protects against oxidative damage and is highly be a predictor of kidney function in diabetes at 72th scietifi c session. We expressed in blood vessels, particularly in arterial walls. We investigated postulated the hypothesis that NT-proBNP might be a common biomarker of association of SOD3 gene variants with the risk of cardiovascular events diabetic vascular complications. 208 t ype 2 diabetes (age 60.2±13.0, mean±SD) (myocardial infarction, stroke, coronary and carotid revascularization) and were enrolled as a hospital cohort and cross-sectional case controlled study all-cause mortality in type 1 diabetic subjects. was designed. The examination included full medical histories, physical Five SNPs in the SOD3 gene region were genotyped in type 1 diabetic examinations, and blood samples. Subjects with DMR (n=60) were similar subjects from GENEDIAB (n=494, 10-year follow-up) and GENESIS (n= 661, in age but had longer duration of diabetes (p<0.001). HbA1c, serum lipids or 5-year follow-up) cohorts. Cox proportional hazards analyses were used to creatinine were similar. Systolic blood pressure and PWV was signifi cantly estimate hazard ratios (HR) during follow-up. Plasma concentration of SOD3 higher in subjects with DMR(p=0.0004). Serum NT-proBNP was higher in DMR was measured by ELISA in GENEDIAB participants at baseline. than those without (69 vs 39pg/ml, p<0.0001). When subjects were stratifi ed The incidence of cardiovascular events and all-cause mortality was by NT-proBNP levels, the highest tertile (>79pg/ml) had signifi cantly higher 8.9% and 11.7%, respectively, in GENEDIAB. It was 5.0% and 4.1% in proportions of DMR (40.9%) than the lowest (<31pg/ml,17.4%). The highest GENESIS. The G-allele of rs2284659 was associated with the incidence of tertile had OR of 3.30 for DMR (p=0.0117). Subjects with DMR had higher cardiovascular events (HR 1.67, 95% CI 1.15-2.45, p=0.007) and all-cause probability of having CVD, microalbuminuria, neuropathy and peripheral mortality (HR 3.00, 95% CI 1.64-7.50, p<0.0001) in GENEDIAB, adjusted for arterial disease (OR=2.72, 3.29, 4.17 and 5.42 respectively). In conclusion, it sex, age, smoking and renal status. The variant was associated with the was confi rmed that the presence of DMR is a CVD risk and was related to incidence of cardiovascular events (HR 1.89, 95% IC 1.14-3.39, p=0.01) systemic vascularture. The higher levels of NT-proBNP in DMR suggests a in GENESIS, but not with all-cause mortality (HR 1.92, 95% IC 0.86-8.19, continuum of vascular involvement from retinal microvessel to macrovessels p=0.13). Associations were confi rmed in pooled analyses of the cohorts (HR such as coronary or peripheral arteries. 1.63, 95% CI 1.22-2.19, p=0.0008 for cardiovascular events and HR 2.64, 95% CI 1.58-5.40, p<0.0001 for all-cause mortality). The GG-risk genotype was 454-P associated with decreased plasma concentration of SOD3 (GG 205±35, GT Glucose Control after CABG 284±37 and TT 266±42 ng/ml, p=0.001). Comparable results were observed PARAS MEHTA, CHARLES L. BAIMBRIDGE, DAOUD DAOUD, SUSAN A. BAIM- with other variants. BRIDGE, GLENN R. CUNNINGHAM, Houston, TX In conclusion, SOD3 polymorphisms were associated with cardiovascular The Center for Medicare and Medicaid Services requires that glucose events and all-cause mortality in type 1 diabetic subjects. Our results levels (G) are to be <200 mg/dL at 06:00 on post-op Days 1 and 2 in cardiac suggest that SOD3 plays a major role in the protection against oxidative surgery patients. SLEH is an open hospital, so management decisions are stress in type 1 diabetic patients. made by individual physicians. We recommend that patients with known diabetes (DM) be treated with an insulin infusion protocol (IIP) when their 452-P post-op G is >120 mg/dl and that non-DM receive IIP when their post-op G Signifi cant Impact of Gender on the Association of HbA1c With An- is >150 mg/dL. giographically Diagnosed Coronary Atherosclerosis The purpose of this study was to retrospectively assess the effects of HEINZ DREXEL, ALEXANDER VONBANK, PHILIPP REIN, DANIELA ZANOLIN, several variables on post-op G after CABG. CORNELIA MALIN, CHRISTOPH H. SAELY, Philadelphia, PA, Triesen, Liechtenstein, Medical records of 462 consecutive CABG patients in 2010 were reviewed. Feldkirch, Austria We noted pre-op diagnosis of DM, post-op G, use of IIP and pressors (P). The association of HbA1c with angiographically determined coronary Forty-nine percent (226/462) of patients had DM, but 17% did not receive atherosclerosis is unclear. In particular, it has not been investigated so far IIP. Thirty-four percent (80/236) of non-DM had post-op G >150 mg/dL, but whether gender modulates the association of HbA1c with angiographically only 34% received the IIP. Comparison of DM vs. non-DM who received diagnosed coronary atherosclerosis. We therefore aimed at clarifying this the IIP indicated that mean G prior to start of infusion was higher, time in issue. starting IIP was less, duration of infusion was longer, time to achieve G <150 We enrolled a large consecutive series of 1449 patients, 484 women and mg/dL was longer, and insulin infusion rate was higher when the target G 965 men, who did not have previously known diabetes and who underwent was reached. G when IIP was stopped were similar, as were mean 6AM G on coronary angiography for the evaluation of stable coronary artery disease. post-op Day 1 and proportion of patients with G >200 mg/dL. DM had higher Signifi cant coronary atherosclerosis was diagnosed in the presence of mean G and more were >200 mg/dL on post-op Day 2 when 36% of DM vs. signifi cant coronary stenoses with lumen narrowing ≥50%. 23% of non-DM were receiving subcutaneous (SC) insulin.

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A115 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

For patients not on IIP, mean 6AM post-op Day 1 G were similar; but more DM 457-P had G > 200 mg/dL. Day 2 G were higher and more DM had G > 200 mg/dL. Intolerance to Metformin: An Unforeseen Phenotype With Reduced Mean 6AM post-op Day 1 G was higher in patients on IIP and pressors (P) Ischemic Heart Disease, Left-Handedness, ABO Imbalance and vs. no pressor (no-P), but the difference in G > 200 mg/dL was only marginally High Ferritin signifi cant. MICHEL P. HERMANS, SYLVIE A. AHN, MICHEL F. ROUSSEAU, Brussels, Belgium To achieve G <200 mg/dL on post-CABG Days 1 and 2, it is essential that Background: Many type 2 diabetes mellitus (T2DM) patients treated with nearly all DM receive IIP and an IIP be started quickly. It may help to start metformin develop severe gastrointestinal (GI) symptoms leading to drug IIP at a higher infusion rate in DM. It is reasonable to use higher SC doses of

POSTERS discontinuation, depriving them of potentially cardioprotective pleiotropic

Complications long-acting insulin to achieve G <200 mg/dL at 6AM on post-op Day 2. effects of this fi rst-line oral agent. At present, it is unclear whether treating Acute and Chronic Supported by: St. Luke’s Episcopal Hospital diabetes without being able to ever use metformin alters the cardiovascular outcomes. 455-P Patients & Methods: From a population of 773 consecutive T2DM Genetic Variability in GLP-1R Gene Is Associated With Risk of Coro- outpatients, the cardiometabolic phenotype of 83 patients who discontinued nary Artery Disease in Type 2 Diabetes in a Chinese Han Popula- metformin due to GI intolerance ([Met-Intol] cases was compared to that of tion 332 age-/gender-matched metformin-tolerant controls ([Met-Tol]), meaning a XIAOWEI MA, RAN LU, XIAOWEI WEI, GE BAI, JIANWEI ZHANG, RUIFEN DENG, case:control ratio of 1:4. NAN GU, NAN FENG, JIANPING LI, XIAOHUI GUO, Beijing, China Results: Mean age (1 SD) was 70 (13) (male:female 46:54). [Met-Intol] more Objective: Previous studies have showed that GLP-1 has benefi cial effects often belonged to blood group A and subgroup ARh+, with 50% and 66% on coronary heart disease through direct/indirect mechanisms mediated by relative increases (p=0.0039 and 0.0005, respectively). Smoking; diabetes glucagon-like peptide-1(GLP-1) receptor (GLP-1R). The aim of our study is to duration; HbA1c; BMI, blood pressure; waist, fat mass; visceral fat; liver investigate whether or not the genetic variability in gene GLP-1R affects the steatosis; and metabolic syndrome were not different between groups. risk of cardiovascular disease in type 2 diabetes in the Chinese population. There were twice as many non-right-handers in [Met-Intol] (18% vs. 9%; Method: Of total 611 unrelated Han subjects with type 2 diabetes,394 p 0.0262). Statins/fi brates were used by 66%/19% [Met-Tol] vs. 48%/18% individuals with coronary artery disease as cases (CAD) and 217 controls [Met-Intol] (p 0.0051 for statins). LDL-C, non-HDL-C and apoB were higher (Non-CAD) were studied. From HapMap phaseII(r2<0.8 and MAF≥0.05, R#27) in [Met-Intol]. There were no differences between groups concerning eGFR, CHB database, 11 haplotype-tagging single nucleotide polymorphisms (SNPs) albuminuria, erectile dysfunction, and microangiopathy. [Met-Intol] had in GLP-1R gene were selected, rs761387 (T>C), rs2268635(G>A), rs769547 signifi cantly higher serum iron and ferritin. Macroangiopathy was markedly (G>A), rs910162 (T>A), rs3765468 (G>A), rs3765467 (G>A), rs3765466 (A>T), reduced in [Met-Intol] (p=0.0486), the decrease being assigned to both a rs10305456 (C>T), rs10305518 (T>G), rs1820 (T>A),and rs4714210 (A>G). sharp lowering in CAD (-34%; p=0.0374) and PAD (-44%; NS). All the subjects for the 11 SNPs were genotyped by the polymerase chain Conclusion: Metformin-intolerant T2DM exhibit markedly reduced rates reaction-restriction fragment length polymorphism assay or directly DNA- of CAD; this unforeseen cardioprotective phenotype also includes left- sequencing. The frequencies of genotypes and alleles at the loci were handedness; ABO imbalance and iron load. compared between the case and control groups tested by chi-square test and multivariate logistic regression analysis adjusted for confounders. Results: 458-P In recessive inheritance mode, the carriers of genotype GG at rs4714210 in HbA1c Is a Signifi cantly Stronger Predictor of Cardiovascular GLP-1R gene had a lower risk of CAD (OR = 0.918, 95%CI = 0.863-0.976, p = Events in Women than in Men among Patients Undergoing Coronary 0.002; OR’ = 0.485, 95%CI = 0.254-0.926, p’ = 0.028, after adjusted for the Angiography other known CAD risk factors, including sex, age, BMI, smoking status and CHRISTOPH H. SAELY, ALEXANDER VONBANK, PHILIPP REIN, DANIELA ZANO- diabetic duration). The SNP rs910162 was found a tendency to be associated LIN, KATHRIN GEIGER, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, with the CAD risk, mainly in males, with carriers of allele A at a higher risk of Philadelphia, PA CAD in dominant inheritance mode (OR= 1.449, 95%CI=1.014-2.071,P 0.046). The association of HbA1c with future cardiovascular events in the clinically Conclusion: In the Chinese type 2 diabetic patients, genetic variations in important high-risk population of patients undergoing coronary angiography GLP-1R genes are associated with cardiovascular risk. has not been investigated so far. In the present study we therefore addressed Supported by: Peking University First Hospital this issue and also tested the hypothesis that gender modulates the impact of HbA1c on cardiovascular event risk. 456-P We prospectively recorded cardiovascular events over a mean follow-up Lipoprotein (a), the Metabolic Syndrome and Vascular Risk in An- period of 4.4 years in a large consecutive series of 1449 patients, including giographied Coronary Patients 484 women and 965 men who did not have previously known diabetes and ALEXANDER VONBANK, CHRISTOPH H. SAELY, PHILIPP REIN, ABDURAHMAN who underwent coronary angiography for the evaluation of stable coronary SAID, HEINZ DREXEL, Triesen, Liechtenstein, Feldkirch, Austria, Philadelphia, PA artery disease. Lipoprotein (a) [Lp(a)] especially in young individuals is an important During follow-up, the incidence of cardiovascular events was 19.5% in cardiovascular risk factor. However, data on the vascular risk conferred by women and 25.6% in men, corresponding to annual event rates of 4.4% Lp(a) in patients with the metabolic syndrome (MetS) are not available. and 5.8%; p = 0.001. Among women, HbA1c strongly and signifi cantly Lp(a) was measured in a cohort of 587 consecutive patients undergoing predicted cardiovascular events (adjusted OR for a 1% increase in HbA1c = coronary angiography for the evaluation of stable coronary artery disease. 1.69 [1.16-2.45]; p = 0.006), whereas the association between HbA1c and The MetS was diagnosed according to International Diabetes Federation cardio vascular events was weaker and statistically non-signifi cant in men (IDF) criteria. Vascular events were recorded over 8 years. (OR = 1.15 [0.95-1.39]; p = 0.147. An interaction term gender x HbA1c was Median Lp(a) was signifi cantly lower in patients with the MetS (n=345) signifi cant (p = 0.024), indicating that HbA1c was a signifi cantly stronger than in subjects who did not have the MetS (12 [interquartile range 0.8-35] predictor of cardiovascular events among women than among men. vs. 17 [0.8-57] mg/dl; p=0.004). Prospectively, 34% of our patients suffered We conclude that HbA1c is a signifi cantly stronger predictor of cardio- vascular events. Lp(a) proved to be a strong and independent predictor of vascular events in women than in men among patients undergoing coronary vascular events in subjects without the MetS (standardized adjusted HR 1.33 angiography. [1.01-1.74]; p=0.029) but not in patients who had the MetS (HR 1.07 [0.84- 1.37]; p=0.543). An interaction term MetS x Lp(a) was signifi cant (p=0.005), 459-P indicating that Lp(a) was a signifi cantly stronger predictor of vascular events Lipid Parameters in Acute Coronary Syndromes versus Stable Coro- in subjects without Mets than in patients with the MetS. nary Artery Disease in Patients With the Metabolic Syndrome and We conclude that Lp(a) in patients with MetS is low and is not associated in Subjects Who Do Not Have the Metabolic Syndrome with the incidence of vascular events. The power of Lp(a) as a predictor DANIELA ZANOLIN, ALEXANDER VONBANK, PHILIPP REIN, CHRISTOPH H. SAE- of cardiovascular events is signifi cantly modulated by the presence of the LY, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Philadelphia, PA MetS. Differences in lipid parameters between patients with acute coronary syndromes (ACS) and patients with stable coronary artery disease (CAD) are unclear and are addressed in the present study. We enrolled consecutive patients with angiographically proven stable CAD (of whom 37.2% had the MetS according to NCEP-ATPIII criteria and 182

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A116 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES consecutive patients with acute coronary syndromes (of whom 33.9% had the MetS). When compared to patients with stable CAD, HDL cholesterol and apolipoprotein A1 were signifi cantly lower in patients with ACS than in those with stable CAD both among subjects with the MetS (38±9 vs. 48±13 mg/ dl; p<0.001 and 139±38 vs.14±25 mg/dl; p<0.001, respectively) and among those without the MetS (52±17 vs. 60±15 mg/dl; p=0.001 and 147±31 mg/dl vs. 157±26 mg/dl; p=0.003, respectively). Analysis of covariance adjusting for age, gender, smoking, BMI, and hypertension confi rmed an independent POSTERS impact of the ACS state on these lipid parameters both among patients with Complications the MetS (F=5.287; p<0.001) and among subjects who did not have the MetS Acute and Chronic (F=6.042; p=0.014). Total cholesterol, LDL cholesterol, apolipoprotein B, and trigylcerides neither in patients with the MetS nor among subjects without the MetS differed signifi cantly between ACS and stable CAD patients. We conclude that both among patients with the MetS and among non- MetS individuals, HDL cholesterol and apolipoprotein A1 are lower in the ACS state than with stable CAD.

460-P Effects of Empaglifl ozin on Oxidative Stress and Endothelial Dys- function in STZ-Induced Type 1 Diabetic Rat MATTHIAS OELZE, SWENJA KRÖLLER-SCHÖN, MICHAEL MADER, ELENA ZINßIUS, PAUL STAMM, MICHAEL HAUSDING, ERIC MAYOUX, PHILIP WENZEL, EBERHARD SCHULZ, THOMAS MÜNZEL, ANDREAS DAIBER, Mainz, Germany, Biberach, Germany In diabetes, cardiovascular complications are associated with endothelial dysfunction and oxidative stress. Empaglifl ozin (Empa), as a selective sodium glucose cotransporter 2 inhibitor in clinical development, offers a Supported by: Social Development Foundation (2011B031800273) promising novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether 462-P treatment with Empa could improve endothelial dysfunction in type I diabetic Activin A Yields Additional Value for Prediction of MACE in Type 2 rats via reduction of glucotoxicity and associated oxidative stress. Diabetes (T2D) on Top of Conventional CV Risk Factors Type I diabetes in Wistar rats was induced by an i.v. injection of ANNE PERNILLE OFSTAD, KÅRE I. BIRKELAND, SVEND AAKHUS, ELSA ORVIK, streptozotocin (60 mg/kg). One week after injection Empa was administered PÅL AUKRUST, THOR UELAND, MORTEN W. FAGERLAND, LARS GULLESTAD, via drinking water for 7 weeks. Treatment with Empa(10 and 30 mg/kg/d), ODD ERIK JOHANSEN, Baerum, Norway, Oslo, Norway showed reduction of blood glucose and a normalization of endothelial Robust cardiovascular (CV) risk markers are needed to identify individuals dysfunction (aortic rings) in diabetic rats and a reduced oxidative stress in with excessive CV risk in T2D. We assessed the prognostic impact of 3 aortic vessels (dihydroethidine staining), in blood (phorbol ester/zymosan novel CV risk markers on major CV events (MACE): death from all causes, A-stimulated chemiluminescence)compared to control. Additionally, the myocardial infarction, stroke or hospitalization for unstable angina pectoris. higher NADPH-oxidase activity in heart tissue of diabetic animals was 135 T2D patients (26% female, 43% albuminuria, TABLE) treated according to normalized by SGLT2i therapy. optimized standard of care were tested at baseline (BL) for the infl ammatory In this study we could demonstrate that Empa improves hyperglycemia marker Activin A and with a max stress ECG test (evaluated by recovery and prevents the development of endothelial dysfunction and oxidative patterns of the ST-segment [pathological if clockwise or 8 loop]) and stress in type 1 diabetic rats. Future studies will investigate the underlying echo (E/Em). Mean±SD observation time was 8.6±2.1 years. We used Cox mechanisms of these antioxidant and anti-infl ammatory effects with proportional hazard modeling, Harrell C-statistic and the net reclassifi cation special emphasis on the activity of NADPH oxidase and the prevention of improvement to assess the additional value of the novel CV markers to uncoupling of the nitric oxide synthase, which contributes to cardiovascular the conventional. 26 patients had a MACE during follow-up and all 3 novel complications. markers were signifi cantly associated with MACE in a univariate Cox Supported by: Boehringer Ingelheim Pharmaceuticals, Inc. model. After adjustment for conventional risk factors, Activin A and pathol. recovery-loop maintained their signifi cance (HR [95%CI] 2.33 [1.44-3.77] 461-P and 3.94 [1.15-13.52], while E/Em lost independent prognostic value. The Serum Cystatin C May be a Predictor of Coronary Heart Disease in addition of Activin A to standard CV panel improved prediction of outcome, Type 2 Diabetes while pathol. recovery loop and E/Em did not (TABLE). In conclusion, the MAN-MAN WANG, PAN-WEI MU, JIONG SHU, YAN-MING CHEN, GUO-CHAO infl ammatory marker Activin A, yielded additional prognostic value on ZHANG, LONG-YI ZENG, Guangzhou, China predicting MACE in T2D on top of conventional CV risk factors. Serum cystatin C, a novel measure of renal function, is reported as a strong predictor of cardiovascular events. Furthermore it has been shown to predict progression of subclinical coronary atherosclerosis in type 1 diabetes. We investigated the association between cystatin C and coronary heart disease (CHD) and evaluated the value of cystatin C in CHD in type 2 diabetes (T2DM). 149 T2DM patients (94 males/55 females, age 59.9±10.8 years, duration 7.8±7.0 years) without microangiopathy were recruited. After being tested by 320-row CT, 68 patients were diagnosed complicated with CHD (T2DM+CHD), 40 patients were found complicated with coronary atherosclerosis but not CHD (T2DM+CA), 41 patients were without macroangiopathy (T2DM+only). Serum cystatin C levels in T2DM+CHD were signifi cantly higher than those in T2DM+CA, which were higher than in T2DM+only signifi cantly. The study population was divided four equal groups according to cystatin C levels. The severity of macroangiopathy tended to increase with the cystatin C levels. This study suggested that elevated cystatin C level was associated with coronary atherosclerosis and it may be a predictor of CHD in T2DM.

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A117 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

463-P pathway. Sestrin2 was reported either as up- or down-stream of KEAP1/ Hyperglycemia and Insulin Resistance in Cardiac Arrest Patients Nrf2 pathway to involve in the protection, in parallel with Nrf2 against Treated With Moderate Hypothermia various oxidative stresses. The present study examined whether SFN MATTHEW ETTLESON, VANESSA ARGUELLO, AMISHA WALLIA, RICHARD A. could protect from Ang II-induced cardiomyopathy through up-regulating BERNSTEIN, MARK E. MOLITCH, Chicago, IL and activating Nrf2 and its newly-found target gene, sestrin2. FVB mice To determine the effects of therapeutic hypothermia (TH) (33oC) on blood were given subcutaneous injection of Ang II for 2 months with or without glucose (BG) levels and insulin resistance in post-cardiac arrest patients and treatment of SFN for 3 months and kept until 6 months. At 3 and 6 months, blood pressure and cardiac function were assessed. Cardiac fi brosis,

POSTERS their effects on mortality and morbidity, we conducted a retrospective chart

Complications review of patients undergoing TH after in-hospital or out-of-hospital cardiac infl ammation, and oxidative damage were also detected by Western Acute and Chronic arrest between September, 2005 and April, 2008 at Northwestern Memorial blotting, real-time qPCR, and immunostaining. SFN signifi cantly prevented Hospital. BG levels from 72 hours before arrest to 48 hours after TH, IV Ang II-induced high blood pressure at 6M and cardiac dysfunction at both insulin infusion rates, and outcomes were recorded and then analyzed. 3 and 6 months. Ang II treatment induced cardiac pathological changes Of 62 post-cardiac arrest patients treated with TH, 16 (26%) had diabetes including myocardial hypertrophy or degeneration, and increased interstitial mellitus (DM), 52 (84%) patients were treated with insulin via drip for and perivascular accumulation of collagen fi ber. These pathological changes hyperglycemia (within 3 days of arrest), while 46 (74%) had the drip initiated accompanied with signifi cant increases in cardiac oxidative damage, shown during hypothermia. All patients had > 1 episode of hyperglycemia (BG by increased 3-NT and 4-HNE accumulation, and the elevated expression >110 mg/dL). Mean BG levels between cardiac arrest and the initiation of of infl ammation factors (TNF-α and PAI-1) and fi brosis factors (TGF-β1 and hypothermia were higher in Non-survivors (253 + 112 mg/dL, n=48) than in CTGF). Those changes and damages were almost completely prevented by Survivors (192 + 69 mg/dL, n=24, p=0.016). However, blood glucose values SFN treatment for 3 months, which was accompanied with an up-regulation during hypothermia, during rewarming, and 24-48 hours after hypothermia of Nrf2 expression and activity that was refl ected by increased Nrf2 were not signifi cantly different between the two groups. There were no nuclear accumulation and phosphorylation as well as expression of Nrf2 signifi cant differences in insulin infusion rates or insulin resistance between downstream antioxidants, especially the newly-found target gene, sestrin2. Survivors and Non-survivors at any time points. 25% of the Survivors and These results suggest that Ang II-induced cardiomyopathy can be prevented 26% of the Non-Survivors were previously diagnosed with DM (p=NS). In the by SFN probably via up-regulation of Nrf2 expression and activation and 21 patients with pre-arrest BG values, the peak BG levels occurred following sestrin2 expression as well. the arrest but prior to the institution of hypothermia. In post-cardiac arrest patients treated with TH, mortality was associated 466-P with increased BG levels after cardiac arrest but before the initiation of Relationship Between Lipid Droplet Proteins and Lipid Storage in hypothermia or an IV insulin drip. Likely, it is the severity of stress from Macrophages in Human Atherosclerotic Plaque the cardiac arrest that causes the hyperglycemia in these patients. More KYU YONG CHO, HIDEAKI MIYOSHI, TAKUMA KONDO, TATSUYA ATSUMI, Sap- studies are needed to differentiate whether decreased survival associated poro, Japan with hyperglycemia is due to stress severity rather than the hyperglycemia Macrophages play a major role in arteriosclerosis. Previously, foam per se. cell lipid droplets have been reported to be covered with the lipid droplet (LD) protein perilipin2 (PLIN2, ADRP). In adipose tissue, immature LDs are 464-P coated with PLIN2, and are replaced by perilipin1 (PLIN1, perilipinA) during Gender Does Not Signifi cantly Modulate the Association between maturation. PLIN1 regulates proteins involved in the storage of triglycerides Markers of Infl ammation and the Metabolic Syndrome Among Pa- in LDs and thereby prevents lipotoxicity by sequestering harmful lipids in tients With Stable Coronary Artery Disease stable LDs. In macrophages, however, the expression and function of PLIN1 CHRISTOPH H. SAELY, ALEXANDER VONBANK, PHILIPP REIN, ANDREAS LEIHER- are poorly understood. We examined the relationship between LD proteins ER, DANIELA ZANOLIN, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, and lipid storage using human specimens. Philadelphia, PA 65 plaque specimens were collected from patients undergoing carotid The metabolic syndrome (MetS) confers a stronger increase in endarterectomy and classifi ed according to symptomatic history. 34 cases cardiovascular event risk among women than among men. The MetS is had been suffering from cerebral infarction (symptomatic) while 31 did associated with elevated markers of infl ammation, and infl ammatory not have symptoms, but surgery was performed due to severe stenosis markers have been linked to cardiovascular event risk in MetS patients. (asymptomatic). In addition, macrophages were derived from healthy human We therefore tested the hypothesis that CRP and leukocyte count are more peripheral blood mononuclear cell and cultured with oxidized LDL (oxLDL) or strongly associated with the MetS among women than among men. very low density lipoprotein (VLDL). Immunostaining and western blotting We measured CRP and leukocyte counts a large high-risk cohort of 1041 revealed PLIN2 was increased in plaques from the symptomatic group while patients with angiographically proven stable coronary artery disease, PLIN1 was identifi ed in plaques from both groups. mRNA expression of PLIN2 enrolling 371 women and 670 men. The MetS was diagnosed according to was increased 3.4-fold in symptomatic plaques. In cultured macrophages, National Cholesterol Education Panel III criteria. Interaction analyses were both PLIN1 and PLIN2 existed on the surface of LDs. Small LDs were covered performed using analysis of covariance models, applying a general linear with PLIN2, while large LDs were coated with PLIN1 and contained mainly model approach. triglyceride. oxLDL increased macrophage number and the expression The prevalence of the MetS was signifi cantly higher among women of PLIN2. VLDL dramatically increased expression of PLIN2 in the fi rst 24 than among men (40.2 vs. 31.2%; p=0.004). Both CRP and leukocyte counts hr and then expression of PLIN1 increased as PLIN2 decreased. In human were signifi cantly higher in patients with the MetS than in those without macrophages, PLIN1 may play an important role in effi ciently storing lipids in MetS among women (0.50±0.58 vs. 0.41±0.83 mg/dl; p=0.001 and 6.9±1.7 stable LDs that consist mainly of triglycerides. vs. 6.3±1.8 G/l; p<0.001, respectively) and also among men (0.47±0.67 vs. 0.40±0.72 mg/dl; p<0.001 and 7.1±1.8 vs. 6.6±1.8 G/l; p<0.001, respectively). 467-P Formal interaction analyses did not show a signifi cant MetS by gender The Relationship Between Morning Blood Pressure Surge and interaction neither with regard to CRP (p=0.788) nor to leukocyte count Vascular Endothelial Dysfunction in Patients With Type 2 Diabe- (p=0.333), indicating that the associations between CRP and leukocyte tes Mellitus count did not differ signifi cantly between women and men. KOICHIRO YODA, SHINSUKE YAMADA, MAKI YODA, KAE HAMAMOTO, MASA- From the data of this large study we conclude that gender does not HIRO TSUDA, KATSUHITO MORI, MASANORI EMOTO, MASAAKI INABA, Osaka, signifi cantly modulate the association between CRP or leukocyte count and Japan the MetS among patients with stable coronary artery disease. Background: Morning blood pressure surge (MBPS) has been established as an independent predictor of cardiovascular event. However, little is 465-P known onthe association between MBPS and glycemic control in patients Prevention of Angiotensin II-Induced Cardiomyopathy by Sul- with type 2 diabetes mellitus (T2DM). foraphane Is Associated With Up-Regulation of Nrf2 and Sestrin2 Objective: The aim of this cross-sectional study was to examine the YING XIN, YANG BAI, YANG ZHENG, LU CAI, Louisville, KY, Changchun, China association between MBPS and glycemic control, and to investigate whether Angiotensin II (Ang II) is thought to play an important role in the patho- MBPS may affect vascular endothelial function in T2DM patients. genesis of diabetic cardiomyopathy. Sulforaphane (SFN) has gained more Design and Methods: We examined 50 T2DM patients (M/F; 25/25, age; attention by its anti-oxidative effect through activation of KEAP1/Nrf2 60 ± 13 years). MBPS and endothelial function were assessed by 24 hours

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A118 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES ambulatory blood pressure monitoring, and the brachial artery fl ow-mediated revealed pronounced differences between diabetic patients and control dilation (FMD), respectively. subjects among the youngest 50% of the participants. Results: In simple regression analysis, HbA1c (ρ 0.373, P 0.009) and Signs of accelerated carotid atherosclerosis assessed by MRI were triglycerides (ρ 0.375, P 0.009) exhibited a signifi cant and positive correlation found at a very early stage of type 2 diabetes, despite good regulation of with MBPS. In multiple regression analysis including age, gender, 24-hour hyperglycemia, hyperlipidemia and blood pressure. There is an effect on systolic blood pressure (24h-SBP), triglycerides, and HbA1c as independent the arterial wall remaining after correction of classical cardiovascular risk variables, HbA1c (β 0.344, P 0.013) alone were associated signifi cantly in factors, suggesting a legacy effect. a positive manner with MBPS. In simple regression analysis, 24h-SBP (ρ POSTERS

-0.359, P 0.012), MBPS (ρ -0.289, P 0.043) and HbA1c (ρ -0.301, P 0.035) Complications were signifi cantly correlated in a negative manner with FMD. In multiple Acute and Chronic regression analysis including age, gender, 24h-SBP, MBPS, LDL-cholesterol, and HbA1c, MBPS (β -0.305, P 0.043) alone emerged as a signifi cant factor associated in a negative manner with FMD. Conclusion: The present study suggested that poor glycemic control might be associated with the occurrence of MBPS, which could be a signifi cant determinant of endothelial dysfunction in T2DM patients.

468-P Nonalcoholic Fatty Liver Disease and Endothelial Dysfunction in 470-P Patients With Type 2 Diabetes Cardiovascular Disease in Patients With Type 1 Diabetes in a Medi- JANG YEL SHIN, JUNG SOO LIM, MI YOUNG LEE, CHOON HEE CHUNG, Wonju, terranean Area Republic of Korea EMILIO ORTEGA, GEMMA ROJO, CONXA CASTELL, MARGARITA GIMENEZ, IG- Some observational studies have suggested that nonalcoholic fatty liver NACIO CONGET, Barcelona, Spain, Málaga, Spain disease (NAFLD) is associated with subclinical atherosclerosis and endothelial Cardiovascular disease (CVD) is the major cause of morbi-mortality dysfunction (ED) in general populations. In this study, we examined the in subjects with diabetes. Although, most of the information on CVD association of NAFLD with ED in patients with type 2 diabetes. epidemiology comes from data in type 2 diabetes (T2D), the age-adjusted Ninety-four patients with type 2 diabetes were consequently enrolled relative risk for CVD is higher in T1D compared to T2D. (mean age: 53.3 ± 8.4 years; body mass index (BMI): 24.9 ± 2.9 kg/m2; We aimed to describe the prevalence of CVD in adult T1D within the duration of diabetes: 9.0 [3.0-15.0] years; HbA1C: 8.4 ± 1.4%). Clinical and Catalan National Public Health insulin pump registry (n=1382, CNPH-CSII), biochemical metabolic parameters were assessed. The severity of NAFLD and, to compare this data with CVD prevalence in non-diabetic (NOND) was determined using liver ultrasound. High-grade (hg) NAFLD was defi ned from a population nationwide study (n=4026, [email protected] Study). CVD as moderate or severe fatty liver disease. Endothelial function was measured information obtained from an interviewer-administered questionnaire (Di@ by brachial fl ow-mediated dilation (FMD). ED was defi ned as less than the bet.es Study) and from a physician reporting form (CNPH-CSII). Differences median value of FMD. in the crude (Chi-square) and adjusted prevalence of coronary heart (CHD), All patients were divided into three subgroups: normal (24.5%), low- cerebrovascular (CNSD), peripheral vascular (PVD), and overall CV (CVD) grade (39.3%), and high-grade (36.2%). Patients with hgNAFLD have higher disease were investigated. values of BMI, waist circumference (WC), triglyceride (TG), apolipoprotein We found differences in age (median 34 vs. 44 years), body mass index B, and homocysteine compared to those with normal. FMD with increasing (median BMI 24 vs. 27 kg/m2), and proportion of females (71 vs. 58 %), the severity of NAFLD showed a signifi cant gradual decrease (8.4±1.2 vs. current smokers (19 vs. 30%), and antihypertensive medication (17.1 vs. 7.4±1.6 vs. 6.6±1.6%; p<0.001). After adjusted for age and gender, FMD 14.3%), (p≤0.01, all), between T1D and NOND groups, respectively. CHD, was negatively correlated with BMI, WC, total cholesterol, homocysteine, CNSD, PVD, and overall CVD in T1D and NOND was (%) 2.97 vs. 2.78 (p=0.7), fi brinogen, and uric acid. In multivariate regression analysis, hgNAFLD and 0.72 vs. 1.24 (p=0.11), 2.61 vs. 0.32 (p<0.0001), and 5.14 vs. 4.02 (p=0.08), fi brinogen were independently associated with FMD. Patients with ED had respectively. However, age-and-sex adjusted multiple logistic regression higher values of BMI, WC, serum Creatinine (Cr), uric acid, fi brinogen, and models showed that T1D had a higher prevalence (OR 95% CI) of CHD (2.6 homocysteine compared to those without ED. Odds ratio (OR) of hgNAFLD [1.7-3.8]), PVD (20.5 [10.3-41]), and CVD (3.3 [2.4-4.5]), than NOND individual predicting the presence of ED after adjusted for age and gender was 3.58 (p< 0.0001, all). Prevalence for CNSD was similar between groups (1.2 (95% CI, 1.42-9.04; p=0.007). OR remained signifi cant after further adjusted [0.6-2.5]). Additional adjustment for smoking, BMI, and treatment with for serum Cr, uric acid, fi brinogen, and homocysteine, but was attenuated antihypertensive medication did not signifi cantly modify these results. after further adjusted for BMI. In a Mediterranean country, subjects with T1D had a higher age-and-sex In conclusion, our fi ndings suggest that hgNAFLD is independently asso- adjusted prevalence of CVD as compared with non-diabetic individuals. ciated with ED in patients with type 2 diabetes. Differences observed in the prevalence of CVD could be in part explained by the intrinsic nature of a registry-based observational investigation. 469-P Supported by: CIBERDEM Signs of Accelerated Carotid Atherosclerosis Assessed by MRI in Newly Diagnosed Type 2 Diabetic Patients 471-P PERNILLE H. HØYEM, ESBEN LAUGESEN, ANDERS F S. MIKKELSEN, BRITT Association of Aortic Pulse Wave Velocity and Cardiovascular Risk CHRISTENSEN, ULLA K. OPSTRUP, PER L. POULSEN, MOGENS ERLANDSEN, BILL Factors in Patients With Non-Insulin Dependent Diabetes KERWIN, SAMUEL THRYSØE, JENS S. CHRISTIANSEN, WON Y. KIM, TROELS K. PINELOPI GRIGOROPOULOU, IOANNA ELEFTHERIADOU, CHRISTOS ZOUPAS, HANSEN, Aarhus, Denmark, Seattle, WA CHRISTOS LIASKOS, DESPOINA PERREA, KONSTANTINOS MAKRILAKIS, AL- Type 2 diabetes is associated with accelerated atherosclerosis, which EXANDER KOKKINOS, NICHOLAS KATSILAMBROS, NICHOLAS TENTOLOURIS, causes macro-vascular complications. We aimed to investigate morpho- Athens, Greece logical differences in atherosclerosis in the carotid arteries assessed by Arterial stiffness represents the link between metabolic syndrome and MRI in newly diagnosed type 2 diabetic patients and non-diabetic control cardiovascular disease. Reduction of the elastic properties of the arteries subjects. has been described in patients with type 2 diabetes. Aortic pulse wave One hundred type 2 diabetic patients diagnosed within the last 5 years velocity (aPWV) has emerged over the last years as an independent predictor and 100 age- and gender-matched non-diabetic control subjects underwent of cardiovascular mortality. magnetic resonance imaging of carotid arteries bilaterally with a dedicated Aim of this study was to assess the association of aPWV and cardiovascular carotid coil. Scans were performed with four different contrast weightings risk factors in patients with type 2 diabetes. and analysed in a software tool to assess atherosclerosis morphology. A total 193 (100 men, 93 women) with type 2 diabetes, with mean Among diabetic patients the minimal lumen area was 17.6% smaller age 63.62 ± 9.12 years and diabetes duration 7 (3-16) years, participated (P<0.001) and maximal normalized wall index was 3.0% higher (P=0.038) in the study. The mail eligible criteria were age between 40-75 years, than in the control subjects. This remained signifi cant after adjustment absence of macrovascular disease and non-smokers. Baseline demographic for LDL-cholesterol and smoking habits (minimal lumen area P<0.001 and characteristics were recorded; plasma glucose, serum lipids, HbA1c, urine maximal normalized wall index P=0.012). Sub-analysis of those measures albumin-creatinine ratio, serum osteoprotegerin and hs-CRP were measured.

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A119 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

Aortic PWV was measured non-invasively using the SphygmoCor® system Twenty fi ve mM glucose (HG) caused upregulation of ECM protein (AtCor, Sydney, Australia). fi bronectin (FN) in the Ecs. Such upregulations were associated with Among the participants, 149 (77%) patients had hypertension, 56 (28%) increased oxidative stress and heme oxygenase 1 expression. MaFGF (20ng/ had peripheral neuropathy and 127 (65.8%) patients had LDL cholesterol ml) prevented such abnormalities. In parallel, HG caused cardiomyocyte levels > 100 mg/dl. After linear regression analysis, aPWV was signifi cantly hypertrophy and increased expression of hypertrophy associated transcripts, associated with age (p<0.001), body mass index (p=0.017), diabetes duration angiotensinogen (ANG) and atrial natriuretic peptide (ANP). Treatment of (p=0.007), urinary albumin excretion rate (p=0.032), estimated glomerular cells with MaFGF prevented such alterations. fi tration rate (p<0.001), serum osteoprotegerin (p=0.014), and the presence Supported by: CDA; NSFC POSTERS

Complications of peripheral neuropathy (p<0.001). No signifi cant association was observed Acute and Chronic between aPWV and glucose, HbA1c, serum lipids, arterial blood pressure 474-P and hs-CRP. Adiponectin Protects Against Metabolic Syndrome by its Modula- In patients with non-insulin dependent diabetes, arterial stiffness is tion of Lipid and Glucose Metabolism associated with the presence of autonomous nervous system dysfunction and ANIZE D. VON FRANKENBERG, CAROLINA DE OLIVEIRA, MÔNICA SOST, PEDRO serum osteoprotegerin levels, independently of most of the other risk factors. SADDI-ROSA, BÁRBARA NEDEL, RAFAELA ZANDAVALLI, DULCINÉIA SIMION- ATO, AMANDA FABBRIN, MANOELLA SANTOS, LUIS CANANI, ANDRÉ REIS, 472-P FERNANDO GERCHMAN, Porto Alegre, Brazil, São Paulo, Brazil Albuminuria Signifi cantly Predicts Cardiovascular Events in Pa- Adiponectin, a hormone expressed in the adipose tissue, has insulin tients With Type 2 Diabetes Independently from the Baseline Coro- sensitizer properties. As a result, hypoadiponectinemia may be related to nary Artery State the development of metabolic syndrome (MS). To examine the relationship of DANIELA ZANOLIN, PHILIPP REIN, ALEXANDER VONBANK, CHRISTOPH H. SAE- adiponectin with MS in patients (n=588; age 58.0±11.0 y, mean±SD, women LY, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Philadelphia, PA 53.1%, MS rates 87.4%) from cardiovascular and metabolism units of two Albuminuria is an important indicator of cardiovascular risk. We have university hospitals: 1. undergoing coronary angiography. 2. referred for recently shown that it is also associated with angiographically determined determination of glucose tolerance status and its management. Glucose coronary artery disease (CAD). Whether albuminuria predicts cardiovascular tolerance status was determined by an oral glucose tolerance test and/ events independently of the baseline coronary artery state in patients with or Hba1c. Lipid panel and plasma adiponectin (µg/mL) were measured. MS type 2 diabetes (T2DM) has not been investigated yet. was defi ned by at least three of the following: hypertension, low HDL and/ We measured urinary albumin and creatinine concentrations in 211 or high triglycerides levels, hyperglycemia and high waist circumference. consecutive patients with T2DM undergoing coronary angiography for Adiponectin levels were lower in patients with MS than in those without MS the evaluation of suspected or established stable CAD. Albuminuria was (8.79 [5.8-13.7] vs 14.1 [9.8-21.3]; median [P25-P75], P<0.001). Adiponectin defi ned as a urinary albumin to creatinine ratio (ACR) of 30 µg/mg or greater. decreased with increasing number of MS criteria (P<0.001). While comparing Prospectively, we recorded vascular events over 3.2±1.4 years. by each MS criteria, adiponectin levels were signifi cantly lower by the During follow up, 24.6% of our patients suffered cardiovascular events. presence of the following: HDL (8.7 [5.7-13.54] vs 12.6 [8.7-19.2]; P<0.001), The cardiovascular event rate was signifi cantly higher in patients with triglycerides (7.6 [5.1-11.8] vs 11.0 [7.4-16.5], P<0.001), waist circumference albuminuria (n=85) than in those with normoalbuminuria (35.3 vs. 17.5%; (9.1 [6.0-13.9] vs 12.4 [7.6-18.6], P<0.001), glucose (9.07 [6.0-13.9] vs 11.0 [7.0- p=0.003). Cox regression analysis adjusting for age, gender, BMI, smoking, 18.6]), P=0.002), and blood pressure (9.1 [6.0-14.1] vs 11.0 [8.1-18.5], P=0.013). systolic and diastolic blood pressure, LDL cholesterol, HDL cholesterol, Adiponectin was positively related with HDL (r=0.419, P<0.001) and inversely eGFR, and use of ace inhibitors/angiotensin II antagonists confi rmed that related with triglycerides (r=-0.326, P<0.001), fasting plasma glucose (r= albuminuria signifi cantly predicted cardiovascular events independently -0.150, P=0.001) and waist circumference (r=-0.257, P<0.001). While adjusting from conventional risk factors (adjusted HR 1.96 [1.11-3.46]; p=0.021). for age and sex high adiponectin levels were inversely related to MS [OR = Further adjustment for the angiographically determined presence of CAD 0.923 (95%CI = 0.898-0.950)]. Protection against MS associated with at baseline did not signifi cantly attenuate the predictive power of the ACR increasing adiponectin levels is not affected by sex and age, being possibly (HR 1.84 [1.04-3.27]; p=0.037). Similar results were obtained when the related to its positive modulation of lipid and glucose metabolism. ACR was entered into the fi nal regression model as a continuous variable Supported by: FIPE; FAPERGS; CAPES; CNPq (standardized adjusted HR 1.30 [1.02-1.65]; p=0.037). Albuminuria signifi cantly predicts cardiovascular events in patients with 475-P T2DM independently of established cardiovascular risk factors and of the Association Between Waist-to-Height Ratio and Cardiometabolic baseline coronary artery state. Risk Factors Among Youth With Diabetes LENNA L. LIU, HENRY S. KAHN, DAVID J. PETTITT, DAVID MAAHS, JENNIFER W. 473-P TALTON, RALPH B. D’AGOSTINO, JR., BETTINA M. BEECH, NANCY A. CRIMMINS, The Effects of a Modifi ed Acidic Fibroblast Growth Factor in Dia- ARCHANA LAMICHHANE, ANGELA D. LIESE, RONNY A. BELL, SEARCH FOR DIA- betic Cardiomyopathy BETES IN YOUTH STUDY GROUP, Seattle, WA, Atlanta, GA, Santa Barbara, CA, SUBRATA CHAKRABARTI, LINBO ZHANG, SHALI CHEN, CHI ZHANG, YI TAN, LU Aurora, CO, Winston-Salem, NC, Cincinnati, OH, Chapel Hill, NC, Columbia, SC CAI, GUANG LIANG, LITAI JIN, XIAOKUN LI, London, ON, Canada, Louisville, KY, The association between central adiposity and cardiometabolic risk Wenzhou, China factors (CMRFs) is well known among adults with diabetes; however, less Diabetes alters function of several growth factors, including fi broblast is known among youth. We examined the association of Waist-to-Height growth factor (FGF), by altering their expression or by glycosylation. ratio (WHtR), an emerging measure of central adiposity, to CMRFs in youth Although, FGF supplementation prevents cardiac ischemia/reperfusion 10-19 years old with type 1 (T1D: N=2,863) or type 2 (T2D: N=531) diabetes injury, it has signifi cant mitotic activity, a potential obstacle for clinical from the SEARCH for Diabetes in Youth Study. WHtR was calculated for each appli ca tion. As mitotic domains of aFGF is separate from its biological active participant from measures of height and waist circumference (WC). WHtR domains, we developed a novel mutant from recombinant human acidic FGF and WC as indicators of central adiposity were compared with BMI z-score (MaFGF), by deleting the nuclear translocation domain (N-terminal 26 amino (BMIz, calculated from height and weight measures using CDC 2000 growth acids). Ma FGF has no mitotic activity and shows protective action against charts). CMRFs included: log-triglyceride (TG), total, HDL- and LDL-cholesterol oxidative stress. As target tissue damage in diabetes, is largely mediated and systolic (SBP) and diastolic (DBP) blood pressure. Linear regression through oxidative stress we investigated protective effects of MaFGF on models with forward selection stratifi ed by diabetes type and adjusted for diabetic cardiomyopathy, both in vivo and in vitro. age, race/ethnicity and sex were used to identify the strongest indicator for To study glucose induced myocyte hypertrophy, we used H9C2 cells and each CMRF. WHtR was the strongest indicator for three CMRFs (Total and freshly isolated neonatal rat cardiomyocytes. To investigate glucose induced LDL-cholesterol, DBP) among T1D youth. WC was the strongest indicator for extracellular matrix overproduction we investigated human endothelial cells log-TGs among T1D and T2D youth and for HDL-cholesterol among T1D youth (ECs). In vivo studies were performed in STZ-induced diabetic mice after 1 and 6 and DBP among T2D youth. BMIz was the strongest indicator for SBP among months of diabetes. The animals were monitored with respect to blood glucose, T1D and T2D youth and for HDL-cholesterol among T2D youth. These data body weight and urine glucose, ketones and volume. Groups of diabetic mice indicate that WHtR may be added as a useful clinical indicator, particularly were injected with MaFGF (10 ug/Kg/day). Echocardiographic analyses were among youth with T1D, for assessing cardiometabolic risk. performed. Both cells and cardiac tissues collected at sacrifi ce, were analysed for specifi c miRNAs, mRNAs and proteins and morphologic changes.

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A120 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

events with clinical manifestations that required assistance. In accordance Table: Linear Regression Results* Examining the Association between Two with the DCCT/ EDIC protocol, hypoglycemia less than severe is defi ned as Measures of Central Adiposity or BMIz and CMRFs, Stratifi ed by Diabetes Type mild (MH). T1D T2D Nonnormally distributed variables were transformed. To analyze the WC WHtR BMIz WC WHtR BMIz association between CAC and hypoglycemia, we applied Relative Risk (RR), Log-TG 0.051 0.022 logistic, Tobit, and nonparametric models. Since signifi cant effects of A1C (<0.0001) (0.0005) levels on hypoglycemia were found, we stratifi ed by A1C levels (good <7.5%; fair 7.5% - <8.5%; poor 8.5% - <9.5%; very poor ≥9.5%). In addition, MH LDL-Cholesterol** 0.018 POSTERS (<0.0001) was stratifi ed by the existence of SH. RR models adjusted for sex and age Complications showed signifi cant RR ratios for the log transformed time averaged weekly Acute and Chronic HDL-Cholesterol 0.072 0.077 rate of MH without SH: RR 1.65, 95% CI: 1.04 - 2.61, p<0.04, A1C 7.5 - 8.5%, (<0.0001) (<0.0001) and the inverse yearly SH rate: RR 0.96, 95% CI: 0.93 - 0.99, p<0.02, A1C Total Cholesterol** 0.008 <7.5%). Each rate is based on total follow up years. Hypoglycemia during the (<0.0001) EDIC but not DCCT study was a signifi cant factor (SH rate: p<0.02, MH rate Systolic Blood Pressure (SBP) 0.047 0.058 without SH: p< 0.03). Adjusting for additional parameters or using different (<0.0001) (<0.0001) models did not change the trends. Diastolic Blood Pressure (DBP) 0.019 0.035 Our analysis seems to link results of contradictory reports. We found that (<0.0001) (<0.0001) hypoglycemia is a risk factor for preclinical arteriosclerosis. However, the *Partial R2 (p value) from forward selection procedure; adjusted for age, role of hypoglycemia is not straightforward. One size doesn’t fi t all. race/ethnicity, sex Supported by: Public accessible DCCT/EDIC data (only) supplied by NIDDK ** With criterion p<0.01 no indicators entered the models among T2D youth Supported by: CDC 478-P Prognostic Importance of Admission versus Persistent Glycemia in Acute Coronary Syndrome 476-P M. SHOAIB ZAHEER, MUHAMMAD UWAIS ASHRAF, M.U. RABBANI, MOHAM- Matrix-Metalloproteinases, a Missing Link between Atherosclero- MAD ZUBAIR, JAMAL AHMAD, Aligarh, India sis and Diabetes, are Associated With Exogenous Insulin Whether persistent hyperglycemia (PG) during hospitalisation has a ALEXANDER WRESSNEGGER, GERFRIED PESAU, KATRIN NAGL, FLORIAN OBEN- greater impact on adverse outcomes in Acute Myocardial Infarction (AMI) DORF, CLEMENS HOEBAUS, RENATE KOPPENSTEINER, GUNTRAM SCHERN- than a single random glucose measurement is not well defi ned. To fi nd out THANER, GERIT-HOLGER SCHERNTHANER, Vienna, Austria the association of admission glycemia (AG) VS PG on outcomes in patients Cardiovascular disease (CVD) is still the primary cause of death in of ACS. Prospective, cohort, hospital-based. We evaluated 200 patients of diabetes patients (≈40%). In addition, atherosclerosis is more frequent, ACS for admission and in-hospital glycemia and their impacts on outcomes. aggressive and accelerated in patients with diabetes than without. Studies AG was defi ned as a plasma glucose >198 mg/dl and PG as a random glucose like ACCORD and VADT suggest a non-benefi cial effect of - especially high - >140 mg/dl at any point during hospitalisation. Demographic & biochemistry insulin supplementation therapy (IST) on CVD. However, a causal relation including risk factors recorded. A multiple regression was done to evaluate of IST and CVD has not yet been proven. We hypothesize that matrix- association of various parameters with worse prognosis. Of the 200 patients metalloproteinases (MMP), which are involved in plaque formation, rupture evaluated, 35 (17.5%) presented with AG. 31 (15.5%) had PG. Males were and thus acute events, are associated with exogenous insulin. predominant and 47 (23%) previously known diabetic patients. 62 (31%) had We included 122 patients with type 2 diabetes and CVD: 32 female, unstable angina, 52 (26%) NSTEMI and 86 (43%) STEMI, between PG and age: 71±8.9 years, diabetes duration: 13.5±10.4 years, fasting glucose: LEF, higher troponin levels and in-hospital mortality and between LEF and 7.7±2.2mg/dl, fasting insulin 18±15µU/ml, fasting C-peptide: 3.7±2.6ng/ml, age (p <0.001), serum creatinine (p 0.023) and mean in-hospital glucose (p HbA1c: 6.7±1.1 rel.%. 32 of those patients received IST. MMP-8 and MMP- 0.005). F- indices were compared with AG for their ability to discriminate 9 were measured by multiplex bead array and Tissue inhibitor of metallo- hospitalisation survivors from non- survivors. All average glucose metrics proteinases-1 (TIMP-1) by ELISA, both together describing the likelihood of performed better than AG. The ability of these models improved as the plaque rupture potential. time window increased (F-indices for admission, mean 24 hours, 48 hours We are the fi rst to report a signifi cant association of MMP-8 (r=0.593, and 72 hours were 2.51, 12.05, 8.3 and 5.72 respectively) The present study p=0.001), MMP-9 (r=0.590, p=0.001) and MMP-8/TIMP-1 (r=0.378, p=0.043) demonstrates that PG is a better discriminator of prognosis than AG in with insulin levels in patients with IST. This correlation, however, was not patients of ACS. found in diabetic patients without insulin treatment. After elimination of patients with albuminuria and female patients, this correlation became even 479-P stronger and in addition a correlation between MMP-9/TIMP-1 (r=0.575, Association between Erectile Dysfunction and Echocardiographic p=0.020) and insulin was found even though TIMP-1 was signifi cantly higher Variables of Ventricular Hypertrophy and Diastolic Function in Dia- in patients with IST (p=0.011). In addition, the association of Insulin with betic Hypertensive Patients MMP-8 and MMP-9 levels in insulin treated patients withstood multivariate BEATRIZ D. SCHAAN, MATEUS D. SEVERO, LIANA F. LEIRIA, PRISCILA S. LE- Regression and thus Insulin was identifi ed the single most independent DUR, ALEXANDRE D. BECKER, FERNANDA M. AGUIAR, DANIELA MASSIERER, predictor of MMP-8 and MMP-9. VALÉRIA C. FREITAS, MIGUEL GUS, Porto Alegre, Brazil Having in mind that these two MMPs have been associated with different A cross-sectional study was conducted in order to assess the prevalence manifestations of CVD, IST may promote CVD in patients with diabetes by of erectile dysfunction (ED) in subjects with diabetes and hypertension and elevating the MMP/TIMP-1 ratio. also the association between ED and cardiovascular risk variables such as echocardiographic changes. 477-P We evaluated 114 men with type 2 diabetes mellitus and hypertension Hypoglycemia in the Diabetes Control and Complications and Epide- selected at a tertiary care teaching hospital in Southern Brazil. Erectile miology of Diabetes Interventions and Complications Trial (DCCT/ dysfunction was assessed by the International Index of Erectile Function EDIC): What Is its Story With Cardiovascular Disease (CVD)? (IIEF-5) score. Clinical and laboratory variables, including C-reactive protein ELKE FÄHRMANN, LAURA ADKINS, CAMERON LOADER, JIM DENVIR, HYOIL (CRP), ambulatory blood pressure monitoring (ABPM), ankle brachial index HAN, HENRY K. DRISCOLL, Huntington, WV (ABI), and transthoracic echocardiography were evaluated. Patients with ED Recently, major attention has been paid to the role of hypoglycemia as (IIEF-5 < 22) and without ED (IIEF-5 ≥ 22) were compared. a risk factor in CVD. While EURODIAB investigators concluded that severe Patients were 56.8 ± 5.7 years-old, systolic and diastolic blood pressure hypoglycemia is not a risk factor in type 1 diabetes, other investigators were 150.7 ± 19.5 mmHg and 85.4 ± 11.4 mmHg, respectively, and HbA1c found the opposite to be true. The role of mild hypoglycemia as a CV risk was 8.0 ± 1.7%. The majority (74.6%) of patients had ED. Presence of factor was not investigated. Our aim is to shed light onto these contradictory microalbuminuria was similar between groups (p = 0.81), as well as of results and additionally assess the role of mild hypoglycemia. cardiovascular autonomic neuropathy (p = 0.84) and 10-year risk of coronary We analyzed the effect of hypoglycemic events on calcifi cation score (CAC) heart disease (UKPDS risk engine; 18.6 ± 12 and 24.3 ± 13% in patients measured during the EDIC study. Severe hypoglycemia (SH) is defi ned by without ED vs. those with ED, p = 0.05). Levels of CRP, ABPM values and

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A121 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

ABI were also similar between groups. Twenty-four mean systolic blood Atorvastatin therapy reduced central arterial stiffness in this diabetes pressure was 131.2 ± 12.1 and 131.7 ± 19.5 mmHg in patients without ED vs. cohort with demonstrable effects at 3 months that persisted at 12 months, those with ED (p = 0.91). with no dose-dependent effect observed. The correlation observed between Echocardiography variables related to cardiac chamber diameters, left reductions in PWV and reductions in concentrations of OPG suggests ventricular hypertrophy and diastolic function were similar between groups, atorvastatin may reduce PWV via direct anti-infl ammatory effects on the except for that there was a slight lower left ventricular ejection fraction in vasculature. men with ED (64.9 ± 7.3 vs.68.1 ± 3.9 %, p = 0.004). In high-risk diabetic hypertensive individuals, ED is highly prevalent, but POSTERS 482-P Complications its presence is not associated neither with echocardiographic variables, nor Association between Diabetes-Related Complications and Health- Acute and Chronic with other cardiovascular risk factors. care Resource Use and Costs in a Large Medicare Advantage Popu- Supported by: CNPq; HCPA lation LESLIE HAZEL-FERNANDEZ, DAMION NERO, JANE STACY, S. LANE SLABAUGH, 480-P NICK PATEL, YUNUS MEAH, JEAN BALTZ, JONATHAN BOUCHARD, Miramar, FL, 5-year Follow-Up Study on Japanese Type 2 Diabetic Patients With Louisville, KY, Princeton, NJ Asymptomatic Coronary Artery Lesions Diagnosed by Multidetector Previous studies suggest that much of the excess burden of care associated Computed Tomography (MDCT) with type 2 diabetes mellitus (T2DM) is linked to T2DM complications, YOKO MATSUZAWA, KAZUHIKO YUMOTO, JUN SAITO, MASAO OMURA, TET- especially among the elderly. This study evaluated healthcare resource use SUO NISHIKAWA, Yokohama, Japan (HRU) and costs of T2DM complications in members of a large Medicare We performed MDCT under written informed consent to 74 type 2 diabetic Advantage with Prescription Drug Coverage plan. patients in 2008. All of the patients had no symptoms or history of coronary Medical and pharmacy claims from 01/01/2010 to 12/31/2011 of 333,576 artery disease (CAD) , and they were negative for exercise-tolerance ECG patients with T2DM were examined. All-cause and T2DM-related claims test. We found signifi cant stenosis (defi ned as >50% stenosis in either of were identifi ed as all medical and pharmacy claims, and medical claims the coronary arteries) in 21cases (28.4%). Cardioangiography (CAG) was with primary ICD-9 CM codes of 250.x0 or 250.x2 and prescription claims performed in 13 patients, and 4 of them needed PCI for the severe stenosis for antidiabetic mediations. HRU and costs were based on claims within a in LAD or RCA. In the current study, we examined the prognosis of those year. T2DM complications, including retinopathy, nephropathy, neuropathy, patients after follow-up of 4.5years in average. 65 patients (84.4%) were cerebrovascular, cardiovascular, peripheral vascular and metabolic successfully followed by medical records or telephone interview. In the complications, were assessed via a modifi ed Diabetes Complications group without signifi cant stenosis (n=45), only 1 patient (2.2%) developed Severity Index (DCSI). Subjects were split into six groups based on DCSI nonfatal angina pectoris. Another one suffered from a reccurence of scores from 0 to ≥5. glucagonoma which had been repeatedly diagnosed before, but other 43 Much of the study population (78.6%) had at least one T2DM complication. patients (95.6%) were free from major life-threatning events. On the other All-cause and T2DM-related resource use and costs and claims for hand, in the patients who had signifi cant stenosis in 2008 (n=20), 3 patients hypoglycemia and depression, showed a positive relationship with DSCI. (15.0%) developed nonfatal CAD, and another 3 (15.0%) died from cancers Medical costs had a stronger relationship with DCSI score than did pharmacy newly diagnosed during the follow-up period. Thus event-free survival costs. Preventing T2DM progression to higher complication severity in the rate in this group was only 70.0%. The odds ratio of the stenosis(+) group elderly with antidiabetic medications could result in signifi cant costs savings compared to the stenosis(-) group was 7.76 (95%CI:0.75-79.91) and 9.21 overall. (1.67-50.95) for CAD events and CAD+mortality, respectively. Multivariate Distribution of Patients with T2DM complications (DCSI scores) and Annual logistic analysis revealed that the stenosis of coronary artery remains a All-cause Healthcare Costs signifi cant predictor of CAD+mortality (p=0.048) after adjustment of age, sex, smoking, HbA1c and baPWV. In conclusion, Japanese type2 diabetic DCSI = 0 DCSI = 1 DCSI = 2 DCSI = 3 DCSI = 4 DCSI = ≥5 patients with asymptomatic CAD were proved to carry high possibility of N= 71,514 N= 45,499 N= 60,657 N= 42,341 N= 41,189 N= 72,376 symptomatic CAD and mortality. Physicans should pay attention to the risk Selected Complications Retinopathy 0% 16.4% 11.7% 19.1% 18.2% 32.3% of malignant diseases as well as to the metabolic risk factors to manage Nephropathy 0% 3.0% 27.1% 39.2% 57.7% 78.0% these patients. Neuropathy 0% 33.0% 17.2% 43.7% 34.4% 62.0% Healthcare Costs (mean) Total $5,247 $7,196 $9,057 $10,812 $13,913 $21,431 481-P Medical $3,398 $4,865 $6,664 $8,056 $11,053 $17,897 The Effect of High versus Low Dose Atorvastatin on Central Arterial Pharmacy $1,970 $2,460 $2,515 $2,878 $2,984 $3,664 Stiffness in Male Patients With Type 2 Diabetes COLIN DAVENPORT, DAVID T. ASHLEY, EOIN P. O’SULLIVAN, CLAIRE M. MCHEN- RY, AMAR AGHA, CHRISTOPHER J. THOMPSON, DONAL J. O’GORMAN, DIAR- MUID SMITH, Dublin, Ireland 483-P Increased central arterial stiffness is associated with hypertrophy of the Analysis of Modulators on Atherogenic CD14+CD16+ Monocytes in left ventricle and increased cardiovascular mortality. Patients with type Type 2 Diabetic Patients 2 diabetes mellitus (T2DM) typically have increased arterial stiffness in KOKA MOTOYAMA, MASANORI EMOTO, TAKASHI MISHIMA, YUKO YAMAZAKI, comparison to age-matched controls. Statin therapy improves atherogenic TOMOAKI MORIOKA, KATSUHITO MORI, SHINYA FUKUMOTO, TETSUO SHOJI, lipid profi les and reduces infl ammation within the vasculature, but its effects MASAAKI INABA, Osaka, Japan on stiffness of the central arteries in T2DM are largely unknown. Monocytes in human blood are heterogeneous. Recent studies have shown The primary aim of our study was to determine whether statin therapy that CD14+CD16+ monocytes predict cardiovascular events. Type2 diabetes reduces central arterial stiffness in a dose-dependent manner in male is one of the risk factors of cardiovascular events. However, modulators of patients with T2DM. Central arterial stiffness was directly measured via atherogenic CD14+CD16+ monocytes in type2 diabetes are still unclear. We carotid-femoral pulse wave velocity (PWV). The secondary aim was to investigated the association between CD14+CD16+ monocytes and physical determine the effects of statin intervention on circulating concentrations of or biochemical parameters in 149 type 2 diabetic patients (age, 62 ± 14 (SD) osteoprotegerin (OPG), an indirect biomarker of atherosclerotic infl ammation years, duration 14 ± 12 (SD) years). and arterial stiffness. Two major monocyte subsets (CD14++CD16- and CD14+CD16+) were Fif t y-one male T2DM patients with microalbuminuria ceased statin therapy identifi ed by fl ow cytometry. Physical parameters such as blood pressure for 6 weeks, followed by randomisation to either 10 or 80mg of atorvastatin. (BP) and body mass index (BMI) and biochemical examinations were PWV and OPG were measured at randomisation, 3 and 12 months. obtained from the patients. In simple regression analysis, number of Groups were comparable at baseline. PWV decreased from 10.5±1.1 to CD14+CD16+ monocytes was signifi cantly associated with age (r= 0.227, 9.7±1.1m/sec (p<0.01 from baseline) at 3 months, and 9.2±1m/sec (p<0.001 p=0.006), systolic BP (r= 0.192, p=0.022) and serum uric acid (r= 0.191, from baseline) at 12 month, indicating a decrease in central arterial stiffness. p=0.021) and negatively correlated with HLD-C (r=-0.174, p=0.036) and OPG also decreased signifi cantly at 3 and 12 months. The reductions in PWV eGFR(r=-0.187, p=0.023). CD14+CD16+/CD14++CD16- ratio was associated and OPG did not differ between the groups. Baseline PWV and OPG values with age (r= 0.268, p=0.001) and systolic BP (r= 0.263, p=0.002). Multiple correlated strongly (r=0,48, p<0.01), as did their response to atorvastatin regression analysis indicated that age (β= 0.301, p=0.001), systolic BP over 12 months (r=0.32 delta-OPG and delta-PWV, p<0.05). (β=0.212, p=0.002), HDL-C (β=-0.175, p=0.011) and number of CD14++CD16-

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A122 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

(β=0.560, p<0.001) were signifi cant independent contributors to CD14+C16+ risk factors in patients with type 1 diabetes, highlighting the increased monocytes. With similarity to CD14+CD16+ number, signifi cant contributors cardiovascular risk in type 1 diabetic patients with increased VAT. to CD14+CD16+/CD14++CD16- ratio were age (β=0.286, p=0.001), systolic BP (β=0.227, p=0.005) and HDL-C (β=-0.163, p=0.049). This study suggests 486-P that age, systolic BP and HDL-C are the possible modulator of atherogenic Complex Interaction between Central Adiposity and Type 2 Dia- monocytes in Type2 diabetes. betes for Prediction of Future Carotid Intima-Media Thickness in Mexican Americans

484-P HEMANT KULKARNI, MANJU MAMTANI, THOMAS D. DYER, MICHAEL C. MA- POSTERS Novel Metabolic Syndrome Index Is a Useful Biomarker for Coro- HANEY, ANTHONY G. COMUZZIE, LAURA ALMASY, JOANNE E. CURRAN, JOHN Complications nary Heart Disease Risk Prediction and Diagnosis of the Metabolic BLANGERO, San Antonio, TX Acute and Chronic Syndrome Carotid intima-media thickness (IMT), a measure of atherosclerosis, is NABILLA ABDELLA, OLUSEGUN A. MOJIMINIYI, Safat, Kuwait an informative predictor of cardiovascular morbidity and mortality. Large There is need to simplify screening tests for metabolic syndrome (MetS) cross-sectional studies have demonstrated that central adiposity and and coronary heart disease (CHD) risk so that high risk patients can be type 2 diabetes (T2D) occupy vital positions in the web of atherosclerosis profi ciently identifi ed early. In routine practice, components of MetS - causation. The aim of this study was to determine the independent and increased waist circumference (WC), high fasting plasma glucose (FPG), interactive associations of central adiposity and T2D with prospectively low high-density lipoprotein cholesterol (HDL-C), high triglycerides (Tg) and measured carotid IMT. hypertension are assessed separately with presence of three components We used the phenotypic data from the San Antonio Family Heart Study being diagnostic. We hypothesized that an index (metabolic syndrome in which the mean common carotid artery IMT (CCAIMT) and mean internal index - MSI), computed as WC x Tg/HDL-C would offer a single diagnostic carotid artery IMT (ICAIMT) was ultrasonographically measured 5 years after number with high sensitivity and specifi city for CHD risk assessment and recruitment in 747 Mexican Americans. Waist-hip-ratio (WHR) was used as the diagnosis of MetS. Fasting insulin, glucose, adiponectin, high-sensitivity a measure of central adiposity. Using polygenic models implemented in the C-reactive protein (hs-CRP) and full lipid profi le were determined in 98 Type SOLAR software and adjusting for age, sex and their interactions we found 2 diabetic (T2DM) subjects and that the heritability of CCAIMT and ICAIMT was 0.28 (p=5x10-7) and 0.49 521 apparently healthy fi rst degree relatives of T2DM subjects. Clinical (p=8x10-22), respectively. The phenotypic correlation between CCAIMT and and anthropometric data were recorded; subjects were classifi ed with ICAIMT was only 0.27. In an interactive polygenic model, T2D at baseline the number of criteria of MetS (IDF). Insulin resistance was assessed with [(beta,p) 0.33, 0.002] and baseline WHR (0.09, 0.015) were independently HOMA-IR. Regression analyses were used to determine the associations of associated with CCAIMT with a non-signifi cant interaction term (-0.14, MSI with other risk factors, CHD and MetS. MSI cut off values for MetS 0.138). In contrast, neither T2D (0.03, 0.770) nor WHR (0.02, 0.559) at were 155 for males and 105 for females. MSI showed signifi cant correlations baseline were independently associated with ICAIMT but their interaction (r) with age (0.2), WC (0.6), glucose (0.2), Tg (0.9), HDL-C (-0.6), Systolic BP was (0.28, 0.005). As a corollary, in diabetics (n=101) WHR was signifi cantly (0.2), Diastolic BP (0.2), adiponectin (-0.4), HOMA-IR (0.4) and hs-CRP (0.4). associated with ICAIMT (0.26, 0.008) while in non-diabetics (n=646) WHR MSI showed stepwise increase with increasing number of MetS criteria. was signifi cantly associated with CCAIMT (0.11, 0.003). Binary logistic regression showed that the odds ratio (OR) of MetS and CHD Our results allow three conclusions: i) although anatomically very close, as predicted by MSI were 2.7 (95% CI: 2.1-3.55) and 3.6 (95% CI: 1.9 -5.3) the CCAIMT and ICAIMT exhibit different levels of heritability; ii) the respectively. ROC analysis showed that MSI had signifi cantly higher area associations of existing T2D and WHR are differential with future CCAIMT under the curve (0.986) compared with WC (0.682), HDL-C (0.790) and Tg and ICAIMT; and iii) central adiposity (quantifi ed by WHR) associates with (0.955) for detection of MetS. We conclude that the MSI offers a single, ICAIMT in diabetics and with CCAIMT in non-diabetics. easily computable risk estimate for CHD and an excellent diagnostic test Supported by: NIH (P01HL045522), (R37MH59490) for MetS. Supported by: KFAS (2004-1302-03) 487-P Serum Pigment Epithelium-Derived Factor Levels are Independently 485-P Correlated With the Presence of Coronary Artery Disease Visceral Fat Is an Important Determinant of Coronary Artery Calcifi - FEIFEI WANG, XIAOJING MA, MI ZHOU, XIAOPING PAN, JIE NI, MEIFANG GAO, cations in Patients With Type 1 Diabetes ZHIGANG LU, JINGYU HANG, YUQIAN BAO, WEIPING JIA, Shanghai, China WOUTER GOOVAERTS, TIM BRITS, KEN CARPENTIER, AN VERRIJKEN, EVELINE Pigment epithelium-derived factor (PEDF) is a multifunctional protein L. DIRINCK, CHRISTOPHE DE BLOCK, LUC F. VAN GAAL, Edegem, Belgium which possesses anti-angiogenic, anti-tumorigenic, anti-oxidant, anti- Despite growing attention to visceral adipose tissue (VAT) as a predictor infl ammatory, anti-thrombotic, and neuroprotective properties. PEDF has of type 2 diabetes and cardiovascular complications, data on the relationship been proved to be closely correlated with metabolic syndrome (MS) and its between coronary heart disease and visceral fat in type 1 diabetes are components that are all risk factors of cardiovascular disease and may play scarce. In this cross-sectional pilot study we investigated whether VAT a protective role against vascular injury and atherosclerosis. The present contributes to an increased risk of coronary artery calcifi cations (CAC) in study was designed to investigate the relationship between serum PEDF and type 1 diabetes. coronary artery disease (CAD). In 105 type 1 diabetic patients (40% female), free of known cardiovascular A total of 312 consecutive in-patients (including 228 with CAD and 197 disease, CAC score (CACS) and VAT (at level L4-L5) was measured using CT with MS) aged 38~86 years who underwent coronary angiography by the scan. Anthropometry, HbA1c and lipids were measured. CACS was calculated standard Judkins technique were enrolled. MS was defi ned according using Agatston scoring method, with 10 as the upper limit of normality. to the 2007 Chinese Joint Committee for Developing Chinese Guidelines Subjects had a mean age of 46±12, a mean duration of diabetes of 25±10 on Prevention and Treatment of Dyslipidemia in Adults. Serum PEDF was years and a mean HbA1c of 7.6±0.8%. Mean BMI, waist circumference and measured by sandwich enzyme immunoassay and used to carry out VAT were 25±4.1 kg/m², 90±13 cm and 106±71 cm2 respectively. A total of multivariate stepwise regression analysis to assess correlation with patient 59 patients (56%) had a VAT <100 cm2 and 46 (44%) had a VAT >100 cm2. demographic and clinical parameters. Multiple logistic regression analysis CACS was >10 in 26 patients of the group with VAT >100 cm2 compared was performed to identify factors independently correlated with CAD. to 19 patients of the group with a VAT <100 cm2 (57% vs 32%, OR=2.74, Patients with MS had signifi cantly higher levels of serum PEDF than p=0.013). HDL was lower in the VAT>100 cm² than in the VAT<100 cm2 group non-MS subjects (11.1(8.2, 14.2) vs. 10.1(7.6, 12.4) µg/mL; P<0.05). Patients (57 vs 68 mg/dl, p= 0.002), but triglycerides, LDL and HbA1c did not differ with CAD also had signifi cantly higher serum PEDF than non-CAD subjects signifi cantly. Logistic regression analysis with HbA1c, VAT, LDL, smoking and (11.0(8.1, 14.2) vs. 10.3(8.1, 12.8) µg/mL; P<0.05). Multivariate stepwise systolic blood pressure as modifi able risk factors and CACS as dependent regression analysis showed that TG, CRP, eGFR, and hypoglycemic therapy variable showed an association with VAT (OR=1.013, p=0.003), systolic blood were independently correlated with serum PEDF levels. Multiple logistic pressure (OR=1.063, p=0.019), and smoking (OR=2.916, p=0.047). When we regression analysis indicated that serum PEDF was independently positively included age and diabetes duration (non-modifi able risk factors), only age correlated with the presence of CAD. was a signifi cant contributor (OR= 1.196, p<0.001). Serum PEDF levels are independently positively associated with the In conclusion, almost half of the patients with type 1 diabetes had excess presence of CAD in a Chinese population. The results help to identify PEDF VAT. Our study demonstrates a link between CACS and age as non-modifi able as a protective response against vascular damage and subsequent CAD. risk factor and VAT, systolic blood pressure and smoking as modifi able

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A123 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

488-P Therapeutic Effect of Zinc on Type 1 and Type 2 Diabetic Cardiomyo- & 490-P pathy Is Associated With Enhanced Akt/GSK3β Insulin Signaling Exendin-4 Counteracts Palmitate-Induced Apoptosis by Inhibiting Pathway De Novo Production of Ceramides in Human Cardiac Progenitor YUEHUI WANG, WEIXIA SUN, LU CAI, Changchun, China, Louisville, KY Cells Cardiac insulin resistance is a key pathogenic factor for diabetic ANNA LEONARDINI, LUIGI LAVIOLA, ROSSELLA D’ORIA, MAURA R. ORLANDO, cardiomyopathy. We have shown the protective role of zinc supplement in MARIA ANGELA INCALZA, VALENTINA ANDRULLI BUCCHERI, ANNALISA NA- TALICCHIO, SEBASTIO PERRINI, FRANCESCO GIORGINO, Bari, Italy

POSTERS the prevention of diabetic cardiomyopathy in STZ-induced type 1 diabetic Complications mice (Circulation 2006). We also showed that the inhibition of GSK-3β in Ceramides have been suggested to be important mediators of Acute and Chronic the heart of metallothionein (MT)-transgenic diabetic mice could prevent lipotoxicity-induced apoptosis in different cell types. Increased apoptosis the development of cardiomyopathy (Diabetes 2009). However, whether of cardiomyocytes and cardiac progenitor cells in response to metabolic zinc have the protective effects on diabetic cardiomyopathy in transgenic and oxidative stressors has been proposed as a mechanism of myocardial OVE26 type 1 and Akt2-KO type 2 diabetic mice and the relationship damage and dysfunction. Glucagon-like peptide-1 (GLP-1) and GLP-1 analogs between zinc and Akt/GSK3β insulin signaling pathway remains unknown. exert prosurvival effects on cardiac cells. To investigate the mechanisms Here we used OVE26 and Akt2-KO diabetic mice and demonstrated that of fatty acid-mediated cell damage, human cardiac progenitor cells (hCPC) zinc have therapeutic effect on diabetes-induced cardiac remodeling, were isolated from right auricle biopsies and exposed to 0.1 to 0.5 mM detected by CTGF and TGF-β1 expression and collagen deposition (Sirius- palmitate up to 24 h. Dose- and time-dependent increase of CPC apoptosis red staining). These pathological changes were accompanied by signifi cant was demonstrated by the evaluation of caspase-3 activation, caspase-3 increases in oxidative damage (3-NT and 4-HNE), and infl ammation (PAI-1 cleavage and cytosolic release of oligosomes, respectively (p<0.05). In and TNF-α) in diabetic hearts, but not in zinc-treated diabetic hearts. In hCPC exposed to palmitate, intracellular ceramide content, evaluated by the heart of both type 1 and type 2 diabetic mice , we found decreased immunofl uorescence, was concomitantly augmented, and palmitate also phosphorylation of Akt and Akt/GSK3β.Correspondingly, cardiac glycogen induced a signifi cant increase in mRNA and protein levels of ceramide synthase phosphorylation, hexokinase II and PGC-1α expression, which all synthase 5, a critical enzyme in ceramide generation. Co-incubation of involve in the regulation of glucose and lipid metabolisms, were signifi cantly CPCs with fumonisin-B1, a specifi c ceramide synthase inhibitor, partially altered. Diabetes also increased Akt negative regulators, TRB3 expression prevented palmitate-induced apoptosis. When cells were pretreated with and PTEN phosphorylation. The above molecule’s changes were completely the GLP-1 analog exendin-4 (20 nM for 16 h), palmitate-induced apoptosis prevented by zinc treatment for 3 months. These results suggest that zinc was prevented (p<0.05), and the increase in ceramide levels was also can increase Akt phosphorylation that inactivates GSK-3β function probably reduced (p<0.05). Furthermore, exendin-4 prevented the increase of via prevention of diabetic up-regulation of TRB3 and PTEN expression, ceramide synthase 5 expression, both at the gene and protein levels, resulting in a therapeutic effect on diabetic cardiomyopathy. that occurred in response to palmitate. In conclusion, de novo ceramide accumulation contributes to palmitate-induced apoptosis of hCPC, and this is counteracted by the GLP-1 analog exendin-4. Amelioration of lipotoxicity COMPLICATIONS—MACROVASCULAR—CELLULAR via ceramide modulation may contribute to the cardioprotective effects of MECHANISMS OF ATHEROGENESIS IN DIABETES GLP-1-based therapies in subjects with type 2 diabetes. Supported by: Fondazione Eli Lilly Italia; FORISID

Guided Audio Tour: Cellular Mechanisms of Cardiovascular Disease in & 491-P Diabetes (Posters: 489-P to 496-P), see page 17. Selective Inhibition of Protein Kinase C Theta Prevents Cardiac Remodeling and Fibrosis in Streptozotocin-Induced Type 1 Diabetic & 489-P Mice Soluble CD14 Is Highly Released by Epicardial Adipose Tissue from ZHAO LI, ZHU-QIU JIN, Brookings, SD Patients With Type 2 Diabetes and Promotes Cardiac Dysfunction Dilated cardiomyopathy is one of the severe complications for patients and Insulin Resistance with diabetes mellitus. T lymphocyte infi ltration and expansion were SABRINA GREULICH, BUJAR MAXHERA, MARCEL BLUMENSATT, DANIELLA observed in diabetic patients with high risk of acute coronary syndromes. HERZFELD DE WIZA, KONSTANTINOS SMIRIS, ARTUR LICHTENBERG, JO- Protein kinase C (PKC) theta plays a critical role in the activation of mature HANNES RUIGE, MARGRIET OUWENS, Düsseldorf, Germany, Ghent, Belgium T cells. We hypothesized that inhibition of PKC theta might protect hearts Adipokines released from epicardial adipose tissue (EAT) can directly through inhibition of T cell stimulation in type 1 diabetic hearts. In this study, affect cardiac function. Soluble CD14 (sCD14) is selectively elevated in type 1 diabetic mice were induced by low-dose streptozotocin (STZ) (50 EAT from patients with type 2 diabetes. However, the function of sCD14 in mg/kg for 5 days) in male C57BL/6J mice (B6) and Rag1 knockout (KO) mice cardiomyocytes is incompletely understood. This study assessed the levels without mature lymphocytes. A cell-permeable selective PKC theta peptide of sCD14 in conditioned media from EAT from patients with and without type inhibitor (PPI) was administered intraperitoneally (0.2 mg/kg/day) for 4 2 diabetes and studied the effect of recombinant sCD14 on cardiomyocyte weeks (fi rst phase) and 2 weeks (second phase) prior to the evaluation of contractile function and insulin signaling. the cardiac function. At the 11th week, cardiac function was evaluated via a Levels of sCD14 were 8-fold increased in conditioned media from patients Langendorff perfusion system. The contractile force was recorded. Cardiac with type 2 diabetes as compared to controls without type 2 diabetes morphology and fi brosis were determined. Total PKC theta and pT538-PKC (2ng/ml vs. 0.25ng/ml; n=20, P<0.001). In vitro, sCD14 reduced sarcomere theta as well as infi ltrated T cells within the hearts were detected with shortening and cytosolic Ca2+-fl uxes in primary rat cardiomyocytes. These immunostaining. Our results indicated that PPI had no effects on high blood changes were paralleled by reduction in the protein expression of Serca2a, a glucose level in both B6 mice and Rag1 KO mice (p=n. s.). STZ induced cardiac key regulator of myocardial Ca2+-metabolism. Furthermore, cardiomyocytes remodeling and fi brosis in B6 mice but not in Rag1 KO mice. Remarkably, incubated with sCD14 showed decreased insulin-mediated phosphorylation PPI reduced cardiac remodeling and LV dilation in B6 mice. The ratio of LV of Akt-Ser473. Finally, we observed that sCD14 induced the phosphorylation lumen area to LV total area was decreased in PPI group. Cardiac fi brosis was of the MAPK family members p38 and ERK1/2, which have been linked to attenuated in PPI group. PPI also improved cardiac contractility in diabetic contractile dysfunction, in cardiomyocytes. mice (0.56 +/- 0.056 g, vs 0.33 +/- 0.12 g in STZ group, P<0.05). PPI decreased These data show that sCD14 impairs cardiomyocyte contractile function pT538-PKC theta expression and reduced the infi ltration of T cells within and insulin sensitivity, suggesting that the increased release of sCD14 from the diabetic hearts. These fi ndings suggest that selective inhibition of PKC EAT from patients with type 2 diabetes could contribute to the induction of theta improves cardiac function and reduces cardiac remodeling and fi brosis cardiac dysfunction in these patients. in STZ-induced B6 diabetic mice. Mature T lymphocytes play a key role in Supported by: German Diabetes Association; German Center for Diabetes Re- pathophysiology of diabetic cardiomyopathy. search

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A124 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

The magnitude of Gc and Mc pro-infl ammatory activation induced by these & 492-P stimuli, though, remains undefi ned. We studied 5 healthy, 5 obese, and 5 High Glucose Induces Apoptosis and Fibrosis via Enzymatic Glyco- T2D subjects in 3 separate occasions, with 4-h i.v. infusions of glucose sylation of RBP4 Receptor in Endothelial Cells (target 220 mg/dL), lipids (target 3x basal FFA values), or glucose+lipids. Gc SHYI J. SHIN, CHAO H. CHEN, KUN D. LIN, Kaohsiung, Taiwan and Mc activation surface markers CD11b, CD14, CD16, CD62L, and CD66b Retinoic acid and retinol-binding protein 4 (RBP4) possess a variety of were measured by fl ow cytometry. I.v. infusion of glucose only caused important biological propertiess. We recently showed that the increase little change in surface makers expression, while lipid infusion, alone or in of free RBP4 can cause apoptosis via its infl uence on STRA6/JAK2/STAT5/ combination with glucose, induced 35-50% increases in Gc and Mc CD11b POSTERS

CRBP-1/RARα pathway. Non-enzymatic glycation is one pathogenetic factor expression. This effect was more pronounced in healthy and obese subjects Complications of diabetic complications, but the role of enzymatic N-glycosylation remains (fi g1.) than in T2D, in which greater variability of baseline values may have Acute and Chronic elusive. We hypothesize that hyperglycemia may affect N-glycosylation of been caused by overlapping pro-infl ammatory stimuli. Our data indicates a RBP4 receptor (STRA 6) and its function. The effect of high glucose on the powerful acute effect of lipids, independent of hyperglycemia, in activating expression of STRA6/CRBP-I/RARα, fi bronectin, collagens and numbers of Gc and Mc surface marker expression, further emphasizing the importance apoptotic cells were investigated in vascular endothelial cells. Whether of careful nutritional choices and weight control. these changes were reversed by using siRNA of UGGT1 (UDP-glucose: glycoprotein glucosyltransferase 1, one important ER protein folding sensor) were investigated. We found that high glucose can reduce the binding activity between RBP4 and STRA6, increase the expression of glycosylated STRA6 and UGGT1, suppress CRBP-I and RARα, and then induce apoptosis and fi brosis in vascular endothelial cells. Surprisingly, UGGT1 knockdown can attenuate the overproduction of glycosylated STRA6, reverse the suppression of CRBP-I, RARα, fi bronectin, collagens, and reduce the increase of apoptotic cells. These results indicate that UGGT1 over-expression, induced by high glucose, can enhance the glycosylation of RBP4 receptor, but decrease CRBP-I/RARα and fi nally lead to apoptosis and fi brosis in endothelial cells.

& 493-P Ceramide Disrupts the Association Between I2PP2A and PP2A and Causes Endothelial Dysfunction In Vivo LEENA PANNEERSEELAN, TING RUAN, ANINDITA RAVINDRAN, YOUYOU LI, QUAN-JIANG ZHANG, E. DALE ABEL, J. DAVID SYMONS, Salt Lake City, UT We hypothesized that ceramide disrupts the interaction between inhibitor 2 (I2) of protein phosphatase 2A (PP2A) and PP2A and evoke endothelial dysfunction. Bovine aortic endothelial cells (BAECs) were treated for 3 h with BSA (V), V + 500 µM palmitate (P), V + ceramide inhibition (10 uM myriocin; M), or P+M. P-induced increases (p<0.05) in ceramide vs. V were prevented (p<0.05) in cells exposed to P+M (n=8). Permeabilized cells treated as above were incubated with 1° antibodies for eNOS, PP2A, I2PP2A, or ceramide, followed by incubation with fl uorescent Supported by: NIH (P01HD048721), (UL1RR031985) 2° antibodies to assess subcellular localization using confocal microscopy. Relative to V-treatment, P: (i) increased ceramide association with I2PP2A & 495-P in the cytosol; (ii) decreased PP2A association with I2PP2A in the cytosol; Receptor for Advanced Glycation End Products (RAGE)-Dependent and (iii) increased PP2A association with eNOS in the membrane (all p<0.05; Transcriptional Regulation of Early Growth Response Gene-1 (Egr-1) n=6 per combination). All responses were negated in P+M -treated cells. in Diabetic Murine Aortic Endothelial Cells In isolated membrane fractions treated as above: (i) P increased (p<0.05) GURDIP DAFFU, YUNLU XU, ANN MARIE SCHMIDT, SHI FANG YAN, New York, PP2A association with eNOS in a ceramide-dependent manner; and (ii) P NY -induced reductions in basal and insulin-stimulated p-eNOS(S)1177 to eNOS The receptor for advanced glycation end products (RAGE) is a multiligand (both p<0.05) were prevented by ceramide inhibition (i.e., P+M; n=8) or PP2A receptor that plays a key role in diabetic complications. Early growth inhibition (4 µM LB1, n=6). Next, mice haploinsuffi cient for dihydroceramide response gene-1 (Egr-1) is a zinc fi nger transcription factor which mediates desaturase (des1+/-) and their wild-type littermates (des1+/+) were infused detrimental cellular responses in acute hypoxia; in contrast, in chronic for 6 h with lard-oil (LO) or vehicle (veh). hypoxia/ischemia, Egr-1 mediates adaptive responses. We previously showed Subgroups were treated with 1.5 mg/kg LB1 or vehicle (IP) for 3 days prior that RAGE regulates Egr-1 expression in hypoxia-exposed macrophages to LO or veh infusion. Endothelium-dependent relaxation of femoral arteries and endothelial cells, and others showed that Egr-1 is highly expressed in was impaired (p<0.05) in LO des1+/+ vs. veh des1+/+ mice. Endothelial adipose tissue of both the diabetic db/db mouse and human type 2 diabetic dysfunction observed in LO des1+/+ mice was less severe (p<0.05) when subjects. Here we demonstrate that RAGE differentially regulates Egr-1 ceramide accrual (i.e. LO des1+/- mice) or PP2A activation (i.e., LO des1+/+ gene and protein expression under acute (15 min) versus chronic (48 hrs) high mice + LB1) was inhibited. Ceramide dissociates PP2A from I2PP2A and glucose exposure (25 mM D-glucose) in primary murine aortic endothelial activates PP2A to an extent that causes endothelial dysfunction in vivo. cells (MAEC). MAEC devoid of RAGE acutely exposed to high glucose (15 Supported by: 2R15HL091493 min) demonstrate markedly lower Egr-1 mRNA expression compared to wild-type (WT) ECs. In contrast, when RAGE null MAEC are chronically & 494-P cultured in highglucose (48 hrs), Egr-1 mRNA and protein expression is Activating Effect of Acute Hyperglycemia and Hyperlipidemia on signifi cantly upregulated when compared to WT ECs. This suggests that a Granulocyte and Monocyte in Healthy, Obese, and Type 2 Diabetic RAGE-dependent molecular switch regulates Egr-1 expression due to length Subjects of high glucose exposure. In addition, transient transfections of deletional/ PETER HORVATH, STACY R. OLIVER, FRANK P. ZALDIVAR, SHLOMIT RADOM- truncated Egr-1 promoter-reporter constructs in WT or RAGE null MAEC AIZIK, PIETRO R. GALASSETTI, Irvine, CA displayed differentialpromoter activity in basal glucose exposure (5.5 mM In obesity and type 2 diabetes (T2D) chronic formation of atherosclerotic D-glucose) vs high glucose (25 mM D-glucose) exposure. Most notably were plaques is mediated, at least in part, by innate immune system cells, such those constructs with truncations of AP-1, Sp1, CRE and/or SRE potential as granulocytes (Gc) and monocytes (Mc). Gc and Mc may become activated binding sites that displayed higher promoter activity in RAGE null MAEC by typical metabolic stimuli, such as hyperglycemia and hyperlipidemia, in high glucose conditions when compared to WT. Studies are underway the latter often associated with a hypercaloric diet in obesity, and with to further probe these promoter sites by electrophoretic mobility shift and the choice of lipid-rich foods in T2D in the attempt to avoid carbohydrates. supershift assays (EMSA) and ChIP assays to establish functionality.

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A125 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

498-P & 496-P The Endothelial Nitric Oxide Synthase Derived Nitric Oxide Regu- Characterization of Persistent Defects in Insulin Activation, Migra- lates Vascular 26S Proteasome Functionality tion, and VEGF Production in Fibroblasts from Type 1 Diabetic Pa- HONGTAO LIU, SHUJIE YU, YU LI, JIAN XU, Oklahoma City, OK tients of Extreme Duration Loss of vascular homeostasis leads to diabetes morbidity and mortality, MOGHER KHAMAISI, I-HSIEN WU, STEPHANIE HASTINGS, HILLARY A. KEENAN, attributable to early loss of endothelial nitric oxide (NO) bioavailability. In GEORGE L. KING, Boston, MA contrast, early diabetes is found to increase 26S proteasome functionality Abnormalities in fi broblastic and angiogenic responses to injury are

POSTERS and boost vascular infl ammatory response. We wonder whether endothelial

Complications associated with poor wound healing in diabetic patients. We have NO synthase (eNOS)-derived NO functions as a physiological regulator of the Acute and Chronic characterized fi broblastic function and cytokine expression in response to 26S proteasome. To test this, we exposed proteasome reporter (UbG76V- ischemia and hyperglycemia using fi broblasts derived from subjects in the GFP)-expressing endothelial cells to NO and monitored proteasome Joslin Medalist Study who have had type 1 diabetes mellitus (T1DM) for 50 functionality. We also generated UbG76V-GFP-expressing eNOS-knockout years or longer and exhibited protection from microvascular complications. mice by breeding and backcrossing between eNOS-knockout and UbG76V- Fibroblast functions from ten Medalists and fi ve controls without diabetes of GFP mice (The Jackson Laboratory). Like the selective NO donors, NO derived similar age were compared. Expression of vascular endothelial growth factor from the activated eNOS (by pharmacological and genetic approaches) (VEGF) mRNA and protein levels in the cells of Medalists were reduced by induced (poly)-UbG76V-GFP accumulation, which was associated with 45% and 54%, respectively, compared to controls. Hypoxia, induced by 5% increased O-GlcNAc modifi cation of the proteasome regulatory complex PO2, increased VEGF mRNA and protein expression by 4.5 and 2.1-fold in (PA700/Rpt2) and reduced proteasome chymotrypsin-like activity (p<0.05, fi broblasts, respectively from controls, but no response was detected in the n=3/group). Conversely, siRNA knockdown of O-GlcNAc transferase (OGT), Medalists’ fi broblasts. Incubation with 25mM (HG) vs. 5mM glucose (NG) for the key enzyme for O-GlcNAcylation, abolished the NO-elicited effects. 72h caused a 90% reduction in VEGF expression in the controls (p=0.01) and Consistently, adenoviral overexpression of O-GlcNAcase, the enzyme 70% in the Medalists (p=0.01). Expression of TGF-β and fi bronectin mRNA responsible for O-GlcNAc removal, mimicked the effects of OGT knockdown. levels at NG conditions were increased in the Medalists by 2 and 3-fold, Finally, compared to the control UbG76V-GFP mice, eNOS-lacking mice respectively, compared to the controls. exhibited accelerated UbG76V-GFP degradation in isolated aortas, in parallel Fibroblast migration was reduced in the Medalist by 2.5-fold compared with increased chymotrypsin-like activity and decreased PA700/Rpt2 to controls. Incubation with HG for 8h caused a signifi cant reduction in cell O-GlcNAcylation, without changing PA700/Rpt2 protein levels. Importantly, migration in both controls (p=0.03) and the Medalists (p<0.05), which was the changes were enhanced in eNOS-depleted mice when rendered diabetic normalized by incubation with PDGF or 10% serum, but not with insulin. with streptozotocin (p<0.05, n=5/group). Collectively, the eNOS-derived NO Similarly, insulin activation of p-AKT, p-ERK and p-focal adhesion kinase (FAK) functions as an OGT-mediated inhibitory regulator of the 26S proteasome, were reduced by 55, 22, and 57% in the Medalists’ fi broblasts, respectively, likely through PA700/Rpt2 O-GlcNAcylation. The lack of eNOS-derived NO vs. controls but PDGF’s effects were not affected. Thus, fi broblasts from might contribute to elevated vascular 26S proteasome functionality in early patients with extreme duration of T1DM retained specifi c defects to insulin diabetes. signaling, which may cause abnormal angiogenesis and fi broblast responses Supported by: NIH; AHA; OCAST to injury, possibly leading to poor wound healing in diabetes. Supported by: JDRF; NIH 499-P DPP-4 Inhibitor Linagliptin Attenuates Vascular Smooth Muscle 497-P Cell Proliferation and Neointima Formation after Vascular Injury FoxO1 Compartmentalization Results in Different Metalobomic Pro- YUICHI TERAWAKI, TAKASHI NOMIYAMA, TAKAKO KAWANAMI, YURIKO fi les in Human Endothelial Cells HAMAGUCHI, TOMOKO TANAKA, KUNITAKA MURASE, TOSHIHIKO YANASE, ROSSELLA MENGHINI, VIVIANA CASAGRANDE, MARTA FABRIZI, ROBERT Fukuoka, Japan STOEHR, MARIA MAVILIO, RENATO LAURO, MASSIMO FEDERICI, Rome, Italy The aim of glycemic control is not only lowering blood glucose level but FoxO1 is a pivotal element in the regulation of endothelial activation. also improving quality of life and mortality through preventing occurrence To learn about the pathways that FoxO1 uses to activate endothelium and progression of vascular complications. According to this opinion, to we pefomed GC/MS and LC/MS/MS metabolomics. We infected human investigate direct vascular protective effect of antidiabetic agents might be umbilical endothelial cells (HUVEC) with adenovirus encoding FoxO1 WT, able to provide important information for the choice of antidiabetic agents. FoxO1 nuclear form ADA, FoxO1 deacetylated form KR and adenovirus-GFP We previously reported that GLP-1 receptor agonist Exendin-4 directly for control. Cell lysates and supernanants as well as mRNA were collected decreases atheroma formation (Arakawa M, Diabetes 2010) and neointima for analysis. ADA showed signifi cantly increased levels of dimethylarginine formation (Goto H, BBRC 2011), independent on glucose lowering effect. In SDMA and ADMA and this observation would be consistent with reduced the present study, we examined whether DPP-4 inhibitor linagliptin could NO production upon ADA expression. The levels of 7-alpha and 7-beta- reduce vascular smooth muscle cell proliferation in vitro and in vivo model. hydroxycholesterol, lipid peroxidation products from cholesterol increased We demonstrated rat aortic smooth muscle cell (RASMC) proliferation assay in the WT and ADA groups in relation to the GFP control group and further with 1~100µM linagliptin treatment. Compared with control, linagliptin elevated in the KR group which would be consistent with increased lipid decreased RASMC proliferation signifi cantly, in dose dependent manner. peroxidation in response to all three alleles of FOXO1 over-expression. Furthermore, we performed a model of guide-wire induced femoral artery 1-Stearoylglycerophosphoglycerol, a lysophosphatidic acid (LPA) showed injury in mice with or without 3mg/kg/day oral linagliptin treatment (n=9 signifi cantly higher levels in the KR group and its levels increased even in each group). 4 weeks after injury, serum GLP-1 concentration was more in the ADA group in relation to the GFP and the WT groups. LPA is a signifi cantly higher in linagliptin-treated mice (CT14.79±0.72pmol/l vs. Lina potent signaling molecule for endothelial cells, activation of which result 25.97±1.99pmol/l), though blood glucose level (CT 69.9±0.41mg/dl vs. Lina in various consequences depending on the context of other concomitant 72.2±1.33mg/dl) and body weight (CT 24.47±0.55g vs. Lina 23.82±0.71g) signaling programs. To test the effi cacy of the metabolomics approach we were not changed. Next, we measured square of neointima formation and analyzed by qPCR in mRNA from the HUVEC infected with the various FoxO1 media, subsequently calculated intima/media ratio. Interestingly, intima/ isoform, the three major enzymes involved in ADMA generation, protein media ratio was decreased with linagliptin-treated mice (CT 0.85±0.28 vs. arginine methyltransferase 1 and 2 (PRMT1, PRMT2) and dimethylarginine Lina 0.46±0.17). dimethylaminohydrolase (DDAH). Interestingly we found that DDAH was These data suggest that DPP-4 inhibitor linagliptin might be able to signifi canlty downregulated in ADA cells, providing a molecular support for attenuate vascular smooth muscle cell proliferation and neointima formation the metabolomics results. after vascular injury independent on glucose lowering effect. Our data suggest that FoxO1 affects endothelial cell function through several metabolic pathways potentially implied in endothelial activation and dysfunction, two processes that are associated to increased incidence of diabetes and atherosclerosis.

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A126 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

500-P 502-P Anti-Infl ammatory Effects of the Phosphodiesterase-5 Inhibitor Accelerated Atherosclerosis in Individuals With the Haptoglobin Tadalafi l on TNF-α-Induced Infl ammation in Human Endothelial 2-2 Genotype and Diabetes Is Mediated by Macrophage Lysosomal Cells Injury and Apoptosis JOSEFIN OLAUSSON, LOVISA SJÖGREN, REZA MOBINI, LENA STRINDBERG, RABEA ASLEH, NINA S. LEVY, ANDREW P. LEVY, Haifa, Israel EMANUEL FRYK, LILLEMOR MATTSSON HULTÉN, PER-ANDERS JANSSON, Background: There exists a common functional allelic polymorphism G o t h e nb u r g , Sweden in the Haptoglobin (Hp) gene which has been associated with the risk of

Impaired insulin action reduces synthesis of nitric oxide (NO) and elevates cardiovascular disease in individuals with diabetes mellitus (DM). In the POSTERS expression of endothelin-1 (ET-1) in vascular endothelial cells, i.e. promotes atherosclerotic plaque, the CD163 macrophage Hp-hemoglobin (Hb) receptor Complications endothelial dysfunction and vascular infl ammation. Tadalafi l (Cialis®) is is responsible for clearing Hb released after intraplaque hemorrhage. In DM, Acute and Chronic a selective PDE-5 inhibitor, which enhances the NO signaling pathway by the Hp 2 allele protein product is defective in its Hb scavenging properties augmenting formation of cyclic guanosine monophosphate (cGMP) through and has been associated with increased iron in the atherosclerotic plaque. reduction of its hydrolysis by cGMP-degrading PDEs. It has been suggested Hypothesis: We proposed that the increased iron accumulation in Hp that PDE-5 inhibitors have anti-infl ammatory properties although the 2-2 plaques and DM was due to an impaired processing of the Hp 2-2-Hb mechanism is not clear. Therefore, our aim was to elucidate which signaling complex within macrophage lysosomes and that this iron was toxic to the pathway tadalafi l may affect to confer this anti-infl ammatory effect. macrophage lysosomal membrane thereby resulting in the loss of lysosomal TNF-α was used to mimic infl ammatory activation in Human Umbilical Vein membrane integrity and cellular injury. Endothelial Cells (HUVEC) and the cells were treated with tadalafi l. Protein Methods and Results: Confocal microscopy using lysotropic pH indicator was extracted after 15 min. Phosphorylated c-Jun N-terminal Kinase (p-JNK) dyes was used to demonstrate that uptake of Hp 2-2-Hb complexes by expression was signifi cantly reduced by 63% while p-NFκB, p-p38 and p-ERK the macrophage CD163 receptor disrupted the lysosomal pH gradient. were not affected by tadalafi l, indicating that tadalafi l is specifi c and exerts Cellular fractionation studies of lysosomes isolated from macrophages its effects by modulating the JNK pathway. Further, tadalafi l attenuated the incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal gene expressions of iCAM (18%), vCAM (30%), E-Selectin (36%), ET-1 (49%), membrane oxidation and a loss of lysosomal membrane integrity. We found and IL-6 (22%) after 12 hours. Moreover, tadalafi l increased gene expression that the lysosomal acidic pH was an essential component responsible for of eNOS (26%) indicating that tadalafi l may amplify NO synthesis. In addition, redox active iron generation and iron induced lysosomal oxidative injury secreted ET-1 in the cell medium was decreased after 12 hours of tadalafi l associated with Hp 2-2-Hb uptake. Additionally, several apoptotic markers treatment (18%). In conclusion, tadalafi l shows anti-infl ammatory properties (including DNA fragmentation and active caspase-3) were signifi cantly by modulating the JNK pathway and should be further tested as an agent to increased in macrophages endocytosing the Hp 2-2-Hb complexes. The ameliorate endothelial dysfunction. oxidative lysosomal injury and macrophage apoptosis associated with Hp Supported by: Eli Lilly and Company 2-2-Hb complexes were dramatically enhanced when using glycosylated Hp- Hb complexes. 501-P Conclusions: Lysosomal injury and subsequent macrophage apoptosis Effects of Pioglitazone on Endothelial Progenitor Cells in Type 2 Dia- secondary to uptake of Hp 2-2-Hb complexes may play a major role in the betic Patients With Vascular Complications: The SPLENDOR Study development of diabetic atherosclerosis. STEFANO DEL PRATO, DANIELA LUCCHESI, LAURA PUCCI, ROSAMARIA BRUNO, Supported by: NIH ELEONORA RUSSO, MONIA GAROFOLO, GIANPAOLO FADINI, VERONICA SAN- CHO BORNEZ, ROSSELLA RUSSO, ROBERTO MICCOLI, ANGELO AVOGARO, 503-P LORENZO GHIADONI, CINZIA DI PIETRO, GIUSEPPE PENNO, Pisa, Italy, Padova, Fetuin-A Regulates the Expression of Angiogenic Proteins via An- Italy, Rome, Italy other Pathway than TLR4 Signaling Endothelial progenitor cells (EPCs) contribute to endothelial repair. EPCs DOROTHEA I. SIEGEL-AXEL, NORBERT STEFAN, SUSANNE ULLRICH, KILIAN RIT- number and function are impaired in T2DM. Pioglitazone (PIO) affects TIG, BIRGIT SCHREINER, ULRIKE SCHMIDT, HARALD STAIGER, HANS-EBERHARD favourably vascular health. In a double-blind, randomized 24-week trial, we SCHALLER, HANS-ULRICH HARING, Tübingen, Germany examined the effect of PIO vs. glyburide (GLY) as add-on to metformin on Purpose: Fetuin-A contributes to lipid disorders and type 2 diabetes but circulating EPCs in thirty-fi ve T2DM with CV disease randomized to 30 mg/ maybe also to cardiovascular diseases. We found, in accordance to Pal et day PIO (n=18; age 62±6, BMI 30.1±3.4 kg/m2, HbA1c 8.0±0.4%) or 10 mg/day al. (Nat Med 2012), that fetuin-A induces IL-6 and IL-8 expression in fat cells GLY (n=17; age 60±9, BMI 31.5±4.1, HbA1c 7.9±0.5%). Mean exposure to study in concert with palmitate (PAL) via TLR4 signaling. In addition we showed drugs was 184.5±8.0 days for PIO and 184.6±10.2 days for GLY; compliance that perivascular fat cells (PVFC) secrete high levels of angiogenic proteins. was 96.9±2.7% and 96.3±5.4%. CD34+/KDR+ EPCs were measured (FACS) Aim of this study was to examine if fetuin-A infl uences the expression and at baseline and at 12-wk and 24-wk along with ISI Matsuda and metabolic/ secretion of proinfl ammatory and angiogenic proteins in PVFC, and if other infl ammatory parameters. Glycemic control improved to a similar extent pathways than TLR4 are involved. by 24-wk (HbA1c: PIO -0.76±0.81%, GLY -0.77±0.75%). Baseline CD34+/ Methods: PVFC and endothelial cells (EC) from human specimens of arm KDR+ cells/106 cytometric events were similar in PIO (32.4±18.4), and GLY arteries were cocultured in transwell systems +/- EC. Cells were treated (28.1±17.6; p=NS). Upon treatment, minor changes in CD34+/KDR+ cells/106 +/- TLR4 inhibitor CLI-095 6 h before addition of PAL and/or fetuin-A (600µg/ occured for both treatments at 12-wk (PIO -2.9±23.5, GLY +8.32±17.9; p=NS), ml), or +/- PI3K inhibitor Ly29 1 h before insulin addition for 30 min. After 6 or and 24-wk (PIO +5.8±33.8, GLY +12.2±37.0; p=NS vs basal and between 24 h, proteins and mRNA were quantitiated. Intracellular FoxO location was groups). The same was for CD34+/CD133+/KDR+ events. PIO resulted in a visualized by immunostaining. greater improvement in insulin sensitivity (ISI Matsuda, baseline: 1.81±0.30 Results: PVFC expressed and secreted IL-6 and IL-8, and the angiogenic vs. 1.85±0.21; 24-wk +0.68±0.18 vs. -0.03±0.20; p=0.022) and adiponectin factors PAI-1, bFGF, PDGF-BB, MCP-1, VEGF, PlGF, HGF. Fetuin-A strongly levels (+2.5±1.0 vs. +0.2±0.4 ng/ml; p=0.0005). On the contrary, there was no upregulated IL-8 and IL-6 expression which was potentiated by PAL and difference in changes of VEGF (-3.22±5.8 vs. -1.9±3.9 pg/ml), EPO (+0.28±0.82 blocked by CLI-095. In contrast, MCP-1 was induced by fetuin-A, without vs. -0.98±0.93 mIU/ml), SDF-1 (-2.05±0.92 vs. -0.87±0.48 pg/ml), MDA increase by PAL and blockade by CLI-095. PlGF was also induced by fetuin-A, (+0.39±0.54 vs. -0.53±0.25 µmol/l), FRAP (-29.5±26.7 vs. +503±476.8 µmol/l), partially by PAL and blocked by CLI-095. However, fetuin-A inhibited HGF and LOOH (+0.44±0.53 vs. -0.61±0.32 µmol/l). Over a short treatment period, expression and release but PAL and CLI-095 had no effect. Insulin stimulation PIO improved insulin sensitivity and increased adiponectin with similar caused FoxO translocation and HGF increase which was inhibited by fetuin-A reduction in HbA1c as compared to GLY. Both treatments were associated or PI3 kinase inhibitor. with minor, not signifi cant changes in the number of circulating EPCs. Conclusions: In contrast to the increase of IL-6 and IL-8 by fetuin-A, effects on other proinfl ammatory and angiogenic proteins were not or only partially dependent on TLR4 signaling. Predominantly the inhibition of HGF by fetuin-A is mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Thus, beside insulin resistance fetuin-A may regulate the expression and secretion of proteins involved in atherosclerosis. Supported by: German Center for Diabetes Research

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A127 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

504-P 506-P Dipeptidyl Peptidase-4 Impairs Microvascular Endothelial-Depen- The DPP-IV Inhibitor Saxagliptin Inhibits Palmitate-Induced Apop- dent Relaxation: The Role of Cyclooxygenase tosis of Human Umbilical Vein Endothelial Cells and Human Cardiac CARLOS F. SÁNCHEZ-FERRER, SUSANA VALLEJO, TANIA ROMACHO, LAURA A. Progenitors VILLALOBOS, NINA WRONKOWITZ, HENRIKE SELL, JUERGEN ECKEL, CONCEP- LUIGI LAVIOLA, MARIA ANGELA INCALZA, MAURA R. ORLANDO, CRISTINA CIÓN PEIRÓ, Madrid, Spain, Düsseldorf, Germany CACCIOPPOLI, ANNA LEONARDINI, ROSSELLA D’ORIA, VALENTINA ANDRULLI Cardiovascular complications are the main cause of morbidity and BUCCHERI, ANNALISA NATALICCHIO, SEBASTIO PERRINI, FRANCESCO GIORG-

POSTERS mortality in obese and diabetic patients. Dipeptidyl peptidase-4 (DPP- INO, Bari, Italy

Complications 4) is a multifunctional glycoprotein playing a major role in the regulation Exposure of endothelial cells and cardiomyocytes to the fatty acid Acute and Chronic of glucose homeostasis. Indeed, DPP-4 inhibitors are currently used to palmitate results in biochemical abnormalities leading to cell dysfunction prolong the insulinotrophic effects of incretins. Recently, DPP-4 has been and apoptosis. DPP-IV inhibitors may exert protective actions on the identifi ed as a novel adipokine over-secreted in obesity where it promotes cardiovascular system. The aim of this study was to investigate the protective insulin resistance and may represent a link between adipose tissue and the effects of the DPP-IV inhibitor saxagliptin on palmitate-induced apoptosis of metabolic syndrome (Lamers et al. Diabetes 60: 1917-25, 2011). The aim of human umbilical vein endothelial cells (HUVEC) and human cardiac progenitor this study was to explore the capacity of DPP-4 to directly impact on vascular cells (hCPC). Expression of DPP-IV in HUVEC and hCPC was confi rmed at function with special focus on vascular reactivity. the mRNA level by qRT-PCR, and at the protein level by immunoblotting Mesenteric microvascular fragments isolated from 3 month-old female and immunofl uorescence, respectively. Exposure of HUVEC to 0.25 mM B57BL/6 mice were mounted on a small vessel wire myograph. Pre- palmitate for 24 h resulted in increased expression of the adhesion molecule incubation with DPP-4 (20 to 500 ng/mL) did not alter the contractility ICAM-1 (p<0.05) and of the pro-apoptotic mediator Bax (p<0.05), and to noradrenaline (1 nmol/L to 1 µmol/L). However, DPP-4 impaired the was associated with a 5- to 7-fold increase in cellular apoptosis (p<0.05), endothelium-dependent relaxation to acetylcholine (1 nmol/L to 1 µmol/L) evaluated by the detection of both cytoplasmic oligosomes and cleavage in a concentration-dependent manner by up to 75%, without modifying of caspase-3. Long-term exposure to palmitate also increased apoptosis of endothelium-independent relaxations to sodium nitroprusside (1 nmol/L hCPC by 6-fold (p<0.05). Pretreatment with the DPP-IV inhibitor saxagliptin to 10 µmol/L). Moreover, the co-incubation of DPP-4 (200 ng/mL) with its (0.05 mM) for short times (5 to 15 min) resulted in a dose-dependent increase enzymatic inhibitor K579 (100 nmol/L) prevented the impaired endothelium- in the phosphorylation of Akt, Erk-1/2 and eNOS in HUVEC, and of Akt in dependent relaxation by DPP-4. Similarly, the cyclooxygenase inhibitor hCPC, respectively (p<0.05). Importantly, saxagliptin almost completely indomethacin (10 µmol/L) and the thromboxane A2 receptor antagonist abrogated palmitate-induced apoptosis of both HUVEC and CPC (p<0.05); SQ29548 (10 µmol/L) did abrogate the impairing action of DPP-4. The however, this response required preincubation of the cells for at least NADPH-oxidase inhibitor apocynin (10 µmol/L) did not restore the relaxation 2 h whereas shorter times were ineffective. Thus, saxagliptin prevents impaired by DPP-4. palmitate-mediated apoptosis of HUVEC and hCPC, thereby augmenting the In conclusion, DPP-4 directly impairs endothelium-dependent relaxation, potential for vessels and heart regeneration under conditions of lipotoxicity. through a mechanism that involves cyclooxygenase activation and likely Even though saxagliptin can induce pro-survival signaling via Akt within the release of a vasoconstrictor prostanoid. Over-production of DPP-4 in minutes, inhibition of cellular DPP-IV for at least 2 h is required to exert the obesity and diabetes might therefore contribute to endothelial dysfunction anti-apoptotic effect, suggesting that this response may occur via increased associated with both metabolic diseases. bioavailability of non-GLP-1 peptides. Supported by: PRI-AIBDE (2011-0811), (SAF2011-28011), (SAF2011-24648); DAAD; Supported by: AstraZeneca/Bristol-Myers Squibb DFG 505-P 507-P Liraglutide Mitigates TNF-Alfa Induced Alterations in HUVEC from WITHDRAWN Diabetic Donors GLORIA FORMOSO, PAMELA DI TOMO, PAOLA LANUTI, BARBARA FARICELLI, MARCO MARCHISIO, ASSUNTA PANDOLFI, AGOSTINO CONSOLI, Chieti, Italy High glucose amplifi es in vitro endothelial cells activation by infl ammatory cytokines and enhances TNF-α induced endothelium adhesion molecules expression (V-CAM1, I-CAM1). Endothelial Micro Particles (EMP) are thought to refl ect endothelial infl ammation, being directly indicative of endothelial cell stress/damage. Liraglutide, a GLP-1 Rx agonist used in type 2 diabetes therapy, reduced PAI-1 and V-CAM1 expression in endothelial cells cultured in high glucose and exposed to TNF-α in vitro. Aim of our study was to evaluate, in endothelial cells harvested from umbilical cords of women affected by gestational diabetes (D-HUVEC) and thus exposed to hyperglycemia in vivo, whether, as compared to control cells (C-HUVEC): 1) TNF-α (5 ng/mL) induces higher V-CAM1 and I-CAM1 expression and exposure on plasma membrane, greater endothelium monocyte adhesion and greater EMP release in culture media. 2) Liraglutide addition (100nM) to the culture media is able to modulate TNF-α induced alterations. As compared to C-HUVEC, D-HUVEC showed higher V-CAM1 and I-CAM1 (western blot and Amnis) expression and exposure on plasma membrane (p<0.05) in response to TNF-α. Endothelial monocyte adhesion (rotational adhesion assay) was also greater in D-HUVEC and EMP release (cytofl uorimetric analysis) was greater in their culture media (p< 0.05 for both). In D-HUVEC (but not in C-HUVEC), Liraglutide exposure signifi cantly reduced TNF-α induced V-CAM1 e I-CAM1 expression and exposure on plasma membrane (p<0.05). In the same cells Liraglutide exposure also reduced endothelial monocyte adhesion and EMP release (p<0.05). In conclusion, chronic exposure to hyperglycemia in vivo amplifi es TNF-α induced pro-atherogenic alterations in endothelial cells. Liraglutide mitigates TNF-α effects and results in reduced cell stress/damage as indicated by reduced EMP release. Liraglutide could thus exert a protective effect against endothelial dysfunction triggered by infl ammatory cytokines and hyperglycemia. Supported by: Novo Nordisk, Inc.

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A128 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

508-P 3.65±0.60%) was greater with PIO (+0.72±0.88 vs. +0.32±0.77%; p=0.574) Kinin B1 Receptor Agonist Overcomes Diabetes-Associated Resis- though not statistically different. There were no differences in GTN%. With tance to Cardioprotective Agents in Myocardial Ischemia PIO, OGIS improved (+36±12 vs. +5±7; p=0.04) and adiponectin increased LOUIS POTIER, LUDOVIC WAECKEL, MARIE-PASCALE VINCENT, FERNAND JU- (+2.5±1.0 vs. +0.2±0.4 ng/ml; p=0.0005) along with a greater reduction in NIOR GOBEIL, CHRISTINE RICHER, MICHEL MARRE, RONAN ROUSSEL, FRAN- triglycerides (-60±20 vs. +3±14 mg/dl; p=0.008) and an increase in HDL ÇOIS ALHENC-GELAS, NADINE BOUBY, Paris, France, Sherbrooke, QC, Canada (+6±2 vs. -3±1 mg/dl; p=0.0002) and Apo-A1 (+0.11±0.04 vs. -0.05±0.05 g/l; Cardiac ischemia is a leading cause of death in patients with diabetes. p=0.029). Compared to GLY, PIO was associated with a slight reduction in hs-CRP (-21±11 vs. +2.74±10 mg/l; p=0.084), PAI-1 (-16.3±9.2 vs. +4.4±18.1 ng/

The diabetic ischemic heart is experimentally resistant to cardioprotective POSTERS treatments. The kallikrein-kinin system (KKS) is involved in cardiac protection ml; p=0.056), with no differences for IL-6, sICAM-1, sVCAM-1, and MCP-1. In Complications during ischemia reperfusion (IR) in non diabetic animals. We hypothesized T2DM with CVD already on metformin, 24 week addition of PIO as compared Acute and Chronic that pharmacological activation of KKS can lead to cardioprotection in IR. to GLY, was not associated with signifi cant changes in endothelial function We tested the effects of new potent selective agonists of kinin B1 (B1R) in spite of improved insulin sensitivity, lipid profi le, and infl ammatory or B2 (B2R) receptors administered before reperfusion in non diabetic and response. diabetic mice (streptozotocin). In non diabetic mice, the B2R agonist reduced infarct size by 47%, similarly to ramiprilat, or ischemic postconditioning. 510-P The B1R agonist had no effect. In diabetic mice, whereas the B2R agonist, Abnormal Hyaluronan Catabolism Is Involved in the Development of ramiprilat or ischemic postconditioning had no cardioprotective effect, Chronic Ulcers in Diabetes Mellitus the B1R agonist reduced signifi cantly infarct size (fi gure). This effect was NATALYA YEVDOKYMOVA, SERGIJ PODPRYATOV, Kyiv, Ukraine abolished by selective B1R antagonist. It is generally accepted that hyaluronan (HA) is implicated in wound The cardioprotective effect of B1R agonist was associated with activation healing regulation, but its role in the formation of chronic diabetic ulcers are of PI3K/Akt and ERK1/2, both leading to GSK-3β inhibition. Differential effect poorly understood. Earlier we demonstrated the excessive accumulation of of the agonists in non diabetic and diabetic mice is linked to inactivation of high-molecular-weight HA by fi broblasts of diabetic (Type-2) patients with B2R signaling and induction of B1R synthesis in diabetic heart. In conclusion, ulcers. We also observed intensifi cation of this process under the action pharmacological activation of B1R overcomes resistance of diabetic heart to of exogenous lactate, the obligatory component of wound healing process cardioprotective treatments. B1R agonist may become novel therapeutics and known modulator of HA metabolism. However, neither the expression for diabetic patients in acute coronary syndromes. of hyaluronan synthases (HAS1,2,3), nor hyaluronidases (HYAL1,2) were examined in diabetic fi broblasts. In this work we aim to compare the expression of HAS1,2,3 and HYAL1,2 by fi broblasts of diabetic patients with and w/o ulcers; and to study the effect of exogenous L-lactate on the above mentioned enzymes. All diabetic fi broblast lines used in this study (three from ulcer-free patients and three from those with ulcers) demonstrated the same levels of HAS1,2,3 mRNAs expression. Exogenous lactate (15 µmole/ml, 24 h) up-regulated the expression of HAS2 and 3 (but not HAS1) in all groups of cells. In contrast, HYAL2 expression in fi broblasts of diabetic patients with ulcers was inhibited (~1.4-fold) both at the level of mRNA and protein as compared to cells of patients without ulcers. In addition the lactate treatment up-regulated the expression of HYAL1 only. We believe that fi broblasts of diabetic patients with ulcers have impaired HA catabolism; and furthermore that the defi ciency of HA short fragments may be the cause of ulcers’ development.

511-P Bone Morphogenic Protein 4 Induced by Glucolipotoxicity in Human Vascular Endothelial Cells HYUK-SANG KWON, OAK-KEE HONG, HYUN-JI CHUN, MEE-KYOUNG KIM, KI- HYUN BAEK, KI-HO SONG, SOON-JIB YOO, BONG-YUN CHA, Seoul, Republic of Korea Objective: Bone mophorgenic protein 4 (BMP4) has been known to be pro-atherogenic molecules in the previous literatures. Here we investigate how glucolipotoxicity affects BMP4 expression in endothelial cells and how 509-P BMP4 plays a role in diabetic vascular complications. Effects of Pioglitazone on Endothelial Function in Type 2 Diabetic Methods: Human umbilical vein endothelial cells (HUVECs) were treated Patients With Vascular Complications: The SPLENDOR Study with high glucose concentration (27.5 mM) and/or palmitate (500uM) for 24 GIUSEPPE PENNO, LAURA PUCCI, DANIELA LUCCHESI, ROSAMARIA BRUNO, hours or 72 hours. Expression of BMP4 in HUVECs treated with high glucose ELEONORA RUSSO, MONIA GAROFOLO, VERONICA SANCHO BORNEZ, ROS- and/or palmitate were examined. Effect on high glucose and/or palmitate on SELLA RUSSO, SAULA DE KREUTZENBERG, ROBERTO MICCOLI, ANGELO AVOG- the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin), ROS, ARO, LORENZO GHIADONI, CINZIA DI PIETRO, STEFANO DEL PRATO, Pisa, Italy, and monocyte binding were examined. Using siRNA for BMP4, how BMP4 Padova, Italy, Rome, Italy affects on the expression of adhesion molecules, ROS and monocyte binding In T2DM, pioglitazone (PIO) reduces CVD risk, likely through improved in HUVECs with high glucose and/or palmitate treatments. insulin sensitivity and benefi cial effects on lipids, BP, and infl ammation. In Results: High glucose increases BMP4 expression in dose dependent a double-blind, 24-week trial, we examined the effects of PIO vs. glyburide manner in HUVECs. Fatty acids (palmitate) potentiates its expression. (GLY) as add-on to metformin on endothelial function in 35 T2DM with Expression of adhesion molecules were increased by treatments with high CVD randomized to 30 mg/day PIO (n=18; age 62±6, BMI 30.1±3.4 kg/m2, glucose and/or palmitate. When BMP4 was knockdowned by siRNA, this HbA1c 8.0±0.4%) or 10 mg/day GLY (n=17; age 60±9, BMI 31.5±4.1, HbA1c expression was blunted. High glucose and/or palmitate increases ROS 7.9±0.5%). Compliance was 96.9±2.7% (PIO) and 96.3±5.4% (GLY). At production in HUVECs, BMP4 knockdown ameliorated this response. In baseline and 24-wk, fl ow-mediated (FMD) and glyceryl trinitrate-mediated monocyte binding assay, high glucose with palmitate induced monocyte dilation (GTN) were determined along with metabolic/infl ammatory markers binding in HUVECs and it was prevented by BMP4 siRNA. and insulin sensitivity (OGIS). HbA1c improved to a similar extent in PIO Conclusion: This study suggests that BMP4 might play a certain role in (-0.76±0.19%) and GLY (-0.77±0.18%). Basal brachial artery diameter (BAD; high glucose and/or fatty acid induced atherogenesis in diabetes 4.3±0.2 vs. 4.3±0.3 mm) increased by 24-wk with PIO (+0.19±0.10 mm) vs. Supported by: National Research Foundation of Korea (2012-007098) no changes in GLY (-0.04±0.10 mm, p=0.098). Maximal BAD in response to reactive hyperemia was greater with PIO vs. GLY (+0.22±0.10 vs. -0.05±0.10 mm, p=0.034). Consensually, FMD increase from baseline (4.04±0.63 vs.

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A129 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE

512-P 7.3±1.0%) and high- (74.4±12.9 mg/dl; N=48, M/F 46/54%; age 65.8±8.3; BMI Glucose and Palmitate Contribute to Endothelial Dysfunction by De- 27.3±5.6; HbA1c 7.3±0.8%) HDL-C. At fasting, venous blood was drawn in EDTA creasing Autophagic Flux tubes and processed within 2-hrs. PBMCs were fractionated using Biocoll KAREN A. WEIKEL, YASUO IDO, NEIL B. RUDERMAN, Boston, MA density-gradient; after 3-day culture, non-adherent cells were discarded The mechanisms by which cellular stress infl uences autophagic fl ux in and adherent cells positive for Dil-ac-LDL/Lectin were identifi ed as EPCs. cardiovascular disease vary between tissues and cellular contexts. The After 5-7-day culture in EBM-2 medium, adherent cells were evaluated for cellular response to glucose and fatty acids is regulated by adenosine viability/proliferation (MTT assay), senescence (beta-galactosidase activity detection), migration (modifi ed Boyden chamber using VEGF as chemo-

POSTERS monophosphate-activated protein kinase (AMPK), but how AMPK infl uences

Complications the effects of hyperglycemia and hyperlipidemia on autophagy in endothelial attractant), adhesion capacity (on fi bronectin-coated culture dishes) and Acute and Chronic cells remains to be established. In human aortic endothelial cells (HAECs), we ROS production (ROS-sensitive probe CM-H2DCFDA). EPCs cells from low- evaluated the effects of glucose and fatty acids on endothelial dysfunction HDL T2DM had mean 26% lower viability as compared to high-HDL (100±30 by measuring transcription of endothelial nitric oxide synthase and adhesion vs. 74±29; p=0.0001). 4-hr H2O2 exposure impaired cell viability to a similar molecules, adhesion of THP-1 monocytes, and cellular ATP and ADP levels. extent in both groups (100±27 vs. 69±25 in high-HDL, p<0.0001; 100±24 vs. Autophagic fl ux was assessed based upon expression levels of p62 and 81±19 in low-HDL, p=0.007). EPC senescence was comparable in the two LC3-II, as well as lysosomal enzyme activity. Compared to HAECs in non- groups (100±28 vs. 98±22; p=0.774) and was not signifi cantly modifi ed by stressed conditions, HAECs exposed to elevated concentrations of glucose H2O2. There was no difference in the migration capacity (100±38 vs. 110±35; and palmitate had a smaller ATP/ADP ratio and showed increased adhesion p=0.442), while cell adhesion was 25% impaired in low- compared to high- of monocytes. Under these hyperglycemic and hyperlipidemic conditions, HDL (100±23 vs. 75±15; p=0.0001). Finally, ROS production was slightly (12%) autophagic fl ux was also decreased. Activation of AMPK attenuated higher in low- (n=27) than in high-HDL (n=18, +12±29; p=0.426). These fi ndings glucose- and palmitate-induced changes in these parameters, and chemical persist after adjustment for gender, age, BMI, HbA1c. In T2DM, HDL-C is a induction of autophagic fl ux diminished glucose- and palmitate-induced determinant of circulating EPCs function contributing to CV protection. changes in ATP/ADP and monocyte adhesion. Up-regulation of autophagic fl ux may be one way in which AMPK can ameliorate glucose- and palmitate- 515-P induced dysfunction in HAECs. Rho-Kinase Inhibition Attenuates Endothelial Infl ammation by Mod- Supported by: NIH/NHLBI (HL068758) ulating ER Stress Signaling DAIJI KAWANAMI, KEIICHIRO MATOBA, RINA OKADA, MASAMI TSUKAMOTO, 513-P JUN KINOSHITA, SHO ISHIZAWA, YASUSHI KANAZAWA, TAMOTSU YOKOTA, Increased Oxidative Injury Associated With the Haptoglobin 2-2 KAZUNORI UTSUNOMIYA, Tokyo, Japan Genotype in DM may be due to an Impaired Ability to Block Heme The process of atherosclerosis is affected by interactions among Transfer from the Haptoglobin 2-2-Hemoglobin Complex numerous biological pathways. Accumulating evidence shows that NINA S. LEVY, ANDREW P. LEVY, Haifa, Israel endoplasmic reticulum (ER) stress plays a crucial role in the development Introduction: Haptoglobin (Hp) 2-2 genotype is associated with increased of atherosclerosis. Rho-kinase is an effector of small GTP-binding risk of CVD and ESRD in individuals with diabetes mellitus (DM). Hp protein protein Rho and has been implicated as an atherogenic factor. Previous binds extracorpuscular hemoglobin (Hb) and thereby prevent the release of study demonstrated that fasudil, a specifi c Rho-kinase inhibitor, exerts heme from Hb. Heme which is released from Hb can readily intercalate into cardioprotective effect by downregulating ER stress signaling in ischemia cell membranes or lipoproteins and promote oxidation. reperfusion heart failure model. However, the molecular link between Hypothesis. We hypothesized that the Hp 2-2 protein is ineffi cient in ER stress and Rho-kinase in endothelial cells has not been elucidated. In blocking the release of heme from Hb. We further hypothesized that glycation this study, we investigated the mechanisms by which fasudil regulates of Hb may affect this transfer. We therefore investigated the possibility endothelial infl ammation during ER stress. Tunicamycin, an established that glycated Hb undergoes increased heme transfer, which would further ER stressor, induced VCAM-1 mRNA expression in endothelial cells. increase oxidative injury in Hp 2-2 DM. Intriguingly, fasudil inhibited VCAM-1 indcution. From a mechanistic stand Methods. We developed a novel quantitative assay to measure the point, fasudil inhibited tunicamycin-mediated inductions of activating transfer of heme between Hb molecules by preparing 55Fe-Hb from mouse transcription factor (ATF)4 and subsequent C/EBP homologus protein erythroleukemia cells incubated in the presence of 55Fe-transferrin and (CHOP), that are important factors in endothelial infl ammation. Furthermore, DMSO. Equimolar (6.25 uM) amounts of purifi ed met 55Fe-Hb and cold, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is biotinylated Hb were incubated overnight at 37oC after which the biotinylated crucial for atherogenic response by ER stress. These fi ndings indicate that Hb was recovered with streptavidin-coated magnetic beads. Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and Results. Hemopexin blocked heme transfer by 50%. Hp 1-1 protein was p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil as effi cient as hemopexin in blocking heme transfer and was fi vefold more would be an important therapeutic approach against atherosclerosis under effective than the Hp 2-2 protein in blocking heme transfer (p<0.05). 55Fe- conditions with ER stress including diabetes. Hb isolated from cells grown in the presence of high glucose resulted in an approximately three fold greater heme transfer compared to 55Fe-Hb isolated from cells cultured under normal glucose. Similarly, cold, biotinylated Hb COMPLICATIONS—NEPHROPATHY—BASIC AND glycosylated in vitro resulted in a two to three fold increase in heme transfer EXPERIMENTAL SCIENCE compared to unglycosylated biotinylated Hb. Conclusions. The Hp 2-2 protein is less effi cient at blocking heme transfer from Hb compared to Hp 1-1. Furthermore, the increase in heme transfer Guided Audio Tour: Mechanisms Revealed by Experimental Models of when Hb is glycosylated may provide a mechanistic explanation for the Diabetic Nephropathy (Posters: 516-P to 523-P), see page 17. increase in CVD seen in Hp 2-2 DM. Supported by: NIH (RO1DK085226-03 to A.P.L.) & 516-P Metabonomics Revealed Xanthine Oxidase-Induced Oxidative 514-P Stress was Involved in the Pathogenesis of Diabetic Nephropathy Infl uence of HDL Cholesterol Levels on Endothelial Progenitor Cells JINGPING LIU, YOUNAN CHEN, SIRONG HE, CHENGSHI WANG, YAN REN, HA- Function in Patients With Type 2 Diabetes Mellitus OMING TIAN, JIE ZHANG, YANRONG LU, JINGQIU CHENG, Chengdu, China DANIELA LUCCHESI, SIMONA GEORGIANA POPA, VERONICA SANCHO BORNEZ, To investigate the metabolic changes in DN, we applied NMR-based LAURA PUCCI, ELEONORA RUSSO, MONIA GAROFOLO, ROSSELLA RUSSO, AN- metabonomics to examine serum, urine and renal extract from control and GELA DARDANO, ROBERTO MICCOLI, GIUSEPPE PENNO, STEFANO DEL PRATO, STZ-induced DN rats. The signifi cantly changed metabolites between control Pisa, Italy, Craiova, Romania and DN group were identifi ed by multivariate statistical methods. Real-time HDL are inversely associated with CHD. Additional atheroprotective PCR, ELISA and immunohistochemical staining was performed to observe actions have been claimed on top of reverse cholesterol transport. Subjects the renal pathophysiologic changes. Compared with control, DN rats showed with high-HDL show increased endothelial progenitor cells (EPCs) colony increased VLDL/LDL, 3-hydroxybutyrate, lactate, N-acetylglycoproteins, forming units and HDL-C is considered to affect EPCs number and function. unsaturated fatty acids, uric acid and allantoin, as well as decreased HDL, We characterized in vitro function of circulating EPCs of T2DM with low- alanine, arginine, glutamate, succinate and citrate. The elevations of uric (34.7±7.9 mg/dl; N=57, M/F 70/30%; age 61.8±7.8; BMI 30.6±4.7; HbA1c acid and allantoin indicated increased generation of ROS through enhanced

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A130 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE xanthine oxidase (XO) pathway in the kidney of DN rats. The mRNA and extraordinary (~24%) GBM thickness decrease/protection seen in OVEJtmt protein level of XO was signifi cantly increased in the kidney of DN rats. The (compared to OVE) mice and the complete obliteration of diabetes-induced levels of 8-OHdG and 8-iso-Prostaglandin F2α were signifi cantly elevated GBM thickening was not predicted. Equally impressive, however, was the in the urine and kidney of DN rats. The mRNA levels of CD14, IL-8, NF-kB, similar CBM thickness pattern observed in myocardium of OVEJtmt mice. ICAM1 and TGF-β1 were signifi cantly increased in the kidney of DN rats. This included signifi cantly increased CBM thickening (10.5%) in OVE mice These observations indicated that XO-induced oxidative stress can further (compared to FVB controls) and the elimination of CBM thickening in OVEJtmt active infl ammation and fi brosis pathways, and together accelerated the mice. Remarkably, both GBM and myocardial CBM thicknesses in severely development and progression of DN. This metabonomic study provides diabetic OVEJtmt animals were reduced/protected to levels well below FVB POSTERS insight to the discovery of potential biomarkers, and understanding the controls (11.4% and 12.9%, respectively). To our knowledge, this is the fi rst Complications molecular mechanisms in pathogenesis of DN. experimental design to consistently eliminate capillary BM thickening in Acute and Chronic severely diabetic animals. Supported by: Lions Clubs of North Dakota

& 518-P Involvement of CX3CL1-CX3CR1 System in the Development of Dia- betic Nephropathy in Streptozotocin-Treated Mice MACHI FURUTA, YUUKI SHIMIZU, AKIKO YAMANA, MINORU UEYAMA, SHUHEI MORITA, ASAKO DOI, YUKO ISHIDA, MIZUHO NOSAKA, TOSHIKAZU KONDO, TOKIO SANKE, Wakayama, Japan We explored the pathophysiological roles of CX3CL1-CX3CR1 system in the development of streptozotocin (STZ)-induced diabetic nephropathy. In C57BL/6 (WT) mice, the intrarenal gene expression of CX3CL1 and its specifi c receptor, CX3CR1 was apparently enhanced at 3 and 6 months after STZ challenge, and CXCR1 was mainly expressed intrarenal macrophages, thus indicating that CX3CL1-CX3CR1 axis might be involved in STZ-induced diabetic nephropathy. When mice lacking CX3CR1 (KO) were treated with STZ (KODM), blood glucose levels were increased to a similar extent, compared with STZ treated WT mice (WDM). Moreover, there was no signifi cant difference in systolic blood pressure between WDM and KODM mice. Although urine albumin excretion, a hallmark of diabetic nephropathy, was elevated in both WDM and KODM mice at 3 and 6 months after STZ challenge, it was signifi cantly higher in WDM mice than in KODM ones. Histopathologically, the PAS-positive area in the glomeruli and interstitial fi brotic changes were less evident in KODM mice than in WDM ones at 6 months after STZ administration. Actually, the gene expression of type IV collagen was signifi cantly attenuated in KODM mice. Additionally, macrophage accumulation in the glomeruli was attenuated in KODM mice, compared with WDM mice. These observations indicated that the absence of CX3CR1 could alleviate STZ-induced diabetic nephropathy, with a concomitant of attenuated macrophage recruitment. Moreover, intrarenal gene expression of TGF-β, ICAM-1, VCAM-1 and iNOS was less enhanced in KODM mice, compared with compared with WDM mice. Collectively, CX3CL1- CX3CR1 system plays critical roles in STZ-induced diabetic nephropathy.

& 519-P Increased Expression of Acetyl-CoA Carboxylase β Is Involved in Podocyte Injury in Diabetic Nephropathy YUKI TANAKA, SHINJI KUME, SHIRO MAEDA, HISAZUMI ARAKI, KEIJI ISSHIKI, Supported by: NSFC (30930088), (30872382), (3120075) SHIN-ICHI ARAKI, ITSUKI OSHIMA, DAISUKE KOYA, MASAKAZU HANEDA, AT- SUNORI KASHIWAGI, TAKASHI UZU, HIROSHI MAEGAWA, Otsu, Japan, Yoko- hama, Japan, Osaka, Japan, Kahoku, Japan, Asahikawa, Japan Acetyl-CoA carboxylase (ACC) β is an enzyme that catalyzes the & 517-P carboxylation of acetyl-CoA to malonyl-CoA, leading to initiation of fatty Targeted Metallothionein Overexpression in Endothelial Cells Elim- acid synthesis and inhibition of β-oxidation in mitochondria. We previously inates Glomerular and Myocardial Capillary Basement Membrane reported that a single nucleotide polymorphism (SNP), rs2268338, within Thickening in Transgenic Diabetic Mice the gene encoding ACCβ was associated with susceptibility to diabetic EDWARD CARLSON, DONNA LATURNUS, KELLEN ALBRECHT, WESTON BOWK- nephropathy in Japanese patients with type 2 diabetes. Although ER, KIM YOUNG, JENNIFER CHHOUN, Grand Forks, ND subsequent functional analyses suggested that increased expression Most capillary basement membranes (CBMs) are thickened in diabetes of ACCβ in the kidney contributed to susceptibility to the disease, its and though the involved molecular mechanisms are elusive, hyperglycemia- pathological signifi cance has not been fully elucidated yet. To know the driven formation of free radicals and their documented mitigation by role of ACCβ in the pathogenesis of diabetic nephropathy, we examined overexpression of antioxidants (e.g. metallothionein, MT), strongly suggests the effect of ACCβ overexpression on podocyte injury using podocyte- that oxidative stress may play a role in this process. We recently reported specifi c ACCβ transgenic (TG) mice and cultured murine podocytes. TG mice that crossing severely diabetic transgenic (OVE) mice (blood sugars >600mg/ showed normal renal manifestation under non-diabetic condition. However, dl and HbA1c ~9.5%) with transgenics (Mt, Nmt,) that overexpress MT under diabetic condition induced by streptozotocin (STZ) injection, TG mice specifi cally in cardiac myocytes and glomerular podocytes, completely developed higher level of urinary albumin excretion along with signifi cant protects their bi-transgenic progeny (OVEMt, OVENmt) from diabetes- reduction in synaptopodin expression in podocytes compared to wild- induced capillary BM thickening. In the current study, our recently developed type mice. In cultured murine podocytes infected with adenovirus vectors transgenic (Jtmt) mice that overexpress MT specifi cally in endothelial encoding ACCβ, the expression of synaptopodin and podocin were decreased cells, were bred to OVE mice, and at 150 days, the OVEJtmt bi-transgenic under high glucose condition, but not under normal glucose condition. progeny and age-matched controls were prepared for TEM analysis of CBM Furthermore, overexpression of ACCβ under high glucose condition resulted thickness by the orthogonal intercept technique. As expected, glomerular in reorganization of stress fi bers, increased production of cytokines such BMs (GBMs) and myocardial CBMs were signifi cantly wider (10.5%, 11.6%) as MCP-1, IL-6, TNF-α and VEGF, and induction of apoptosis in the murine in OVE diabetics than in non-diabetic, Jtmt, or FVB controls. However, the podocytes. From these observations, it is suggested that excess of ACCβ

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A131 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE

contributes to exacerbation of podocyte injury in diabetic nephropathy, and We also performed studies in cultured human podocytes to determine the regulation of ACCβ function may be a new therapeutic option to prevent direct renal specifi c effects of TGR5 activation. Treatment with INT-777 podocyte injury in patients with diabetic nephropathy, although further prevented glucose induced podocyte apoptosis and increased podocyte studies are required to prove the precise molecular mechanisms for ACCβ- SIRT1, p-AMPK, and PGC-1α protein abundance. These effects were induced podocyte injury. associated with increased mitochondrial number, decreased mitochondrial ROS production, and increased SOD2 gene expression. In addition, INT- & 520-P 777 decreased SREBP-1 protein and increased PPARα, LCAD, and CPT-1β expression, resulting in decreased lipid accumulation in podocytes. These POSTERS Blockade of CCL2/CCR2 Signalling Ameliorates Diabetic Nephropa- Complications thyin db/db Mice studies indicate a novel role for TGR5 in inducing energy metabolism, Acute and Chronic EUN YOUNG LEE, SU JIN SEOK, EUN SOO LEE, JI-HYE LEE, HONG MIN KIM, YEO mitochondrial biogenesis, and fatty acid oxidation by activating AMPK, JOO KIM, SHELDON CHEN, CHOON HEE CHUNG, Cheonan, Republic of Korea, SIRT1, PGC-1α, and SIRT3, which lead to prevention of oxidative stress Wonju, Republic of Korea, Chicago, IL and lipid accumulation; fi rmly establishing an important role for TGR5 in CCL2/C-C chemokine receptor 2 (CCR2) signalling is suggested to play a preventing diabetic kidney disease. signifi cant role in various kidney diseases including diabetic nephropathy. We investigated the renoprotective effect of a CCR2 antagonist, RS102895, & 522-P on the development of diabetic nephropathy in a type 2 diabetic mouse SGLT2 Inhibition Prevents the Development of Nephropathy in Dia- model. Six-week-old diabetic db/db and non-diabetic db/m mice were fed betic Mice either normal chow or chow mixed with 2 mg/kg/day of RS102895 for 9 LIRU QIU, XIAOXIN WANG, VERONICA HOGG-CORNEJO, CHERELLE PARKER, weeks. We investigated the effects of CCR2 antagonism on blood glucose, GERALD SHIOSHITA, JIMMY REN, YIN LIANG, MOSHE LEVI, Aurora, CO, Raritan, blood pressure, albuminuria and the structure and ultrastructure of the NJ kidney. Diabetes-induced albuminuria was signifi cantly improved after Inhibition of the renal sodium glucose co-transporter, SGLT2, lowers blood CCR2 antagonist treatment, and glucose intolerance was improved in the glucose in patients with diabetes and in rodent diabetes models. The effects RS102895-treated diabetic mice. RS102895 did not affect blood pressure, of SGLT2 inhibition on the progression of diabetic renal disease have not been body weight or kidney weight. Mesangial expansion, glomerular basement fully established. We determined the effects of a selective SGLT2 inhibitor membrane thickening and increased desmin staining in the diabetic kidney (JNJ-39933673) on diabetic nephropathy in db/db mice for 12 weeks. JNJ- were signifi cantly improved after RS102895 treatment. The up-regulation 39933673 is a potent SGLT2 inhibitor with IC50 values of 230 and 1.4 nM, of vascular endothelial growth factor mRNA expression and the down- respectively, against human SGLT1 and SGLT2 expressed in CHOK1 cells. It regulation of nephrin mRNA expression were markedly improved in the increases urinary glucose excretion and decreases blood glucose levels in kidneys of RS102895-treated diabetic mice. Increased renal CD68 and diabetic rodent models (doses of 0.1-10 mg/kg). In the present study, db/db arginase II and urinary malondialdehyde in diabetes were effectively mice were fed with JNJ-39933673 mixed chow (0.07 g/kg) or regular chow. attenuated by RS102895 treatment. Blockade of CCL2/CCR2 signalling by The body weight, food intake, and blood glucose levels were monitored RS102895 ameliorates diabetic nephropathy not only by improving blood weekly. After 12 weeks of treatment, we found that SGLT2 inhibition glucose levels but also by preventing CCL2/CCR2 signalling from altering caused a signifi cant reduction in A1c in treated db/db mice compared with renal nephrin and VEGF expressions through blocking macrophage infi ltration, control group, and marked decreases in urinary albumin (745±36 mg/g in infl ammation and oxidative stress in type 2 diabetic mice. db/db vs. 207±5 mg/g in treated db/db, p<0.001) and urinary thiobarbituric Supported by: National Research Foundation of Korea (2010-0005071), acid-reacting substances (TBARS) (1.09±0.13 mmol/g in db/db vs. 0.48±0.10 (2012R1A1A2044121) mmol/g in treated db/db, p<0.01), an indicator of oxidative stress. JNJ- & 521-P 39933673 treatment also prevented mesangial expansion, accumulation of extracellular matrix proteins as determined by fi bronectin and type IV G Protein Coupled Receptor TGR5 Activation Prevents Diabetic Kid- collagen quantitative immunofl uorescence microscopy, and podocyte loss ney Disease in db/db Mice and in Human Podocyte Cells as determined by WT1 and synaptopodin quantitative immunofl uorescence MOSHE LEVI, XIAOXIN WANG, LIRU QIU, VERONICA HOGG-CORNEJO, CHERELLE microscopy (all p<0.05). In addition, in JNJ-39933673-treated mice, SGLT2 PARKER, LUCIANO ADORINI, MARK PRUZANSKI, MOIN SALEEM, JEFFREY KOPP, inhibition prevented renal accumulation of macrophages as determined by RADU MOLDOVAN, Aurora, CO, New York, NY, Bristol, United Kingdom, Bethesda, CD68 immunofl uorescence microscopy, and renal neutral lipid accumulation MD as determined by Oil Red O staining. In summary, our study results showed The purpose of the present study was to determine the role of the G that SGLT2 inhibitor treatment prevents the development of nephropathy in Protein Coupled Receptor TGR5 in modulation of diabetic nephropathy. db/db mice. Treatment of db/db mice with the selective TGR5 agonist INT-777 decreased proteinuria, podocyte injury, mesangial expansion, tubulointerstitial fi brosis, and CD68 macrophages in the kidney. & 523-P In line with these results, it increased renal protein levels of p-AMPK, B7.1 on Podocytes as a Novel Therapeutic Target for Diabetic Neph- SIRT1 mRNA, PGC-1α and ERRα, and SIRT3 and Nrf-1 mRNA, which are ropathy master regulators of mitochondrial biogenesis, inhibitors of oxidative ANDREA VERGANI, ROBERTO BASSI, MONIKA NEWCZAS, MARCUS PEZZOLESI, stress, and inducers of fatty acid β-oxidation. It also increased Nrf-2 mRNA, MELISSA CHIN, MARIA PIA RASTALDI, ANNA SOLINI, JOCHEN REISER, JORDAN KREIDBERG, ANDRZEJ S. KROLEWSKI, PETER MUNDEL, MOHAMED SAYEGH, decreased mitochondrial H2O2 generation, increased superoxide dismutase 2 (SOD2) activities leading to decreased protein carbonylation and decreased PAOLO FIORINA, Boston, MA, Milan, Italy, Pisa, Italy, Chicago, IL Glomerular podocytes, damaged during the progression of diabetic urinary H2O2 and TBARS levels, and increased PPARα, UCP2, and CPT-1β, which mediate fatty acid β-oxidation resulting in decreased renal lipid nephropathy, express under certain stress conditions the immune-related accumulation. In contrast, conditional deletion of TGR5 in podocytes led to a molecule B7.1 (or CD80). We thus explored if podocytes up-regulate under marked and signifi cant increase in urinary albumin and nephrin excretion. hyperglycemia B7.1 and if its targeting with CTLA4-Ig, a clinically available drug, is benefi cial to protect podocytes from hyperglycemia-induced damage and apoptosis. Our data show that B7.1 is upregulated in vitro in podocytes cell-line cultured in high-glucose (30mM) and in vivo in glomerular podocytes during the development of diabetic nephropathy in three different murine models: db-db, streptozotocined C57BL/6 and OVE26 mice. The treatment with CTLA4-Ig reduces in vitro high glucose-mediated podocyte abnormalities (loss of synaptopodin and phalloidin-paxillin structures and reduction of the activated/total β1-integrin ratio); in vivo CTLA4-Ig (500 µg of antibody at day 0, and then 250 µg at day 2, 4, 6, 8, and 10 and thereafter twice a week) prevents urinary albumin excretion (UAE) increase (db-db mice:

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A132 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE

UAE: untreated 7wks=59.7±8.5, 25wks=110.1±13.4 µg/24h; n=15, p=0.003; These monkeys also presented with increased mesangial matrix expansion, CTLA4-Ig-treated 7wks=58.6±8.8, 25wks=49.4±7.1 µg/24h: n=15, p=ns) and glomerular basement membrane (GBM) thickening and glomerulosclerosis. glomerular morphological abnormalities (mesangial expansion, deposition of Global messenger RNA from kidneys from 6 diabetic NHPs with nephropathy collagenen IV, and loss of nephrin and ZO-1). (T2DN), 3 diabetic NHPs without nephropathy (T2D) and 3 non-diabetic Similarly, in kidney biopsies obtained from patients with type 2 diabetes, NHPs (C) were hybridized on Affymetrix GeneChip® Rhesus Macaque B7.1 is upregulated in podocytes compared to their not-diabetic counterparts. Genome Array. Comparison between T2DN and T2D and T2DN and C At GWAS (Genome-Wide-Association-Study), one SNP (single-nucleotide respectively revealed signifi cant differential expression of 28 and 829 polymorphism) mapping in the B7.1 gene is associated with diabetic genes. The regulated genes in T2DN include CCL2, ACE2, HAVCR1 and SPP1. POSTERS nephropathy development [progression to end renal stage disease (ESRD): DAVID (Database for Annotation, Visualization and integrated Discovery) Complications

SNP rs2629396 OR=1.56, p=0.008], and the soluble CD28 (B7.1 ligand) levels analysis also showed that the most common type of enriched pathway Acute and Chronic in the serum of type 2 diabetic patients predict the progression to ESRD. was the ‘complement and coagulation cascade’. Other pathways included These data and the presence of a clinically available compound candidate ‘extracellular matrix (ECM)-receptor interaction’ and ‘renin-angiotensin B7.1 as a new therapeutic target for diabetic nephropathy. system’. Our DN model mimics results found in human at the physiological and molecular level, further demonstrating the value of naturally occurring 524-P diabetic cynomolgus model. The identifi cation of the unique gene signature Senescence Marker Protein-30 Defi ciency Accelerates Diabetic of monkeys naturally developping DN may also facilitate the development of Renal Injury Through Tubulointerstitial Fibrosis in a Mouse Model new mechanism-based diagnostics and therapies. of Type 1 Diabetes HIROSHI OKADA, TAKAFUMI SENMARU, GOJI HASEGAWA, Kyoto, Japan 526-P Aging is a risk factor for the progression of diabetic nephropathy leading Oleanolic Acid and N-Acetylcysteine Ameliorates the Progression to end-stage renal failure. of Diabetic Nephropathy in Type 2 Diabetic Rat Model However, its mechanism has not been elucidated. Senescence marker EUN SOO LEE, HONG MIN KIM, MI RI HYUN, EUN YOUNG LEE, MI YOUNG LEE, protein-30 (SMP30) is abundantly expressed in renal tubular cells and CHOON HEE CHUNG, Cheonan, Republic of Korea, Wonju, Republic of Korea decreases with aging. Our studies using SMP30 knockout mice have revealed Diabetic nephropathy is a leading cause of end-stage kidney failure and that a reduction in SMP30 expression may contribute to age-associated increasing morbidity and mortality in diabetic patients. Renal fi brosis is a hall deterioration of cellular function and the enhanced susceptibility to harmful mark of renal disease and characterized by tubular atrophy, accumulation stimuli in aged tissue. In this study, we investigated the effects of SMP30 of fi brosis, and increased interstitial matrix deposition. Oleanolic acid (OA) defi ciency on the pathogenesis of diabetic nephropathy. Diabetes (DM) was is a pentacyclic triterpene which is present in fruits and vegetables. It is induced using streptozocin in male SMP30 Y/- mice (KO) and wild-type, known as anti-infl ammatory, anti- hyperlipidemic, and anti-oxidant effects. SMP30 Y/+, mice (WT) at 7 weeks of age. Twelve weeks after the induction N-acetylcysteine (NAC) is a pharmaceutical drug and nutritional supplement of diabetes, the mice were sacrifi ced for the study. Urinary albumin excretion used primarily as a mucolytic agent and it has been shown to exert a was increased in KO compared to WT both in DM and non-DM condition protective effect in the β-cell of diabetic mouse. In this study, we categorized (P<0.05). The proportions of cortical tubulointerstitial fi brosis area (Sirius LETO as a normal group 1, OLETF as a diabetic group 2, diabetic treated with red stain) were increased in KO compared to WT (1.02±0.20 vs 3.00±1.38%, OA (100 mg/kg/day) group 3 and diabetic treated with NAC (300 mg/kg/day) P<0.05). In DM, this increase was markedly enhanced in KO (2.02±0.34 vs group 4. We treated them from the age of 25 to 45 weeks. After 20weeks of 19.34±6.10%, P<0.05). On the other hand, SMP30 deletion did not affect treatment, body weight was not difference between diabetic and diabetic mesangial expansion (PAS stain). The changes in tubulointerstitial fi brosis treated group. 24-hour urinary albumin and albumin creatinine ratio (ACR) were well associated with the expression of HIF-1α, not 4HNE, as assessed were signifi cantly reduced in OA-treated group and NAC-treated group when by immunohistchemistry. However, increase of HIF-1α mRNA was observed compared to OLETF group. Also, HOMA-IR was decreased in the diabetic only in KO of non-DM condition (P<0.05). treated group but HOMA-β shown elevated in the same group respectively. Similar to the HIF-1α protein expression, the mRNA expression of CTGF Urinary SOD was increased in OA and NAC treated group as compared with and MCP-1 were increased in KO both in non-DM and DM condition. These diabetic group. The GBM thickness and the glomerular volume were found results indicate that SMP30 defi ciency deteriorates diabetic nephropathy to be decreased in OA and NAC treated group when compared with diabetic through tubular injury. Stabilization of HIF-1α and following signaling may be group. Moreover, we seen decreased VEGF and α-SMA expression in OA and mainly involved in these changes. The decrease of SMP30 could be a factor NAC treated group when compared with diabetic group. Also, Decreased which explains the cross-talk between pathogenesis of various chronic nephrin expression increased in OA, NAC treated group whencompared kidney diseases, including diabetic nephropathy, and aging. with diabetic group. In conclusion, the above fi nding suggested that OA and NAC have protective and therapeutic effects on diabetic nephropathy by 525-P decreasing the expression of VEGF, α-SMA along with increase in nephrin Gene Expression Profi ling in the Kidneys from Non-Human Primates expression and plasma SOD activity. With Diabetic Nephropathy MICHAEL BENZINOU, FENGLAI DU, BINGDI WANG, XIAOLI WANG, YUPENG FANG, FRANCINE M. GREGOIRE, YI-XIN (JIM) WANG, Santa Clara, CA, Taicang, China, Kannapolis, NC Diabetic nephropathy (DN) complication in patients with type 2 diabetes is the most common cause of chronic kidney disease and end-stage renal disease. Hyperglycemia is the driving force of DN. To understand how to prevent DN progression and reverse its pathophysiology, it is necessary to explore the molecular mechanisms that cause the disease in a model exposed to persistent hyperglycemia. Unlike conventional experimental rodent models, which relevance to human in vivo physiologic and metabolic kinetics remains unclear, diabetes and its associated complications naturally develop in non-human primates (NHP). Among 68 monkeys with long history of hyperglycemia, 8 presented with glycosuria, microalbuminuria, elevated urinary cystatin C-to-creatinine ratio and N-acetyl-beta-D-glucosaminidase.

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527-P 529-P High Glucose Induces Sumoylation of Smad4 via SUMO2/3 in Glom- Nuclear Hormone Receptor Expression in Mouse Kidney and Renal erular Mesangial Cells Cell Lines YONG XU, XUEQIN ZHOU, WEI HUANG, MAOJUN YANG, Luzhou, Sichuan, China NAOTO TERAMI, DAISUKE OGAWA, JUN EGUCHI, HIROMI TACHIBANA, CHIK- Transforming growth factor-β(TGF-β)signaling pathway is an important AGE SATO-HORIGUCHI, TAKASHI HATANAKA, ATSUKO NAKATSUKA, JUN pathway which mediated renal fi brosis in diabetic nephropathy(DN) . Recent WADA, HIROFUMI MAKINO, Okayama, Japan researches showed that SUMOylation(Small ubiquitin-related modifi er, Although several nuclear hormone receptors (NHRs) have shown a

POSTERS SUMO) is a post-translational modifi cation involved in the regulation of renoprotecitve effect in the context of diabetic nephropathy, the expression Complications TGF-β signaling pathway through smad4, the common Smad mediator of and role of NHR in kidney are still unclear. In this study, we have analyzed the Acute and Chronic TGF-βsignaling. SUMO family consists of SUMO1,SUMO2/3 and SUMO4 expression of all 49 of the NHR superfamily in mouse kidney and cell lines in and studies demonstrated that sumoylation of smad4 can enhance TGF-β both normal and high glucose condition. We used normal and STZ-induced signaling. However, the role of sumoylation of signaling molecules on the diabetic C57BL/6 mice, db/m (control) and db/db (diabetic) mice, and renal cell diabetic nephropathy is unclear. To explore sumoylation of smad4 in Rat lines including mesangial cells, podocytes, proximal tubular epithelial cells, Glomerular Mesangial Cells (GMCs) induced by high glucose, we divided and collecting duct cells. RNA was isolated from tissue samples or cultured cultured rat GMCs into 5 groups: normal glucose group(5.6 mmol/L); cells. To evaluate the mRNA expressions of NHR in the kidney and cultured high glucose groups(10, 20 and 30mmol/L); mannitol group was used as cells, quantitative RT-PCR was performed using TaqMan® Array Fast Plates. osmotic control. The expression of SUMO1 and SUMO2/3 was measured 25 NHR members were expressed in the kidney of normal C57BL/6 and db/m by Western Blot and RT-PCR, the sumoylation between SUMO and smad4 mice (Fig. 1), and 6 NHRs were not expressed in mouse kidney. In addition, was detected by co-immunoprecipitation and immunofl uorescence confocal 5 NHRs were upregulated in diabetic C57BL/6 and db/db mice. In cell lines, laser microscopy. Our results showed that, compared with normal group, the PPARδ was highly expressed in mesangial and proximal tubular epithelial expression of SUMO1 and SUMO2/3 was increased in high glucose groups cells, and COUP-TF II/III was expressed in podocytes and collecting duct in a dose-dependent manner(p<0.05). There was no expression of SUMO4 cells. The expression profi le of NHR was different between normal and high in both normal and high glucose group. Analyses of co-immunoprecipitation glucose conditions. NGFI-B was upregulated in mesangial and collecting and confocal laser scanning showed that smad4 interacted with SUMO2/3 duct cells by high glucose stimulation. These fi ndings identify NHR present but not SUMO1 in every group and the sumolyation (SUMO2/3) of smad4 in in mouse kidney and cell lines and suggest potential therapeutic targets in high glucose were strongly enhanced compared with normal group(p<0.05). kidney for the treatment of diabetic nephropathy. Our data indicate that sumolyation of samd4 by SUMO2/3 is involved in the regulation of TGF-β signaling in diabetic nephropathy. Supported by: Affi lated Hospital of Luzhou Medical College

528-P Study of Early Renal Damage in Diabetic Rhesus Monkeys DAN WANG, JINGPING LIU, SIRONG HE, CHENGSHI WANG, JIUMING ZHAO, YOUNAN CHEN, LICHAUN YANG, YAN REN, HAOMING TIAN, GUANGNENG LIAO, LAN LI, FANG LIU, YUJIA YUAN, GUANG YANG, JINGQIU CHENG, YAN- RONG LU, Chengdu, China To identify diagnostic or predictive biomarkers of diabetic kidney damage and investigate the pathophysiology of diabetic nephropathy. Diabetic monkeys were induced by STZ, and regulated blood glucose (BG) by administration of exogenous insulin. Monkeys were divided into four groups, including the normal group (n=3), group A ( BG < 10 mmol/L, n=3), group B (BG 15-20 mmol/L, n=5), and group C (BG 15-20 mmol/L and intake salt and peanuts, n=3) (Fig A, B, C). The following parameters were evaluated in the different course of disease: 1) Blood biochemistry and urine routine; 2) HbAlc; 3) Color Doppler ultrasound; 4)Angiography; 5) Renal biopsy; 6) Renal fi brosis-related gene expression levels. The expression of Smad2/3 530-P in group B and C became high at 24 months (M) after STZ injection(Fig H, Dual Activation of FXR and TGR5 Protects from Diabetic Nephropa- I, J, K), while only few monkeys from group C displayed mild pathological thy and Retinopathy in Mouse Model of Type 1 Diabetes changes (Fig O). Group C manifested a certain extent pathological changes XIAOXIN WANG, LIRU QIU, SUGATA HAZRA, QIUHONG LI, LUCIANO ADORINI, and abnormity at 36 M (Fig L, M, N), meanwhile, group B and C began to MARK PRUZANSKI, MARIA B. GRANT, MOSHE LEVI, Aurora, CO, Gainesville, FL, appear microalbuminuria. Then group B and C showed kidney dysfunction New York, NY (Fig D, E, F, G) and obvious pathological changes at 42 M. The results of Bile acids are agonists for the nuclear hormone receptor, farnesoid X fundus examination were normal. Poor glycemic control, sodium and fat receptor (FXR), and the G protein-coupled receptor TGR5. We examined the intake aggravated kidney injury. Increasing renal fi brosis-related gene effect of their combined activation on diabetic nephropathy and retinopathy expression and pathological changes appeared before microalbuminuria and in a model of type 1 diabetes mellitus (T1D). Using the DBA/2J streptozotocin renal function changes. (STZ)-treated mouse as a model of T1D, we examined the effect of a novel FXR/TGR5 dual agonist, INT-767, on kidney and retinal pathology and its impact on endothelial progenitor cell (EPC) function, known to promote vascular repair. Treatment of T1D mice with the dual FXR/TGR5 agonist improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fi brosis. INT-767 reduced the number of acellular capillaries and reduced infl ammatory infi ltration in the retina and corrected diabetes-associated EPC dysfunction. These results indicate novel signaling pathways for the bile acid receptors FXR and TGR5 in the control of diabetic microvascular complications, with benefi cial effects via a number of mechanisms, including activation of the protective arm of the renin- angiotensin system, anti-infl ammatory effects and enhanced endothelial progenitor function promoting vascular repair.

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A134 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE

531-P 533-P TGF-Beta1 Inhibits Sodium Glucose Cotransporter 2 (SGLT2) Ex- Aldose Reductase and RAGE: Key Mechanisms in the Pathogenesis pression in Human Renal Proximal Cells of Diabetic Nephropathy in Murine Models KUMIKO AGATSUMA, YUMI TAKIYAMA, JUN HONJO, YUKIHIRO FUJITA, MICHAELE B. MANIGRASSO, ANJALI GANDA, ROSA ROSARIO, CARMEN HUR- TSUYOSHI YANAGIMACHI, HIROYA KITSUNAI, HIDEMITSU SAKAGAMI, YUICHI TADO DEL POZO, FEI SONG, RADHA ANANTHAKRISHNAN, VIVETTE D’AGATI, MAKINO, MASAKAZU HANEDA, Asahikawa, Japan RAVICHANDRAN RAMASAMY, ANN MARIE SCHMIDT, New York, NY SGLT2 represents a novel target for normalizing glycemia. SGLT2 is Aldose reductase is the rate-limiting enzyme in the polyol pathway and

responsible for reabsorption of ~90% of fi ltered glucose in the S1 segments mediates generation of advanced glycation endproducts. ARinhibitors POSTERS of proximal tubule. Strangely, SGLT2 inhibitors in clinically development prevent mesangial expansion and glomerular basement thickening (GBM). Complications inhibit only 30-50 % of renal glucose reabsorption. To address the issue of We tested the hypothesis that receptor for advanced glycation end-products Acute and Chronic the effi cacy of the SGLT2 inhibitor, we investigated the regulation of SGLT2 gene deletion in t1DM mice with human levels of AR (TghAR+) protect expression in diabetic milieu. Initially, we examined the gene expression of against DN. SGLT2 in human renal proximal tubular epithelial cells (HRPTEC) by RT-qPCR. Wild-type (WT) and TghAR+ male mice bred into the RAGE(+) or RAGE- HRPTEC constitutively expresses SGLT2 mRNA, not SGLT1. Neither high null (RKO) background were made diabetic and sacrifi ced at 26 wks and glucose (25.5 mM) nor hypoxia (1%) affected the expression of SGLT2 mRNA. mesangial sclerosis, GBM and podocyte effacement were measured. Gene Angiotensin II (10-7 M) signifi cantly increased SGLT2 mRNA (132.3 ±15.7%, markers were measured using renal cortex. p<0.05), and insulin (100 nM) slightly increased SGLT2 mRNA expression by Table 1. TghAR-RKO have a reduced GBM, foot process effacement and ~120% of control. Profi brotic TGF-beta1 markedly decreased SGLT2 mRNA sclerosis. expression (48.3±10.4 %, p<0.01). Although MAPK inhibitors did not restore TGF-beta1-repressed SGLT2 mRNA, SGLT2mRNA expression was inhibited Genotype by SB203580 (p38 MAPK inhibitor). Intriguingly, Hepatocyte Nuclear Factor WT TghAR+ TghAR-RKO (HNF)-1alfa, a modulator of SGLT2 expression, not HNF-1beta, was drastically (n=3) (n=4) (n=2) decreased by TGF-beta1 treatment, thereby providing a mechanism by which GBM (nm) 276 ± 35 271 ± 11 200 ± 9 TGF-beta1 represses proximal tubular cell SGLT2 mRNA expression through Foot Process Effacement (%) 18.3 ± 5.8 25.0 ± 0.0 11.5 ± 5.0 decreased HNF-1alfa. Finally, by immunohistochemistry, remnant cortical proximal tubules showed intense SGLT2 staining in brush border membrane Mesangial Sclerosis 1.7 ± 0.3 2.4 ± 0.3 2.0 ± 0.4 of Zucker diabetic fatty rats (ZDF) at 40 weeks of age, compared to those of Zucker lean rats. However, because tubulointerstitial fi brosis was evident Table 2. Ratio of target genes normalized to b-actin. in ZDF, the extent of positive immunoreactivity for SGLT2 in diabetic kidney Genotype was reduced. WT TghAR+ TghAR-RKO Taken together, SGLT2 expression was subject to a complex regulation (n=3) (n=4) (n=2) by variable pathway. Especially, our data, for the fi rst time, indicated that TGF beta 1.0 0.7 0.3 TGF-beta1 repressed SGLT2 expression in human renal proximal cells, which might limit the effi cacy of the SGLT2 inhibitor. Col IV-1a 1.0 1.1 1.3 MCP-1 1.0 1.6 0.1 532-P TNF alpha 1.0 1.8 0.7 Diabetes-Associated SORCS1 Gene Regulates Water and Electro- lyte Balance This study shows that RAGE deletion in TghAR+ mice results in protection MELKAM KEBEDE, ANGIE OLER, JHARNA SAHA, KATHRYN L. SCHULER, BREN- from DN. Studies are underway to see if the actions of AR are mediated solely DAN J. FLOYD, JADWIGA MARCINKIEWICZ, ANNIK PRAT, NABIL G. SEIDAH, through the RAGE pathway or by RAGE-independent mechanisms. ARJANG DJAMALI, FRANK BROSIUS, ALAN D. ATTIE, Madison, WI, Ann Arbor, Supported by: JDRF (39-2009-645) MI, Montreal, QC, Canada Body water homeostasis is maintained by the prominent vasopressin- 534-P regulated water channel aquaporin-2 (AQP2). Upon vasopressin stimulation, Paricalcitol Prevents the Development of Diabetic Nephropathy by AQP2 translocates from intracellular storage vesicles to the apical membrane Suppressing Infl ammation of principal cells in renal collecting ducts. This translocation renders the JI RYANG KIM, SANG SOO KIM, IN JOO KIM, BO HYUN KIM, YUN KYUNG JEON, apical membrane permeable to water and allows water reabsorption. WON JIN KIM, SANG MI KIM, MIN YOUNG OH, YONG KI KIM, SEONG SU MOON, Impaired sorting and traffi cking of AQP2 causes diabetes insipidus, a Busan, Republic of Korea, Gyeongju, Republic of Korea disease characterized by a massive loss of water through the kidney. SorCS1 Vitamin D might have a potential anti-infl ammatory role in chronic kidney is a member of the vacuolar protein sorting 10 family of receptors and plays a disease. Recent evidence indicates that infl ammation has emerged as being role in protein traffi cking and sorting. Several reports link the Sorcs1 gene to a key pathophysiological mechanism in the development of diabetic kidney diabetes and its complications, however the direct role of Sorcs1 in diabetes disease. We investigated whether synthetic vitamin D analogue paricalcitol susceptibility and complications is unknown. We therefore generated a might have protective role in development of diabetic nephropathy by whole-body Sorcs1 knockout (KO) mouse on a C57BL/6 background. One of inhibiting renal infl ammation in streptozotocin (STZ)-induced diabetic mice. the phenotypes observed in female Sorcs1 KO mice was a reduction in net Two diabetic groups were treated with STZ, one of which was cotreated water retention following an acute water challenge. Under basal conditions, with paricalcitol (0.1 µg/kg, 3 times/week) for 12 weeks. The control group Sorcs1 KO mice have similar urine output as their wild-type (WT) littermates was treated with vehicle only. The administration of paricalcitol effectively despite a tendency for a lower water intake. When challenged with an acute restored the amount of albumiuria induced by diabetes. However, the water load, both the WT and KO mice increased their 24-hr urine output serum calcium level did not signifi cantly differ among 3 groups. Paricalcitol equally. However, the KO mice decreased their water intake by approximately administration effectively restored the increased infi ltration of T cells and 50%. Thus, the net water retention was reduced by approximately 80% in macrophages in the kidney of diabetic model. In addition, paricalcitol also the KO mice. Further urine analysis revealed that urine from the KO animals suppressed the increased RANTES protein expression in renal tubular has elevated osmolarity, [Na+], [K+], and [Cl-] after an acute water load. epithelium of diabetic model. The treatment of paricalcitol effectively Finally, immunofl uorescence labeling of AQP2 revealed a mixture of apical suppressed renal mRNA expression of RANTES and TNF-α, but not MCP- and sub-apical localization in kidneys of WT mice. In contrast, in the KO 1. Finally, paricalcitol restored the suppression of vitamin D receptor (VDR) kidneys, AQP2 was distributed randomly throughout the cell without distinct expression and suppressed the activation of NF-κB in kidney by induced apical localization. Taken together, our data suggest that SorCS1 is essential diabetes. In conclusion, the active vitamin D analogue prevented albumin for proper regulation of water and electrolyte balance and is required for excretion rate in diabetic mice model and has anti-infl ammatory properties, appropriate AQP2 apical localization. Studies are now underway to identify supporting the hypothesis of a relationship between its renoprotective and the mechanism(s) of such regulation. anti-infl ammatory activity.

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A135 COMPLICATIONS—NEPHROPATHY—BASIC AND EXPERIMENTAL SCIENCE

535-P 537-P Effect of Cooked Meat Meal on Creatinine and eGFR in Diabetes Re- Effects of Sevofl urane on Src and FAK Expression in Kidney of Dia- lated Kidney Disease betic Rats With Renal Ischemia/Reperfusion Injury SUNIL NAIR, SARAH O’BRIEN, KEVIN J. HARDY, JOHN P. WILDING, Liverpool, HONGYUN LU, QIONG YANG, YINGJUAN ZENG, FANG HU, YING SUN, SHUNKUI United Kingdom, Prescot, United Kingdom LUO, YU ZHANG, LIAO SUN, Zhuhai, Guangdong Province, China Fasting is not routinely recommended for renal function tests, despite the Ischemia / reperfusion (I/R) injury is the main cause of acute renal failure, known effects of cooked meat on creatinine. We therefore studied variation in in diabetic condition, kidney is more sensitive to I/R injury. This study is

POSTERS creatinine and estimate glomerular fi ltration rate (eGFR) following a standardised aimed to observe whether the sevofl urane pretreatment can be protective

Complications cooked meat meal in 80 subjects: healthy volunteers, diabetes patients with to the renal functions and expression of Src and FAK in kidney of diabetic Acute and Chronic chronic kidney disease (CKD) stages 1 & 2, 3A, 3B and 4 (n=16/group). rats with renal I/R injury. SD rats were randomly divided into non-diabetic The interventions were a standardised cooked meat and a non-meat meal, sham-operation control group and non-diabetic rats with renal I/ R injury each providing approximately 54g protein, together with 250 mls of water, on group. Diabetic SD rats which induced by STZ were randomly divided into separate days. Fasting and post-prandial blood samples at 1, 2, and 4 hours diabetic rats sham-operation control group, diabetic rats with renal I/ R injury were drawn for creatinine measurement using kinetic alkaline picrate assay group and sevofl urane pretreatment diabetic rats with renal I/ R injury group. on an Olympus AU640 analyser. The modifi ed 4-variable MDRD equation Kidney I/R model were set up with ligaturing bilateral renal artery. Bilateral traceable to isotope dilution mass spectrometry creatinine was used to renal arteries were blocked 45 min by artery clip and opened after 24 hours; calculate eGFR. Shifts in creatinine and eGFR were compared using Wilcoxon Sevofl urane preconditioning treatment was exposed to 2.5% sevofl urane for signed rank test and results presented (Table) as median and interquartile 30 min, and then exposed to fresh air for10 min; Serum levels of Cr and BUN ranges of creatinine and eGFR before and after the standardised meat meal; were determined after 24 hours; Expression of kidney tissue Src and FAK p values compare fasting vs. peak creatinine and nadir eGFR respectively. were determined by immunohistochemistry. Our results showed that whether in non-diabetic group, renal I/ R injury can obviously increase serum Cr and Volunteers (n=16) CKD 1 & 2 (n= 16) CKD 3a (n=16) CKD 3b (n=16) CKD 4 (n=16) BUN level and down-regulate the expression of Src and FAK in kidney tissue CREATININE (BUN, Src, FAK:P< 0.01,Cr:P<0.05), and diabetic rats were more sensitive to (µmoles/L) renal I/ R injury (Cr, BUN, Src, FAK:P< 0.01). Serum Cr and BUN of sevofl urane Fasting 74 98 128.5 152.5 238 preconditioning diabetic rats with renal I/ R injury group were obviously lower, (68.2 - 84.25) (84 – 107) (113 – 139) (133 – 166) (191 – 303) but the Src and FAK of nephridial tissue were higher than that in diabetic rats Post-prandial 79 105.5 150.5 165 256 with renal I/ R injury group (P < 0 .01). From these data, we can conclude that (73.5 - 89.5) (93.5 – 109) (131 - 163) (147 – 169) (193 -325) sevofl urane preconditioning can protect renal function with renal I/R injury of P=0.002 P=0.001 P=0.001 P=0.009 P=0.007 diabetic rats, through up-regulating Src and FAK expression. Supported by: Zhuhai Planning Project of Science and Technology eGFR (ml/min/1.73m2) (2010B04102028) Fasting 102.8 80.4 52 41.9 23.1 (85.3 – 130.4) (68.6 – 97.2) (47.1 – 63.6) (38.2 – 48.5) (17.8 – 29.4) 538-P Post-prandial 93.5 71.6 42.8 38.8 21.55 Activating Transcription Factor 3 Plays as a Novel Potent Regulator (76.9 – 132.9) (67.5 – 83.3) (38.6 – 51.5) (36.4 – 41.4) (14.5 -27.5) of Renal Fibrosis and Macrophage Infi ltration in Streptozotocine- P=0.125 P=0.002 P=0.001 P=0.001 P=0.038 Induced Diabetic Nephropathy WON HO KIM, JI YEON KIM, KEON JAE PARK, Chungbuk, Republic of Korea Signifi cant increases in serum creatinine were seen in all groups, probably Diabetic nephropathy (DN), an end-stage disease of diabetic complication, due to absorbed creatinine from cooked meat. This resulted in signifi cant fall is characterized by fi brosis of renal glomerulus and tubulointerstitial region. in eGFR in all stages of CKD studied; 6 of 16 CKD 3a patients were misclas- However, the exact molecular mechanisms by which diabetes may initiate sifi ed as CKD 3b. This could impact management as threshold for commencing or exacerbate chronic kidney disease remain elusive. Here, we investigated and withdrawing certain medications and expensive investigations is defi ned whether ATF3 affects the development of diabetic kidney failure and by eGFR. especially, expression of infl ammatory factors and macrophage or monocytes infi ltration involved in renal fi brosis. To this, Diabetic kidney failure rats were 536-P induced by injection of streptozotocine (STZ, 60 mg/kg, i.p.) and maintaining SUMOylation of NF-κB Signaling Molecules in Cultured Rat Mesan- for 8 weeks. The STZ-induced DN rats exhibited renal dysfunction, as gial Cells Under High Glucose evidenced by increased volume of renal glomerulus, thickened basement YONG XU, WEI HUANG, XUEQIN ZHOU, MAOJUN YANG, Luzhou, China membrane, and increased mesenterium mass, which are consistent with Post-translational modifi cation of proteins by the small ubiquitin-like higher levels of albuminuria, serum creatine, blood glucose, and serum modifi ers (SUMO) has emerged as an important regulatory mechanism MCP-1 or TNF-α. The CD68+ cells were also signifi cantly increased in the for alteration of protein activity, stability, and cellular localization.The glomerulus and tubulointerstitial region of DN group. In quantitative RT2- latest research demonstrated that SUMO were extensively involved in PCR profi ling assay, MCP-1 was signifi cantly increased in STZ-treated renal the regulation of NF-κB pathway,which play critical roles in regulating tissues, along with marked induction of ATF3. In microRNA chip assay, we infl ammation and contribute to diabetic nephropathy.However,the roles have identifi ed 11 microRNA upregulated in STZ-treated rats and one of of SUMOylation in regulating NF-κB signaling on the diabetic nephro- them is specifi cally regulated by ATF3-dependent manner. STZ-induced renal pathy is unclear. This study observed the change of interaction between nephropathy and fi brosis were remarkably attenuated by in vivo delivering of SUMO proteins (SUMO1,SUMO2 / 3) and NF-κB pathway related signaling ATF3 siRNA using in vivo-jetPEI system. Also, in vivo injection of the target molecules (IκBα, NEMO) in cultured rat Glomerular Mesangial Cells microRNA for ATF3 ameliorates renal fi brosis and the accumulation of ECM (GMCs) stimulated by high glucose. We divided cultured rat GMCs into 5 molecules. Also, ATF3 acts as a direct transcriptional activator on MCP-1 groups: normal glucose group (5.6 mmol/L), high glucose groups (10, 20 gene expression by direct interaction with MCP-1 promoter. Collectively, and 30mmol/L), Mannitol group was used as osmotic control group, the we know that ATF3 may play as an important regulator of renal fi brosis expression of SUMO1,SUMO2/3, IκBα, NEMO was measured by Western by enhancing ECM accumulation and macrophage infi ltration and thereby blot and RT-PCR, interaction between SUMO1,SUMO2 / 3 and IκBα, NEMO reveals a new aspect of the therapeutic mechanism of DN. was observed by co-immunoprecipitation and immunofl uorescence confocus Supported by: Korea National Institute of Health microscope. The results showed that the expression of SUMO1,SUMO2/3 was enhanced in high glucose group in a dose-dependent manner(p<0.05). 539-P The expression of the SUMOylation (SUMO1,SUMO2/3) of IκBα, NEMO in mTORC1-Mediated Autophagy Insuffi ciency Exacerbates Proteinu- high glucose and Mannitol group were signifi cantly decreased compared ria-Induced Proximal Tubular Cell Damage in Obese Diabetic Mice with normal glucose group (p<0.05) .In high glucose group ,the expression KOSUKE YAMAHARA, SHINJI KUME, DAISUKE KOYA, YUKI TANAKA, HISAZU- of IκB was decreased whereas the expression of NEMO without change MI ARAKI, KEIJI ISSHIKI, SHIN-ICHI ARAKI, MASAKAZU HANEDA, ATSUNORI .Our study support the hypothesis that high glucose maybe take part in the KASHIWAGI, TAKASHI UZU, HIROSHI MAEGAWA, Otsu, Japan, Kahoku, Japan, pathogenesis of diabetic nephropathy via impacting SUMOylation of IκB and Asahikawa, Japan activation of NF-κB signaling. Obesity is an independent risk for declining kidney function in diabetic Supported by: Affi liated Hospital of Luzhou Medical College patients with proteinuria, although the mechanism has not been elucidated.

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A136 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

Autophagy is an intracellular degradation system to maintain homeostasis ESRD incidence was computed by quartiles of baseline TNFR1 and TNFR2 by removing damaged proteins and organelles, and its insuffi ciency is likely distributions. Age-sex-adjusted incidence rate ratios (IRR) were computed to be associated with the pathogenesis of obesity-related diseases. We by Mantel-Haenszel stratifi cation. Cox regression analysis was used to thus examined whether autophagy is involved in the mechanism underlying assess the hazard ratio of ESRD per one quartile increase in the distribution obesity-related exacerbation of proteinuria-induced proximal tubular of each TNFR after adjusting for baseline age, sex, diabetes duration, HbA1c, epithelial cell (PTEC) damage using mice models. In non-obese mice, overt albumin-to-creatinine ratio (ACR) and GFR. At baseline, median ACR was 72 proteinuria induced by intraperitoneal albumin-overload led to mild PTEC mg/g (IQR 19-502 mg/g) and mean iothalamate measured GFR was 134 ± damage and apoptosis determined by histological analysis and cleavages 59 ml/min. ESRD developed in 51 participants during follow-up. TNFR1 and POSTERS of PARP, along with autophagy activation in PTECs reabsorbing albumin. TNFR2 correlated with each other (r=0.76). The incidence rate of ESRD was Complications

In contrast, high fat diet-induced obese diabetic condition suppressed signifi cantly higher among participants in the highest vs. lowest quartiles of Acute and Chronic proteinuria-induced autophagy activation in PTECs and exacerbated TNFR1 or TNFR2 (age-sex-adjusted IRR=6.1, 95% CI 2.5-14.7, and IRR=13.0, proteinuria-induced PTEC damage and apoptosis. In addition, PTEC-specifi c 95% CI 4.5-37.8, respectively). TNFR1 was associated with a 2-fold higher autophagy-defi cient mice by Atg5 gene deletion developed severe PTEC risk of subsequent ESRD (HR=2.1, 95% CI 1.4-3.1), and TNFR2 with a nearly damage in albumin-overload model, suggesting that proteinuria-induced 3-fold increase in ESRD (HR=2.9, 95% CI 1.8-4.8), after adjustment for autophagy was renoprotectively elicited. Complex 1 of the target of baseline covariables. Elevated serum concentrations of TNFR1 or TNFR2 rapamycin(mTORC1), a potent signal complex to inhibit autophagy, was predict ESRD in American Indians with T2D. In contrast with Caucasians activated in PTECs of obese diabetic mice. Obesity-mediated autophagy with T2D, TNFR2 predicted ESRD more strongly than TNFR1. insuffi ciency was also restored by treatment with rapamycin, an mTORC1 specifi c inhibitor. In conclusion, these results suggest that hyperactivation of & 542-P mTORC1 signaling is involved in the mechanism underlying obesity-mediated Regulation of Leukocyte Expression and Soluble from Concentra- autophagy insuffi ciency in PTECs, and that a restoration of autophagy may tions of TNFR1 and TNFR2 in Type 1 Diabetes be a new therapeutic option to improve the renal outcome in obese diabetic MONIKA A. NIEWCZAS, JUNG EUN LEE, KIM MIJEONG, WILLIAM WALKER, patients with persistent proteinuria. ADAM M. SMILES, KEVIN MCDONNELL, RITA HOLAK, ROBERTO BASSI, PAOLO FIORINA, ANDRZEJ S. KROLEWSKI, Boston, MA 540-P Elevated levels of circulating sTNFRs were associated with high risk of Hyperactivation of Akt/mTOR and Defi ciency in Tuberin Increased kidney complications in diabetes in the large cohort Joslin Kidney Study. the Oxidative DNA Damage in Kidney Cancer Patients With Diabetes The receptors correlated with each other, which suggest mutual systemic SAMY L. HABIB, TIFFANIE SALAS, SITAI LIANG, San Antonio, TX cellular source and/or exosomes involvement. We have measured cell Patients with diabetes are at a higher risk than the general population surface (CS) and intracellular (IC) expression of TNFRs and TACE in leukocyte of developing cancer in several organs including kidney. Recent study from subsets together with exosomal (CD63+) and free fraction of soluble TNFRs our laboratory showed that 25.4% of kidney cancer patients have diabetes in 30 subjects with type 1 diabetes, normal renal function and no proteinuria. indicting that diabetes is a major contributing factor in increasing the risk of Results: Circulating sTNFR1 correlated strongly with circulating sTNFR2 kidney cancer. In the current study, we have explored one of the mechanisms (r=0.9). Exosomal fraction accounted for 42% of circulating sTNFR1 and for by which diabetes can accelerates tumorigenesis in kidney. Kidney cancer 78% of sTNFR2, respectively. Proportion of the cells expressing TNFRs is tissue from patients with diabetes showed higher activity of Akt, inactivation presented in the Table. of tuberin and loss in total protein expression of tuberin compared to kidney cancer patient without diabetes or diabetes alone. In addition, signifi cant increase in phospho-Akt/tuberin expression was associated with increase in Ki67 expression, and activation of mTOR (measured by phosphorylation of p70S6K) in kidney tumor with or without diabetes compared to diabetes alone. These data indicate that activation Akt/mTOR and inactivation as well as defi ciency in tuberin resulted in increase cell proliferative protein Ki67. Increased tuberin phosphorylation and decreased total tuberin expression resulted in signifi cant decrease in protein expression of OGG1 and increase TACE was expressed on the cell surface of the 70% monocytes. in oxidative DNA damage, 8-oxodG. Importantly, these data show that the Circulating sTNFRs correlated moderately with neutrophil and monocyte majority of staining of Akt/tuberin/p70S6K phosphorylation was more number (r>0.3 for each). Circulating exosomal fraction of sTNFRs correlated prominent in the tubular cells. In addition, accumulation of oxidative DNA with IC neutrophil expression of the respective receptor (for TNFR1: r=0.34, damage is localized only in the nucleus of tubular cells within the cortex for TNFR2: r=0.39). This is the fi rst systematic study of TNFRs expression region. In summary, these data suggest that Akt/tuberin/mTOR pathways in diabetic subjects. We have shown that neutrophils and monocytes play an important role in the regulation DNA damage and repair pathways highly express TNFRs proteins and that intracellular expression correlate that may predispose diabetic kidney to pathogenesis of renal cell carcinoma. with exosomal TNFRs fraction. Our data suggest that those two leukocyte Supported by: U.S. Dept. of Veterans Affairs subsets may be involved in the regulation of circulating levels of sTNFRs. Supported by: P&F DERC (to M.A.N.)

COMPLICATIONS—NEPHROPATHY—CLINICAL AND & 543-P TRANSLATIONAL RESEARCH Gas Chromatography as a New Opportunity of Diabetic Nephropa- thy Diagnostics? PRZEMYSłAW MIARKA, BEATA GRABOWSKA-POLANOWSKA, JACEK FABER, Guided Audio Tour: New Learning’s from the Clinical Side of Diabetic MONIKA SKOWRON, AGATA PIETRZYCKA, MALGORZATA WALUS-MIARKA, Nephropathy (Posters: 541-P to 548-P), see page 19. PAWEł ZAGRODZKI, IRENEUSZ SLIWKA,´ MAREK STE˛PNIEWSKI, WłADYSłAW SUłOWICZ, Kraków, Poland & 541-P Diabetic nephropathy is the main cause of renal replacement therapy. Circulating TNF Receptors 1 and 2 Predict End-Stage Renal Disease Breath tests seem to be a promising diagnostic device offering early non- in American Indians With Type 2 Diabetes invasive disease detection. Trimethylamine (TMA), dimethyl sulfi de (DMS), MEDA E. PAVKOV, ROBERT G. NELSON, WILLIAM C. KNOWLER, ANDRZEJ S. carbon disulfi de (CS2) are mentioned in literature as potential markers of KROLEWSKI, MONIKA A. NIEWCZAS, Atlanta, GA, Phoenix, AZ, Boston, MA chronic kidney disease (CKD) and diabetic nephropathy. The aim of our study In Caucasian patients with type 2 diabetes (T2D) circulating TNF receptor was to determine breath composition in persons suffering from CKD. 1 (TNFR1) and, to a lesser degree, TNF receptor 2 (TNFR2) predict end- Breath samples were collected from 14 CKD patients (6 with type 2 stage renal disease (ESRD) as demonstrated in the Joslin Kidney Study. We diabetes) and 7 healthy volunteers (control group - CG). Exhaled air samples examined the relationship between serum concentrations of TNFR1 and were analyzed by gas chromatography equipped with mass spectrometer TNFR2 and ESRD in American Indians with T2D. (GC-MS). Samples were enriched using solid ESRD was defi ned as dialysis, kidney transplant, or death attributed to phase microextraction (SPME). The mean age in whole group was 64.6 ± diabetic nephropathy. Participants (n=194, mean age 46 ± 10 years) were 15.8 years, average duration of CKD 11 years. The mean values of serum followed for a median of 7.1 years [interquartile range (IQR) 5.3-9.1 years]. creatinine and blood urea in CKD patients were 171.2 ± 74.2 µmol/l and 11.9 ±

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A137 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

4.0 mmol/l respectively. eGFR in the CKD group averaged 37.4 ± 18.9 ml/ 1). Our study demonstrated that haptoglobin is a putative biomarker that min/1.73 m2, albuminuria 700 mg/day. TMA was detected in 11 CKD patients correlates with ERFD. only and ranged from 0.19 to 4.03 nmol/l (4.78 - 98.61 ppb). Other detected breath components were DMS, CS2, aceton and potential markers of oxidative stress: propane, butane, pentane, 2-methylpentane, hexane. The DMS was present in breath of all participants: in CG ranged from 0.05 to 0.39 nmol/l (1.26 - 9.00 ppb), in CKD from 0.01 to 1.02 nmol/l (0.26 - 24.85 ppb) p<0.05. Carbon disulphide was detected in all CKD patients (range: POSTERS

Complications 0.009 - 0.15 nmol/l (0.24 - 3.80 ppb), and in 4 persons from CG (range 0.01 to

Acute and Chronic 0.02 nmol/l (0.25 - 0.58 ppb) p<0.05. Diabetic patients were characterized by signifi cantly higher TMA and aceton level in breath samples than other patients (p<0.05). TMA correlated with glucose (r=0.60 p=0.02) and fi brinogen (r=0.33 p=0.02) concentration. The results suggest that current diagnostic procedures may be supplemented by detection of TMA, DMS and CS2 concentration in human breath. & 546-P & 544-P Diabetic Nephropathy: A Complication of Insulin Resistance in Type AQP5 Is a New Potential Urinary Biomarker of Diabetic Nephropathy 1 Diabetes WENZHENG ZHANG, YIYANG LU, HONGYU WU, LIHE CHEN, QIAOLING ZHOU, PETTER BJORNSTAD, JANET K. SNELL-BERGEON, MARIAN J. REWERS, DIANA Houston, TX, Changsha, China JALAL, MICHEL CHONCHOL, RICHARD JOHNSON, DAVID MAAHS, Aurora, CO No biomarkers have been used for diagnostic, prognostic and therapeutic Diabetic nephropathy (DN) is a major cause of mortality in type 1 diabetes purposes in the management of diabetic nephropathy (DN), possibly due (T1D). Insulin resistance is well documented in T1D, and is thought to to various limitations. Water channel AQP5 is important for generation of contribute to both the initiation and progression of DN, but limited data saliva, tears, and pulmonary secretions. AQP5 is undetectable and thus may exist. play little role in normal kidney. We recently reported immunofl uorescence We assessed the relationship between insulin resistance at baseline and staining of AQP5 in 17/17 kidney biopsies of DN patients, but in 0/15 patients development of two early phenotypes of DN; microalbuminuria (albumin/ with minimal change disease studied. Here, we hypothesize that AQP5 is a creatinine ratio [ACR] > 30mg/g) and rapid renal function decline (glomerular new potential urinary biomarker of DN. To this end, we measured plasma and fi ltration rate [GFR] loss > 3mL/min/1.73m2 per year) over 6 years. Subjects urinary AQP5 with an AQP5-specifi c enzyme-linked immunosorbent assay with T1D (n=449) and controls (n=565) in the Coronary Artery Calcifi cation kit. The kit has a detection range of 0.156-10 ng/ml, sensitivity of 0.059 ng/ in Type 1 Diabetes (CACTI) study had insulin sensitivity index (ISI) estimated ml and no signifi cant cross-reactivity or interference between human AQP5 at baseline and 6-year follow-up. ISI is a model used to estimate insulin and analogues. Although plasma AQP5 was detected in all samples tested sensitivity derived from waist circumference, triglycerides and diastolic blood (Fig. 1A), urinary AQP5 was recorded in 1/24 of normal controls (Con), 2/15 pressure and has been validated in a previous euglycemic hyperinsulinemic of diabetes mellitus (DM), and 15/25 of DN. The average level of urinary clamp study from the same cohort. AQP5 was signifi cantly higher in DN than in the other two groups (Fig. 1B). ISI was lower in subjects with T1D, compared to those without (T1D: Urinary AQP5 is apparently independent of fast blood glucose, and modestly, 4.4 ± 1.6, controls: 15.4 ± 6.7; P < 0.0001). GFR declined over 6 years in the but signifi cantly correlated with plasma AQP5, serum creatinine and urinary entire cohort estimated both by CKD-EPI creatinine (P < 0.0001) and CKD-EPI albumin excretion (Fig. 1C). The age ranges of the three groups were 25-60, Cystatin C (P < 0.0001) equations. Higher ISI at baseline predicted lower 29-74, and 35-80, respectively. Our preliminary study suggests that AQP5 risk of developing ACR > 30mg/g (OR: 0.65, 95% CI 0.49 - 0.85, P = 0.003) is a new potential urinary biwomarker of DN. More studies with larger and univariately and after adjusting for HbA1c (OR: 0.69, 95% CI 0.51 - 0.93, P = younger test populations are required. 0.01). Higher ISI at baseline conferred protection from rapid decline of GFR by CKD-EPI Cystatin C (OR: 0.77, 95% CI 0.64-0.92, P= 0.004), and remained signifi cant after adjusting for HbA1c and age (OR: 0.80 95% CI 0.67 - 0.97, P = 0.02). We found no such relation between ISI and rapid GFR decline estimated by CKD-EPI creatinine (P= 0.38). In conclusion, higher insulin sensitivity appears to be an independent predictor of lower risk of developing ACR > 30mg/g and rapid GFR decline - early phenotypes of DN. Supported by: NIDDK; NHLBI

& 547-P The Association of Diabetic Retinopathy With Renal and Cardiac & 545-P Outcomes Shotgun Proteomics Identifi ed Proteins Specifi c for Early Renal AMY K. MOTTL, NICHOLAS PAJEWSKI, CRAIG GREVEN, VIVIAN FONSECA, FAR- Function Decline in Patients With Type 2 Diabetes were Verifi ed in AMARZ ISMAIL-BEIGI, WALTER AMBROSIUS, EMILY CHEW, JOHN B. BUSE, Cha- Retrospective and Prospective Cohorts pel Hill, NC, Winston-Salem, NC, New Orleans, LA, Cleveland, OH, Bethesda, MD JIN-KUI YANG, JIAN-PING FENG, ZHONG XIN, Beijing, China We sought to examine whether in the high risk population from the The predictive value of MA is limited for predicting early renal function ACCORD trial, diabetic retinopathy (DR) would strongly associate with renal decline (ERFD) in patients with type 2 diabetes (T2DM).This study investigated and cardiac outcomes. the hypothesis that proteins in the urine of patients with sight-threatening ACCORD was a randomized trial of people with type 2 diabetes, age proliferative diabetic retinopathy (PDR) is a strong indicator for ERFD in ≥ 40yrs, hemoglobin A1c >7.5% and cardiovascular disease. A subgroup of T2DM patients. We fi rst analyzed urine specimens by shotgun proteomics participants had baseline stereoscopic fundus photographs graded centrally. obtained from our sight-threatening PDR case-control study. Of all patients Serum creatinine, random urine albumin:creatinine ratio and assessment with T2DM screened, those with diabetic duration longer than 15 years, with for incident cardiovascular events were obtained every 3 months for four microalbuminuria (MA) and free of diabetic retinopathy (NDR) were served as years. The referent group (no or mild DR) was compared to participants with control group. Meanwhile, those patients with MA and proliferative diabetic moderate-severe nonproliferative DR (NPDR) or proliferative DR. Multivariate retinopathy (PDR) were served as case group. The urinary proteins of the Cox proportional-hazards regression analyses estimated the hazard ratio two groups were isolated and performed with a Shotgun proteomic study. (HR) for the main composite outcome of doubling serum creatinine, end- At least 120 proteins were found to have a higher ratio in the PDR group. stage renal disease (ESRD) or death. Other outcomes included 1) the primary Then, top three proteins (haptoglobin, α-2-Macroglobulin and complement trial cardiovascular outcome of incident myocardial infarction, stroke, or component 3), that increased in the urine from the PDR group were further cardiovascular death and 2) a renal outcome of incident macroalbuminuria, verifi ed by immunoassay. Last, urinary haptoglobin excretion rate (UHER) doubling serum creatinine, ESRD, or death. was tested and used to predict renal function decline in an 8-year follow- At baseline, 1,692 / 621 / 1,014 / 42 participants had no, mild, moderate up retrospective T2DM cohort and a 3.5-year prospective T2DM cohort (Fig NPDR, or severe NPDR/proliferative DR, respectively. The adjusted HR in

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A138 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH those with at least moderate DR for the main composite outcome was 1.3 are associated with increased risk of hypoglycemia (HYPO) and as renal (95% CI: 1.0-1.7). The HR for the primary cardiovascular outcome was 1.6 function declines, the risk of HYPO may be further increased due to reduced (1.1, 2.2). Excluding individuals with baseline macroalbuminuria, the HR for clearance of these agents. DPP-4 inhibitors improve glycemic control and are the composite renal outcome was 1.6 (1.3, 2.0). The outcomes most greatly associated with a low risk of HYPO. A post hoc analysis of pooled data from impacted included incident macroalbuminuria (HR= 2.3, 95% CI: 1.4-3.8) and 3 double-blind studies compared 25- to 30-wk treatment effects of SITA 100 incident cardiovascular events (HR=2.0, 95% CI: 1.5-2.8). mg/day and SU (in titrated doses) on change from baseline in A1C, fasting Consistent with previous population-based studies, moderate-severe DR plasma glucose (FPG), body weight (BW) and incidence of symptomatic HYPO is modestly associated with cardiac and renal outcomes, even in the high- in patients who meet National Kidney Foundation criteria for mild renal 2 POSTERS risk population of the ACCORD trial. Further research is needed to elucidate insuffi ciency (eGFR = 60 to 89 mL/min/1.73 m ). Both A1C and FPG decreased Complications the environmental factors and pathogenetic mechanisms that differentiate similarly with SITA and SU (Table). A lower incidence of HYPO was observed Acute and Chronic these diabetic complications. with SITA. BW decreased with SITA, compared to an increase with SU. More Supported by: NHLBI patients on SITA than SU achieved the composite endpoint of >0.5% A1C reduction with no HYPO or BW gain at 30 wks. In conclusion, SITA provided similar glycemic effi cacy, with less HYPO and with BW loss compared to BW & 548-P gain with SU in T2DM patients with mild renal insuffi ciency. Multifactorial Intervention in Advanced Diabetic Nephropathy (ADN): Renal Outcomes Sitagliptin Sulfonylureas LEON FOGELFELD, PETER HART, JADWIGA MIERNIK, JOCELYN KO, DONNA CAL- N = 584 N = 596 VIN, BETTINA TAHSIN, ANWAR ADHAMI, RAJEEV MEHROTRA, Chicago, IL Baseline A1C, % 7.6 ± 0.8 7.6 ± 0.9 A multifactorial-multidisciplinary trial aimed to delay progression of CKD Δ A1C, % -0.62 (-0.68, -0.56) -0.68 (-0.74, -0.62) 3-4 to ESRD through improved coordinated care. Low-income CKD pts were ‡ randomized into usual care control (CNT) or intervention (INT). The 2-year A1C <6.5%, n (%) 180 (30.8) 223 (37.4) INT, led by a team of renal and DM MDs, APNs, and RDs who saw pts A1C <7.0%, n (%) 348 (59.6) 384 (64.4) together, targeted intensive DM-renal control. Baseline FPG, mg/dL 154.7 ± 35.6 156.8 ± 40.3 At baseline (BL), 60 INT and 60 CNT pts had no signifi cant differences for Δ FPG, mg/dL -16.6 (-19.1, -14.0) -18.0 (-20.6, -15.5) age (56 vs 58), eGFR (38 vs 37), albumin creatinine ratio (ACR) in mg/g (1963 vs 1246), SBP (142 vs 144), A1C (8.2 vs 8.3), and BMI (34 vs 34). Among all Patients with HYPO AE, n (%) 40 (6.8) 156 (26.2)* study pts, 58% were male, 55% African American, and 23% Hispanic. Baseline BW, kg 85.7 ± 16.5 86.4 ± 16.8 Fewer INT (13%) than CNT (28%) developed ESRD, p<0.05 (fi gure). In both Δ BW, kg -0.9 (-1.2, -0.7) 1.4 (1.1, 1.6)* groups, ESRD occurred most in BL CKD 4 but more in CNT (33% vs 57%). INT Composite†, n (%) 235 (40.6) 99 (16.8)* had higher ACR decrease (62% vs 42%, p<0.05), A1C <7% attainment (50% vs 30%, p<0.05) and trended towards better lipid/BP control (p=NS). Data are mean ± SD, LS mean change (Δ) from baseline (95% CI), or counts Signifi cant differences between 25 ESRD and 95 ESRD-free pts were BL (proportion of patients). *p<0.001, ‡p<0.05 for difference between SITA and SU; GFR (28 vs 40), annual GFR decline (15 vs 3), BL ACR (2362 vs 1139), last ACR †Composite = patient experienced an A1C decrease >0.5% with no HYPO and (2896 vs 1201), and last A1C (6.9 vs 7.8). no BW gain In multivariate Cox analysis, being in INT reduced the hazard ratio to develop ESRD (0.096, CI 0.028-0.33) as did higher BL GFR (0.85, CI 0.79- Supported by: Merck, Sharp & Dohme 0.91). A greater annual decline in GFR increased the hazard ratio (1.25, CI 1.16-1.36). 550-P In conclusion, INT delayed ESRD. Improved A1C and ACR plus not yet Ambulatory Blood Pressure (ABP) and Glomerular Structure in identifi ed variables may have infl uenced better outcomes. Multifactorial- Normoalbuminuric (NA) Normotensive (NT) Type 1 Diabetic (T1D) multidisciplinary care may serve as an ADN treatment paradigm. Patients (pts). MICHAEL C. TRESSLER, LUIZA CARAMORI, MICHAEL MAUER, Minneapolis, MN Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) in the US. Hypertension is a known DN risk factor. However, the Renin Angiotensin System Study (RASS) demonstrated that treatment with enalapril or losartan did not change the rate of development of DN lesions in NA NT T1D pts. Several studies investigated the associations between ABP and DN. None, however, looked at renal structural in relation to ABP. Therefore, we studied the relationships between baseline ABP measurements and renal structure in RASS participants. The RASS was a parallel, double-blind, placebo-controlled, multicenter, DN primary prevention clinical trial. 248 out of the 285 pts who met the inclusion criteria had baseline ABP measurements. Renal structure was assessed by electron microscopy morphometry of kidney biopsy material and ABP assessed by SpaceLabs 90207 monitor before randomization. Linear regression showed multiple signifi cant associations between ABP variables and renal structure (mesangial matrix fractional volume and glomerular basement membrane (GBM) width). Adjusted for age, T1D duration, offi ce BP, and HbA1C, no ABP variable was independently associated with renal structure. There were also no differences in structural parameters in pts in the lowest and highest quintile for night to day ratio of SBP, DBP, MAP, or pulse. Even though Supported by: Sanofi ABP has been shown to be predictive of development of microalbuminuria in other studies, ABP parameters were not independently associated with 549-P renal structure in these NA NT T1D pts. Analyses of longitudinal data will Comparison of Treatment With Sitagliptin (SITA) or Sulfonylurea determine if baseline ABP predicts changes in renal structure over 5 years. (SU) in Patients With Type 2 Diabetes Mellitus (T2DM) and Mild Re- nal Insuffi ciency 551-P SAMUEL S. ENGEL, LEI XU, GREGORY T. GOLM, EDWARD A. O’NEILL, KEITH D. Renal Histological Heterogeneity and Functional Progress in Normo KAUFMAN, BARRY J. GOLDSTEIN, Whitehouse Station, NJ and Microalbuminuric Japanese Type 2 Diabetic Patients Patients with T2DM frequently develop progressive impairment in renal TATSUMI MORIYA, YOSHIKI SUZUKI, MASAYUKI IWANO, SHIGEKI INOMATA, function, and chronic kidney disease is present in more than 40% of adults MASAKAZU HANEDA, Sagamihara, Japan, Niigata, Japan, Fukui, Japan, Akita, with diabetes. The magnitude and duration of hyperglycemia are both Japan, Asahikawa, Japan associated with progression of kidney disease, and improved glycemic Renal structure and function in normo- and microalbuminuric type 2 control may slow it. SUs are commonly used antihyperglycemic agents, but diabetic patients with low GFR remains to be elucidated. Histological

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A139 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

patterns of renal injury in type 2 diabetes were heterogeneous, but 553-P whether or not renal function in atypical injury patterns is becoming worse Exenatide Suppresses Keap-1 and Stimulates Expression of Nrf-2 remains obscure. Therefore, we examined 104 type 2 diabetic patients with Depended Genes percutaneous renal biopsy (73 males, 52±11 years old, 58 normoalbuminuria, HUSAM GHANIM, AJAY CHAUDHURI, SANDEEP DHINDSA, SANAA ABUAY- 46 microalbuminuria) from four Japanese institutes and followed up 35 SHEH, KELLY GREEN, NITESH KUHADIYA, CHERIE VAZ, ANUPAM OHRI, PARESH cases for 9 years. DANDONA, Buffalo, NY Renal injury patterns revealed by light microscopy of tissues are Nuclear factor erythroid 2-related factor (Nrf)-2 is an anti-oxidant categorized as: Category I (C1), normal or near-normal renal structure;

POSTERS transcription factor that when released from its inhibitor, Keap-1, translocates

Complications Category II (C2), typical diabetic glomerulopathy; and Category III (C3), to the nucleus and binds to anti-oxidant response elements (ARE) of several Acute and Chronic atypical injury patterns (disproportionately severe tubulointerstitial/ anti-oxidant genes. It has been shown recently that Nrf-2 might be involved vascular damage with no/mild glomerulopathy). There were 29 C1, 57 C2, in protection from the development of diabetic nephropathy in mice with and 18 C3 patterns among all the patients; however, the distribution of injury streptozotocin induced diabetes. Since we have previously demonstrated patterns was not different between the albuminuria stages (Chi-square, ns). that exenatide exerts rapid and potent anti-oxidant and anti-infl ammatory Renal function was signifi cantly decreased in C2 and C3 compared to initial effects in diabetic patients, we investigated the effect of exenatide eGFR while the GFR decline rate was faster in C2 compared with the other 2 treatment on Nrf-2/Keap-1 system. Twenty four obese type 2 diabetics groups (Table 1). In normo-microalbuminuric type 2 diabetic patients, urinary were randomized to receive either exenatide 10µg twice daily (n=12, mean biomarkers more accurately identifying histological patterns of renal injury HbA1c:8.6±0.4%) or placebo twice daily (n=12, mean HbA1c:8.5±0.3%) for are necessary since GFR decline rates were different according to injury 12 weeks. Fasting blood samples were obtained at baselines and at 3, 6 patterns during 9 years of observation. and 12 weeks later. Mononuclear cells (MNC) were isolated and tested for Table 1 expression of related genes. Following exenatide, HbA1c fell to 7.4±0.5% (P<0.05) and there was no weight loss. Exenatide treatment also suppressed Category Initial eGFR Final eGFR Matched pairs Slopes of GFR (ml/min/1.73m2)(ml/min/1.73m2) (Initial-Final) decline Keap-1 protein by 21±8% and increased the mRNA expression of NQO-1, (ml/min/1.73m2/yr) GST-1P and HO-1 by 51±15%, 42±10% and 39±10%, respectively (P<0.05). Exenatide also increased expression of p21 (a competitor of Keap-1 that C1 (n=12) 88.3±15.1 79.9±23.6 ns -0.61 stabilizes Nrf-2) by 68±26%. In addition, exenatide suppressed TGF-1β (a C2 (n=13) 93.9±27.0 60±22.3 0.0001 -4.18 fi brogenic cytokine involved in the pathogenesis of diabetic nephropathy) C3 (n=10) 91.9±16.7 74.9±12.4 0.0058 -1.67 concentrations by 20±7% (from12.93±0.57 to 10.37±1.04ng/ml, P<0.05). We ANOVA ns ns — 0.02 conclude that exenatide treatment in type 2 diabetics suppresses Keap-1 and (3 groups) stimulates the expression of Nrf-2 regulated anti-oxidant enzymes. Although these observations are in MNC and not in the kidney it might be relevant to renal pathophysiology since the monocyte/macrophage has an important 552-P pro-infl ammatory role in the pathogenesis of diabetic glomerulopathy. In Patients With Type 1 Diabetes and Proteinuria Sustained Reduc- Further clinical investigations on the effect of exenatide and other GLP-1 tion of Glycemia Prevents Risk of End-Stage Renal Disease mimetics on diabetic nephropathy are clearly needed. JAN SKUPIEN, JAMES D. WARRAM, ADAM M. SMILES, ANDRZEJ T. GALECKI, ROBERT C. STANTON, ANDRZEJ S. KROLEWSKI, Boston, MA, Ann Arbor, MI 554-P No strong evidence exists that in patients with type 1 diabetes (T1D) Effect of Roux-en-Y Gastric Bypass on the Development and Pro- and proteinuria improved glycemia can reduce the risk of end-stage renal gression of Diabetic Nephropathy disease (ESRD). ALI AMINIAN, HELEN M. HENEGHAN, ESAM BATAYYAH, HECTOR ROMERO-TAL- 350 patients with T1D and proteinuria were followed for 5-18 years to AMAS, ANDREA ZELISKO, JOHN P. KIRWAN, SANGEETA R. KASHYAP, STACY A. ascertain ESRD (n=112, incidence rate 3.9/100 person-years). HbA1c before BRETHAUER, PHILIP R. SCHAUER, Cleveland, OH study entry (5-year pre-baseline period) was compared with follow-up values Favorable effects of bariatric surgery on glycemic status and blood (median 3.4 years after baseline), and changes were examined for effects on pressure have been shown. However, the ultimate results of these changes risk of ESRD during follow-up. on end-organ complications of type 2 diabetes mellitus (T2DM) are not Median HbA1c was 9.3% in pre-baseline period and decreased during well characterized. The primary aim of this study was to assess the long follow-up to 8.8%. HbA1c was categorized into good, fair and poor (≤8, >8- term renoprotective effects of bariatric surgery. Data of fi fty obese ≤9.5, and >9.5%, respectively). 220 patients (63%) remained in the same patients with T2DM who had complete clinical and laboratory evaluations category (Table diagonal), in 35 (10%) HbA1c category worsened (below including glycated hemoglobin (A1C), fasting blood glucose (FBG), serum diagonal) and in 95 (27%) it improved (above diagonal). The incidence rate creatinine, and urinary albumin/creatinine ratio (uACR) at least 5 years after of ESRD decreased dramatically with improving HbA1c, for example, with laparoscopic Roux-en-Y gastric bypass (RYGB) were retrieved from an IRB improvement from poor to fair HbA1c, the incidence of ESRD dropped from approved database. Complete remission (A1C <6% and FBG <100 mg/dL 7.5 to 3.3/100 patient-years). off diabetic medications) and partial remission (A1C 6-6.4% and FBG100- 125 mg/dL off medications) of T2DM were determined. The progression of Incidence rates of ESRD (per 100 patient-years) according to pre and post- diabetic nephropathy (DN) was defi ned as an increase of one or more of baseline category of HbA1c the 3 stages of albuminuria: normo- (uACR <30 mg/g), mirco- (uACR =30- Pre-baseline HbA1c Pre-baseline HbA1c 299 mg/g), and macro-albuminuria (uACR ≥300 mg/g). At a median follow- Post-baseline HbA1c good fair poor up of 6 years (range 5-8) after surgery, a mean excess BMI loss of 58% was associated with a mean reduction in A1C and FBG of 1.5% and 56.1 good 1.8 (n=59) 3.0 (n=35) 0.0 (n=14) mg/dL, respectively. Long-term complete remission, partial remission, and fair 1.9 (n=12) 3.7 (n=78) 3.3 (n=46) improvement rate of T2DM was 28%, 22%, and 36% respectively. High poor (n=0) 5.7 (n=23) 7.5 (n=83) blood pressure was improved in 73% of hypertensive patients. Of the 33 normo-albuminuric patients at baseline, only 2 developed albuminuria (6.1%) In Cox regression relative hazard of ESRD per 1% post-baseline HbA1c at follow-up. Of the 17 albuminuric patients at baseline, DN regressed in improvement was 0.72 (95% CI: 0.61, 0.89; p<0.001). This effect was inde- 9 (52.9%) and remained stable in 8 (47.1%) patients without any case of pendent from pre-baseline HbA1c and not confounded by sex, age, urinary progression. The follow-up creatinine of the cohort was signifi cantly lower albumin/creatinine, baseline renal function, blood pressure and antihyper- than the baseline values with a mean difference of 0.1±0.2 mg/dL (p=0.02). tensive treatment. The fi ndings of this study indicate that the antidiabetic and antihypertensive In T1D patients with proteinuria sustained improvement of glycemic control effects of RYGB result in a low incidence or progression and high regression is an effective treatment that reduces long-term risk of ESRD. rate of DN in bariatric surgical patients. Supported by: JDRF (1-2008-1018), (3-2009-397); NIH (DK41526)

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A140 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

555-P Twelve healthy young males were examined in a randomized, controlled, Plasma Levels of Very Long Chain Ceramide Species are Associated double-blinded, single-day, cross-over trial to evaluate the effects of two With Decreased Odds of Macroalbuminuria hours GLP-1 infusion on kidney functions. Glomerular fi ltration rate (GFR) and NATHANIEL L. BAKER, KELLY J. HUNT, RICHARD L. KLEIN, SAMAR M. HAMMAD, renal plasma fl ow (RPF) were assessed with 51Cr-EDTA and 123I-hippuran, MOHAMMED AL GADBAN, GABE VIRELLA, MARIA F. LOPES-VIRELLA, DCCT/ respectively, using a constant infusion renal clearance technique based on EDIC RESEARCH GROUP, Charleston, SC timed urine sampling. We measured levels of sphingoid bases and their phosphates and multiple GLP-1 had no signifi cant effect on either GFR [+1.9%, 95% CI(-0.8 ; 4.6%)] or RPF [+2.4%, 95% CI(-3.6 ; 8.8%). Fractional urine excretion of lithium ceramide species in plasma obtained from 497 type 1 diabetic patients at POSTERS the time of entry into the Diabetes Control and Complications Trial (DCCT) increased 9% (p=0.013) and renal sodium clearance increased 40% (p=0.007). Complications and studied their association with the development of abnormal albuminuria Angiotensin II decreased 19% (p=0.003) while renin, aldosterone, and the Acute and Chronic after 14-20 years of follow up in Epidemiology of Diabetes Intervention urinary excretion of angiotensinogen showed no signifi cant changes. Cohort (EDIC). These subjects had normal albumin excretion rates (AER The results indicate that although GLP-1 markedly reduces proximal tubule <40 mg/24 h) at the time of plasma sampling (DCCT Baseline). While the sodium reabsorption, the acute effects on GFR and RPF are very limited majority of patients (N=291; 59%) continued with normal AER levels in healthy humans. The fi nding of GLP-1’s ability to reduce angiotensin II throughout follow-up, 141 patients (28%) progressed to AER ≥40 mg/24 concentration is novel and may play a part in the possible kidney protective h, but <300 mg/24 h (microalbuminuria), and 65 (13%) progressed to AER properties of GLP-1. ≥300 mg/24 h (macroalbuminuria). To test the association of log transformed Supported by: Novo Nordisk A/S; Danish Agency for Science Technology and sphingolipid levels with the development of albuminuria, multinomial logistic Innovation regression models were used with normal, micro-and macro-albuminuria as the outcomes of interest. Increased levels of very long chain ceremide 557-P species measured at DCCT baseline were associated with decreased odds Renin Angiotensin System Blockade Increases Soluble Klotho in of developing macroabluminuria from baseline to EDIC year 8 (Table). Low Patients With Type 2 Diabetes, Systolic Hypertension, and Albu- plasma levels of select ceramide species may precede the development of minuria nephropathy in Type 1 diabetes, likely by altering the function of glomerular JANAKA KARALLIEDDE, GIUSEPPE MALTESE, GIANCARLO VIBERTI, LUIGI GNU- membranes. DI, London, United Kingdom Objective: Soluble-Klotho is a phosphaturic anti-ageing protein and putative mediator of cardio-renal disease. The role of soluble-Klotho in diabetic kidney disease (DKD) is unknown. Methods: Soluble-Klotho was measured in a secondary analysis of 76 patients with Type 2 diabetes, systolic hypertension, albuminuria at baseline and at 24-weeks following randomisation to valsartan/hydrochlorothiazide (n=37) or amlodipine (n=39) treatment. Aortic-pulse wave velocity (Ao-PWV) and albuminuria were also measured. Results: Despite similar attained blood pressures (BP), valsartan/hydro- chlorothiazide signifi cantly increased, soluble-Klotho mean±SD, from 432.7±179 to 506.4±226.8 pg/ml, p=0.01 and reduced serum phosphate 1.05±0.38 to 0.84±0.31 mmol/l, p=0.04 compared to amlodipine (430.1±145.8 to 411.9±157.6 pg/ml and 0.95±0.18 to 0.87±0.49 mmol/l). There was a signifi cant between treatment group difference, mean (95% confi dence interval), in soluble Klotho, 91.9 (19.9 to 162) pg/ml and serum phosphate levels -0.22 (-0.05 to -0.43) mmol/l with Valsartan/hydrochlorothiazide treatment, p<0.05 for both. In the valsartan/hydrochlorothiazide group change in soluble-Klotho was negatively correlated with change in phosphate (Pearson correlation R2 -0.41 p=0.07). Soluble-Klotho was not associated Ao-PWV and albuminuria which fell signifi cantly only with valsartan/hydrochlorothiazide. Conclusions: Treatment with a Renin-Angiotensin-System (RAS) blocker is associated with an increase in soluble-Klotho which may explain the BP- independent cardio-renal benefi ts of these drugs in DKD.

558-P Correlation Between Neuropathy and Progression of Other Diabetic Complications in Type 1 Diabetic Patients Aged 40 Years of Age or Younger CHIAKI SEO, RIMEI NISHIMURA, YOSHIKO ONDA, DAISUKE TSUJINO, KIYOTAKA ANDOU, KAZUNORI UTSUNOMIYA, Tokyo, Japan In general, it is reported that in diabetic polyneuropathy, nerve conduction velocity is fi rst affected, followed by sensory nerve function, then autonomous nerve function, and fi nally motor nerve function. However, very Supported by: NIDDK (R01-DK081352) few studies addressed the issue of how neuropathy may correlate with other complications in diabetes. This study was conducted to investigate 556-P whether or not the extent of sensory and autonomic nerve damage may Glucagon-Like Peptide-1 (GLP-1): Effect on Kidney Hemodynamics correlate with the presence or absence of retinopathy or nephropathy. A and Renin-Angiotensin-Aldosterone System in Healthy Men total of 42 type 1 diabetic patients participated in the current study. Mean JEPPE SKOV, ANDERS DEJGAARD, JØRGEN FRØKIÆR, JENS J. HOLST, THOMAS vibration perception thresholds (VPT) on bilateral medial malleoli were JONASSEN, SØREN RITTIG, JENS S. CHRISTIANSEN, Aarhus, Denmark, Bags- used as a measure of sensory neuropathy, and coeffi cients of variation of vaerd, Denmark, Copenhagen, Denmark R-R intervals (CVR-R) in resting echocardiograms were used as a measure Glucagon-like peptide-1 (GLP-1) is an incretine hormone with multiple of sensory neuropathy. The absence of retinopathy was defi ned as normal, actions besides control of glucose homeostasis. GLP-1 is known to cause and the presence of retinopathy as simple retinopathy or higher in Davis’s natriuresis in humans but the effects on basic renal physiology are still classifi cation, and urinary albumin less than 30 mg/day was defi ned as partly unknown. the absence of nephropathy, and 30 mg or higher as the presence of This study therefore aimed to demonstrate the effects of GLP-1 on kidney nephropathy. VPT and CVR-R values were examined for their ability to predict hemodynamics, electrolyte handling, and the renin-angiotensin-aldosterone for the presence or absence of diabetic retinopathy or nephropathy by using system in healthy men. a logistic regression model. The subjects’ median age was 35 years of age,

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A141 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

their median duration of diabetes 6.5 years, their median BMI 21.3 kg/m2, 561-P and their median HbA1c value 8.8%. VPT and CVR-R were not signifi cant Impact of Vitamin D on Diabetic Nephropathy and Other Kidney predictors of the presence or absence of retinopathy. In contrast, CVR-R, Diseases in Pre-Dialysis Patients: A Meta-Analysis of Randomized but not VTP, was a signifi cant predictor of the presence or absence of Controlled Trials nephropathy (odds ratio, 0.386; 95% CI, 0.185-0.807) (P = 0.011). Among the LIJUAN XU, XUESI WAN, ZHIMIN HUANG, DONGHONG FANG, WANPING DENG, type 1 diabetic patients, VPT and CVR-R did not predict for the progression YANBING LI, Guangzhou, China of retinopathy. However, while the decreases in VPT did not predict for Recent studies have supported a role for both newer and more established progression of nephropathy, the decreases in CV were found to be useful

POSTERS R-R vitamin D compounds in improving proteinuria, although systematic evaluation

Complications in predicting if their urinary albumin values were in excess of 30 mg/day. is lacking. Furthermore, concerns remain regarding the infl uence of vitamin Acute and Chronic D on the progression of renal function. We analyzed the effi cacy and safety 559-P of vitamin D in pre-dialysis patients with diabetic nephropathy and other Macroalbuminuria Predicts the Incidence of Acute Heart Failure in kidney diseases and compared the use of newer versus established vitamin Japanese Patients With Type 2 Diabetes Mellitus D compounds by performing a meta-analysis of randomized controlled MAYU TOCHIYA, HISASHI MAKINO, YOKO OHATA, TAMIKO TAMANAHA, AYAKO trials. A literature search of PubMed (1975 to September, 2012), EMBASE. TANAKA, RYO KOEZUKA, ICHIRO KISHIMOTO, Suita, Japan com (1966 to September, 2012) and Ovid EBM Reviews (through September, Albuminuria is a major risk factor for cardiovascular diseases. However, 2012) was conducted. Eighteen studies were eligible for fi nal inclusion in this the relationship between the degree of albuminuria and the incidence of analysis and of these patients more than half suffered diabetes. Compared heart failure (HF) is poorly understood. In the present study, the association to the placebo or no interference, both the newer and established vitamin D between baseline urinary albumin excretion and a risk of hospitalization for sterols reduced proteinuria to a similar extent (RR, 2.00; 95% CI, 1.42 to 2.81). incident HF was examined in patients with type 2 diabetes. Among patients No decrease in the glomerular fi lter rate was observed (SMD, -0.10; 95%CI, admitted to our department, 998 type 2 diabetes patients who were -0.24 to 0.03), and the risk for dialysis initiation was 1.48 (95% CI, 0.54 to asymptomatic and underwent screening 24-hour urinary albumin excretion 4.03) with vitamin D treatment. Additionally, there was an increased risk of were selected and the effect of baseline albuminuria on a HF event rate was hypercalcemia for patients treated with either newer or established vitamin analyzed at a mean follow-up of 5.6 years after discharge. Multivariable Cox D compounds as compared to the controls (RR, 4.78; 95% CI, 2.20 to 10.37). regression models were performed to examine the independent association The head-to-head studies showed no differences in the effects of treatment between baseline albuminuria and HF incidence during follow-up. Among with either the newer or established vitamin D compounds on proteinuria all subjects (mean age 66.5 years; 66.5% men; HbA1c 8.7%), 127patients or the risk of hypercalcemia. Thus both newer and established vitamin D (12%) were hospitalized for HF. When the study population was categorized therapy appears to decrease proteinuria and have no negative infl uence on to normal group (urinary albumin creatinine ratio (ACR) < 30 mg/day), renal function in pre-dialysis patients with diabetic nephropathy and other microalbuminuria group (30 ≤ ACR < 300 mg/day) and macroalbuminuria kidney diseases. But the occurrence of hypercalcemia should be evaluated group ( 300 mg/day ≤ ACR), Kaplan-Meier estimation shows that the risk when vitamin D is provided. No superiority for newer versus established of future HF hospitalization was signifi cantly increased in patients with vitamin D analogues is found. macroalbuminuria (p<0.001; log-rank test). Cox regression analysis revealed that the association between macroalbuminuria and incident HF was 562-P independent of age, gender, hypertension, smoking, estimated glomerular Glucose Profi le in Patients With Type II Diabetes and End Stage fi ltration rate, HbA1c and left ventricular ejection fraction at baseline Renal Disease: Comparison between Hemodialysis Sessions and (p=0.03). It is, therefore, concluded that, in patients with type 2 diabetes, Inter-Hemodialysis Periods macroalbuminuria is tightly associated with incident HF independently of AGNES SMAGALA, HENRIETTE SISSOKO, JEAN-PIERRE LEFLOCH, LAURENCE cardiorenal function and glucose control. KESSLER, FRANÇOIS CHANTREL, Colmar, France, Mulhouse, France, Paris, France, Strasbourg, France 560-P The main diffi culty to obtain adequate glycemic control in diabetic patients Skin Autofl uorescence Refl ects Microvascular Impairment in Pa- with end stage renal disease (ESRD) treated by hemodialysis is due to the tients With Diabetes consecutive dialysis and inter-dialysis periods. In this population, glucose JAN SKRHA JR., JAN SOUPAL, GABRIEL LONI EKALI, MARTA KALOUSOVA, profi le is not well documented. MARTIN PRAZNY, JAN SKRHA, Prague, Czech Republic, Ayos, Cameroon We analyzed the glucose profi le by continuous glucose monitoring (CGM) Advanced glycation endproducts (AGEs) are involved in the pathogenesis during dialysis and inter-dialysis periods in patients with type II diabetes of diabetic complications. Their accumulation in the skin augments skin and ESRD. autofl uorescence (AF). The aim of this study was to evaluate skin AF in CGM (Navigator, Abbott) was performed in 33 diabetic patients (14 patients with diabetes in respect of their microvascular impairment. female/19 male, age: 66±8yrs, diabetes duration: 23±11yrs, hemodialysis Skin AF was measured in 60 diabetic patients (34 Type 1 /T1DM/, 26 duration: 3.8±2.6yrs) during 54 hours, including 2 consecutive hemodialysis Type 2 /T2DM/; aged 54 ± 11 yrs) and 19 healthy controls (aged 46 ± 15 sessions at 0, 1 and 3 months with determination of the mean CGM glucose yrs) on forearm by AGE-Reader (Diagnoptics BV, Groningen, Netherlands). value, maximal glucose excursion, MAGE during consecutive dialysis and Results were expressed as arbitrary units (AU) and compared with age, inter-dialysis periods. Comparisons were done using ANOVA for repeated glycated hemoglobin HbA1c (expressed in IFCC units), diabetes duration and measures. parameters of microangiopathy and neuropathy. Albuminuria was expressed Mean glucose value of 78 CGM was 171±36 mg/dL with coeffi cient of as albumine-creatinine ratio (ACR) and neuropathy as vibratory perception variability: 34±9% and MAGE: 104±42 mg/dL. During dialysis sessions, threshold (VPT) by Biothesiometer. mean glucose value was signifi cantly lower: 136±46mg/dL vs. 171±35 mg/ Skin AF was signifi cantly higher in both T1DM and T2DM as compared to dL between two dialysis sessions (p< 0.001). Standard deviation, coeffi cient healthy controls (2.35 ± 0.47 AU, 2.57 ± 0.60 AU vs 1.96 ± 0.33 AU, ANOVA of variability and MAGE signifi cantly decreased by 35±5mg/dL (p<0.001), p=0.0002). Mean HbA1c was similar in both types of diabetic patients (74 ± 20±1% (p<0.001) and 58±7mg/dL (p<0.001) respectively. Percentage of 14 vs 71 ± 21 mmol/mol, NS). Interestingly, patients with normal albuminuria glucose value < 60mg/dL increased signifi cantly during dialysis: 4.4±9.6 % (ACR<2.5 g/mol creatinine) had signifi cantly lower AF in comparison to vs. 2.1±7.9% between two dialysis sessions (p<0.001) with a mean glucose patients with positive (micro)albuminuria (T1DM: 2.28 ± 0.43 vs 2.63 ± 0.54 value signifi cantly higher: 49±10mg/dL vs. 37±24 mg/dL (p< 0.001). AU, p=0.043; T2DM: 2.45 ± 0.53 vs 3.00 ± 0.69 AU, p=0.019). Similarly, Conclusions: During hemodialysis sessions, mean glucose CGM value patients without neuropathy (VPT<25 V) had lower AF as compared to and glucose variability are improved in patients with type II diabetes and patients with neuropathy (T1DM: 2.30 ± 0.40 vs 2.80 ± 0.87 AU, p=0.05; ESRD. Hypoglycemic risk increases but low glucose values are less severe. T2DM: 2.49 ± 0.68 vs 2.53 ± 0.59 AU, p NS). Signifi cant positive relationship The use of 100mg/dL glucose dialysate solution in all dialysis sessions may was found between AF and both HbA1c (r=0.45, p<0.001), (micro)albuminuria contribute to the decrease of glycemic variability. CGM allows documenting (r=0.55, p<0.001) and VPT (r=0.31, p<0.02) in patients with diabetes. the glycemic status in this group of patients with high cardiovascular risk. Skin autofl uorescence is signifi cantly elevated in patients with both diabetic nephropathy and neuropathy. Therefore it could be a convenient tool for easy evaluation of microvascular impairment risk in patients with diabetes. However, long-term follow-up studies are warranted.

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563-P 565-P Estimation of Glomerular Filtration Rate in Type 1 Diabetic Patients Pitavastatin Improves the Estimated Glomerular Filtration Rate With Normal Renal Function (eGFR) in Patients With Type 2 Diabetes and Hypercholesterolemia FOTIOS ILIADIS, ALEXANDRA NTEMKA, CHARALAMBOS MARGARITIDIS, ARETI Treated With Sitagliptin MAKEDOU, EUSTRATIOS MORALIDIS, MARIA DIVANI, ANNA GOTZAMANI- SOICHI KURIOKA, YASUO OHYAMA, AKIRA ICHIBANGASE, HIDEHO MURATA, PSARAKOU, APOSTOLOS HATZITOLIOS, DIMITRIOS GREKAS, TRIANTAFILLOS NOBUHIKO KATAOKA, HIROAKI SHIMOMURA, HIDEAKI SAWAKI, TAMAKI IMAI, DIDANGELOS, Thessaloniki, Greece SHINICHIRO OHASHI, Osaka, Japan

The MDRD equation was compared with the CKD-EPI equation and with Previous studies have shown that statins improve renal function in patients POSTERS a cystatin C based formula (TAN equation) for glomerular fi ltration rate with chronic kidney disease. In this study, we examined the renal effect of Complications estimation (eGFR) in type 1 diabetic patients with normal renal function (GFR pitavastatin in patients with type 2 diabetes and hypercholesterolemia, who Acute and Chronic ≥ 90 ml/min per 1.73 m2 without micro- or macroalbuminuria). had already been treated with or without the DPP-4 inhibitor sitagliptin. We studied 78 type 1 diabetic patients (44 were men) with mean (SD): The subjects in this study were 81 patients with type 2 diabetes and age 33.6 (8.8) years, BMI 26.1(4.3) Kg/m2, HbA1c 6.6 (1.7)%, ACR (albumin hypercholesterolemia. Twenty-nine patients were treated with sitagliptin creatinine ratio) 5.6 (4.2) mg/gr creatinine. GFR was measured using plasma (SITA) and 52 patients were not (no-SITA). The patients were treated with clearance of 51Cr-EDTA (mGFR). GFR was estimated using MDRD (MDRDGFR), 2mg pitavastatin for 6 months, and we evaluated the effects on eGFR, lipid CKD-EPI (CKD-EPIGFR) and TAN (cystCGFR) equations. All calculations were profi le, and glycemic control. Age (SITA 62±12 vs. no-SITA 63±10 years, performed using SPSS 17.00 and p<0.05 was taken to indicate statistical p=0.71), male gender (55 vs. 56 %, p=0.96), body mass index (26.2±5.0 vs. signifi cance. 26.1±3.9 kg/m2, p=0.90), fasting plasma glucose (FPG; 178±57 vs. 151±57 MGFR, MDRDGFR, CKD-EPIGFR and cystCGFR were 106.2 (10.3), 123.0 mg/dl, p=0.05), HbA1c (7.5±1.4 vs. 7.9±1.4%, p=0.21), total cholesterol (TC; (26.2), 120.3 (12.5) and 105.2 (22.5) ml/min per 1.73 m2 respectively (p<0.05 254±39 vs. 243±34 mg/dl, p=0.20), triglyceride (TG; 189±93 vs. 178±101 for difference between mGFR and MDRDGFR - CKD-EPIGFR). Bias (mean mg/dl, p=0.62), HDL-C (54±13 vs. 56±16 mg/dl, p=0.68), LDL-C (164±36 vs. difference between estimated GFR and mGFR) was 16.8, 14.1 and -0.9 ml/min 155±27 mg/dl, p=0.22), apolipoprotein B-100 (ApoB; 134±26 vs. 126±20 mg/ per 1.73 m2 for MDRDGFR, CKD-EPIGFR and cystCGFR respectively (p<0.05 dl, p=0.15), serum creatinine (Cr; 0.83±0.27 vs. 0.79±0.25 mg/dl, p=0.57), and for difference in bias between MDRDGFR and cystCGFR and between CKD- eGFR (71.3±23.2 vs. 73.3±20.1 ml/min/1.73m2, p=0.69) were not signifi cantly EPIGFR and cystCGFR). Precision (SD of the bias) was 25.9, 14.4 and 23.3 different between the two groups. A signifi cant increase of the eGFR was ml/min per 1.73 m2 for MDRDGFR, CKD-EPIGFR and cystCGFR respectively observed in the SITA group (p<0.001), but not in the no-SITA group (p=0.15), (p<0.05 for difference in precision between MDRDGFR and CKD-EPIGFR and the changes of Cr (-0.13±0.16 vs. -0.03±0.15 mg/dl, p=0.0079) and eGFR and between cystCGFR and CKD-EPIGFR). Accuracy 30% (proportion of (+12.9±16.3 vs. +2.8±14.2 mg/dl, p=0.0049) were signifi cantly different in estimated GFR results within 30% of mGFR) was 74.2%, 87.9% and 83.3% the two groups after 6 months of pitavastatin treatment. Changes of lipid for MDRDGFR, CKD-EPIGFR and cystCGFR respectively (p<0.05 for difference profi le and glycemic control were not signifi cantly different between the in accuracy 30% between CKD-EPIGFR and MDRDGFR). two groups. These fi ndings suggest that combination therapy of pitavastatin TAN cystatin C based formula was less biased than MDRD and CKD-EPI and sitagliptin may exhibit a renal protective effect in patients with type 2 equation. On the other hand CKD-EPI equation was more precise than MDRD diabetes and hypercholesterolemia. and TAN equation. Moreover, CKD-EPI equation presented higher accuracy 30% than MDRD equation. These results support the superiority of CKD-EPI 566-P and cystatin C based equations over MDRD equation for GFR estimation in Time Course of Antiproteinuric Effect of Aliskiren in Arterial Hy- type 1 diabetic patients with normal renal function. pertension Associated With Type 2 Diabetes and Microalbuminuria ROBERTO FOGARI, AMEDEO MUGELLINI, ANNALISA ZOPPI, PAOLA PRETI, PA- 564-P MELA MAFFIOLI, TIZIANO PERRONE, GIUSEPPE DEROSA, Pavia, Italy A Novel Therapeutic use of Liraglutide for the Prevention of Pro- The aim of this study was to compare the antiproteinuric effect of gression of Overt Diabetic Nephropathy in Patients With Type 2 aliskiren and ramipril in hypertensive patients with type 2 diabetes and Diabetes microalbuminuria. SHIGEKI IMAMURA, KEIJI HIRAI, AIZAN HIRAI, Togane, Japan One hundred and thirty eight patients were treated with aliskiren 300 mg Diabetic nephropathy (DN) is the leading cause of end-stage renal once a day or ramipril 10 mg once a day for 12 weeks and checked after 1, 2, disease worldwide. When a patient develops overt diabetic nephropathy 4, 8, 12 weeks and 2 and 4 weeks after treatment withdrawal. with persistent macroalbuminuria, kidney function starts to decline. After We evaluated clinic and ambulatory blood pressure, urinary albumin the introduction of antihypertensive therapy including blockade of the renin- excretion rate (UAER), plasma aldosterone. angiotensin system (RAS), the mean rate of decline in GFR was improved We observed that both aliskiren and ramipril induced a similar lowering from 10-20 ml/min per year to 2.0-10 ml/min per year. However still further in clinic and ambulatory blood pressure (p<0.001 vs baseline). However, clinical trials are performed for the improvement in clinical outcome. We such a lowering persisted longer after stopping aliskiren than after stopping demonstrated that 6 months administration of liraglutide to overt DN in ramipril regimen. Both treatments reduced UAER, but the decrease in UAER type 2 diabetic patients decreased proteinuria in the ADA’s 72nd scientifi c associated with aliskiren was more pronounced, the difference vs ramipril sessions. We studied the effect of long term administration of liraglutide on being maximal at week 12 (-42% vs -15%, p<0.01). Two weeks after stopping proteinuria and the progression of overt diabetic nephropathy (DN) in 23 type therapy, UAER remained below baseline values with aliskiren, but not with 2 diabetic patients (male:13 and female:10), who were already treated with ramipril. Plasma aldosterone decreased in the aliskiren group, whereas combining dietary sodium restriction and blockade of RAS. Patient education in the ramipril group it decreased until week 8 and thereafter increased for salt restriction was performed with EHR-based intensive coaching towards baseline values. program “TOGANE”. In the present study, the primary endpoint is the rate We can conclude that aliskiren has a greater and more prolonged of decline in renal function, while a reduction in proteinuria is a surrogate antiproteinuric effect than ramipril; it might partly be related to a higher endpoint. Liraglutide were administered over 12 months. Administration of degree of intrarenal RAAS blockade. liraglutide caused a signifi cant decrease in HbA1c levels from 7.45 ± 0.21 % to to 6.88 ± 0.25 % (p<0.05), and in BMI from 27.6 ± 0.85 kg/m2 to 26.5 ± 567-P 0.8 kg/m2 (p<0.001). Systolic blood pressure (SBP) and estimated glomerular Time Difference of Glycemic Control on Renal Function Changes in fi ltration rate (eGFR) did not change. UP was signifi cantly decreased from Type 2 Diabetic Outpatients 2.39 ± 0.49 g/g to 1.38 ± 0.27 g/g after 12 months (p<0.01). The administration CHIA-LIN LEE, TSAI-CHUNG LI, SHIH-YI LIN, JUN-SING WANG, I-TE LEE, LI-NIEN of liraglutide caused a signifi cant improvement in the rate of the decline in TSENG, YUH-MIN SONG, WAYNE H.H. SHEU, Taichung, Taiwan estimated GFR from -6.58 ml/min/1.73m2/year to 0.33 ml/min/1.73m2/year Background: Benefi ts of glycemic control have been widely reported for (p<0.01). To our knowledge, the present study demonstrated for fi rst time diabetic nephropathy. However, previous studies demonstrated opposing that liraglutide may have a possible role in the prevention of the progression correlation between HbA1C and GFR in cross-sectional and longitudinal of overt DN in type 2 diabetic patients. condition. This study aims to establish a model that explains the controversial effects of glycated hemoglobin (HbA1C) on GFR. Methods: This retrospective cohort study followed subjects with type 2 diabetes mellitus, enrolled between June 2006 and December 2006, for four years. The effects of HbA1C on estimated GFR (eGFR) were examined both

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A143 COMPLICATIONS—NEPHROPATHY—CLINICAL AND TRANSLATIONAL RESEARCH

cross-sectionally and longitudinally. The dual effects of HbA1C on eGFR, and decrease of estimated glomerular fi ltration rate (eGFR) was mainly related how renal function interferes with these effects, were investigated. to that of CV (r=0.821 p=0.000) than CEC (r=0.469 p=0.03), indicating renal Results: Of the 1992 subjects enrolled, 1699 completed the follow-up. atrophy mainly occurs in the cortex due to glomerular degeneration. We HbA1C was positively correlated with eGFR in the cross-sectional study (β analyzed the sensitivity of cystatinC, ICVR and sCr to detect eGFR <60 on the coeffi cient=1.44, 95% CI: 0.71 to 2.17, p=0.0001). In the longitudinal study, ROC curve. The area under the curve was 0.979 (CI: 0.958-0.998) in cystatin higher baseline HbA1C resulted in a greater decline in eGFR. The annual C, 0.918 (CI: 0.866-0.921) in ICVR, 0.858 (CI: 0.794-0.921) in sCr, respectively. eGFR decline rate was -1.89, -1.29, and -0.68 ml/min/1.73m2/year for Thus, both cystatin C and ICVR are more useful markers than sCr in diagnosing baseline HbA1C of >9%, 7% to <9%, and <7%, respectively. The eGFR value CKD. In stage2 CKD, positive microalbuminuria were signifi cantly related to POSTERS

Complications was simultaneously affected by concurrent (β coeffi cient=0.78, 95% CI: 0.48 cystatin C in Spearman’s rank correlation coeffi cient but not to ICVR or sCr, Acute and Chronic to 1.08, p<0.0001) and preceding HbA1C (-0.52, -0.82 to -0.23, p<0.0001). The suggesting Cystatin C may be a useful marker to detect early glomerular positive effects of concurrent HbA1C on eGFR reached statistical signifi cance dysfunction. In summary, we could evaluate diabetic nephropathy more at all chronic kidney disease (CKD) stages; however, the negative effects of accurately in combination with cystatin C and ICVR, instead of sCr. preceding HbA1C only applied to CKD stages 3 and 4. Conclusions: We developed a new model that demonstrates how 570-P preceding and concurrent HbA1C simultaneously affect eGFR in opposing Comparison of Different Estimating Equations for Glomerular Filtra- ways. The dynamic effects varied among different CKD stages. The tion Rate in Type 2 Diabetic Patients deterioration of eGFR at CKD stages 3 and 4 may be postponed by intensive XUANRONG CHEN, MANMAN WANG, LONGYI ZENG, Guangzhou, China glycemic control. There are several equations based on serum cystatin C(sCysC) or serum creatinine(Scr) used to calculate GFR. To determine the appropriate equations 568-P to estimate GFR in type 2 diabetes mellitus(T2DM), we evaluated different Obstructive Sleep Apnea and Diabetic Nephropathy: A Cohort GFR prediction equations widely used in patients with T2DM. From October Study 2008 to February 2011, a total of 279 type 2 diabetic patients admitted in ABD A. TAHRANI, ASAD ALI, NEIL T. RAYMOND, SAFIA BEGUM, KIRAN DUBB, Q. the 3rd Affi liated Hospital of Sun Yat-Sen University were investigated. The A. ALTAF, MILAN K. PIYA, ANTHONY H. BARNETT, MARTIN J. STEVENS, Birming- sCysC was measured by particle enhanced turbidmetric immunoassay; the ham, United Kingdom, Coventry, United Kingdom Scr was determined enzymatically and GFR was estimated by radiological Diabetic nephropathy (DN) is a leading cause of end-stage renal disease marker 99mTc-DTPA. The GFR was estimated using the Cockroft-Gault (CG) (ESRD). Obstructive sleep apnea (OSA) is common in patients with type 2 equation, the Modifi cation of Diet in Renal Disease (MDRD) equations, the diabetes (T2DM). OSA is associated with increased oxidative and nitrosative Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, the stress and impaired microvascular regulation. Hence, it is plausible that OSA Stevens’ equation and the Rule’s equation. The consistence and accuracy promotes the development and progression of DN. within 50% between equations based on sCysC and 99mTc-GFR were A cohort study assessing the relationship between OSA and DN in compared. The participants were 53% male, had mean age 59.9±10.79 years, patients with T2DM. Patients with known respiratory disorders (including Scr 104.13±45.48umol/L, sCysC 1.38±0.43mg/L and 99Tc-GFR 69.8±20.98 ml/ OSA) or ESRD were excluded. DN was defi ned as the presence of albuminuria min. Receiver operating characteristic (ROC) curves were plotted to evaluate (urinary albumin creatinine ratio >3.4 mg/mmol) or an estimated glomerular accuracy of predicting moderate renal damage(GFR<60ml/min). The area fi ltration rate (eGFR) <60 ml/min/1.73 m2. DN progression was based on under curve(AUC) of EPI equation and RuiJin equation were larger than those eGFR measurements. of equations based on sCysC, MDRD equation and CG equation(EPIAUC0.995, 224 patients were included. The prevalence of OSA and DN at baseline RuiJinAUC0.994, sCysCAUC0.961, MDRDAUC0.969, CGAUC0.976, p<0.0001). was 64.3% (n=144) and 40.2% (n=90), respectively. DN prevalence was By using Bland-Altman analysis, Stevens’ equation showed the least higher in patients with OSA (OSA+) compared to those without OSA (OSA-) mean bias(-0.8) while Rule’s and Stevens’ equation showed the best 50% [49.3%, n=71 vs. 23.8%, n=19, p<0.001]. Following adjustment, OSA (OR 2.37, accuracy(88.9% and 89.6% respectively). In different stages of renal 95%CI 1.07-5.21, p=0.03) remained independently associated with DN. function, equations based on sCysC provide better accuracy estimates After an average follow up of 2.5 (0.7) years, eGFR decline was greater in predicting GFR than others based on Scr. In all, the best correlation is in OSA+ compared to OSA- patients (median (IQR)) (-6.8% (-16.1% to 2.2%) Rule’s equation. No advantage has been proven by using equations based vs. -1.6% (-7.7% to 5.3%), p=0.002). After adjusting for baseline eGFR and on combining sCysC with Scr, age, sex and race in predicting GFR when other confounders, baseline OSA (B=-3.8, p=0.044) and AHI (B=-4.6, p=0.02) comparing to the equations including sCysC only. remained independent predictors of study-end eGFR. Baseline Serum nitrotyrosine abundance (B=-0.24, p=0.015) was an 571-P independent predictor of study-end eGFR after adjustment. Nephropathy in Australian Aboriginal and Anglo-Celt Patients With OSA is independently associated with DN in patients with T2DM. eGFR Type 2 Diabetes: The Fremantle Diabetes Study Phase II declined faster in patients with T2DM complicated by OSA. Nitrosative TIMOTHY M.E. DAVIS, KERRY HUNT, DANIEL MCAULLAY, WENDY A. DAVIS, Fre- stress may provide an important pathogenetic link between OSA and mantle, Australia, Perth, Australia DN. Interventional studies to assess the impact of OSA treatment on the Nephropathy frequently complicates type 2 diabetes in Aboriginal patients development and progression of DN are needed. but there has been no detailed comparison of its prevalence and predictors Supported by: National Institute for Health Research between racial groups in a multi-cultural Australian setting. We studied 105 Aboriginal type 2 patients (mean±SD age 54.2±11.9 years, 34.3% males) and 569-P 827 Anglo-Celts (ACs; aged 67.5±10.6 years, 51.4% males) from the community- Inverse of Renal Cortical Volume Ratio Is a New Index to Detect based Fremantle Diabetes Study Phase II. All had valid data including serum Glomerular Impairment in Type 2 Diabetic Nephropathy creatinine (from which estimated glomerular fi ltration rate (eGFR) was TOSHIMASA FUJIWARA, Chiba, Japan calculated using the Chronic Kidney Disease (CKD) Epidemiology Collaboration To estimate the relationship between renal atrophy and the function, we equation) and urinary albumin:creatinine. The percentage of Aboriginal patients measured renal cortical volume by echosonography and assessed its relation with an eGFR <60 ml/min/1.73m2 (26.9%) was similar to that in the AC group to renal markers. Consecutive 200 outpatients (male; 118, female; 82) with (32.2%; P=0.31), but the Aboriginal patients were more likely to have albuminuria type 2 diabetes were recruited (except stage 4~5 CKD). Their backgrounds (normo-/micro-/macro- 37.6/36.6/25.8 % vs 60.2/34.5/5.4 %, respectively, were as follows; Age:67.8±9.5 years old, HbA1c: 7.31±0.71%, and treatment P<0.001). Based on the Kidney Disease Improving Global Outcomes prognostic duration:13.1±7.1 years. We assumed a kidney as an ellipsoidal body, and its 3 CKD categories, 19.6% of Aboriginal patients were at very high risk (eGFR 30- semi-principal axes were measured. Renal volume (RV) was calculated by the 59 and at least microalbuminuria, eGFR <30, or renal replacement) vs 10.0% of ellipsoidal body formula and divided by body surface area. Mean calculated ACs (P<0.001). In a logistic regression model adjusting for potential confounders RV is almost similar to previuos reported one measured in dissected kidneys. including age and diabetes duration, Aboriginality was associated with an odds The central echo complex volume (CEC: assumed as a composite of renal ratio of 6.72 (95% CI 2.86-15.78) for very high risk of CKD (P<0.001). In individual pelvis and collecting duct) was also calculated, and Cortical volume (CV: logistic regression models in the two groups, a very high risk of CKD was assumed as a composite of glomerulus, renal tubules and arterioles) was independently and positively associated with retinopathy and systolic blood defi ned as RV minus CEC, and CVratio (CV divided by RV) was calculated. pressure in the Aboriginal group (P≤0.012), and with age, diabetes duration Then, inverse of CVratio (ICVR) was calculated to be compared with serum and serum HDL-cholesterol (inversely) in the AC group (P<0.001). Intermittent creatinin (sCr) and cystatinC and its relationship was investigated. The claudication was a signifi cant associate in both (P≤0.003). The substantially

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A144 COMPLICATIONS—NEUROPATHY increased risk of CKD in type 2 indigenous Australians may refl ect an increased COMPLICATIONS—NEUROPATHY susceptibility to microvascular complications in general and to the effects of systolic hypertension in the case of nephropathy. Supported by: NHMRC (Australia) Guided Audio Tour: Neuropathy—Novel Tests and Novel Therapeutic Targets (Posters: 574-P to 581-P), see page 19. 572-P Urinary Podocyte Excretion Predicts Worsening of Albuminuria in Patients With Type 2 Diabetes & 574-P POSTERS MASAO TOYODA, HAN MIYATAKE, MASUMI KONDO, EITARO TANAKA, MASAA- Corneal Confocal Microscopy Is as Profi cient as Electrophysiology Complications KI MIYAUCHI, MORITSUGU KIMURA, TOMOYA UMEZONO, MASANORI HARA, and Skin Biopsy in Detecting Neuropathy in Subjects With Type 1 Acute and Chronic DAISUKE SUZUKI, MASAFUMI FUKAGAWA, Isehara, Japan, Niigata, Japan Diabetes Mellitus Recent studies reported that the presence of podocytes in the urine UAZMAN ALAM, IOANNIS N. PETROPOULOS, HASSAN FADAVI, OMAR ASGHAR, refl ects severe injury of these cells in glomeruli. Therefore, examination of ANDREW MARSHALL, GEORGIOS PONIRAKIS, AHMED AL-AHMAR, AHMAD the relationship between urinary podocyte excretion and the development KHEYAMI, MARYAM FERDOUSI, SHAZLI AZMI, MITRA TAVAKOLI, ANDREW J.M. and progression of diabetic nephropathy is clinically important. Research on BOULTON, RAYAZ A. MALIK, Manchester, United Kingdom the relationship between urinary albumin excretion and urinary podocyte We have compared nerve conduction studies (NCS), Intra-epidermal nerve excretion in diabetic patients is limited to cross-sectional studies. fi bre density (IENFD) and Corneal confocal microscopy (CCM) to detect nerve In the present longitudinal study, we investigated how well urinary fi bre damage in Type 1 diabetic patients with subclinical neuropathy. podocyte excretion refl ects changes in urinary albumin excretion rate and The neuropathy symptom profi le (NSP), neuropathy disability score (NDS), progression of nephropathy. vibration perception threshold (VPT), warm and cold thresholds (WT, CT), For this purpose, we measured the number of podocytes in urine samples from NCS, IENFD and CCM were assessed in 75 age-matched (43.6±14.5yrs) in 150 patients with variable severity of type 2 diabetic nephropathy, ranging controls (C) and 87 subjects with Type 1 Diabetes (T1DM) (47.2±15.7yrs, from normoalbuminuria to end-stage renal failure. We also measured serial Duration of Diabetes 32.0±16.6yrs). changes in urinary albumin in patients with microalbuminuria [urinary albumin Compared to controls, subjects with T1DM had higher HbA1c (%) (8.3±1.4 excretion (UAE) rate: 30-300 mg/g Cr] during a follow-up period of 6 months. vs 5.6±0.3, p<0.0001), systolic BP (mmHg) (131±19 vs 124±19, P=0.02) and A signifi cant increase in UAE was noted during follow-up in micro- triglycerides (mmol/l) (1.5±0.7 vs 1.2±0.6, P=0.004). NSP (3.7±5.1 vs 0, albuminuric patients (UAE: 30-300 mg/g Cr) with podocyteuria, but not in p<0.0001), NDS (3.3±3.2 vs 0.5±0.9, p<0.0001), VPT (Volts) (14.4±12.6 vs those without podocyteuria. 5.5±4.5, P<0.0001) were increased and sural nerve amplitude (µV) (9.7±6.7 The results suggest that measurement of the number of podocytes in vs 21.0±9.6, P<0.0001), sural nerve conduction velocity (m/s) (43.5±5.6 vs urine samples seems useful for assessment of podocyte injury and the 51.0±4.4, p<0.0001), peroneal nerve amplitude (mV) (4.1±6.4 vs 5.4±1.9, risk of overt albuminuria in microalbminuric type 2 diabetic patients. The P<0.0001) and peroneal nerve conduction velocity (m/s) (40.3±7.2 vs 48.9±3.6, results also suggest that urinary podocyte excretion may be a marker for the p<0.0001) were reduced, although they fell within the normal range. CT (ºC) development of albuminuria in microalbuminuric type 2 diabetic patients. (25.2±5.8 vs 28.4±2.1, P<0.0001) and WT (39.7±4.2 vs 36.7±2.9, P<0.0001) Long-term observation of this group and further studies are required to were increased and IENFD(mm2) was reduced (5.6±4.2 vs 8.4±4.4, P=0.03) elucidate the clinical and predictive value of podocyteuria as a marker for compared to controls. the development of albuminuria. Using the rapid, non-invasive test of CCM we found a reduction in corneal nerve fi bre density (no.mm2) (25.1±9.4 vs 37.4±6.1, P<0.0001); nerve fi bre 573-P length (mm/mm2) (18.9±6.7 vs 26.2±5.2, P<0.0001); nerve branch density A Mathematical Model of Renal Disease Progression and Its Valida- (55.6±32.0 vs 90.5±36.1, P<0.0001) and an increase in nerve fi bre tortuosity tion (17.0±6.9 vs 14.8±3.2, P=0.003) in T1DM vs controls. KENNY SHUM, STUART SAMUEL, San Francisco, CA We demonstrate evidence of signifi cant small fi bre neuropathy in subjects The Archimedes nephropathy model was updated to incorporate with T1DM and subclinical neuropathy, which was readily detected using the new evidence on chronic kidney disease (CKD). It models the chances of novel rapid reiterative technique of Corneal Confocal Microscopy. developing (i) long-term persistent microalbuminuria, (ii) long-term persistent Supported by: JDRF macroalbuminura, (iii) CKD Stage3 and (iv) ESRD, as well as the evolution of the urinary albumin to creatinine ratio (UACR) and the glomerular fi ltration & 575-P rate (GFR). The model was built using results from the Chronic Kidney Disease In-Vivo Corneal Confocal Microscopy as a Novel Non-Invasive Tool Prognosis Consortium and data from NHANES 1999-2008 and the United to Study Autonomic Nerve Function in Type 1 Diabetes States Renal Data System (USRDS). Risk variables are: gender, race/ethnicity, ERNESTO MADDALONI, FRANCESCO SABATINO, ROSSELLA DEL TORO, STEFA- age, diabetes, smoking, HbA1c, systolic blood pressure, UACR and GFR. We NIA GRANDE, ANDREA PALERMO, ANGELO LAURIA, ANNARITA MAURIZI, SIL- validated the model against NHANES III, EPIC-Norfolk, the Indian Medical VIA MANFRINI, STEFANO BONINI, PAOLO POZZILLI, Rome, Italy Registry, the Pathways Study, MESA and PREVEND (results not shown). The Cardiac Autonomic Neuropathy (CAN) is a serious diabetes-related validations for ESRD (See Table) highlight two challenges. First, the defi nition complication due to small (A , B and C) fi bre nerves damage. Its early of ESRD in many research settings tends to be broader than the one in USRDS, δ detection seems essential to stratify patients for risk of premature death. leading to a lower predicted rate. Second, there are large variations in ESRD Tests currently available to study small fi bres status, such as the ex vivo incidences in different countries. Calibration by country could lead to better confocal microscopy of skin biopsy, could result too invasive to be performed predictions. However, after adjusting for baseline risk factors, the Archimedes in routine practice. Model was still unable to predict the ESRD rate in ACCORD, indicating that In-Vivo Corneal Confocal Microscopy (IVCCM) is an innovative and non- either ACCORD is an outlier or there are inherent characteristics of the ACCORD invasive technique proved as a good marker for diabetic sensorimotor population that the model fails to capture. When ACCORD data becomes neuropathy, allowing earlier diagnosis. The peculiar feature of IVCCM is its publicly available, we shall be able to analyze this issue in more detail. ability in detecting presence and grade of C and Aδ fi bres damage. Validation results for ESRD incidence The aim of this study is to investigate the correlation between corneal small nerve fi bre degeneration signs detected with IVCCM and cardiac Study Arm / Subgroup N Duration Observed Predicted Ratio autonomic function in subjects with type 1 diabetes (T1D). (year) Incidence (%) Incidence (%) (Pred/Obs) IVCCM was performed to assess corneal innervation pattern in terms of ACCORD Standard control 5115 5.0 3.0 0.6 0.18 beedings number, nerve refl ectivity, length and tortuosity; clinical tests of ADVANCE Placebo 5571 4.3 0.4 1.1 2.73 cardiac autonomic function were performed to assess heart rate variation ALLHAT Chlorthalidone 15255 6.0 1.8 1.3 0.72 with deep breathing (E:I ratio), blood pressure and heart rate (30:15 ratio) response to standing. ARIC All 15321 15.1 1.6 0.9 0.59 Of the 18 T1D subjects enrolled in the study (age: 36.2±11.5 years; BMI CHS GFR 1268 8.9 4.7 4.4 0.94 23.7±3.5 Kg/m2; disease duration 19.7±10.8 years; HbA1c 7.4±1.1%), 22% (n=4) RENAAL Placebo 762 3.4 30.9 24.0 0.78 showed pathological results at least one of the autonomic test performed, RENAAL Lorsartan 751 3.4 23.1 20.4 0.88 suggesting an early impairment of autonomic function. In the whole study population the number of nerve beedings in 100 µm was signifi cant related

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A145 COMPLICATIONS—NEUROPATHY

with both 30:15 ratio (r=0.470, p=0.04) and E:I ratio (r=0.501; p=0.03). No signifi cant correlations were found between the other variables studied. & 578-P Results of this study show that beedings number counted with IVCCM Identifi cation of Factors Associated With Sural Nerve Regeneration is related with the autonomic response to provocative tests, supporting and Degeneration in Diabetic Neuropathy IVCCM as a new non-invasive tool to accurately quantify nerve morphology JUNGUK HUR, KELLI A. SULLIVAN, RODICA POP-BUSUI, BRIAN C. CALLAGHAN, and hence as an aid in defi ning the autonomic nerve function in T1D. EVA L. FELDMAN, Ann Arbor, MI Diabetic neuropathy (DN) is the most common debilitating and costly complication of diabetes. Patients with DN demonstrate variable degrees

POSTERS & 576-P

Complications of nerve regeneration and degeneration; however, risk factors affecting Transplantation of Induced Neural Crest Cells from Spheroidal Der- Acute and Chronic these processes are not clearly understood. To identify risk factors mal Stem Cells Improves Diabetic Polyneuropathy in Mice closely associated with sural nerve regeneration in patients with DN, we MASAKI KONDO, HIDEKI KAMIYA, TATSUHITO HIMENO, TETSUJI OKAWA, JIRO retrospectively examined the demographic, anthropometric and biochemical KATO, ATSUSHI HUJIYA, YOJI HAMADA, KEIKO NARUSE, NAOMI NISHIO, SA- data of diabetic subjects from a double-blind, placebo-controlled, 52-week CHIKO ITO, YUTAKA OISO, KEN-ICHI ISOBE, JIRO NAKAMURA, Nagoya, Japan, trial of acetyl-L-carnitine. Based on the change of sural nerve myelinated Nagakute, Japan fi ber density ( MFD%), subjects were divided into three groups: Regenerator Background and Aims: We have identifi ed and established spheroidal Δ (top 16 percentiles, n=67), Degenerator (bottom 16 percentiles, n=67), and dermal stem cells (sDSCs) from skin that possess partial pluripotency, Intermediate (n=290), with dramatically increased, decreased, and steady and succeeded to induce neural crest cells (NCs) from sDSCs. Here we MFD%, respectively. Fisher’s exact test, ANOVA, and multifactorial investigated the effects of NCs transplantation on diabetic polyneuropathy Δ logistic regression analyses were performed to identify signifi cant risk (DPN) in mice. factors. MFD%s were 35.6±17.4 (Regenerator), -4.8±12.1 (Intermediate), Materials and Methods: We cultured dermal cells from 5-week old EGFP- Δ and -39.8±11.0 (Degenerator). HbA1c at baseline was the only risk factor C57BL/6 male mice and formed 2x104 dermal cells into spheroids (sDSCs). signifi cantly different between Regenerator (8.3±1.6%) and Degenerator NCs were induced from sDSCs by co-culturing with PA 6 for 10 days. Diabetes (9.2±1.8%), p=0.01 with other factors adjusted. Support Vector Machine was induced by intraperitoneal injection of STZ to 8-week old C57BL/6 male classifi er using HbA1c alone demonstrated 62.4% accuracy of classifying mice. Then, NCs (1x103 cells per cluster) or saline were injected into the right subjects into Regenerator or Degenerator in a 20-fold cross-validation. or left hindlimb muscles (10 clusters/limb) of 12-week STZ diabetic (D) and A preliminary microarray experiment based on a subset of these nerve age-matched normal (N) mice, respectively. Four weeks later, thermal planter samples revealed that the up-regulated genes in Regenerator are enriched test (TPT), nerve conduction velocities (MNCV and SNCV) and histological in cell cycle and myelin sheath functions, while the down-regulated genes image analysis in hindlimb tissue were evaluated. are enriched in immune/infl ammatory responses. In conclusion, these data, Result: sDSCs expressed several Embryonic stem cell (ESC)- specifi c based on the largest available cohort with MFD information, suggest that transcription factors, the pluripotency markers. Induced NCs expressed AP2, HbA1c level predicts myelinated nerve fi ber regeneration and degeneration Foxd3, Snail and Sox10 that are neural crest cell markers. Four weeks after in patients with DN and that maintaining optimal blood glucose control is transplantation, NCs resided in the injected muscles and some showed CD34 essential in patients with DN to prevent continued nerve injury. or αSMA positive. And we did not fi nd teratoma or other tumors formation. D Supported by: JDRF showed impaired thermal sensation (TPT; N: 5.5±1.7 s, D: 9.6±3.6) indicating hypoalgesia, and delayed MNCV (N: 53.6±6.9 m/s, D: 36.9±6.7) and SNCV (N: 32.9±4.9 m/s, D: 21.9±2.7), which were signifi cantly ameliorated by NCs & 579-P transplantation (TPT: 5.2±1.1, MNCV: 48.1±5.5, SNCV: 33.7±5.8). Prevalence and Risk Factors of Peripheral and Cardiovascular Au- Conclusion: These results indicate that transplantation therapy with tonomic Neuropathy in Newly Diagnosed Diabetic Patients: The induced NCs from sDSCs would be safe and effective for DPN. GDS Cohort STEFANIE NOWAK, MARSEL SCHEER, BETTINA NOWOTNY, KARSTEN MÜSSIG, & 577-P MICHAEL RODEN, DAN ZIEGLER, GDS GROUP, Düsseldorf, Germany There is Little information on the prevalence and risk factors of diabetic Metabolomic Analysis of Glycolytic Pathway Responsible for Dia- distal symmetric sensorimotor polyneuropathy (DSPN) and cardiovascular betic Neuropathy beyond Polyol Pathway Hyperactivity autonomic neuropathy (CAN) in newly diagnosed diabetic patients using HIROKI MIZUKAMI, KENTARO TSUBOI, KAZUNORI SANGO, SOOKJA K. CHUNG, comprehensive phenotyping. We aimed to evaluate the prevalence and risk SOROKU YAGIHASHI, Hirosaki, Aomori, Japan, Setagaya, Japan, Hong Kong, China factors of DSPN and CAN in subjects with type 1 and type 2 diabetes (T1D, Clinical application of aldose reductase (AR) inhibitor disclosed its effi cacy T2D) with a diabetes duration <1 year participating in the German Diabetes on diabetic neuropathy (DN) but not under poor glycemic control. Similarly, Study (GDS) at baseline. The sample studied comprised 154 subjects with AR-defi cient mice (ARKO) are protected from the development of neuropathy T1D (age: 34.5±11.6 (mean±SD) years, 63% male, HbA1c: 6.9±1.6%) and 348 in diabetic condition but not under prolonged period. It is therefore likely that subjects with T2D (age: 52.6±10.5 years, 64% male, HbA1c: 6.5±1.1%). DSPN metabolic pathway beyond polyol pathway operates in the pathogenesis was assessed by motor and sensory nerve conduction velocity (MNCV, of DN. To explore a new mechanism of DN, comprehensive metabolomic SNCV) in 4 nerves, vibration perception threshold (VPT), thermal detection analysis was conducted on the sciatic nerve in diabetic ARKO. ARKO and thresholds (TDT), Neuropathy Symptom Score (NSS), and Neuropathy control C57BL/6J mice (WT) 6 wks of age were made diabetic (DM) by Disability Score (NDS), while CAN was determined by autonomic function streptozotocin (i.p.). Over 16 wk diabetic duration, motor nerve conduction tests (AFT). Normal limits adjusted for age, sex, height, and BMI were velocity (NCV) was monitored and at the end of experiment sciatic nerve defi ned at the 2.5th or 97.5th percentiles in 148 (NCV) and 88 (VPT, TDT, was procured for comprehensive metabolomic analysis by capillary AFT) healthy subjects. The most frequently abnormal nerve function tests electrophoresis with mass spectrometer. At 12 wk diabetic duration, there were: sural SNCV: T1D: 33%, T2D: 31%; peroneal MNCV: T1D: 17%, T2D: was a signifi cant delay of NCV in diabetic WT but not in diabetic ARKO. At 19%; malleolar VPT T1D: 17%, T2D: 24%; and cold TDT (foot): T1D: 38%, T2D: 16 wk diabetes, both diabetic WT and ARKO showed a signifi cant NCV delay. 21%. The prevalence of DSPN (Toronto criteria, 2011) was: subclinical (stage By metabolomic analysis, there was marked accumulation of glucosamine 1a) DSPN: T1D: 27%, T2D: 19%; asymptomatic (stage 1b) DSPN: T1D: 2% (>10x vs non-diabetic ARKO and non-diabetic WT), galacturonic acid (>10x), T2D: 7%; symptomatic (stage 2) DSPN: T1D: 3%, T2D: 7%. Asymptomatic sarcosine (x4), and 2-deoxyglucose 6-phosphate (x3) in both diabetic ARKO CAN was observed in 8% in T1D and 13% in T2D. Risk factors signifi cantly and WT. On the other hand, there was a marked decrease in carnitine (0.6x associated with DSPN in multivariate models included age and height, while vs non-diabetic ARKO and non-diabetic WT) and 2-aminoadipic acid (0.7x) in age and male sex were related to CAN. In conclusion, polyneuropathy is both diabetic ARKO and WT. There were no signifi cant differences in these encountered in one third of type 1 and type 2 diabetic patients within 1 metabolites between diabetic ARKO and diabetic WT. As oxidative stress year after diagnosis of diabetes, but remains subclinical or asymptomatic in pathway, both reduced and oxidized glutathione were increased in both the majority of the cases. Effective measures to timely prevent these early diabetic groups and the ratio of reduced/oxidized form was kept constant. nerve alterations should be adopted. There was no signifi cant change in the ratio of intermediates in TCA cycle Supported by: German Federal Ministry of Education and Research between diabetic and non-diabetic groups. Under hyperglycemia, collateral glycolytic pathway is activated to produce several candidates independent of polyol pathway. Among those, glucosamine may be one of possible targets for the cause of DN.

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A146 COMPLICATIONS—NEUROPATHY

582-P & 580-P Mitochondrial DNA Deletion Burden Correlates With Diabetic Neu- Rho GTPase-Activating Protein DLC2 Defi ciency in Mice Leads to ropathy (DPN) Risk Factors and Predicts DPN Progression More Severe Painful Diabetic Neuropathy MADHAVI JINKA, Baltimore, MD LEE S. TIRRELL, CHI WAI CHEUNG, SOOKJA K. CHUNG, Hong Kong, China Objective: Mitochondrial (mt) dysfunction has been linked to numerous DLC2, a Rho GTPase-activating protein with specifi c activity for RhoA, has diseases including neuropathy. We hypothesized that mtDNA damage been shown to play a role in pain response. Mice defi cient for this protein burden is associated with known DPN risk factors and that baseline mtDNA (DLC2 KO) are hyperalgesic compared to wild type in acute pain tests,

deletion burden (mtDNA_db) would predict DPN progression POSTERS showing higher response to noxious thermal and infl ammatory stimuli, while Methods: MtDNA_db was assessed from distal leg biopsy samples Complications also showing an increase in RhoA activity in peripheral nerves. Because in two well-characterized DPN populations. First, a cross sectional study Acute and Chronic of the increased acute pain response in DLC2 KO mice, and since RhoA of 59 subjects with a broad range of DPN severities was studied. Linear is known to be involved in the development and maintenance of diabetic regression was used to assess correlations between mtDNA_db and known complications, we used them to study chronic pain in a model of diabetic DPN risk factors. Second, 22 subjects mild DPN and 1-year follow-up. neuropathy. Wild type (DLC2 WT) and DLC2 KO mice were subjected to Worsening or stable NCV and IENFD respectively defi ned ‘progressors’ or multiple low dose streptozotocin (MLDS) treatment to induce diabetes. ‘non-progressors’. Logistic regression was used to assess whether baseline After 8 weeks, their pain response was tested. From the hot plate test, mtDNA_db predicted progression of DPN. The 4977bp common mtDNA diabetic DLC2 KO mice (DLC2 KO; STZ) were found to be hyperalgesic to deletion product was normalized to the internal control HVR2, using the thermal stimuli compared to diabetic DLC2 WT mice (DLC2 WT; STZ) as 2-Δct method. well as vehicle-treated controls of both genotypes (DLC2 KO; Veh and DLC2 Results: DM duration (p=0.03), epidermal denervation (p=0.003) were WT; Veh). DLC2 KO; STZ also displayed mechanical allodynia compared to signifi cantly associated with mtDNA_db in the cross-sectional study. In DLC2 WT; STZ and both control groups from the von Frey fi lament test. A a multivariate modeling a composite score of DM duration, smoking, and qPCR screen was then conducted using dorsal root ganglia (DRG) from these HTN was also signifi cant (p=0.01). There was no association between age mice. Gene expression of the voltage-gated sodium channel Nav 1.9 was or sural amplitude and mtDNA_db. Baseline A1C and mtDNA_db were shown to be signifi cantly higher in DLC2 KO; STZ compared to DLC2 WT; signifi cantly associated with progression of neuropathy in a multivariate STZ, while there was a trend of increased levels in DLC2 KO; Veh versus logistic regression (p=0.05). DLC2 WT; Veh. This channel, located mainly in nociceptors of the DRG, is Interpretation: These results show a strong correlation between DPN risk known to lower the threshold for action potentials, thus playing a role in factors and mtDNA deletion burden. Also, mtDNA deletion burden predicted sensitizing neurons and possibly leading to the observed heightened pain neuropathy progression among a small sample of subjects. Together, these response. Taken together, diabetic DLC2 KO mice have more severe painful x-sectional and longitudinal study results are complementary and support diabetic neuropathy, with thermal hyperalgesia and mechanical allodynia. a causal relationship between mtDNA_db and DPN and imply that small This may be explained by higher expression of Nav 1.9 mRNA. Future studies caliber sensory nerve fi bers are particularly vulnerable to mt dysfunction. will further examine changes in sodium channels and other pain mediators that lead to this altered pain response. Supported by: Research Grants Council of Hong Kong (to S.K.C.) 583-P Effects of Antioxidant Alpha-Lipoic Acid on Heart Rate Variability in Type 2 Diabetic Patients With Cardiac Autonomic Neuropathy & 581-P CHONG HWA KIM, SU JIN JUNG, KYUNG WAN MIN, BONG YUN CHA, Bucheon, Patient Perspectives on Pain Severity in Diabetic Peripheral Neu- Republic of Korea, Seoul, Republic of Korea ropathy Diabetic cardiac neuropathy, which is characterized by reduced heart ALESIA SADOSKY, JOSEPH HOPPER, BRUCE PARSONS, New York, NY, Evanston, IL rate variability (HRV), frequently coexists with peripheral neuropathy. We An online survey was conducted in 2012 in the United States to aimed to evaluate the effi cacy of oral treatment with the antioxidant alpha- characterize the patient perspective of diabetic peripheral neuropathy (DPN) lipoic acid (ALA) in type 2 diabetics with cardiac autonomic neuropathy and its painful symptoms. Patients with Type 1 or 2 diabetes experiencing (CAN), assessed by heart rate variability (HRV).In a randomized, double-blind any symptoms of DPN were recruited via the Survey Sampling national placebo-controlled multicenter trial, type 2 diabetic patients with reduced consumer research panel. The survey included questions on DPN symptoms HRV were randomly assigned to treatment with daily oral dose of 1200 mg and patient-physician discussion of the patient’s DPN. Of 1,004 respondents, ALA (n = 46) or placebo (n = 45) for 6 months. CAN was assessed by the fi ve 83% reported painful DPN symptoms and 77% reported it impacted daily tests according to the Ewing’s protocol and the time and frequency domain activities. However, only 41% of patients with painful symptoms were of the heart rate variability (HRV) was evaluated.All the baseline measures diagnosed with DPN. 47% of the respondents who reported pain (393 of were similar between groups, except for the low-frequency band (LF). After 832; 52% female, average age 56 years) were recontacted to obtain further 6 months of treatment with ALA, some HRV parameters showed some information on pain severity (numerical rating scale; 0 = no pain, 10 = most improvement. The standard deviation of normal-to-normal RR intervals pain); approximately half (49%) of these patients reported severe pain, and (SDNN; ms) in standing position increased from baseline to 6months by 1.9 13% and 38% reported mild or moderate pain, respectively. At increasing ms (-27.9 to 33.8) in the group given ALA and decreased by -4.0 ms (-35.6 pain severity, greater proportions of patients reported symptoms typical of to 24.0) in the placebo group (p=0.06 for ALA vs. placebo). Power spectrum neuropathic pain. Among the patients with severe pain, 47% reported these in the LF band in standing position increased by 15.8 ms2 (-74.6 to 210.5) in symptoms as always being painful, and 67% reported experiencing them ALA, whereas it declined by -15.0 ms2 (-175.0 to 139.0) in placebo (p= 0.08 “all or most of the time.” Impact of pain on function was greater at higher for ALA vs. placebo). Furthermore, there was a trend toward a favorable pain severity levels; signifi cantly (p<0.05) more patients with severe pain effect of ALA versus placebo for high frequency(HF) band power spectrum (46%) reported “a lot” of impact on work relative to those with mild (4%) and in supine position (P = 0.091 for ALA vs. placebo). No differences between moderate (16%) pain, and sleep was affected “a lot” in 45% of patients with the groups were noted regarding the rates of adverse events.High doses of severe pain relative to mild (2%; p<0.05) and moderate (15%; p<0.05) pain. ALA not only well-tolerated oral dose of 1200 mg/day for 6 months but also Although discussion of DPN symptoms with their physician was reported may slightly improved heart rate variability in type 2 diabetic patients with more often with increased pain severity, less than one-third (32%) of patients cardiac autonomic neuropathy. with severe pain discussed their symptoms in detail with their physician. These fi ndings suggest a need to improve the patient/physician dialogue for discussing DPN that is distinct from the underlying diabetes, and a need for better approaches to manage the painful symptoms associated with DPN.

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584-P 586-P Early Non-Progressive Small Fibre Neuropathy in Type 2 Diabetes Prevalence of Diabetes in Patients Admitted to the Hospital With SHAZLI AZMI, UAZMAN ALAM, HASSAN FADAVI, OMAR ASGHAR, IOANNIS N. Primary Diagnosis of Orthostatic Hypotension PETROPOULOS, GEORGIOS PONIRAKIS, ANDREW MARSHALL, MARYAM FER- ADITI GUPTA, JANICE GILDEN, Chicago, IL DOUSI, AHMAD KHEYAMI, AHMAD ALAHMAR, MITRA TAVAKOLI, ANDREW Autonomic neuropathy is a chronic complication of long standing Diabetes J.M. BOULTON, RAYAZ A. MALIK, Manchester, United Kingdom (DM). Orthostatic Hypotension (OH) which can result from autonomic We have undertaken a longitudinal study utilising neurological assessment neuropathy is a signifi cant cause of morbidity in the elderly population.

POSTERS (Neuropathy Disability Score (NDS), QST (VPT, cold and warm temperature Therefore we looked at the prevalence of DM in patients admitted to the

Complications threshold (CT and WT)), electrophysiology and in particular corneal confocal hospital with the primary diagnosis of OH. Acute and Chronic microscopy (corneal nerve fi bre density (FD), branch density (BD), fi bre length We analyzed data from the discharge weighted Nationwide Inpatient (FL) and tortuosity (FT) to explore the natural history of diabetic neuropathy Sample (NIS) from the year 2010. Patients with the primary discharge in relation to risk factors in 25 type 2 diabetic patients and 17 age and sex diagnosis of Orthostatic Hypotension were identifi ed using ICD9 code 4580. matched controls assessed at baseline, 12 and 24 months. Differences in demographics which included age and sex as well as the There was no signifi cant difference for age (63.8±7.2 v 61.7±5.3, prevalence of Hypertension (HTN), Hypoglycemia and autonomic neuropathy P=0.36), BP mmHg (132.4±18.4/72.9±9.9 v 137.5±17.3/77.1±10.4, P=0.6) were compared between patients with and without DM. and triglycerides (1.9±1.3 v 1.7±0.7, P= 0.82) but a signifi cant difference in The discharge weighted data showed 69,615 patients were admitted with cholesterol (4.0±0.9 v 5.3±0.9, P<0.0001) and HDL-C (1.2±0.3 v 1.6±0.4, P= the primary diagnosis of orthostatic hypotension , of which 31.4% (21,859) 0.0005). HbA1c(%) (7.7±1.4 v 5.8±0.2, P=0.0001) was increased in diabetic were diabetic. The demographic data shows that diabetic patients were patients with duration of diabetes (15±10yrs). With regards to neuropathy younger (Median age 73 yrs vs 77 yrs) and had a higher percentage of male. assessment: NDS (2.9±2.4 v 1.4±1.4, P=0.03), WT (41.8±3.4 v 37.7±3.8, In addition, while the prevalence of HTN (77.1% vs 66.6%) and autonomic P=0.0007) were increased and CNFD (no.mm2) (27.5±10.3 v 37.3±7.2, neuropathy (4.9% vs 1.4%) was higher in diabetic patients with Orthostatic P=0.002) was decreased, but there was no difference for VPT (13.1±7.7 v hypotension, the prevalence of hypoglycemia (0.1% Vs 0.4%) was lower in 9.2±6.0, P=0.076), CT (26.5±2.1 v 27.2±2.1, P=0.028), sural nerve amplitude these patients. In addition peripheral neuropathy was present in 12% of (µV) (10.5±5.8 v 12.8±6.1, P=0.26), sural nerve velocity (NV) (m/s) (46.7±0.1 v diabetic patients with OH. 49.2±0.1, P=0.56), CNBD (no.mm2) (74.7±36.1 v 96.8±35.5, p=0.06) or CNFL Our analysis shows a signifi cant percentage of patients admitted to the (mm/mm2) (22.3±8.1 v 26.8±4.7, p=0.06). At 24 months, no signifi cant change hospital with primary diagnosis of orthostatic hypotension also have DM. was noted in NDS (0/10) (3.8±2.8), VPT(14±6.7), CT (26.2±1.9), WT (41.9±2.9), sural NV(m/s) (44.2±3.9), or amplitude (11.2±5.9). There was no signifi cant 587-P change in corneal FD (28.0±8.2), BD(74.0±31.2) or FL (22.36±6.3). Effect of Gastric Bypass Surgery on Diabetes Neuropathy This longitudinal study shows thermal threshold testing and CCM detects LING L. CHUAH, NAJAH BAQAI, CHRISTOPHER GRAHAM, ALEX D. MIRAS, ALES- early small fi bre damage in Type 2 diabetic patients without an abnormality of SIA NICOTRA, NOFAL M. KHALIL, CAREL W. LE ROUX, London, United Kingdom, vibration perception or neurophysiology. There is no signifi cant deterioration Dublin, Ireland in neuropathy over 24 months, likely refl ecting overall good control of Roux-en Y gastric bypass surgery(RYGB) is known to improve glycaemic glycemia, blood pressure and lipids. control in obese patients with Type 2 diabetes (T2DM); however, its effects Supported by: NIH; JDRF on diabetes related peripheral neuropathy are not known. In previous studies, rapid improvements in glycaemia through medical therapy resulted in 585-P paradoxical deterioration in microvascular complications. Induction of rapid Novel Phosphodiesterase-5 Inhibitor Avanafi l Resorted the Erectile glycaemic control by RYGB may therefore have similar harmful effects. Dysfunction in the Neonatal Non-Insulin Dependent (Type 2) Dia- We investigated the impact of RYGB on peripheral neuropathy in obese betic patients with T2DM. The primary endpoint was the presence of neuropathy SERAP GUR, DIDEM YILMAZ, NUR BAYATLI, OZGE UN, Ankara, Turkey and the secondary outcomes were body mass index (BMI) and HbA1c. Type 2 diabetes mellitus (T2DM) is a chronic disease, and the prevalence Thirty-two patients underwent nerve conduction studies (NCS) and of erectile dysfunction (ED) is three times higher in this population. The thermal threshold testing (TTT) to assess large and small nerve fi ber function streptozotocin (STZ)- induced T2DM rats in the neonatal period develop preoperatively and at 1 year. the hyperglycemia, abnormal glucose tolerance and insulin resistance in Of these, 5 had peripheral neuropathy preoperatively; and 7 at 1 year adulthood. Although diabetic ED in T1DM animal models has been studied (p=0.75). Compared to baseline, 29 patients had no change, and 3 deteriorated extensively, ED in the neonatal T2DM model was not examined. The aim of at 1 year. Eighteen patients also underwent TTT. Of these, 8 had abnormal this study to investigate the success of novel PDE5 inhibitor avanafi l on in TTT preoperatively; 4 with normal pre-operative TTT developed abnormal vivo erectile function and in vitro isolated corpus cavernosum smooth muscle TTT postoperative (p=0.31). RYGB achieved signifi cant reduction in BMI and (CCSM) responses in T2DM model. T2DM was induced by the administration HbA1c at 1 year. (p<0.0001) of 90 mg/kg STZ (i.p.) in two-day-old rats. Male Sprague Dawley rats were In conclusion, our preliminary results do not show signifi cant deterioration divided into two groups: age matched-control (6 mo-old) and T2DM (6 mo- in large and small fi ber function in obese T2DM patients at one year old) at 12 weeks after inducing diabetes. The functional activities were post surgery. RYGB appears to be safe for diabetes related peripheral tested in vivo by intracavernous pressure (ICP) recording and in vitro via neuropathy. organ bath contractility studies after avanafi l (10µM). T2DM in rats signifi cantly attenuated in vivo erectile response to cavernous nerve stimulation, which was returned after intracavernous administration of avanafi l. Diabetic CCSM displayed decreased ACh and EFS responses after precontraction with phenylephrine as compared controls. In the presence of avanafi l (10µM), diminished responses to neurogenic and endothelium-dependent-relaxations were normalized. Our study validates the effect of intracavernous administration of novel PDE5 inhibitor avanafi l for diabetes-associated ED in an T2DM animal model. The present data also give support to the in vitro observation that diabetes impairs neurogenic and endothelium-dependent responses, precluding the complete recovery of erectile function with PDE5 inhibitors and explaining the relatively poor clinical response of diabetic men with ED to PDE5 inhibition. Supported by: Ankara University

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A148 COMPLICATIONS—NEUROPATHY

588-P glucose fl uctuation may be more dangerous than constant high glucose (HG). Relationship between Toe-Brachial Index and Peripheral Neuropa- The aim of this study was to explore the protective effects of Salvianolic thy in Patients With Diabetes Mellitus acid B (Sal B, one of the major water-soluble compounds of Radix Salviae XIAOYING HE, ZHIMIN HUANG, AILING CHEN CHEN, WEIJIAN KE, YANBING LI, Miltior r hiz ae) on inter mit tent high glucose (IHG) and HG induced mito chondrial Guangzhou, China pathway activation and Schwann cells (SCs) apoptosis. SCs were were Aims: Ankle-brachial index(ABI) and Toe-brachial index(TBI) are very treated for 48 hrs consistently with 5.6mM of glucose, with 50mM of useful clinical test to assess the arterial blood supply to the foot. Diabetic glucose as HG, with 5.6 and 50mM glucose altering every 8 hrs as IHG, with IHG or HG in the presence of 0.1 µM, 1 µM and 10 µM of Sal B respectively. peripheral neuropathy (DPN) is a common microvascular complication(6), but POSTERS little is known about the association of DPN with TBI and ABI. This study Intracellular ROS generation was detected using fl uorescent probe DCFH- Complications was conducted to investigate the diagnostic value of TBI and ABI in DPN. DA and DHE. 8-OHdG was determined using ELISA kit as oxidative stress Acute and Chronic Methods: A retrospective cross-sectional study was conducted involving marker. RT-PCR was performed to analyze the expression levels of Bax and 213 patients with diabetes mellitus who had undergone ABI and TBI( BcL-2. Western blot were performed to analyze the expression levels of measured by Doppler ultrasound) from July 2011 to December 2012. The some important transcription factors. Our data indicated that IHG and HG correlation of decreased TBI and ABI with DPN was analyzed. DPN was induced high levels of ROS production and mitochondrial dysfunction, which diagnosed on the basis of neuropathic symptoms, insensitivity to a 10-g triggered high frequency of SCs apoptosis.The cytotoxic effect of IHG was monofi lament, and abnormal neurophysiological examinations. Data were signifi cantly more potent than that of HG and is a direct effect of glucose on expressed as means±standard deviation for normally distributed data and as the cell. Treatment with Sal B inhibited the IHG-induced oxidative stress by median(interquartile range) for non-normally distributed data. Independent reducing ROS production and 8-OHdG levels, mitochondrial depolarization t-test or rank sum test was used to compare demographic and clinical and apoptosis in both the caspase-dependent and the AIF-mediated caspase- parameters. Receiver operating characteristic(ROC) curve analysis was used independent pathway. More importantly, Sal B inhibited the activation of to evaluate the sensitivity and specifi city of TBI and ABI in diagnosing DPN. molecular pathways of apoptosis in a dose-dependent manner.Our fi ndings Results: Of 213 total patients with diabetes, 68.4% had DPN. Patients provide remarkable evidence that Sal B exhibits a protective effect on SCs with DPN had higher TBI than those without DPN in rank sum test(0.626 exposed to IHG and HG. vs.0.784, p=0.034); however, ABI was not signifi cantly different between patients with or without DPN. ROC analysis determined an area under the 591-P curve equal to 0.736, with an optimal cut-off value of 0.608 that yielded a Severe Symptomatic Diabetic Gastroparesis: Comparison of Three sensitivity of 72.7% and a specifi city of 84.0%. Gastric Emptying Methods Applied Simultaneously to Nine Type 1 Conclusion: The results of this study suggested that TBI may be a valuable Diabetes Patients tool for evaluating diabetic peripheral neuropathy. NIELS EJSKJAER, HENNING GLERUP, GERDA VILLADSEN, KARIN HJORTHAUG, HENRIK BLUHME, JENS FREDERIK DAHLERUP, Aarhus, Denmark 589-P Objective: Diabetic gastroparesis is highly prevalent and is believed to Macrophages Promote a Pro-Infl ammatory Microenvironment in greatly impact glycaemic control. A better understanding of the clinical the Peripheral Nervous System in a Mouse Model of Type 2 Diabetic relevance of delayed gastric emptying requires accurate, accessible and Neuropathy inexpensive methods. This study compares three methods for assessing DIANE E. BENDER, JACQUELINE R. DAUCH, WILSON HSIEH, BRANDON M. YA- gastric emptying: The paracetamol absorption test, the 13C-acetate breath NIK, ZACHARY A. KELLY, YU HONG, HSINLIN T. CHENG, EVA L. FELDMAN, Ann test and the scintigraphy gastric emptying method. Methods: Nine type 1 Arbor, MI diabetes patients suffering severe symptoms of diabetic gastroparesis Diabetic peripheral neuropathy (DN), one of the most common complica- were consecutively recruited. Demographic, clinical data, symptom scores tions associated with diabetes, will ultimately affect 50% of the 18.8 and medication was recorded. After an overnight fast, gastric emptying was million people in the United States with diabetes. Therapies for DN have measured simultaneously by the three methods in each subject. A liquid meal demonstrated little promise and effectiveness. We believe that infl ammation containing 5 mg/ml paracetamol, 75 mg 13C-natrium acetate and 20 MBq drives the development and progression of DN; however information is 99mTc-DTPA was ingested and subject was placed in the gamma camera and lacking regarding the major cellular immune mediator of this process and blood samples for paracetamol and breath tests were obtained. A gamma its functional role. A murine model of type 2 diabetes and DN, the leptin camera was used until no radioactivity was traced in the gastric region. receptor-defi cient C57BKS-db/db mouse, develops allodynia at 8-10 wks of Blood samples were analysed for serum paracetamol concentration. Breath age followed by sensory loss and nerve conduction defi cits at 21 to 24 wks samples were analysed using an isotope-selective infrared spectrometer. of age. We used this model to test our hypothesis that in type 2 diabetes Results: The data will be presented as percentage retained ventricular macrophages populate the peripheral nervous system, resulting in nerve contents as a function of time. A gastric emptying time-retention curve is damage and the signs and symptoms of DN. Quantitative comparisons of drawn for each technique and the results are compared at the 75%, 50% and sciatic nerve sections from non-diabetic control mice (db/+) and diabetic mice 25% retention quartiles demonstrating. The 50% quartile shows superiority (db/db) processed for macrophage markers F4/80 and cell differentiation 68 for the paracetamol test compared to the breath test. Only the scintigraphy (CD68) revealed no signifi cant changes in macrophage numbers per area in test reveals retention at the end of the test. Conclusion: The scintigraphy sciatic nerves at 5, 8, 16, 21, and 24 wks of age. Previous studies implicate test detects meal-retention in the stomach, whereas the paracetamol test the pro-infl ammatory cytokine tumor necrosis factor alpha (TNFα) as a major and the breath test do not. The paracetamol test is superior to the breath mediator of pain and neuronal injury. Western blot and RT-PCR analysis test in this study. In this study we found no fi rm association between gastric indicate TNFα is elevated in diabetic but not control sciatic nerves as early emptying rate and symptoms of diabetic gastroparesis. as 16 wks and remains signifi cantly increased through 24 wks. In parallel, by 21 wks, diabetic sciatic nerve macrophages expressed enhanced TNFα, 592-P suggesting a functional difference in diabetic macrophages and not their total Glucose Fluctuation, More Than Constant High Glucose, Induce number, may contribute to DN progression. We conclude that macrophages Apoptosis of Schwann Cells and Inhibitory Effects of Alpha Lipoic in the sciatic nerve produce excess TNFα, promoting a pro-infl ammatory Acid microenvironment that results in nerve injury. We are currently assessing JU M. LU, LIAN Q. SUN, Beijing, China the therapeutic effi cacy of blocking TNFα in the treatment of DN. The precise pathogenesis of diabetic peripheral neuropathy remains Supported by: NIH (2T32NS007222-31) unclear and hyperglycemia-induced overproduction of ROS may be the unifying link to diabetes complications. This study was to explore the effects 590-P of Alpha lipoic acid (ALA) on intermittent high glucose (IHG) and constant high Salvianolic Acid B Reduces Apoptosis of Schwann Cells Induced by glucose (HG) induced mitochondrial pathway activation and Schwann cells Intermittent or Constant High Glucose via Oxidative Stress (SCs) apoptosis.SCs were treated consistently with 5.6mM glucose, 50mM SUN L. QING, Beijing, China glucose(HG), alternating 5.6 mM or 50 mM glucose (IHG), with IHG or HG Diabetic peripheral neuropathy (DPN) is one of the most common and in the presence of ALA.ROS and 8-OHdG was detected as oxidative stress costly microvascular complications of diabetes and no effective therapy marker. Apoptosis was confi rmed by TUNEL method and some transcription exists for the treatment of DPN. Brownlee has pioneered the concept that factors were analyzed.Our data indicated that IHG and HG induced SCs hyperglycemia could stimulate high levels of reactive oxygen radical (ROS) apoptosis in both caspase-dependent and caspase-independent pathways. production in the mitochondria,which can lead to neuronal injury.Notably, More importantly, the cytotoxic effect of IHG was signifi cantly more potent

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A149 COMPLICATIONS—NEUROPATHY

than that of HG,which is not related to osmolarity.Treatment with ALA 594-P inhibited the HG and IHG-induced oxidative stress and apoptosis in SCs. Assessing Sudomotor Function to Screen for Microvascular Com- Furthermore, ALA down-regulated the release of cytochrome c, AIF nuclear plications in Type 2 Diabetes translocation and Bax expression, but up-regulated Bcl-2 expression. In VIJAY ERANKI, ISABELLE DUFAURE, JEAN-HENRI CALVET, Hyderabad, India, addition, ALA attenuated the activation of caspase-9 and caspase-3 and Paris, France minimized the cleavage of PARP. These fi ndings suggest that variability in Sweat glands are innervated by small C-fi bers, and sudomotor dysfunction glycemic control could be more deleterious than HG and ALA antagonized is one of the earliest abnormalities to manifest in distal small fi ber the IHG-induced activation of mitochondrial pathway and apoptosis in SCs.

POSTERS neuropathy. This study aimed to evaluate SUDOSCAN, a non invasive, quick,

Complications simple and quantitative method to measure sweat function, as a screening Acute and Chronic tool for microvascular complications in type 2 diabetes. 309 patients were evaluated for microvascular complications including peripheral neuropathy using a biothesiomether (Vibration Perception threshold > 15 V), nephropathy through measurement of creatinine clearance and calculation of Modifi cation of Diet in Renal Disease (MDRD < 60 ml/ mnx1.73 m2), and retinopathy through fundus of the eye examination. Small C-fi ber status was assessed through sudomotor function by measurement of hand and foot Electrochemical Sweat Conductance (ESC) and calculation of an autonomic risk score using SUDOSCAN. Hand and foot ESC were lower in patients with at least one microvascular complication as compared to patients without: 49 ± 20 vs 62 ± 17 µS, p <0.001 and 59 ± 21 vs 69 ± 15 µS, p<0.001 respectively. Receiver Operating Characteristics (ROC) curve for detection of at least one microvascular complication is displayed in Figure. Sensitivity and specifi city of autonomic risk score were 82% and 61% respectively. SUDOSCAN could be used for the screening of microvascular complications in type 2 diabetes and may aid in adhering to follow-up guideline recommendations which are currently unfulfi lled.

593-P Assessing Autonomic Dysfunction Using Survey of Autonomic Symptoms (SAS) Score at South Korean Diabetes SUNHEE KIM, HYE RYOUNG YUN, CHO OK BAEK, KYUNG AE LEE, HEUNG YONG JIN, HONG SUN BAEK, TAE SUN PARK, Jeonju, Republic of Korea Autonomic symptoms may occur frequently in diabetes. But, autonomic examinations are not done because of their diversity and complexity. Previously, various instrument to measure autonomic symptoms developed. Among them, the Survey of Autonomic Symptoms (SAS) is an easily administered instrument to measure autonomic symptoms in early diabetic neuropathy. Therefore, we studied the relationship with SAS (translation for Korean) and a series of autonomic tests at South Korean diabetes. The SAS consists of 11 items in women and 12 in men. Each item is rated by an impact score ranging from 1 (least severe) to 5 (most severe). SAS was tested in 30 healthy controls and 35 diabetic patients with neuropathy in Chonbuk National University Hospital at South Korea. The SAS was compared to a series cardiac autonomic tests respectively. Diabetic patients were mostly type 2 diabetes (94.3%) and duration was 595-P 10.3±8.8 year. SAS score increased at diabetic group compared to normal Hyperglycemia-Induced Tau Cleavage In Vitro and In Vivo: A Pos- group (p=0.001), especially vasomotor dysfunction (p<0.05). An increased sible Link between Diabetes and Alzheimer’s Disease SAS symptom score and total impact score were detected at diabetes with BHUMSOO KIM, CAREY BACKUS, SANGSU OH, EVA L. FELDMAN, Ann Arbor, MI cardiac autonomic neuropathy group, compared without them signifi cantly Multiple lines of evidence link the incidence of diabetes to the development (p=0.006, p=0.028 respectively). Among cardiac autonomic neuropathy of Alzheimer’s Disease (AD). Patients with diabetes have a 50 to 75% examinations, valsalva ratio item associated with SAS symptom score and increased risk of developing AD. In parallel, AD patients have a higher than total impact score signifi cantly (p=0.047, p=0.034 respectively). Among SAS normal tendency to develop type 2 diabetes or impaired fasting glucose. Tau symptom score, sudomotor dysfunction mostly affected at the diabetes with is the major component of neurofi brillary tangles (NFT), one of the hallmarks cardiac autonomic neuropathy group (92%). of AD pathology. The current study examined the effect of hyperglycemia The SAS is a valid, easily administered instrument to measure autonomic on tau modifi cation. Glucose treatment of rat embryonic cortical neurons symptoms at South Korean diabetes and would use of screening tool for results in concentration-dependent apoptosis and caspase-3 activation. assessing diabetic autonomic neuropathic symptoms. These changes are well correlated with glucose time- and concentration- dependent tau cleavage. Aβ treatment induces tau cleavage and when added together with glucose there is an additive effect on caspase activation, apoptosis and tau cleavage. Tau cleavage is partially blocked by the caspase inhibitor, ZVAD. Cleaved tau displays a punctate staining along the neurites and colocalizes with cleaved caspase-3 in the cytoplasm. Both type 1 and

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A150 COMPLICATIONS—NEUROPATHY type 2 diabetic mice display increased tau phosphorylation in the brain. In associated with SDNN (P <0.01), only 7.7% of subjects with CAN had agreement with the effects of glucose on tau modifi cations in vitro, there is abnormal CPT value at 5 Hz in C7 level. increased tau cleavage in the brains of ob/ob mice; however, tau cleavage is In conclusion, CPT at each frequency was signifi cantly associated not observed in type 1 diabetic mouse brains. Our study demonstrates that with neuropathic symptom or sign corresponding nerve fi ber stimulated. hyperglycemia is one of major factors that induce tau modifi cation in both However, current reference range of CPT should be re-evaluated due to its in vitro and in vivo models of diabetes. We speculate that tau cleavage in low sensitivity to detect diabetic neuropathy. diabetic conditions (especially in type 2 diabetes) may be a key link for the increased incidence of AD in diabetic patients. 598-P POSTERS Supported by: Michigan Diabetes Research and Training Center Cardiac Autonomic Neuropathy Is Not Associated With QTc Inter- Complications vals But Improved With Glycaemic Control Acute and Chronic 596-P KATHERINE A. VALEROS, CHIN MENG KHOO, Singapore, Singapore Diagnostic Performance of Sural Nerve Conduction Amplitude Is Cardiac autonomic neuropathy (CAN) is associated with higher mortality Maintained in the Elderly in diabetic patients. We examined whether CAN, as measured by the non- XUAN KONG, BONNIEJEAN BOETTCHER, KENNETH SNOW, SHAI GOZANI, invasive method, SUDOSCAN (Impeto Medical Paris France) was associated Waltham, MA with abnormal cardiac conduction (long QTc intervals). We also examined The clinical detection of diabetic peripheral neuropathy (DPN) in the the clinical predictors of CAN and determined the longitudinal change in elderly is challenging because of normal age related nerve degeneration that CAN with glycaemia. We recruited 73 patients with type 2 diabetes mellitus leads to false positives, such as with vibratory sensation testing. Sural nerve (DM) without history of ischemic heart disease (IHD). The mean(SD) age was conduction amplitude is a sensitive and specifi c biomarker for DPN, which 47.5(9.7) years, BMI 28.5(6.1) kg/m2, duration of diabetes 8.8(6.7) years and has recently become available as a point-of-care test. This study evaluated HbA1c 8.63(1.9)%. There were 13 (18%) patients with signifi cant CAN (i.e CAN the hypothesis that the diagnostic performance of sural amplitude is age score ≥35%). The CAN score was signifi cantly correlated with age (r=0.70), BMI independent. (r=0.44), SBP (r=0.47), triglyceride (r=0.25) and eGFR (r=-0.43) (all Ps<0.05). Age, The study was a retrospective cross-sectional comparison of sural BMI and SBP were independent predictors of CAN score (stepwise regression; amplitude in two population-based cohorts. The normal cohort consisted R2=0.82, p<0.001). There was no signifi cant correlation between CAN score of 527 subjects without clinical evidence of DPN. The diabetes cohort and QTc intervals. In a subset of patients (n=28), we examined whether the consisted of 1091 subjects, primarily with Type 2 diabetes, extracted from a CAN score changes with HbA1c at 3 month and 6 month of follow-up. There data registry (77 contributing clinics). All measurements were obtained using was a signifi cant effect of time on CAN score (p= 0.025). The interaction term identical methodology. The cohorts were divided into three age matched between CAN score and glycemic status was signifi cant (p<0.001) (Fig 1). groups (<45, 45-64, ≥65 years). In each group, the area under the ROC curve In summary, there is a high prevalence of CAN among DM patients without served as a measure of diagnostic performance. history of IHD. CAN appears to improve with a reduction in glycaemia. Greater The sural amplitude decreased from the youngest to oldest age groups in age and BMI, and higher SBP are associated with greater CAN score. both cohorts (42% in normal, 67% in diabetes). Sural amplitude discrimination in the ≥65 group was only slightly worse than the 45-64 group (0.85 vs. 0.88) and better than the <45 group (0.85 vs. 0.77). Although diabetes compounds normal age related nerve degeneration, sural amplitude enables evaluation of nerve function in all age groups.

Age (Years) Amplitude in Microvolts (N) ROC (95% CI) Normal Diabetes <45 19.7 ± 8.2 (257) 12.0 ± 7.8 (107) 0.77 (0.71 - 0.83) 45-64 16.2 ± 8.0 (155) 5.8 ± 5.8 (324) 0.88 (0.85 - 0.91) ≥65 11.5 ± 7.6 (115) 4.0 ± 4.5 (660) 0.85 (0.81 - 0.88)

597-P Low Sensitivity of Current Perception Threshold in the Assessment of Diabetic Neuropathy BO KYUNG KOO, JUNG HUN OHN, EUN KY KIM, EUN ROH, TAE JUNG OH, MIN KYEONG KIM, SU MIN HONG, MIN KYONG MOON, Seoul, Republic of Korea Current perception threshold (CPT) can be quantifi ed by stimulating Aβ fi bers at 2,000 Hz, Aδ fi bers at 250 Hz and C fi bers at 5 Hz. C fi bers primarily conduct temperature and pain sensation and also serve a role in autonomic nervous system. We aimed to evaluate the usefulness of CPT for diagnosis of distal polyneuropathy (DPN) and cardiovascular autonomic neuropathy 599-P (CAN) in diabetic patients. Effects of Alogliptin on Peripheral Sympathetic Nerve Activity in CPT was measured with Neurometer® (Neurotron Inc.) in index fi nger (C7 Streptozotocin-Induced Diabetic Rats level) and middle toe (L5 level). At each frequency, a CPT below or above HIROYUKI SASAKI, SATOMI KONDO, MAI KODAMA, DAISUKE SATO, MASATAKA the reference range provided by manufacturer considered abnormal. We KUSUNOKI, TAKAO NAKAMURA, Yamagata, Japan, Yonezawa, Japan, Nagoya, assessed DPN by the Neuropathy Total Symptom Score - 6 (NTSS-6) and Japan 10-g monofi lament pressure sensation. CAN was evaluated by spectral Incretin and incretin-related drugs for the treatment of type 2 diabetes analysis of heart rate variability in addition to heart rate and blood pressure may have a neuroprotective function. However, the effects of incretin on response to Valsalva maneuver, deep breathing, standing-up and exercise. nerve function have not been documented well. Finally, 337 diabetic patients aged 30 - 69 years were included in In the previous study, we reported that action potential (AP) rate of the our analysis after excluding subjects suspicious to have other cause of peripheral sympathetic nerve activity in streptozotocin-induced diabetic neuropathy. Among them, 5.3 % of subjects had NTSS-6 score > 6 and 12.8 (STZ) rats was lower than that in normal rats at steady state, but that burst % showed abnormal 10-g pressure sensation. Subjects with NTSS-6 > 6 rate was not. In addition, the AP rate was increased after intravenous showed signifi cantly higher CPT than subjects with NTSS-6 ≤ 6 at 250 Hz (P = glucose administration (IVGA) in normal rats but not in STZ rats. 0.027) and 5 Hz (P = 0.044) in L5 level. Abnormal 10-g monofi lament test In the present study, we evaluated the effects of alogliptin, a DPP-4 results was signifi cantly associated with CPT at 2,000 Hz both in C7 (P = inhibitor, on the peripheral sympathetic nerve activity in STZ rats. 0.006) and L5 level (P= 0.019). However, only 7.0% and 23.3% of subjects Streptozotocin (80 mg/kg) was intraperitoneally injected into male Wistar with abnormal 10-g monofi lament test had abnormal CPT at 2,000 Hz in C7 rats (8 weeks of age) to cause the pancreatic β-cell destruction. STZ rats and L5 level, respectively. Furthermore, although CPT at 5 Hz in C7 level were divided into two groups (ALO and Control): alogliptin (5 mg/kg) was was signifi cantly higher in subjects with CAN (P <0.01) and signifi cantly orally administered once a day for 4 weeks in ALO group. At the end of the

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A151 COMPLICATIONS—NEUROPATHY

4-week period, we microneurographically detected compound sympathetic 601-P signal in the unilateral sciatic nerve, and recorded it on PC before and after Euglycemic Therapy Restores Endothelial and Autonomic Function IVGA (0.25 ml of 50% glucose solution) for 60 min. The APs in sympathetic in Diabetic Rats signal were manually detected after a denoising procedure using wavelet AN-BANG LIU, HSIEN-TSAI WU, CYUAN-CIN LIU, YU-CHENG LIN, CHEUK-KWAN transform approach. SUN, Hualien, Taiwan, Taipei, Taiwan, Kaohsiung, Taiwan The results showed that: (1) blood glucose was more than 400 mg/dl in Background: Diabetes-associated endothelial and autonomic dysfunctions both groups; (2) plasma insulin was about 0.2 ng/ml in each group; (3) AP are major causes of mortality and morbidity in diabetic patients. In this study, the fi ring rate was signifi cantly higher in ALO group (706±466 ~ 745±491 spikes/

POSTERS impact of blood sugar control on endothelial and autonomic functions at acute

Complications min, n=6) than in Control one (215±84 ~ 264±129 spikes/min, n=7) throughout and chronic stage was assessed in streptozotocin (STZ)-induced diabetic rats. Acute and Chronic the recorded period (P < 0.05) whereas elevation of AP rate after IVGA was Methods: Five eight-week-old male Winstar Kyoto rats received not observed. intraperitoneal STZ and nicotinamide (NA), followed by weekly check of blood These results suggest that alogliptin may prevent severe peripheral sugar. Endothelial and autonomic functions were respectively assessed by neuropathy caused by hyperglycemia, and that alogliptin could insulin- reactive hyperemia-induced dilatation index (DI) and SD1/SD2 ratio (SSR) of independently enhance the peripheral sympathetic nerve activity in the Poinecaré plotting of R-R intervals from ECG at age of 8 weeks, 24 weeks, and hyperglycemic condition. 32 weeks. Five age-matched controls receiving intraperitoneal physiological saline only followed the same protocol. Effect of euglycemic therapy was 600-P assessed after subcutaneous insulin at age of 32 weeks. Subcutaneous Administration of Liraglutide Ameliorates Alzheim- Results: Diabetes mellitus (blood sugar >250 mg/dL) occurred 16 weeks er-Associated Tau Hyperphosphorylation in Rats With Type 2 Dia- after STZ-NA administration. Blood sugar of STZ-treated rats was much betes higher than that of age-matched littermates at age of 24 weeks (P<0.001). YANG YAN, MA DELIN, YUAN GANG, Wuhan, China There were also signifi cant differences in DIs (1.97±0.67 vs. 3.64±1.40, Type 2 diabetes (T2D) increases the risk for developing Alzheimer’s P<0.05) and SSRs (0.29±0.11 vs. 1.18±0.18, P<0.001) between these two disease (AD. Brain insulin resistance contributes to the pathogenesis groups. Two months latter, blood sugar in diabetic rats were still remarkably of AD, and abnormal hyperphosphorylation of tau protein is crucial to higher than that of age-matched controls before insulin treatment (P<0.001). neurodegeneration. Here we studied whether liraglutide, an agonist of There were also signifi cant differences in DIs (0.93±0.12 vs. 2.89±1.31, glucagon-like peptide-1 (GLP-1) and a new anti-diabetic drug, can promote P<0.05) and SSRs (0.39±0.10 vs. 0.91±0.32, P<0.05). After insulin treatment, brain insulin signaling and inhibit tau hyperphosphorylation in the brains of diabetic rats attained euglycemic status with similar blood sugar compared T2D rats. A rat model of T2D was treated with subcutaneous administration to that of normal controls. There was no signifi cant difference in DIs and of liraglutide (0.2 mg/kg body weight) twice a day for up to four weeks. We SSRs between these two groups. found decreased CSF insulin, hyperphosphorylation of tau at AD-associated Conclusions: We demonstrated that hyperglycemia caused vascular phosphorylation sites, and decreased phosphorylation of protein kinase endothelial and autonomic dysfunctions by measuring DIs and SSRs, B (AKT) and glycogen synthase kinase-3β (GSK-3β) in the brains, which respectively. Resuming a euglycemic status signifi cantly reversed these indicated decreased insulin signaling leading to over-activation of GSK-3β, dysfunctions even after chronic hyperglycemia. a major tau kinase, in T2D rats.Liraglutide treatment not only ameliorated Supported by: NSC (100-2221-E-303-001) hyperglycemia and peripheral insulin resistance, but also reversed these brain abnormalities in a time-dependent manner. Our results indicated that 602-P liraglutide not only restores peripheral insulin sensitivity, but also restored Dynamic Infrared Imaging to Quantitate Thermoregulatory Function impaired brain insulin signaling and ameliorates tau hyperphosphorylation in in Individuals With Diabetes for Preclinical Detection of Peripheral rats with T2D.These fi ndings support the potential use of liraglutide for the Neuropathy prevention and treatment of AD in individuals with T2D. MARK BURGE, KATHLEEN COLLERAN, EDUARDO S. BARRIGA, VIKTOR CHEKH, ELIZABETH MCGREW, ANNE EDWARDS, PETER SOLIZ, Albuquerque, NM This study examined recovery patterns of the plantar foot after a cold stimulus using functional infrared images to detect clinical and pre-clinical diabetic peripheral neuropathy (DPN). We hypothesized loss of neural thermoregulatory function would result in a reduced rate of recovery compared to controls. We enrolled N=12 with diagnosed DPN, N=16 with diabetes (DM, no DPN), and N=13 controls. One foot was cooled for 5 min in a water bath of 13° to 14° C. Thermal video of the plantar foot was captured for 15 min after cold stimulus to measure rate and pattern of recovery. Three regions of interest were used to measure rate of recovery after fi ve minutes. The average rate of temperature recovery for controls was 29.4%±18.6%. DPN subjects had an average of 19.8%±17.2%. A two-sample t-test exhibited a signifi cant difference in temperature recovery (p=0.04). Irregular patterns of temp recovery were observed in DPN and some DM, no DPN subjects over the plantar foot during the full 15 min of recovery; these included hypothermic regions (< 22° C) and confi ned areas of hyperthermia (> 28° C). Clinical data, including monofi lament and tuning fork tests, did not correlate with hypo- or hyperthermia to a specifi c level of DPN severity. Functional infrared imaging is capable of detecting temperature recovery irregularities in DM subjects. Further study will allow us to develop a model of DPN progression based on plantar foot temp recovery.

Supported by: NSFC (81100582) Supported by: 1R43DK093192-01

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A152 COMPLICATIONS—NEUROPATHY

603-P of this explorative study was to evaluate whether this new technique can be Transplantation of Dental Pulp Stem Cells Improves Long-Term Dia- used to collect mucosal biopsies from well-defi ned parts of the entire length betic Polyneuropathy in Streptozotocin-Induced Diabetic Rats of the small and large bowel in patients with type 2 diabetes and matched, MAIKO OMI, KEIKO NARUSE, MASAKI HATA, YASUKO KOBAYASHI, NOBUHISA healthy subjects. 12 subjects with type 2 diabetes and 12 body mass NAKAMURA, SHOGO OZAWA, HIDEKI KAMIYA, JIRO NAKAMURA, YOSHINOBU index and age-matched subjects underwent anterograde and retrograde TANAKA, TATSUAKI MATSUBARA, Nagoya, Japan, Nagakute, Japan double-balloon enteroscopy under nurse-administered propofol sedation We have previously shown that the transplantation of cultured progenitor on 2 separate days. In all subjects we attempted to collect 2 mucosal biopsies from every 30 cm from pylorus to rectum. A mean of 21 biopsy or stem cells improved diabetic polyneuropathy. However, the effects of POSTERS long-term diabetic polyneuropathy have not been investigated. Dental pulp sites were sampled in the type 2 diabetes group vs. 25 in the healthy group. Complications stem cells(DPSCs) which are a sort of mesenchymal stem cells located in In 4 out of 24 patients (2 from each group, 17%) sampling from the entire Acute and Chronic the dental pulp cavity are expected as a souce of therapeutic agents, since gastrointestinal system was possible. The mean depth of maximal insertion DPSCs can be located from wisdom tooth extraction or premolar extraction (anterograde) was 478±32 cm in patients with type 2 diabetes vs. 465±44 for orthodontic reasons. In this study, we have investigated whether cm in healthy subjects (P=0.81). The mean depth of maximal insertion with transplantation of DPSCs ameliorated long-term diabetic polyneuropathy retrograde access was 230±36 cm in patients with type 2 diabetes and in streptozotocin(STZ)-induced diabetic rats. DPSCs were isolated and 207±26 cm in healthy subjects. This study demonstrates that double-balloon cultured from 6wk old male Sprague-Dawley rats as previously described. enteroscopy is a valid minimally-invasive way of collecting biopsies from the Identifi cation of DPSCs was analyzed by a fl uorescence activated cell sorter entire human gastrointestinal tract in single individuals and, thus, provides (FACS) analysis. Forty eight weeks after STZ injection, DPSCs(1×106cells/ research and clinical communities with a new possibility to gain access to rat) were injected into the unilateral hindlimb skeletal muscles. Saline hitherto unexplored human anatomy and physiology. was injected into the contra-lateral side. Four weeks after the DPSC transplantation, current perception threshold (CPT), sciatic motor/sensory 606-P nerve conduction velocity (MNCV/SNCV), sciatic nerve blood fl ow (SNBF), Neurological Complications of the BTBR OB/OB Mouse capillary density in skeletal muscles and intraepidermal nerve fi ber density PHILLIPE D. O’BRIEN, NICK J. ROBELL, JUNGUK HUR, JOHN M. HAYES, SANG SU (IENFD) were assessed. STZ-induced diabetic rats showed signifi cant OH, JACQULINE R. DAUCH, YU HONG, EVA L. FELDMAN, Ann Arbor, MI increase in CPTs, delay MNCV/SNCV and decrease in SNBF, capillary density Diabetic neuropathy (DN) is the most common complication associated in skeletal muscles and IENFD in the saline-injected side compared with the with type 2 diabetes mellitus (T2DM). Confounding the issues associated normal rats, all of which were ameliorated in the DPSC transplantation-side. with DN, is the absence of treatment options. To gain a more comprehensive These results suggest that DPSC transplantation could have the therapeutic understanding of disease progression with the aim of discovering effects on long-term diabetic polyneuropathy. new therapies, novel animal models are required to fully explore DN pathophysiology. Ideally, these models should mimic the hallmarks of the 604-P human condition presenting with both the T2DM phenotype and the asso- Association between Cardiovascular Autonomic Neuropathy and ciated complications. Cystatin C in Type 2 Diabetes Mellitus The leptin-defi cient BTBR mouse (BTBR ob/ob) has recently been HONG SEOK LEE, WOO HO BAN, YEON JI KIM, BO KYUNG KIM, WON CHUL HA, identifi ed as an excellent model to study diabetic complications as it has SU JIN OH, HYUN SHIK SON, TAE SEO SOHN, Uijeongbu, Republic of Korea the capacity to develop the characteristics found in the human condition. Cardiovascular autonomic neuropathy (CAN) in type 2 diabetes mellitus Moreover, an additional advantage of using a leptin-defi cient model, is that (T2DM) is associated with substantial morbidity and mortality. Cystatin C leptin administration has been shown to correct the diabetic phenotype and (CC) has emerged as an accurate marker of renal function and is known to restore organ function. be associated with arterial stiffness. The aim of this study is to investigate Here, for the fi rst time, we present data characterizing the neuropathic the association between CAN and serum CC, serum creatinine (Cr), CC- phenotype of the BTBR ob/ob mouse. Using 24 week old mice, we have based GFR, and MDRD-GFR in patients with T2DM. CAN was assessed observed defi cits in sural and sciatic nerve conduction velocities (NCVs), by performing the fi ve standard refl ex tests; heart rate responses to deep increased thermal hyperalgesia and small nerve fi ber loss. Upon determining breathing, to standing, and to Valsalva maneuver, and blood pressure neuropathy onset, we also observed signifi cant NCV defi cits as early as 9 responses to standing and sustained handgrip. The severity of autonomic weeks. The speed and severity at which the neuropathy develops in model neuropathy is quantifi ed by a score composed of the results of each of the offers distinct advantages over other murine models of DN. fi ve tests, where each test was given a value of 0, 0.5, or 1 if it yielded In addition to characterizing the neuropathic phenotype, we have isolated normal, borderline, or abnormal values. According to the sum of the each RNA from dorsal root ganglia and sciatic nerve from both juvenile and results, low risk group for cardiovascular disease is defi ned if the score is adult BTBR ob/ob mice. Using DNA microarray technology, we are currently ≤ 0.5, intermediate group if 1, high risk group if ≥1.5. A total of 147(68 men working to identify pathways dysregulated at different stages of DN, and and 79 women) patients with age of 60.7 ± 11.8 years old, with HbA1c 9.93± recently, we have identifi ed that immune response-related pathways are 2.52% and with diabetes duration 10.9 ± 9.4 years were recruited. Among highly perturbed in both young and mature mice. Future work comprises of 147 patients, the number of low, intermediated, and high risk group was 48 further exploration and analysis of the microarray data with the ultimate patients (33%), 34 patients (23%), 65 patients (44%), respectively. Serum CC goal of identifying novel therapeutic approaches to alleviate/prevent DN. showed signifi cant difference among three groups (low, intermediate and Supported by: NIH (U01-DK076160); Program for Neurology Research and Dis- high), which were 0.96±0.4 mg/L, 1.09 ± 0.5 mg/L and 1.2 ± 0.5 mg/L (P<0.01), covery however, serum Cr was not different among groups.CC-based GFR also had signifi cant difference between three groups (95.8 ± 38.2, 82 ± 33.2 and 71.5 607-P ± 30.5 mL/min/1.73m2respectively) (P<0.01), on the contrary, MDRD-GFR Antioxidant Enzymes and Diabetic Distal Symmetric Polyneuropa- didn’t. In multivariable analysis, decreased CC-based GFR, but not MDRD- thy (DSPN): A Genetic Association Analysis GFR, was signifi cantly associated with CAN (P<0.01). In conclusion, serum CC JACEK KASZNICKI, JOZEF DRZEWOSKI, AGNIESZKA SLIWINSKA, MARCIN KOS- level and CC-based GFR is related to CAN and could be a surrogate marker of MALSKI, ANNA MERECZ, IRENEUSZ MAJSTEREK, Zgierz, Poland, Lodz, Poland cardiovascular complications in patients with T2DM. Oxidative stress associated with chronic hyperglycemia participates in the development of diabetic complications, including DSPN. The aim of the 605-P study was to examine the association between the plasma level/activity The Use of Double-Balloon Enteroscopy in Retrieving Mucosal Biop- of antioxidant enzymes and single nucleotide polymorphisms of glutathione sies from the Entire Human Gastrointestinal Tract—A Methodologi- peroxidase GPx1, superoxide dismutase SOD1, and catalase CAT and the risk cal Study in Patients With Type 2 Diabetes and Healthy Subjects of DSPN. We investigated the genotype and allele frequencies of Pro197Leu NICOLAI A. RHEE, PETER VILMANN, HAZEM HASSAN, JAKOB W. HENDEL, TINA of Gpx1, +35 A/C of SOD1, and -262 C/T in the promoter region of CAT from VILSBØLL, FILIP K. KNOP, Hellerup, Denmark, Herlev, Denmark 110 Polish Caucasian type 2 diabetic (T2DM) patients with DSPN, 135 T2DM With the hitherto existing enteroscopic techniques it has not been patients without DSPN and 156 subjects with normal glucose metabolism. possible to extract biopsies from the entire human gastrointestinal tract. A PCR-RFLP assay was used to identify the distribution of genotypes and In recent years a new enteroscopic procedure, double-balloon enteroscopy, allele frequency. The plasma levels and activity of GPx1, SOD1 and CAT were has made it possible to visualize and collect biopsies from every part of the measured in duplicate by the ELISA immunoassay test. The genetic analysis small intestine in live subjects without the need for open surgery. The aim of Pro197Leu of Gpx1, +35 A/C of SOD1, and -262 C/T of CAT did not reveal

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A153 COMPLICATIONS—OCULAR

any differences in the examined polymorphic variants between studied Primary rat astrocytic cultures were treated with different MG groups. We found signifi cant decrease in the plasma level/activity of GPx1, concentrations (100 µM, 300 µM, 500 µM, 800 µM and 1000 µM) for 24 SOD1, and CAT in T2DM with DSPN in comparison to healthy subjects (table - hrs. Cellular viability was determined by MTT assay. Protein expression of * p<0.05, **p<0.01 compared to healthy subjects). The plasma level/activity astrocytic culture markers (i.e. GFAP), insulin receptor signaling molecules of studied enzymes did not differ signifi cantly between healthy subjects and including insulin receptor substrate (IRS), insulin growth factor receptor T2DM patients, except for GPx1. The results of our study confi rm the role of (IGF-R) and Akt were determined by immunoblotting. After 24 hrs MG antioxidant enzyme defense disorders in the pathogenesis of DSPN. exposure, astrocyte cell viability was decreased and the decrease exhibits a dose-dependent manner with increasing MG concentrations. At the same POSTERS

Complications The plasma level of GPx1, SOD1, and activity of CAT in T2DM patients with or time, up-regulation of GFAP expression was observed in astrocytic cultures

Acute and Chronic without DSPN with 500 µM or above MG treatments. In line with these results, MG also Healthy subjects T2DM patients T2DM patients with DSPN induced insulin resistance in astrocytic cultures treated with 500 µM or GPx1 [ng/mL] higher concentrations of MG,as demonstrated in down-regulations of IRS, IGF-R and Akt. These results therefore imply that MG may lead to astrocytic Mean 15.033 10.852 11.014 SEM 1.377 0.597** 0.977** dysfunction and insulin resistance in astrocytic cultures, suggesting MG may play a role in neuronal degeneration in diabetic neuropathy. SOD1 [pg/mL] Mean 24479.370 24923.960 20309.050 SEM 1375.071 1461.051 904.789* COMPLICATIONS—OCULAR CAT [U/L] Mean 525.513 478.115 444.003 SEM 25.098 22.972 25.394* Guided Audio Tour: Neurovascular Disease of Diabetic Retinopathy (Post- ers: 610-P to 617-P), see page 15. Supported by: Polish Ministry of Science and Higher Education (NN402375838) & 610-P Upregulation of NADPH Oxidase 4 Promotes Aberrant Retinal Neo- 608-P vascularization via Activation of the Endothelial VEGF Receptor 2 Pathway Cardiac Autonomic Dysfunction Is Associated With a Higher Rate SARAH X. ZHANG, L.I. JINGMING, JOSH J. WANG, Buffalo, NY of Metabolic and Blood Pressure Disorders in Obese People at Risk Oxidative stress plays a central role in the pathogenesis of neovascular of Diabetes retinal diseases including diabetic retinopathy. Previously we demonstrated ISABELA BANU, MINH TUAN NGUYEN, ELIANE HAMO-TCHATCHOUANG, EM- that NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidase in MANUEL COSSON, PAUL VALENSI, Bondy, France retinal endothelial cells, and is responsible, at least partially, for retinal Cardiac autonomic dysfunction (CAD) is highly prevalent in obese patients. vascular leakage in type 2 diabetes. However, the role of Nox4 in retinal The present study aimed to examine the role of CAD in metabolic and blood neovascularization (NV), a hallmark of proliferative diabetic retinopathy, pressure disorders in obese people at risk of diabetes. remains unknown. This study is aimed to investigate the function and We included 428 overweight or obese patients without known diabetes. mechanisms of Nox4 in retinal NV and angiogenesis. Oxygen-induced CAD was defi ned as ≥1 out of 3 tests analysing heart rate variations mostly retinopathy (OIR) was utilized as an in vivo model for retinal NV and human dependent on vagal control (Valsalva, deep-breathing, lying-to-standing). retinal endothelial cells (HREC) were used for in vitro studies. Temporal Dysglycemia (diabetes, fasting hyperglycemia and/or glucose intolerance) expression and cellular localization of Nox4 in the retina was determined was detected by an OGTT (WHO criteria). The 10-year risk of diabetes was during OIR. We found that Nox4 expression was mainly localized in estimated by the Findrisk score. retinal vasculature and dynamically correlated with retinal NV formation. CAD was found in 198 patients; 124 patients had dysglycemia and 183 Over-expressing Nox4 in HREC signifi cantly increased extracellular H O had a metabolic syndrome (MS, IDF criteria). The Findrisk score was ≥12 2 2 generation, promoted endothelial tube formation and aggravated VEGF- in 227 patients. The population was separated into 4 groups according to induced VEGFR2 phosphorylation and activation of its downstream Findrisk score ≥12 or <12 and the presence or absence of CAD. Among the pathways ERK1/2 and P38. Conversely, knockdown of Nox4 or scavenging patients with Findrisk≥12, 48.5% had CAD. The patients with Findrisk≥12 H O by catalase in HREC inhibited endothelial migration and suppressed and CAD had higher plasma glucose both at fasting and 120 minutes after 2 2 the activation of VEGFR2 signaling pathway. Interestingly, overexpressing glucose challenge than those with Findrisk<12 and free of CAD (respectively Nox4 also drastically enhanced protein level of ATF4, a transcription factor 5.06±0.13 vs 4.66±0.06 mmol/l, p=0.01 and 7.78±0.28 vs 6.56±0.18 mmol/l, that induces VEGF in retinal cells. In line with these results, knockdown p<0.001). The prevalence of hypertriglyceridemia (≥1.7 mmol/l) (46.0% vs of Nox4 in mouse retina, achieved by periocular injection of adenovirus 30.1%, p<0.02) and elevated blood pressure (SBP≥130 and/or DBP≥85 mmHg) expressing Nox4 siRNA, signifi cantly attenuated retinal NV formation in (65.5% vs 49.1%, p<0.02) was higher among the patients with Findrisk≥12 and OIR mice. Taken together, our results indicated that Nox4 is a key regulator CAD than in those with Findrisk≥12 and free of CAD, with a non signifi cant of endothelial cell angiogenic response. Upregulation of Nox4 contributes trend for dysglycemia (40% vs 29.9%) and MS (64.4% vs 52.4%). retinal NV formation, and targeting retinal Nox4 may present a promising The present data show that in overweight or obese patients at high risk of therapeutic approach for neovascular retinal diseases. diabetes, the presence of CAD is associated with a higher rate of metabolic Supported by: NIH (EY019949); OCAST (HR10-060) and blood pressure disorders, which suggests the role of vagal defects and sympathetic predominance in these disorders. & 611-P 609-P Pioglitazone Increases the Risk of Diabetic Macular Edema in Japa- nese Patients With Type 2 Diabetes Insulin Resistance in In Vitro Astrocytic Cultures under Methylgly- KANTA FUJIMOTO, YOSHIYUKI HAMAMOTO, SACHIKO HONJO, HISATO TAT- oxal Treatment SUOKA, ATSUKO MATSUOKA, YOSHIHARU WADA, HIROKI IKEDA, JUN FUJIKA- MAN TAK CHU, KEN K.L. YUNG, CHRISTOPHER H.K. CHENG, KEVIN K. YUE, Kow- WA, ISAO SAITO, HIROYUKI KOSHIYAMA, Osaka, Japan loon, Hong Kong, Sha Tin, Hong Kong There has been a concern about the potential link of pioglitazone with Diabetes mellitus (DM) is characterized by hyperglycemia and diabetic adverse effects, including an increased incidence of bone fractures, edema, complications. Recently it has been reported that diabetic patients have heart failure and bladder cancer. Recent studies have raised a possibility higher risk of developing neurodegenerative diseases. Astrocyte which that pioglitazone, which generally causes fl uid retention, may develop constitutes the blood-brain-barrie plays an important role in monitoring diabetic macular edema (DME). DME is one of the main causes of visual neuronal activities and astrocytic dysfunction is accompanied with neuronal impairment in patients with diabetic retinopathy. We investigated whether cell death. Methylglyoxal (MG) is a reactive intermediate by-product in the use of pioglitazone may increase the risk of DME in Japanese patients glycolytic pathway and abnormal concentration of MG was observed in with type 2 diabetes. plasma of DM patients. MG caused damages in cells such as endothelial We retrospectively examined the incidence of DME in patients with type cells and cardiomyocytes. In this study, the deleterious effects of MG on 2 diabetes in relation to pioglitazone administration during 12 years from astrocytes were investigated. More specifi cally, the toxicity of MG, MG- 2000 to 2011 using the database in our institute. Subjects included a total of induced astrocytic dysfunction and the disturbance of insulin signaling 22,115 patients with type 2 diabetes. The incidence of DME was calculated. activities in astrocytes after MG treatment were determined.

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A154 COMPLICATIONS—OCULAR

A total of 99 subjects with type 2 diabetes were found to have DME, and cells or pericytes. A non signifi cant reduction of migrating pericytes by 23% the total prevalence of DME in type 2 diabetes was 0.45 % at 12 years of respectively 34% was found. EPO treatment protected neurons, shown by follow-up. Among 953 patients taking pioglitazone, 11 patients were found decreased thinning of the central retina by 10% (p<0.05). EPO treatment to have DME (1.15 %). The unadjusted hazard ratio (HR) of pioglitazone decreased CD74 by 67% (p<0.01) on mRNA level. Heterozygous PKD had a for development of DME was 7.76 [95%CI: 4.00-15.07]. After Cox multiple one third lower CD74 expression, which was further reduced due to EPO- regression analysis (adjusted for age; sex; HbA1c levels; and use of insulin, like peptide by 36% (n.s.). pAkt, which is downstream of the receptors, was antiplatelets, or renin-angiotensin system inhibitors), pioglitazone use localized by immunohistochemistry to the inner plexiforme layer. Quantitiy was associated with an increased risk of DME at 2 years of follow-up (HR and intensity of pAkt increased with treatment. In summary, EPO and EPO- POSTERS

4.71 [95%CI: 1.38-16.10]) and 12 years of follow-up (HR 5.12 [95%CI: 2.61- like peptide reduce neurodegeneration, vasoregression and activation of Complications

10.04]). Furthermore, combination therapy with pioglitazone plus insulin was microglial cells in a transgenic model of retinal degeneration. Thus, EPO and Acute and Chronic associated with a higher risk for DME (HR 9.21 [95%CI: 4.06-20.89]). EPO-like peptide can be suitable to prevent or decelerate vasoregression in In this study, the association was observed between pioglitazone and diabetic retinopathy. DME, suggesting an increased risk of DME by pioglitazone in Japanese Supported by: Deutsche Forschungsgemeinschaft (GRK 880 Vascular Medicine) patients with type 2 diabetes. & 614-P & 612-P Superoxide Generation by Retina in Diabetes; Photoreceptors, Mi- Endothelial Specifi c Loss of BMAL1 Results in Vascular Phenotype tochondria, and NADPH Oxidase Similar to that in Diabetes TIMOTHY S. KERN, YUNPENG DU, Cleveland, OH ASHAY D. BHATWADEKAR, YANPENG DIAO, JUDE AL-SABAH, SERGIO CABAL- Prior work by us and others has provided strong evidence that oxidative LERO, CHOOGON LEE, MARK S. SEGAL, MARIA B. GRANT, Gainesville, FL, Tal- stress plays an important role in the development of the vascular lesions lahassee, FL of early diabetic retinopathy. We have investigated the contribution of Dysregulation of circadian clock plays a pivotal role in glucose homeostasis photoreceptors to the retinal oxidative stress, and the sources of that and development of type 2 diabetes. The transcription factor BMAL1 (brain superoxide generated in diabetes. In wildtype C57Bl/6J mice diabetic for & muscle ARNT-like protein) constitutes a positive arm of circadian clock 2 mos, dichlorofl uorescein or DHE stains indicated that the majority of and mice defi cient in BMAL1 possess a range of disorders like reduced oxidative stress was localized in the photoreceptor layer. Degeneration of lifespan, premature ageing, monocytosis and development of cataracts. We photoreceptors (using rhodopsin knockout or iodoacetic acid) signifi cantly recently reported that a mutation in clock gene Per2 causes retinal vascular inhibited the diabetes-induced increase in superoxide (measured with defects similar to those observed in diabetes; in this study we explored the luciginen) observed in wildtype C57Bl/6J mice. In wildtype diabetic mice, 2 impact of endothelial specifi c deletion of Bmal1 on the retinal vasculature mos administration of either methylene blue (acts to bypass abnormalities in (microvasculature) and the femoral artery (macrovasculature). electron transport system) or apocynin (NADPH oxidase inhibitor) inhibited Endothelial-specifi c mutant mice (Bmal1fx/fx; Tek-cre) were generated the diabetes-induced increase in superoxide generation, indicating that and retinal mRNA expression of nitric oxide synthase isoforms (iNOS, eNOS) both mitochondria and NAPDH oxidase participate in this oxidative stress. superoxide dismutase (SOD1, SOD2), and glutathione peroxidase (GPX-1) Photoreceptors play a critical role in the diabetes-induced oxidative stress were measured by qRT-PCR. In parallel, retinal microvascular injury was in the retina. performed using the ischemia-reperfusion model, while macrovascular Supported by: U.S. Dept. of Veterans Affairs injury was achieved by inducing endoluminal damage of the femoral artery with a 0.015 mm-diameter angioplasty guidewire. & 615-P The retinal expression levels of eNOS and iNOS remained unchanged in Novel 5-Lipoxygenase Inhibitor Reduces Capillary Degeneration in Bmal1fx/fx;Tek-cre mice when compared to wild type controls, however the Mouse Model of Diabetic Retinopathy there was a marginal increase in GPX-1 mRNA levels. Interestingly, levels of REENA BAPPUTTY, RAMAPRASAD TALAHALLI, JOHN HUTCHINSON, ROSE A. hypoxia inducible factor 1α and Per2 mRNA were increased 1.2 and 1.5-fold, GUBITOSI-KLUG, Cleveland, OH, San Diego, CA respectively. Trypsin digestion evaluation of retinas for number of acellular Degeneration of retinal capillaries in diabetes may result from a chronic capillaries showed a 3-fold (p<0.05) increase in the Bmal1fx/fx;Tek-cre infl ammatory process that damages the endothelium. Pro-infl ammatory retinas. Quantifi cation of neo-initmal thickness of injured femoral arteries leukotrienes have been implicated as mediators of diabetes-induced retinal showed a 20% increase in neo-intimal hyperplasia as compared to similarly infl ammation in the diabetic mouse model. We investigated the effects of injured wild type controls. a novel 5-lipoxygenase inhibitor AM-679 on early features of retinopathy In conclusion, our study highlights the importance of the circadian in diabetic mice, including 1) retinal superoxide production, 2) infl ammatory clock system in maintaining vascular homeostasis and that endothelial marker expression (e.g., NFkB and ICAM-1), 3) leukocy te-mediated endothelial specifi c deletion of Bmal1 results in accelerated injury of both micro and cell death in culture, and 4) retinal capillary degeneration. macrovasculature beds. Immediately following chemical induction of diabetes, we administered Supported by: Thomas H. Maren Junior Investigator Award (to A.D.B.); DK090730; AM-679 (5 mg/kg body weight/day dissolved in drinking water) to diabetic EY00773 (to M.B.G.) mice. At 16 weeks of diabetes duration, retinas from diabetic mice demonstrated a characteristic rise in superoxide production, NFkB p65 & 613-P subunit expression, and ICAM-1 expression compared to retinas from non- Protection of the Neurovascular Unit in a Model of Retinal Neurode- diabetic mice. Notably, AM-679-treated diabetic mice developed signifi cantly generation by Epo and Epo-Like Peptide less of each of these diabetes-induced changes. (35-75% reductions, p<0.05 STEPHANIE BUSCH, AIMO KANNT, FREDERICK PFISTER, YUXI FENG, SIGRID compared to diabetic mice) Previously we demonstrated that leukocytes, HOFFMANN, NORBERT GRETZ, HANS-PETER HAMMES, Frankfurt, Germany, which express 5-lipoxygenase, are the primary source for the rise in Mannheim, Germany leukotriene generation in the diabetic mouse model. When co-cultured Polycystic kidney disease (PKD) rats develop photoreceptor degeneration, with retinal microvascular endothelial cells, leukocytes from diabetic mice followed by an activation of retinal microglia and Müller cells, shown by CD74 killed retinal microvascular endothelial cells. AM-679-treatment reduced and GFAP upregulation, and subsequent vasoregression. This normoglycemic generation of leukotriene metabolites by the leukocytes and reduced the model is homologous to nonproliferative diabetic retinopathy and is used to endothelial cell death in the co-cultures. In agreement with these in vitro study vasoregression. Given thae damage of the neurovascular unit, neuro- assays and after a diabetes duration of 9 months, the retinal capillaries from and vasoprotective EPO was chosen as a therapeutic agent. Four weeks old AM-679-treated mice were protected from diabetes-induced degeneration. male heterozygous PKD rats were treated 3 times a week with EPO 256IU/kg In summary, the novel 5-lipoxygenase inhibitor AM-679 inhibits the body weight i.p.. Likewise homozygous PKD rats received EPO-like peptide, early biochemical and histological changes of diabetic retinopathy in the which lacks proerythropoietic effects by signaling via the heterodimeric mouse and requires additional study as a potential treatment for diabetic tissue protective receptor (McVicar et al., 2011), 5 times a week (10µg/kg retinopathy. body weight). The animals were sacrifi ced after 4 weeks. Quantifi cation Supported by: Panmira Pharmaceuticals, LLC. of neurovascular morphology, gene analysis and immunohistochemistry were performed. Retinal digest preparations revealed a reduction in the number of acellular capillaries by EPO and EPO-like peptide of 49% (p<0.001) respectively 40% (p<0.05). There was no change in number of endothelial

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618-P & 616-P How Does Diabetic Eye Disease Affect the American Worker? Retinal Dependence on Glycolysis for Biosynthesis: Implications RICHARD A. BROOK, NATHAN L. KLEINMAN, SUNIL PATEL, JIM E. SMEEDING, for Development of Diabetic Retinopathy IAN A. BEREN, BRYCE MILLER, ADAM TURPCU, Newfoundland, NJ, Cheyenne, PATRICE E. FORT, MANDY K. LOSIEWICZ, SUBRAMANIAM PENNATHUR, MI- WY, Abilene, TX, Dallas, TX, San Francisco, CA CHAEL D. DENNIS, SCOT R. KIMBALL, LEONARD S. JEFFERSON, STEVEN F. AB- This study assessed the impact of diabetic macular edema (DME) and COUWER, THOMAS W. GARDNER, Ann Arbor, MI, Hershey, PA diabetic retinopathy (DR) on the absenteeism of US employees and, more Purpose: The retina is a highly glycolytic tissue so we tested the hypothesis

POSTERS specifi cally, commercial drivers, whose profession is dependent on good

Complications that glycolysis is important for retinal anabolic metabolism, notably protein vision. Employee data on absence days due to sick leave, short- and long- Acute and Chronic synthesis, and contributes to diabetic retinopathy. term disability, and workers’ compensation was collected from 1/1/2001- Methods: Protein synthesis was assessed by the fl ooding dose of 6/30/2012 in employees ≥18 yrs of age with continuous eligibility for ≥1 yr phenylalanine method in STZ-induced diabetic rats and Ins2Akita mice, from the Human Capital Management Services (HCMS) database, which and by 35S-Met labeling in ex vivo retinas. Hexokinase activity was contains anonymous data from >20 geographically dispersed US employers inhibited by 2-deoxyglucose (2-DG) or 2-fl uoro-2-deoxy-D-glucose, and Akt (1.7 million+ employees) including manufacturing, insurance, retail, isoforms were inhibited by small molecules; oxidative phosphorylation transportation, telecommunications. Two-part regression models controlled was inhibited by antimycin A. Retinal metabolite levels were analyzed by for demographics and job-related characteristics. Results were compared HPLC and tandem MS. with two employee control groups, one with and one without diabetes Results: Diabetic rats and mice exhibit reduced protein synthesis (neither control group had DR or DME). 466,251 employees met the inclusion concomitant with reduction of the Akt/mTOR pathway. Diabetes progressively criteria, 39,702 of whom were commercial drivers. Employees with DME and reduces Akt-1, Akt-3 and mTOR activity, but 4EBP1 phosphorylation reduction DR missed signifi cantly more work versus the control group without diabetes is transient and S6 phosphorylation is unaltered. Retinal metabolite analysis (Figure 1). Differences between disease groups and controls were generally reveals increased BCAA levels, alterations of TCA cycle and increased larger in commercial drivers. For DR and DME patients, the most common pentose phosphate pathway intermediates. Both glycolysis and growth cause for missing work was sick-leave among non-drivers and short- and long- factor signaling reduce protein synthesis in normal ex vivo retinas but only term disability among drivers. This analysis shows the detrimental impact of glycolytic inhibitors reduced 4EBP1 phosphorylation and mTORC2 complex DME and DR on employee absenteeism, particularly those employees who activation demonstrating only partially common mechanisms. Further, depend on eyesight for their jobs, such as commercial drivers. D-fructose and D-mannose overcame this inhibition and 2-DG, but not antimycin A, reduced retinal mTOR activity and 4EBP1 phosphorylation. Conclusions: These data demonstrates that the retina has a strong dependence on glycolysis and the Akt/mTOR complex with specifi c and different effects on mTORC1 and mTORC2 complexes for essential anabolic metabolism. Alteration of glycolysis and the Akt/mTOR pathways impairs anabolic functions providing novel insights on retinal vulnerability to diabetes. Supported by: NEI (EY020582), (DK094292); Taubman Institute

& 617-P Proliferative Retinopathy in Type 1 Diabetes Is Associated With Ce- rebral Microbleeds and Decreased Skin Capillary Density JORN WOERDEMAN, EELCO VAN DUINKERKEN, MIKE P. WATTJES, FREDERIK BARKHOF, FRANK J. SNOEK, RICHARD G. IJZERMAN, ERIK H. SERNÉ, MICHAELA DIAMANT, Amsterdam, Netherlands Small vessel disease (SVD) accounts for most of the strokes in type 1 diabetes mellitus (T1DM). Retinal microvascular changes appear to refl ect cerebral SVD, but whether diabetic proliferative retinopathy (PDR) is associated with cerebral SVD is unknown. Moreover, it is unclear whether SVD is limited to the brain or part of a generalized microvascular disorder. We investigated whether PDR in T1DM is characterized by cerebral SVD, represented by magnetic resonance imaging (MRI) measured cerebral 619-P microbleeds (CMBs), and hypothesized that if cerebral SVD is part of a Vildagliptin in Addition to Metformin Improves Retinal Arteriolar generalized disorder, then pathological changes might be detected in the Wall to Lumen Ratio and Erythrocyte Flexibility in Patients With easily accessible skin microcirculation. Type 2 Diabetes Mellitus T1DM patients with (n=33; PDR+) and without (n=34; PDR-) proliferative CHRISTINE BERNDT-ZIPFEL, GEORG MICHELSON, INGA KLAMP, MICHAEL MIT- retinopathy and healthy controls (n=33) underwent MRI to study CMBs. RY, ANDREAS PFÜTZNER, THOMAS FORST, Mainz, Germany, Erlangen, Germany Capillary microscopy was used to assess baseline capillary density (BCD) Numerous rheological and microvascular alterations characterize the and capillary density during post-occlusive peak reactive hyperaemia (PRH). vascular pathology of type 2 diabetes mellitus (T2DM). This study investigated Capillary recruitment was calculated by dividing the difference between vascular effects of Glimepiride compared to Vildagliptin in T2DM. BCD and PRH by BCD. Correction for age, gender and hypertension was Forty-four patients with T2DM (28 male, 16 female, age: 59±8 years; performed to determine difference in prevalence of CMBs between the Hb1Ac 7.4±0.7%; T2DM duration: 7.1±7.1 years [mean±SD]) on Metformin groups. monotherapy were included in the study. Patients were randomized to add CMBs were more prevalent in PDR+ (21%) compared to PDR- patients (3%; either Vildagliptin (50 mg twice daily) or Glimepiride individually titrated up p=0.01) and healthy controls (9%; p=0.02). A signifi cant trend was found to 4 mg for a period of 6 months. WLR was measured by Scanning Laser towards lower baseline capillary density (healthy controls 48.4 ± 10.3 vs. Doppler Flowmetry (Heidelberg Engineering, Heidelberg Retina Flowmeter PDR- 46.2 ± 8.8 vs. PDR+ 44.8 ± 7.3 n/mm², respectively; P-for-trend=0.02) HRF, Germany). In addition, measurement of erythrocyte deformability from and capillary density during PRH (controls 67.1 ± 17.0 vs. PDR- 65.2 ± 15.9 fresh peripheral blood draws was performed using laserdiffractoscopy vs. PDR+ 61.8 ± 12.3 n/mm²; P-for-trend=0.04) across study groups. Capillary applying different shear stresses (Rheodyn SSD, Myrenne GmbH, Roetgen, recruitment was signifi cantly lower in participants with CMBs compared to Germany). For comparison between the groups the area under the curve participants without CMBs (28.8 ± 15.0 vs. 40.3 ± 16.4%; p=0.04) (AUC0.3- 6 Pa) was calculated. Patients with T1DM with PDR+ are characterized by a more frequent Both treatments signifi cantly improved HbA1c (Glimepiride: -0.68±0.49; presence of CMBs, which seem to be part of a generalized microvascular Vildagliptin: -0.67±0.49 %; p<0.05 vs. baseline) and fasting glucose levels disorder. (Glimepiride: -1.21±1.4; Vildagliptin: -1.02±1.29 mmol/L; p<0.05 vs. baseline). Supported by: Dutch Diabetes Research Foundation (2005.00.006); EFSD; Dutch Vildagliptin, but not Glimepiride reduced the arterial WLR by 0.05±0.08 Heart Foundation (2010T041) (p<0.05 vs. baseline). The AUC0.3- 6 Pa for the erythrocyte elongation index (EI) increased during both treatments (Glimepiride: 146±151, Vildagliptin: 54±144

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A156 COMPLICATIONS—OCULAR

EI; p< 0.05 vs. baseline). A linear correlation could be observed between bovine retinal pericytes causes 2.6-fold increase in apoptosis (p=0.029) at improved glycemic control (HbA1c) and the increase in EI (r²= 0.275; p<0.0001) 5.5 mM glucose. Co-culturing of pericytes overexpressing SHP-1 with rat but not with the reduction in the retinal WLR. In T2DM, Vildagliptin exerts Müller cells caused 6-fold increase in TGFβ1 mRNA in rat Müller cells which benefi cial effects on the retinal microvascular wall, which are found beyond was not observed in co-cultured retinal endothelial cells. the effects of glucose control. In contrast, the augmentation in erythrocyte These results suggest that overexpression of SHP-1 due to hyperglycemia elasticity seems to be a correlate of improved glycemic control. in the pericytes may indirectly induce the production of infl ammatory Supported by: Novartis Pharmaceuticals Corporation cytokines such as TGFβ1, TNFα and IL1β from Müller cells. The resulting local infl ammation could partly contribute to the initial pericyte apoptosis POSTERS 620-P observed in DR. Complications VEGF Receptor Inhibitor Inhibits High Glucose-Induced the Changes Supported by: JDRF; NIH Acute and Chronic of Hyaloid-Retinal Vessels in Zebrafi sh Larvae SEUNG-HYUN JUNG, YOUNG SOOK KIM, YU-RI LEE, YONG TAE KIM, JIN SOOK 622-P KIM, Daejeon, Republic of Korea Awareness of Diabetic Retinopathy and Eye Exams in the U.S. Based Although a variety of animal models have been used to test drug on NHANES 2005-2008 candidates and study the pathogenesis of diabetic retinopathy (DR), ROHIT VARMA, NEIL M. BRESSLER, QUAN DOAN, JULIE BOWER, ELIZABETH timesaving and inexpensive models are still needed to test the increasing SELVIN, MICHELLE GLEESON, MARK DANESE, CHANTAL M. DOLAN, JENNIFER number of therapeutic approaches. We introduce a model for DR using the FINE, SHOSHANA COLMAN, ADAM TURPCU, Chicago, IL, Baltimore, MD, West- early stage of zebrafi sh transgenic (Danio rerio, fl k:EGFP) by treating embryos lake Village, CA, San Francisco, CA with 30 mM glucose for 5 days. Isolation of retinas from zebrafi sh larvae was The 2012 ADA guidelines recommend patients with diabetes have a developed without forceps using 3% trypsin, and the changes in hyaloid- comprehensive eye exam, including pupil dilation, soon after diagnosis and retinal vessels were investigated. The diameter of the hyaloid-retinal at least annually thereafter. This study estimated the proportion of people vessels was increased by immersion in 30 mM glucose and expression of the with diabetic retinopathy (DR) unaware that diabetes had affected their tight-junction protein ZO-1 was disrupted as shown by immunofl uorescence eyes, the proportion reporting they had not had a dilated eye exam in the staining. Dilated hyaloid-retinal vessels displayed mild vascular leakage past year, and factors associated with these outcomes. after treatment with fl uorescent dye (tetramethylrhodamine) and expression Data were from the National Health and Nutrition Examination Survey of vegf165 mRNA was upregulated. Moreover, we confi rmed that a vascular (NHANES) 2005-2008, a nationally representative, cross-sectional survey endothelial growth factor (VEGF) receptor inhibitor phenotypically rescued conducted by the National Center for Health Statistics. Diagnosis of DR was dilated hyaloid-retinal vessels. In this study, we suggest that 30 mM based on grading of fundus photographs by trained readers. Proportion of glucose-treated zebrafi sh larvae may have application for screening and drug patients reporting prior awareness of diabetic eye disease and dilated eye discovery for DR, especially for tight-junction and VEGF-related pathology exams in the past year were based on patient responses to the following found in retinal vessel disorders. questions: “Have you been told by a doctor that diabetes has affected your eyes or that you had retinopathy?” and “When was the last time you had an eye exam in which the pupils were dilated?” Multivariable logistic regression was used to determine factors associated with each indicator. Of the 286 participants with DR, 70% (95% confi dence interval [CI]: 64- 77%) were not aware that diabetes had affected their eyes and 33% (95% CI: 26-40%) reported not receiving a dilated eye exam in the past year. Hispanic ethnicity (odds ratio [OR] = 0.45, 95% CI: 0.20 to 0.99, P=0.05) appeared associated with being unaware that diabetes affected one’s eyes; having insurance (OR = 7.55, 95% CI: 3.30 to 17.27, P<0.001) appeared associated with reporting a dilated eye exam. These results suggest that many patients with diabetes are not aware it has affected their eyes and many may not be meeting ADA guidelines to receive at least an annual dilated eye exam.

623-P Exploring Leptin Antagonism as Potential Treatment in Diabetic Eye Disease Supported by: KIOM (K11040), (K12040) EVA SURMACZ, CRISTINA PARRINO, LAURA SCOLARO, LASZLO OTVOS, RO- BERTA CORONITI, Philadelphia, PA 621-P Emerging in vitro and in vivo evidence suggests that angiogenic and pro- infl ammatory cytokine leptin might be implicated in the pathogenesis of Overexpression of SHP-1 in Retinal Pericytes Causes Early Pathol- diabetic eye disease. However, the potential of inhibiting leptin function in ogy Changes diabetic eye models has never been explored. WEIER QI, CHONG WEE LIEW, ALLEN CLERMONT, QIAN LI, EDWARD YU, JONG- We investigated mitogenic, angiogenic and signaling activities of leptin SOON LEE, JENNIFER SUN, TIMOTHY S. KERN, GEORGE L. KING, Boston, MA, in relevant ophthalmic cell models, and explored the potential of our leptin Chicago, IL, Cleveland, OH receptor (ObR) antagonist peptide (Allo-aca) to inhibit these functions. Leptin We have reported that increased expression of SHP-1, a tyrosine at 50-250 ng/mL stimulated the growth of monkey retinal (RF/6A) and bovine phosphatase, induced by hyperglycemia can inhibit actions of critical growth/ corneal (BCE) endothelial cells in a dose-dependent manner. The maximal survival factors to cause pericyte apoptosis and initiate the development mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was of diabetic retinopathy (DR). To test this hypothesis, we have targeted the noted at 24 h of 250 ng/mL leptin treatment. Leptin-dependent proliferation overexpression of SHP-1 to the capillary pericytes by breeding Lox-stop-Lox was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A SHP-1 mice with SM22 cre mice. α cells, respectively. In both cell lines, leptin induced angiogenic response, Expression of SHP-1 mRNA was increased by 16-fold in the whole retina i.e., formation of tube-like structures. The maximal increase of angiogenesis and 11-fold in isolated retinal blood vessels which was accompanied by a (163±10 and 133±8% in RF6A and BCE cells, respectively) was observed with 3-fold increase in SHP-1 protein in SM22 -SHP-1 Tg mice (all p<0.05). SHP-1 α a 250 ng/mL leptin treatment for 4 h, and this effect was totally blocked in mRNA and protein levels were also increased in cultured brain pericytes and the presence of 100 nM Allo-aca. In RF/6A and BCE cells, 250 ng/mL leptin the whole brain. Interestingly, mRNA levels of TGF 1, TNF and IL1 mRNA β α β modulated several signaling pathways controlling cell growth, infl ammatory were increased by 6.7, 1.8 and 1.52-fold respectively (all p<0.05) in the whole activity and angiogenesis, including the phosphorylation of STAT3, Akt, retina of Tg compared to Wt mice, suggesting elevation of infl ammation in and ERK1/2 at 15 min, and expression of COX2 and NF-kB at 6-12 h. All the retina. NF B activation was also increased by 1.4-fold (p<0.05) in the κ these leptin responses were signifi cantly inhibited with Allo-aca at 100 whole retina, but it was not changed in the brain. Evan blue permeation was nM concentration. An unrelated control peptide did not demonstrate any increased in the retina by 2.18-fold in Tg mice at 17-20 weeks old (p=0.03) antagonistic activity in all the above assays. compared to Wt mice, but no leakage was observed in the brain. Mean At present, anti-VEGF based therapeutics represent the only biologics circulation time (MCT) measured by fl uorescein angiography was increased approved for treatment of diabetic eye disease, however, limitations of by 1.39-fold (p=0.008) in SHP-1 Tg mice. Overexpression of SHP-1 in cultured

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A157 COMPLICATIONS—OCULAR

these strategies have been noted. Our data provide new insights into the role of leptin in ocular endothelial cells and offer basis for development of ObR antagonists as therapeutics for diabetic microvascular complications affecting the eye. Supported by: Novo Nordisk, Inc.

624-P

POSTERS Adiponectin-Induced Dilation of Isolated Porcine Retinal Arterioles Complications via Production of Nitric Oxide from Endothelial Cells Acute and Chronic TAIJI NAGAOKA, TSUNEAKI OMAE, ICHIRO TANANO, AKITOSHI YOSHIDA, Asa- hikawa, Japan Purpose: Adiponectin has anti-infl ammatory and atheroprotective effects on vascular tissue via the adiponectin receptor (adipoR). However, the action of adiponectin in the retinal microcirculation is unknown. We examined the direct effect and underlying mechanism of the vasomotor action of Supported by: KIOM (K11040), (K12040) adiponectin in porcine retinal arterioles. Methods: Porcine retinal arterioles (internal diameter, 60-90 µm) were 626-P isolated, cannulated, and pressurized (55 cmH2O) without fl ow in this in Risk Factors for Diabetic Retinopathy Severity vitro study. Videomicroscopic techniques were used to record changes in ADAM TURPCU, RICHARD M. BERGENSTAL, NEIL M. BRESSLER, ROHIT VARMA, diameter in response to adiponectin. MICHELLE GLEESON, JULIE BOWER, ELIZABETH SELVIN, MARK DANESE, CHAN- Results: The retinal arterioles dilated in a dose-dependent (0.125-7.5 µg/ml) TAL M. DOLAN, JENNIFER FINE, SHOSHANA COLMAN, QUAN DOAN, San Fran- manner in response to adiponectin. The vasodilation decreased signifi cantly cisco, CA, Minneapolis, MN, Baltimore, MD, Chicago, IL, Westlake Village, CA after removal of the endothelium. NG-nitro-L-arginine methyl ester (a nitric This study compared diabetic retinopathy (DR) prevalence with other oxide [NO] synthase inhibitor), 1H-1,2,4-oxadia-zolo[4,3-a]quinoxalin-1-one (a common diabetes-related complications and examined risk factors asso- soluble guanylyl cyclase inhibitor) but not wortmannin (a phosphatidylinositol ciated with more severe DR in a representative sample of US adults aged 3-kinase inhibitor) inhibited the effect of adiponectin-induced vasodilation ≥40 yrs. 5391 participants in NHANES had exams and fundus photographs comparable to that of denudation. Pretreatment with compound C, an from 2005-2008. Weighted frequencies and proportions were estimated, activated protein kinase (AMPK) inhibitor, partially but signifi cantly reduced accounting for study design, sampling technique and survey non-response. vasodilation. Incubation with GW6471, a peroxisome proliferator-activated Factors associated with more severe DR were evaluated using logistic receptor blocker, did not signifi cantly inhibit vasodilation by adiponectin. regression. 1038 subjects had diabetes; 270 had DR. DR prevalence (all AdipoR1 and adipoR2 immunoreactions were observed in the endothelium severity types) was higher in patients with longer diabetes duration (Fig 1a) of retinal arterioles. and more common than diabetic nephropathy (UACR>300mg/g) and self- Conclusion: Adiponectin elicits mainly endothelium-dependent dilation of reported cardiovascular disease among persons with diabetes for ≥10 yr (Fig the retinal arterioles. Endothelium-dependent vasodilation likely induced by 1b). Non-Hispanic blacks and persons with diabetes for ≥10 yr have greater adiponectin results from NO via activation of guanylyl cyclase that is partially odds of more severe DR (Table 1). Data from NHANES confi rm attention dependent on AMPK activity. Understanding the activity of adiponectin on should be paid to these patients at increased risk of diabetic eye disease. the retinal vasculature may help improve potential therapies for retinal vascular disorders, especially diabetic retinopathy in patients with type 2 diabetes mellitus.

625-P AGEs Modulate Proliferation and Apoptosis in Human Retinal Mi- crovascular Endothelial Cell via Gas6/Axl Signaling DONG HO JUNG, YOUNG SOOK KIM, JIN SOOK KIM, Daejeon, Republic of Korea Advanced glycation end products (AGEs)-mediated endothelial cell (EC) dysfunction plays a pivotal role in the development of diabetic complications. Signaling of Axl and its ligand, growth arrest specifi c 6 (Gas6), is implicated in cell growth, survival, adhesion, and migration. However, interaction between AGEs and Gas6 is unclear in human retinal microvascular endothelial cell (HRMEC). In this study, we investigated the effect of various concentration of AGEs (10, 50, 100, 250 and 500 µg/ml) on Gas6 expression and cell viability in HRMEC. Low concentration of AGE-BSA (10, 50 and 100 µg/ml) induced cell proliferation and increased expression of Gas6 in a dose-dependent manner. However, high concentration of AGE-BSA (500 µg/ml) induced apoptosis and decreased expression of Gas6. Neutralizing of receptor for AGEs antibody inhibited AGE-BSA (500 µg/ml)-induced apoptosis by TUNEL assay. Moreover, Gas6 siRNA also inhibited expression of Bcl-2 in AGE-BSA (500 µg/ml)-treated HRMEC. AGEs (500 µg/ml) promoted the expression of TNF-alpha by Western blot and immunostaining. p-JNK and p53 also increased in AGE-BSA (500 µg/ml)-HRMEC. TNF-alpha siRNA also inhibited the expression of p53 and increased the expression of Gas6 in AGE-BSA (500 µg/ml)-HRMEC. In conclusion, AGEs might participate in the pathophysiology of retinal endothelial cell viability via the RAGE-TNF-alpha- Gas6/Axl signaling pathway.

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A158 COMPLICATIONS—OCULAR

Conclusions: A signifi cant proportion of DM patients had progression of DR one year after surgery. DM patients undergoing bariatric surgery require close retinopathy surveillance. There is a need for a prospective study to identify those at greatest risk of DR progression. Supported by: NIHR-CLAHRC (UK)

629-P

TOX and CDKN2A/B Gene Polymorphisms were Associated With POSTERS Decreased Risks of Microvascular Complications of Type 2 Diabetes Complications FENGJIANG WEI, LIMING CHEN, JINGNA LIN, HONG ZHANG, HONGLING HAN, Acute and Chronic JIA LV, CHUNYOU CAI, WENTAO SHI, CHAO LING, PING YU, HONGXIAO JIAO, FUHUA YANG, MINGSHAN LI, WEI-DONG LI, Tianjin, China Microvascular complications of diabetes (MVCD), including diabetic nephropathy (DN) and diabetic retinopathy (DR), are among the leading 627-P causes of diabetes related blindness and renal failure. Decades of research High Cystatin C Level Predicts Retinopathy in Type 2 Diabetic showed that genetic factors played important roles in the susceptibility of Patients MVCD. In this study, we have collected 1329 Han Chinese type 2 diabetes FANG LIU, RUI HE, JING SHEN, HUI ZENG, LIANXI LI, WEIPING JIA, Shanghai, (T2DM) patients in Tianjin, China: numbers of cases for DN, DR, and China proliferative diabetic retinopathy (PDR) were 615, 381 and 91, respectively; Objective: To investigate the relationship between cystatin C and diabetic 165 T2DM patients were collected as controls: they all had been diagnosed retinopathy (DR) and identify the predictive value of cystatin C for diabetic as T2DM for more than 10 years, without microabluminuria or abnormal ophthalmopathy. under opthalmoscope/fundus photography. We selected 22 candidate Methods: A total of 765 type 2 diabetes mellitus outpatients were recruited genes based on our previous studies and published results of genome wide and divided into quartiles with respect to their cystatin C levels (≤0.8 mg/L, association studies (GWAS) for T2DM and/or complications. Binary case- 0.9-1.0 mg/L, 1.1-1.2 mg/L, ≥1.3 mg/L). The severity of DR was grouped from control association studies were performed for DN, DR, PDR, and MVCD by a bilateral retinal photograph as: no diabetic retinopathy (NDR), non-sight- the PLINK program. Results showed that T2DM candidate gene CDKN2A/B threatening diabetic retinopathy (NSTDR) and sight-threatening diabetic was associated with lower risks of PDR (rs10811661, P=0.0054, OR=0.47); retinopathy (STDR). TOX gene SNP rs1526167 was associated with DN (P=0.0029, OR=0.65), Results: There were signifi cant differences in diabetes duration and preva- DR (P=0.0080, OR=0.66), and MVCD (P=0.0023, OR=0.64); SNPs in genes lence of DR among cystatin C quartiles (all P<0.05). The prevalence of DR and SLITRK5, SMAD3, and LPHN3 yielded moderate associations with increased STDR in the fourth quartile (cystatin C≥1.3mg/L) was signifi cant higher than risks of DN, DR, and/or MVCD. We also carried out pair-wise genotype other three quartiles(all P<0.01) There were also signifi cant differences in interaction assays, the interaction between SNPs rs2295251 (SFI1) and diabetes duration, blood glucose and cystatin C levels among 3 DR groups rs1581498 (SNORD93 gene region) was associated with DN, DR, and MVCD (all P<0.05). The mean value of cystatin C in STDR group was signifi cantly (P=0.00011, 0.0012, 0.00014, respectively). The CDKN2A/B was the top type higher than both NSTDR and NDR groups (both P<0.01). Spearman correlation 2 diabetes associated gene in Han Chinese (including our study), however, analysis showed that the severity of DR was positively associated with the association of CDKN2A/B and MVCD was much less documented. diabetes duration, glycemic control, blood urea nitrogen and cystatin C (all Obviously, the MVCD “protective” alleles of the CDKN2A/B and TOX genes P<0.05). Stepwise multiple regression analysis further revealed that cystatin need to be tested in a larger sample sized study. C (beta = 0.242, P = 0.000), diabetes duration (beta = 0.194, P = 0.000) and Supported by: NSFC (81070576 to W.D.L.) glyco syl ated hemoglobin (beta = 0.114, P = 0.005) were independent risk factors for DR. The receiver operating characteristic (ROC) curve indicated 630-P that cystatin C higher than 1.3mg/L predicts high risk of severe retinopathy Relationship between Sleep Disordered Breathing and Diabetic [odds ratio (OR) =4.96, 95%confi dence interval (CI) =3.014-8.152]. Retinopathy: Analysis in 136 Diabetic Cases Conclusions: There is a close association between cystatin C and the severity AKIHIRO NISHIMURA, TAKATOSHI KASAI, KENICHI SAKAMOTO, KIMIKO ISHIG- of diabetic retinopathy. Cystatin C higher than 1.3mg/L predicts the 4 times URO, KAORU NAGASAWA, MINORU OKUBO, KOJI NARUI, YASUMICHI MORI, increasing morbidity of sight-threatening diabetic retinopathy. Tokyo, Japan Supported by: NSFC (81270397) Whether sleep disordered breathing (SDB) contribute diabetic retinopathy (DR) is unknown. Aim of this study is to assess the relationship between SDB and DR. 628-P Consecutive diabetes (DM) patients who underwent sleep study from The Progression of Diabetic Retinopathy Post Bariatric Surgery 2010 to 2012 were investigated. Seventeen patients were excluded due WEN B. LEONG, SHAHRAD TAHERI, Birmingham, United Kingdom to undergoing hemodialysis, heart failure or chronic pulmonary disease. Purpose: To examine the impact of bariatric surgery on diabetic retinopathy Thus, 136 patients are divided into two groups according to the presence (DR) progression in type 2 diabetes mellitus (DM). or absence of DR. Design: Anonymised data from DM patients undergoing bariatric surgery Patients’ characteristics and results of sleep study are summarized in table at a UK regional bariatric surgery center were collected. Data included age, 1. In the univariable logistic regression analysis including presence of DR as gender, ethnicity, pre- and post-surgical bodyweight and body mass index a dependent variable, age, minimum SO2, eGFR, HbA1c, LDL-cholesterol, DM (BMI), DM duration, HbA1c (Hemoglobin A1c), and retinopathy screening duration, hypertension, cardiovascular disease (CVD), insulin and statin use results pre- and 1y post- surgery. were the signifi cant (i.e. P<0.15) variables. In the multivariable backward Results: Seventy-one eligible patients were included. Mean age was 51±8y, logistic regression analysis including them as independent variables, the 87% were white Europeans, and 55% female. Mean pre-surgical weight and fi nal model included minimum SO2 (odds ratio (OR), 0.89; p=0.001), HbA1c BMI were 138±25Kg and 50±7Kg/m2 and mean post-surgical weight and (OR 1.40; p=0.021), DM duration (OR 1.23; p<0.001) and history of CVD (OR BMI were 111±23Kg and 40±8Kg/m2, respectively. The majority of patients 8.96; p=0.008). received either laparoscopic adjustable gastric band (LAGB) or Roux-en-Y In conclusion, minimum SO2 value was associated with DR independent gastric bypass (RYGB) surgery. Median DM duration was 6y (Interquartile from glycemic control level, duration of diabetes, and history of CVD. This range [IQR]:3,11y) with a difference in pre- and post-surgical HbA1c of suggests that SDB may contribute to the development of DR through not 7.17±19.09mmol/mol. Thirty per cent of patients had DR at baseline compared frequency but degree of intermittent hypoxia. to 28% post-surgery. Maculopathy was present in 9% and 7% of patients at baseline and post-operatively, respectively. Ten patients (14%) had worsening DR. Univariate logistic regression showed that LAGB was protective against DR (odds ratio[OR]:0.224,95% confi dence interval[95%CI]:0.068-0.731) while RYGB resulted in greater DR (OR4.023,95%CI:1.253-12.912). After adjustment for other variables, only male gender was associated with better DR outcome (OR:0.111,95%CI:0.016-0.789).

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A159 COMPLICATIONS—OCULAR

Joint Vascular Society Council (JVSC). For each patients we obtained a calf Patients’ characteristics and results of sleep study and a foot score that we have correlated with the grading of retinopathy. We Non-DR group DR group p value Non-DR group DR group p value observed a direct correlation between PAD and DR severity (p<0.05). (N=99) (N=37) (N=99) (N=37) In conclusion, we observed a high prevalence of DR in type 2 diabetic Age (yrs) 53.9±12.3 58.1±12.4 0.080 Body mass index 27.4±4.6 27.4±5.1 0.949 patients with foot complications (70% of these patients have some degree Male-to- 84.8 81.1 0.595 HbA1c (%) 9.5±2.2 10.1±1.8 0.106 of DR). Furthermore, severity of DR correlates, in our patients, with the female ratio severity of PAD.

POSTERS (%) Complications Diabetes 5.7±6.4 16.7±8.7 0.000* eGFR (ml/ 83.8±26.4 75.2±32.1 0.115 633-P Acute and Chronic duration (yrs) min/1.73m2) Serum Sclerostin, an Inhibitor of Wnt/β-Catenin Signaling Path- Hypertension 56.6 89.2 0.000* Minimum SO2 81.8±7.4 78.2±9.6 0.047* way, Is Associated With the Presence of Diabetic Retinopathy in (%) (%) Type 2 Diabetic Patients Cardiovascu- 6.1 32.4 0.000* SO2 4.6±10.5 4.9±7.9 0.867 MASAHIRO YAMAMOTO, Izumo, Japan lar disease Aims: Recently, it has been reported that Wnt/β-catenin signaling pathway (%) is involved in the etiology of diabetic retinopathy (DR) in rat model. Sclerostin Insulin ther- 42.4 59.5 0.077 Apnea hypopnea 15.1±13.6 17.8±15.1 0.325 (SCL), which is secreted from osteocyte and associated with fracture risk, is apy (%) index (AHI) an inhibitor of this pathway by preventing the binding of Wnt to its receptor. To clarify whether this pathway is involved in pathogenesis of DR in human, we Statin use (%) 39.4 56.8 0.070 4% oxygen 15.4±14.7 17.1±15.2 0.544 desaturation examined the relationship between serum SCL levels and DR in 136 Japanese index (4% ODI) postmenopausal T2DM women (DMw) and 175 T2DM men over 50 years old (DMm), whose creatinine (Cr) levels were within normal range. Methods: The severity of DR of the participants was classifi ed into three groups based on Early Treatment Diabetic Retinopathy Study (ETDRS) 631-P classifi cation as follows: no apparent DR, minimal or mild non-proliferative Infl ammatory Factors and STAT3 Expression of PBMC in Type 2 Dia- DR (mild NPDR group), and moderate NPDR to advanced PDR (PDR group). betic Retinopathy The parameters including SCL levels measured by ELISA, bone metabolic YAN-MING CHEN, YA-WEI QIU, PAN-WEI MU, QIU-JU LV, MAN-MAN WANG, markers and bone mineral density (BMD) were compared between those LONG-YI ZENG, Guangzhou, China patients with and without the presence of DR. Endoplasmic reticulum stress (ERS) has been widely implicated in chronic Results: SCL levels were signifi cantly higher in DMm than DMw (1.8 ± infl ammatory diseases, such as diabetes and its complications. This study 0.7 and 1.1 ± 0.4 ng/ml, P < 0.01). Stepwise regression analysis showed aimed to investigate the role and signalling pathways of activator of that SCL was signifi cantly and positively correlated with Cr level and neck transcription 3 (STAT3) in ERS-associated endothelial infl ammation and BMD in DMw as well as with age, HbA1c, Cr, and neck BMD in DMm group. diabetic retinopathy. Thirty-six T2DM patients complicated with retinopathy Bone metabolic markers were signifi cantly and inversely related with SCL in (DR group), 42 T2DM patients without retinopathy (DM group), and 40 DMm. The trend of severe DR grading was associated with decrease in BMI healthy persons (NC group) were enrolled. Peripheral blood mononuclear as well as BMD, prolonged duration of T2DM, and increase in HbA1c as well cells (PBMC) were collected from all subjects. Protein were extracted from as SCL levels. Multiple logistic analyses adjusted for those variables, age, heparinized PBMC. Western blotting was employed to assess glucose- Cr, calcium, and phosphate revealed that SCL levels were associated with regulated protein 78 (GRP78), intercellular adhesion molecule-1 (ICAM-1), the presence of mild NPDR [DMw, 2.8 (95% CI 1.3-6.1), P < 0.05] and PDR tumor necrosis factor (TNF ), phosphorylated STAT3 (p- STAT3) and total α α [DMw, 9.6 (3.0-30.7), P < 0.01; DMm, 1.8 (1.1-2.9), P < 0.05]. STAT3. The level of GRP78 in DR group was signifi cantly higher than that of Conclusion: These fi ndings suggested that Wnt/β-catenin signaling in DM group (P=0.03), and they were signifi cantly higher than that of in NC pathway was involved in the pathogenesis of DR in T2DM patients. group (P<0.01). The trend could also be found in ICAM-1, TNFα and p-STAT3. However, as for total STAT3, there was not obviously different among these 3 groups. Taken together, our data showed that ERS, infl ammation 634-P and p-STAT3 were activated in the PBMC of T2DM patients especially in Changes of Diabetic Retinopathy in Type 2 Patients: A Five-Year those who were complicated with Retinopathy, which suggesting that the Follow-Up Study at Diabetes Clinics in Japan immune cells in the peripheral circulation may affect DR progress by the YUKO WATANABE, HITOMI FUJII, TAKAICHI MIYAKAWA, Tama, Japan above mechanisms. We conducted a 5-year observational cohort study to reveal changes in Supported by: Science and Technology Development Program of Guangdong diabetic retinopathy (DR) and factors related to the development. A total of (2011B031800155) 206 consecutive adult type 2 diabetic patients seen at diabetes clinics in Tokyo were enrolled in 2007. Eligible patients had regular eye examinations by ophthalmologists, and had spot urine albumin to creatinine ratio (ACR) 632-P measured at least two times in each year. An estimated glomerular fi ltration Prevalence of Diabetic Retinopathy and its Correlation With Sever- rate (eGFR) was calculated by the equation for Japanese. ity of Peripheral Artery Disease in Type 2 Diabetic Patients With We excluded patients with kidney failure. We categorized DR in three Foot Lesions strata: no DR; mild to moderate non-proliferative DR (NPDR); and pre- ROSA GIANNARELLI, ALBERTO COPPELLI, ELISABETTA IACOPI, LOREDANA RIZ- proliferative and proliferative DR (PDR). Progression was determined if DR ZO, MICHELE ARAGONA, DARIO FASANO, IRENE BARGELLINI, ROBERTO CIONI, increased more than one stratum after 5 years; regression was determined ALBERTO PIAGGESI, STEFANO DEL PRATO, Pisa, Italy vice versa. Patients had a mean age (SD), 60.4 (10.5); female, 42%; BMI, Diabetic retinopathy (DR) is the most common microvascular complication 24.8 kg/m2 (4.2); HbA1C, 7.51% (1.27); eGFR 79.9 ml/min/1.72m2 (19.9); and of diabetes and one of the major causes of blindness worldwide. ACR 61.9 mg/g creatinine (215.5). 41.6% of patients had diabetic duration We evaluated the prevalence of DR in 295 type 2 diabetic patients (age: more than 10 years. Developmental patterns of DR were divided in 5 groups 69±10 years; males/females 216/79, BMI: 27.4±4.5 Kg/m2, HbA1c: 8.1±1.9%, (% of patients): group I, persistent no DR (62.2); group II, regression (6.3); diabetes duration: 20±12 years) admitted in our Department for diabetic foot group III, progression (19.5); group IV, persistent mild to moderate NPDR (7.5); problems from 2008 until 2011. All patients were examined with indirect and group V, persistent pre-PDR and/or PDR (4.5). Among 5 groups, there and direct retinoscopy and two non-stereoscopic 45° retinal photographs was no difference in age, sex, duration of diabetes, BMI, eGFR, and HbA for each eye. 1C at start; however, group II tended be older and having lower eGFR. HbA According to the Eurodiab Study classifi cation, 90 patients (30%) had no 1C remained high in group III, whereas decreased in groups I, II, and IV (P = retinal lesions; 77 patients (24%) had non-proliferative retinopathy (mild, 0.002) after 5 years. ACR was high in groups IV and V at start (P <0.001), and moderate or severe), and 128 patients (46%) patients had proliferative increased in group III after 5 years (P <0.001). Insulin users were higher in or laser-treated retinopathy. Patients with retinopathy showed a poorer groups III and V (P =0.03). ARBs and anti-lipids use tended higher in group metabolic control (HbA1c 8.3±2 vs 7.5±1.5%, p<0.05), a longer diabetic II. Conclusions: Though our cohort had longer diabetes duration and high duration (22±11 vs 16.5±11.2 years, p<0.05) and a lower mean age (67.7±9.7 volume of insulin users, 62% of patients remained without having DR, and vs 73.1±11.2 years, p<0.05). regression of DR occurred in 6% by treating diabetes vigorously. Changes in In 228 patients (77.3%) was performed lower-extremity angiography. ACR had signifi cant impact on DR fate. Peripheral artery disease (PAD) was graded according to the Classifi cation of

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A160 DIABETIC DYSLIPIDEMIA

635-P A Genome-Wide Association Study of Risk for Progression to Dia- & 637-P betic Macular Edema Non-HDL Cholesterol Is Related to the Number of Cysteine Residues HETAL S. SHAH, JAN SKUPIEN, JASON NOBLE, AHMED Z. SOLIMAN, GABRIEL D. per Mole in apoE in Type 2 Diabetes POZNIK, ADAM M. SMILES, JENNIFER K. SUN, ANDRZEJ S. KROLEWSKI, ALES- NACIDE ERCAN-FANG, ESRA TOKAR, KIRANJOT GUJRAL, AREEF ISHANI, SANDRO DORIA, Boston, MA NANCY GREER, APOSTOLOS GEORGOPOULOS, ANGELIKI GEORGOPOULOS, Min- Much of the visual morbidity of diabetic retinopathy (DR), a leading neapolis, MN Six genotypes of apoE have been described since the 1970s. These are

cause of vision loss in developed countries, is attributable to its severe POSTERS manifestations: diabetic macular edema (DME) and proliferative diabetic encoded by E2, E3, E4 alleles which differ by the number of cysteine-arginine Complications retinopathy (PDR). Although susceptibility to severe DR is likely infl uenced by substitutions. As a result of these amino acid interchanges, apoE2 has two Acute and Chronic heritable factors, the genes involved are still unclear. We aimed to identify cysteine residues per mole, apoE3 has one, and apoE4 has none. Thus the genetic variants associated with the development of DME alone, given the number of cysteine residues per mole in apoE genotypes (i.e. combinations fact that the underlying pathophysiology of DME and PDR may differ. We of 2 alleles) are: zero (apoE4/4), one (apoE3/4), two (apoE2/4, apoE3/3), three conducted a genome-wide association study of time from diabetes onset (apoE2/3), and four (apoE2/2). The purpose of this study was to investigate to DME, as obtained from the medical records of 974 White T1D subjects: the association between apoE genotypes and non-HDL cholesterol levels in 352 with normoalbuminuria, 296 with microalbuminuria, and 326 with type 2 diabetic patients. A total of 539 patients were enrolled in the study. proteinuria. Our GWAS array consisted of a total of 2.5 M single nucleotide The frequency of occurrence of the 6 apoE genotypes was: E4/4: 16 (3.0%); polymorphisms (SNPs), genotyped (Illumina 300K or 1M, or Affymetrix E3/4: 104 (19.3%); E2/4: 18 (3.3%); E3/3: 335 (62.2%); E2/3: 64 (11.9%); 500K chip) or imputed (MACH 1.0). Data were analyzed by means of a Cox E2/2: 2 (0.4%). We found that a higher number of cysteine residues in apoE proportional hazards model stratifi ed by kidney status, applying a robust genotypes was associated with lower non-HDL levels. This relation was variance estimator to account for the clustering of events between the two highly statistically signifi cant (P = 0.009) and an excellent quadratic fi t (R2 = eyes. A joint 2 d.f. test of genetic marginal association and gene-environment 0.991; see attached fi gure). Our fi ndings uncover a specifi c relationship of interaction, with glycemic control (HbA1c below or above the median, 8.4%) apoE genotype and non-HDL cholesterol, based on the number of cysteine as the interactor, was employed. Although no SNPs reached genome-wide residues per mole in the apoE genotype. Our analysis transformed the signifi cance, 7 SNPs on three loci approached the threshold, with p=3 x 10-7 categorical scale of 6 apoE genotypes into a quantitative scale of number for all three loci. These loci were 14q31.3, near genes GALC, SPATA, KCKN10 of cysteine residues per mole, thus enabling a rigorous assessment of apoE and GPR65; 5q34 near ODZ2; and 7q21.11 near GNAI1. Genetic effects of effects using regression analysis. the SNPs on 14q31.3 and 5q34 were exclusively observed in the high HbA1c stratum, with hazard ratios (HRs) of 1.8 and 1.6, respectively, as compared to HRs of 1.0 and 0.9 in the low HbA1c stratum. In conclusion, using a unique methodological approach, we identifi ed suggestive genetic associations for time to onset of DME in T1D subjects, although none attained genome-wide signifi cance.

DIABETIC DYSLIPIDEMIA

Guided Audio Tour: Diabetic Dyslipidemia (Posters: 636-P to 643-P), see page 17.

& 636-P Acetyl-CoA Carboxylase Inhibition by NDI-630 Inhibits Fatty Acid Synthesis, Stimulates Fatty Acid Oxidation, Reduces Body Weight, Improves Insulin Sensitivity, and Modulates Dyslipidemia in Rats GERALDINE HARRIMAN, JEREMY GREENWOOD, SATHESH BHAT, ROSANA KAPELLER, H. JAMES HARWOOD, Cambridge, MA, New York, NY Simultaneous inhibition of the acetyl-CoA carboxylase isozymes, ACC1 & ACC2, results in concomitant inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation (FAOxn) and may favorably affect obesity, diabetes, and fatty liver disease. Our efforts to discover ACC inhibitors & 638-P have focused on interaction with the subunit dimerization site on the Association between Serum Amyloid A and Scavenger Receptor biotin carboxylase (BC) domain of the enzyme to which the phosphopeptide Class B Type I-Mediated Cholesterol Effl ux to Serum in Type 2 Dia- of phosphoACC binds to prevent dimerization and to which the fungal betes metabolite Soraphen A interacts. Using state-of-the-art structure-based J.G.S. TSUN, S.W.M. SHIU, Y. WONG, S. YUNG, T.M. CHAN, K.C.B. TAN, Hong drug design and crystal structures of human ACC2 BC domain, we identifi ed Kong, China a unique series of allosteric inhibitors that bind to the Soraphen binding Serum amyloid A (SAA) is an acute phase response protein and has site, inhibit enzymatic activity, exhibit functional activity in cultured cells, apolipoprotein properties. Recent evidence from animal studies has shown and exhibit favorable drug-like properties and in vivo effi cacy. For example, that SAA impairs reverse cholesterol transport during the acute phase NDI-630 inhibits human ACC1&2 (IC50 2.0nM), inhibits HepG2 cell FASyn response in vivo. Since type 2 diabetes is associated with chronic subclinical (EC50 66nM), stimulates C2C12 cell FAOxn (2-fold 200nM), inhibits rat liver infl ammation, the objective is to investigate the changes in SAA level in FASyn (ED50 0.14mg/kg), and stimulates rat whole body FAOxn (MED 3mg/ type 2 diabetic patients and to evaluate the relationship between SAA and kg). When administered orally 1x/day for 14 days to high fat-fed diet-induced the capacity of serum to induce cellular cholesterol effl ux via the two known obese (DIO) rats at doses 3-30 mg/kg, NDI-630 reduced weight gain without cholesterol transporters, scavenger receptor class B type I (SR-BI) and affecting food intake, reduced the hyperleptinemia and hyperinsulinemia adenosine triphosphate binding cassette transporter G1 (ABCG1). produced by the high fat diet without altering plasma glucose, improved 264 diabetic patients not on lipid lowering agents and 275 non-diabetic insulin sensitivity (oral glucose tolerance testing), and reduced hepatic controls were recruited. Diabetic patients were subdivided into those with triglycerides to chow-fed control levels. When administered as above for 28 normoalbuminuria (NA, n=110), microalbuminuria (MA, n=79) and proteinuria days to high sucrose-fed DIO rats, in addition to the above effects, NDI-630 (P, n=75). SAA was measured by ELISA. SR-BI-mediated and ABCG1-mediated also reduced plasma triglycerides and free fatty acids to chow-fed control cholesterol effl ux to serum was determined by measuring the transfer of 3 levels and markedly reduced plasma cholesterol. These observations suggest [ H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from that our allosteric ACC inhibitors may favorably affect obesity, diabetes, and human ABCG1-transfected CHO-K1 cells to the medium containing the tested fatty liver disease. serum respectively. SAA was increased in MA and P (p<0.01) compared to controls. Both SR-BI-mediated and ABCG1-mediated cholesterol effl ux to serum was signifi cantly impaired in all 3 groups of diabetic patients (p<0.01)

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A161 DIABETIC DYSLIPIDEMIA

with the proteinuric group having the largest magnitude of reduction. These that ATGL plays an important role in mediating effects of FoxO1 on hepatic changes remained signifi cant even after adjusting for plasma HDL-C level. triacylglycerol turnover, lipogenic gene expression and VLDL secretion. SAA inversely correlated with SR-BI-mediated cholesterol effl ux (r=-0.36, p<0.01) but not with ABCG1-mediated cholesterol effl ux. On linear regression & 641-P analysis, plasma HDL-C, SAA and eGFR were independent determinants of High-Dose Resveratrol Treatment for 2 Weeks Inhibits Intestinal SR-BI-mediated cholesterol effl ux. and Hepatic Lipoprotein Production in Overweight/Obese Men SAA was increased in type 2 diabetic patients with incipient or overt SATYA DASH, CHANGTING XIAO, CECILIA MORGANTINI, LINDA SZETO, GARY F. nephropathy and associated with impairment of SR-BI-mediated cholesterol POSTERS LEWIS, Toronto, ON, Canada Complications effl ux to serum. Preclinical and some human data have suggested benefi cial effects of Acute and Chronic Supported by: RGC (HKU776709M) resveratrol (RSV), a SirT1 activator, in improving insulin resistance (IR) and hypertriglyceridemia (HTG). Overproduction of hepatic ApoB-100-containing & 639-P VLDL particles and intestinal ApoB-48 containing chylomicrons contribute A Role for the Lysosome in Mediating β-Cell Lipotoxicity to the HTG of IR. No prior studies have evaluated the effects of RSV on AARON C. BALDWIN, NANCY M. DAHMS, JOHN A. CORBETT, Milwaukee, WI lipoprotein turnover. Here we assessed lipoprotein turnover after 2-week Elevated levels of plasma FFA may provide a mechanistic link between treatment with RSV (500 mg bid week 1, 1 g bid week 2) and placebo (PB) in increased fat mass, insulin resistance, β-cell dysfunction, and ultimately 8 overweight/obese individuals with mild HTG (mean BMI 31 kg/m2, fasting type 2 diabetes. Previous work in our laboratory has demonstrated that triglyceride 2.1 mmol/L) in a randomised double blind crossover study. elevated levels of palmitic acid drive increased acylation of β-cell proteins. Steady state lipoprotein kinetics was assessed in a constant fed state Aberrant palmitoylation of proteins induces ER stress, and if unresolved, cell with constant primed infusion of deuterated leucine. RSV did not affect death ensues. Studies measuring cell viability have revealed a signifi cant insulin sensitivity (HOMA-IR PB=1.8 vs RSV=1.5, P = 0.3) or fasting plasma difference in sensitivity to lipotoxicity between RINm5F and INS832/13 triglyceride (PB=1.95 vs RSV=2.0 mmol/L, P=0.6). RSV signifi cantly reduced β-cell lines. Our comparisons have pointed towards RINm5F cells’ reduced triglyceride rich lipoprotein (TRL) ApoB-48 production rate (PR) (PB= 1.4 vs expression of the cation-independent mannose 6-phosphate receptor (CI- RSV=1.1 mg/kg/day, P = 0.01) with no signifi cant effect on fractional catabolic MPR) as a major contributor to their increased sensitivity. CI-MPR is important rate (FCR) (PB=2.4 vs RSV=2.1 pools/day, P = 0.35) and a non-signifi cant trend for delivery of nascent hydrolases from the Golgi to the lysosomal system, towards a reduction in TRL ApoB-48 concentration (PB=12.9 vs RSV=10.5 and a reduction in CI-MPR expression would compromise lysosome function. mg/L, P=0.15). RSV reduced TRL-ApoB-100 PR (PB=46.8 vs RSV 34.3 mg/ The lysosomal protease cathepsin D is dependent on CI-MPR for delivery kg/day, P = 0.02) and FCR (PB=4.4 vs RSV=3.3 pools/day, P = 0.04), with no to the lysosome, and absent suffi cient CI-MPR, cathepsin D retention is in signifi cant difference in TRL-ApoB-100 concentration (PB=251 vs RSV=245 fact diminished in RINm5F cells. Lysosome-associated membrane protein mg/L, P=0.8). These results indicate that high-dose RSV reduces intestinal 1 (LAMP1) sorts independently of CI-MPR, and accumulates in RINm5F and hepatic lipoprotein particle production, independent of changes in insulin cells consistent with compromised lysosomes. Data from fl uorescent acid sensitivity or triglyceride concentration. Longer-term studies are necessary hydrolase activity assays confi rm diminished lysosomal function in RINm5F to assess the potential signifi cance of these fi ndings in the management of cells. By interfering with lysosomal acidifi cation, chloroquine treatment of dyslipidemia. INS832/13 cells renders them as sensitive to palmitate as the RINm5F cells. Supported by: CIHR Early experiments with shRNA knockdown of CI-MPR in INS832/13 cells demonstrate decreased hydrolase activities and increased sensitivity to & 642-P lipotoxic death similar to that observed in RINm5F cells. High concentrations The Role of Peripheral and Central GLP-1 Receptor Signaling in Reg- of FFA may stimulate degradative cellular pathways in an effort to reduce ulation of Hepatic Triglyceride Homeostasis and VLDL Production the aberrantly palmitoylated protein load. The CI-MPR dependent lysosomal JENNIFER SACCO, CHRIS BAKER, MARK NAPLES, KHOSROW ADELI, Toronto, ON, proteolytic activity appears to play an important role in mediating the Canada lipotoxic effects of palmitic acid as demonstrated by the discrepancies in Glucagon-like peptide-1 (GLP-1) is an important drug target in the treatment sensitivity between INS832/13 and RINm5F cells. of type 2 diabetes. It is produced by both the intestinal mucosa and brainstem Supported by: R01-DK52194 in response to nutrient ingestion and subsequently stimulates pancreatic insulin release to reduce blood glucose levels. GLP-1 has been previously & 640-P shown to regulate intestinal lipoprotein metabolism and may also play a Adipose Triacylglycerol Lipase and G0S2 Mediate Effects of FoxO1 similar role in the liver. We postulate that GLP-1 receptor signaling regulates on Hepatic Triacylglycerol Turnover, Lipogenic Gene Expression hepatic lipid homeostasis by controlling very low density lipoprotein (VLDL) and VLDL Secretion production in insulin resistance states. Insulin resistance may lead to impaired WENWEI ZHANG, SO YOUNG BU, MARA T. MASHEK, INSUG O-SULLIVAN, C. GLP-1 receptor signaling thus contributing to impaired VLDL production and RONALD KAHN, DOUGLAS G. MASHEK, TERRY G. UNTERMAN, Chicago, IL, Bos- induction of hepatic steatosis. To test these hypotheses, experiments were ton, MA, St. Paul, MN conducted in vivo in diet induced insulin resistant Syrian golden hamsters FoxO proteins are major targets of insulin action and regulate gluco- and C57BL/6 mice. Chronic peripheral treatment with the GLP-1 receptor neogenic, glycolytic and lipogenic gene expression in the liver (JBC agonists, exendin-4 (Byetta) or liraglutide (Victoza), prevented fructose- 281:10105, 2006). FoxO1 promotes lipid mobilization in adipose tissue induced dyslipidemia and decreased VLDL production in both hamster and through regulation of adipose triacylglycerol lipase (ATGL), and ATGL also mouse models as shown by signifi cantly reduced plasma and VLDL-TG, regulates lipid turnover in liver. We asked whether FoxO1 also regulates -cholesterol and -apoB100 levels. This was accompanied by decreased expression of ATGL and its inhibitor, the G0/G1 switch gene protein 2 (G0S2), hepatic lipid content. Intriguingly, acute central administration of exendin-4 in liver. Studies in liver-specifi c transgenic mice show that FoxO1 stimulates via intracerebroventricular injection in hamsters also induced a signifi cant ATGL and suppresses G0S2 expression, and studies in FoxO knockout mice reduction in postprandial VLDL-TG and -cholesterol levels, suggesting that confi rm that endogenous FoxO proteins contribute to regulation of ATGL and the observed effects on hepatic VLDL production may be due to central G0S2 in liver. Studies in liver-specifi c insulin receptor knockout (LIRKO) mice, GLP-1 receptor signaling. We suggest that central GLP-1 receptor activation and LIRKO mice in which FoxO1 also was inactivated in the liver indicate may ameliorate hepatic VLDL production in insulin resistant states. GLP-1 that FoxO1 plays an important role in mediating effects of insulin on hepatic receptor agonism may be an effective tool in ameliorating hepatic steatosis expression of ATGL and G0S2 in vivo. Studies utilizing adenoviral vectors in and hepatic lipoprotein overproduction. isolated hepatocytes confi rm that FoxO1 stimulates ATGL and suppresses Supported by: Heart and Stroke Foundation of Ontario G0S2 expression, and metabolic labeling studies demonstrate that FoxO1 promotes triacylglycerol (TAG) turnover and catabolism through an ATGL- & 643-P dependent mechanism. Interestingly, suppressing hepatic ATGL expression Cinnamon Polyphenols Reduce the Secretion of Apolipoprotein and/or function in FoxO1 transgenic mice increases hepatic TAG content B48-Containing Lipoproteins by Increasing Sirtuins Expression in and also raises circulating TAG levels, refl ecting increased lipogenic gene an Intestinal Porcine Epithelial Cell Line expression and secretion of VLDL, based on analysis by quantitative PCR, BOLIN QIN, HARRY D. DAWSON, RICHARD ANDERSON, Beltsville, MD FPLC fractionation of circulating lipoproteins and inhibition of VLDL turnover Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate key with with Tyloxapol. Together, these results demonstrate that FoxO proteins aspects of lipid metabolism. We have reported previously that cinnamon regulate hepatic expression of ATGL and its inhibitor, G0S2, and indicate polyphenols (CP) improve intestinal derived apolipoprotein (apo) B48-

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A162 DIABETIC DYSLIPIDEMIA containing lipoproteins production in insulin resistant animals. In the current glucose alternating every 12 h). In the presence of PA, SHG induced higher study, we investigated whether the benefi cial effects of CP on apoB48 apoptosis rate (P<0.05 vs NG+F) with concomitant increases in the apoptosis secretion are mediated via SIRTs using an in vitro model of the intestinal protein cleaved caspase-3, 8 and PARP compared with cells in NG; IHG porcine epithelial cell line, IPEC-1. Differentiated postconfl uent IPEC-1 cells, further augmented the apoptosis (P<0.05 vs SHG+PA). Exposure to SHG with grown and treated with CP (10 µg and 100 µg/mL) for 24 h or 48 h, were shown PA also induced higher ROS production compared with those in NG with PA by immunoblotting to inhibit chylomicron-size apoB48 secretion into media (P<0.05), with even higher ROS levels detected in cells treatment with IHG in a dose-dependent manner. CP were shown to enhance cellular SIRT1, 2, with PA (P<0.05 vs SHG+PA). Furthermore, IHG with PA resulted in more 3, 5, 6 and 7 protein expression by immunofl uorescence and immunoblotting swollen mitochondria with more translucide matrix and less cristae, with POSTERS analyses. We also investigated the effects of CP on infl ammatory and highest release of cytochrome c (cyt c) among cells treated with different Complications lipogenic factors, which have been linked recently with the SIRTs signaling glucose concentrations in the presence of PA (P<0.05). Co-treatment with the Acute and Chronic cascade. There was a signifi cant decrease in the infl ammatory factors, TNF-α mPTP inhibitor CsA (1.5µmol/L) prevented both IHG and SHG with PA induced and phospho-NF-kb p65 protein expression in CP treated IPEC -1 cells. CP also ultrastructural abnormalities, decreased cyt c release and ROS production, as downregulated sterol regulatory element binding protein 1c, protein tyrosine well as hepatocytes apoptosis.These fi ndings suggest that glucose fl uctuation phosphatase 1B, and microsomal triglyceride transfer protein which is required together with lipotoxity are reckoned to be the most important factor in for the assembly and secretion of apo B-containing lipoproteins. CP treatment causing hepatocyte apoptosis, and mPTP may act as a “central executioner” also upregulated ATP-binding cassette sub-family G member 5 and scavenger of hepatocytes subjected to the insult of PA and glucose fl uctuation. receptors class B 1 protein levels. Taken together, a novel fi nding of this study Supported by: NSFC (81000364) was the observation that cinnamon polyphenols could improve intestinal apoB48 metabolism through activation of the sirtuins signaling cascade. 646-P Supported by: Integrity Nutraceuticals International; U.S. Dept. of Agriculture Glucagon-Like Peptide-1 Reduces Intestinal Lipid Availability and (58-3K95-7-1184) Lipoprotein Production via a Gut-Brain Axis SARAH L. FARR, CHRIS BAKER, MARK NAPLES, KHOSROW ADELI, Toronto, ON, 644-P Canada Evidence for Several Genetic Variants Affecting Lipoprotein (a) Postprandial dyslipidemia is a component of the metabolic syndrome Level and insulin resistant states, and involves the intestinal overproduction of MAO FU, WENSHEN LU, YU-CHING CHENG, QUINCE GIBSON, XIALIAN SHI, atherogenic chylomicron (CM) particles. Recent evidence indicates that the KEITH TANNER, JEFFREY R. O’CONNELL, BRAXTON D. MITCHELL, ALAN R. hormone glucagon-like peptide-1 (GLP-1) can reduce CM secretion and that its SHULDINER, Baltimore, MD production is impaired with insulin resistance. There is evidence that GLP-1 Lipoprotein (a) [Lp(a)] is an independent risk factor for atherosclerosis- may hinder hepatic lipoprotein assembly by reducing tissue lipid availability; related events. Genetic variation in the LPA gene may be responsible for however, the mechanism of action of GLP-1 on intestinal CM production and between 42% and >90% of variation in circulating Lp(a) concentrations. whether these effects are mediated centrally remains unclear. To test this, We performed a genome-wide association scan for genetic variants that chow-fed hamsters were given an oral fat load containing 3H-triolein and an associated with serum Lp(a) levels in the Old Order Amish. Using the intraperitoneal injection of the GLP-1 receptor (GLP-1R) agonist exendin-4 Affymetrix 500K chip, we successfully genotyped 382,935 single-nucleotide (Ex4, 5nmol/kg) (Byetta). Postprandial lipid and apoB48 accumulation was polymorphisms (SNPs) in 1,200 subjects, and imputed all autosomal SNPs assessed over 8 h in the triglyceride (TG)-rich lipoprotein (TRL) fraction of in HapMap by MACH. We identifi ed two loci signifi cantly associated with the plasma, and the distribution of 3H between jejunal tissue and luminal Lp(a) on chromosome 6q25-q26 and chromosome 11q23 (P < 5 × 10-8) . The contents was assessed. We found that Ex4 signifi cantly lowered TRL-TG, locus on chromosome 11 is close to the APOA5-APOA4-APOC3-APOA1 cholesterol and apoB48 levels; these effects remained even 8 h post fat gene cluster. We identifi ed 137 common variants (P = 5 × 10-8 to 3.91 × load. Jejunum from Ex4-treated hamsters displayed reduced TG and 3H 10-19) spanning ~5.3 Mb region on chromosome 6q25-26 that were within levels at 2 h, with heightened 3H levels in jejunal contents suggesting fat or fl anking 26 genes including LPA. Common variants in six genes (SNX9, malabsorption. By 6 h, jejunal activity of microsomal TG transfer protein SERAC3, TAGAP, FNDC1, PLG, and AGPAD4) were signifi cantly associated (MTP) was reduced with Ex4 treatment, consistent with the notion of limited with Lp(a) levels. To further investigate the effect of LPA variants on Lp(a) TG availability. To determine whether these effects were mediated by neural levels, we sequenced the 40 exons, all intron-exon boundaries and 2 kb of signalling, fat-loaded hamsters were given an intracerebroventricular (ICV) the promoter region of the LPA gene in 24 Amish subjects. We identifi ed 23 injection of Ex4. Similar to peripheral administration, central Ex4 caused variants including 6 missense variants in exons 26, 32, 37, 39 and 40. Rare prolonged (up to 6 h) and signifi cant reductions in TRL-TG and apoB48 levels. genetic variants (rs 3798220, rs10455872) were signifi cantly associated with These effects were negated by co-administration of the GLP-1R antagonist Lp(a) levels (P value: 1.07 × 10-14, and 1.85 × 10-12; minor allele frequency: exendin9-39. In conclusion, Ex4 impairs jejunal TG absorption inducing 0.009 and 0.022, respectively). We also measure kringle IV-2 (KIV-2) numbers prolonged reductions in CM lipidation and assembly. These effects appear using qPCR. The KIV-2 numbers were signifi cantly associated with Lp(a) to be mediated centrally and merit further study. levels (P = 2.28 ×10-9). Conditional association revealed that rs3798220 and Supported by: CIHR rs10455872 associations with Lp(a) levels were independent from one other, and KIV-2 numbers were partially dependent the two SNPs. In conclusion, 647-P we have confi rmed that several genetic variants are signifi cantly associated The Effect of Isolated Hyperglycemia on VLDL Kinetics with Lp(a) levels. Studies characterizing further variation in the genes in this RAKEL F. JOHANSEN, ESBEN SØNDERGAARD, LARS PETER SØRENSEN, JENS S. region and their functional consequences on Lp(a) levels are warranted. CHRISTIANSEN, SØREN NIELSEN, Aarhus, Denmark Supported by: AHA An increased production of very low density lipoprotein-triglyceride (VLDL- TG) plays a key role in diabetic dyslipidemia. The reason for such an increase 645-P is not fully understood, but insulin and glucose levels, insulin resistance, and Intermittent High Glucose Potentiates Palmitate Induced Apoptosis free fatty acid (FFA) availability is believed to play a role in VLDL regulation. of Hepatocytes via Mitochondrial Permeability Transition The aim of this study was to determine the isolated effect of hyperglycaemia XUEYAO YIN, QIANQIAN PAN, SAIFEI ZHANG, FENPING ZHENG, HONG LI, Hang- on VLDL kinetics. zhou, China VLDL-TG kinetics was examined in 8 type 1 diabetic men (no endogenous Mitochondrial dysfunction and oxidative stress, regarded as key insulin production). A 3 hour basal period (glucose 5 mmol/l) was followed mediators in fatty acid-induced hepatocyte apoptosis are thought to be by a 4 hour hyperglycaemic period (glucose 16 mmol/l). An initial insulin core abnormalities responsible for the pathogenesis of nonalcoholic fatty infusion (1.0 mU/kg/min) was administered to normalize plasma glucose liver disease. Our recent data have shown that intermittent high glucose during the initial 30 min of the basal period, followed by a replacement dose (IHG) plays a more signifi cant role in the mitochondria oxidative stress than of a 0.15 mU/kg/min for the remaining study period. Steady state VLDL-TG sustained high glucose (SHG). We sought to clarify the effect of glucose kinetics (VLDL-TG secretion, clearance and oxidation) were assessed by the fl uctuation on the hepatocytes apoptosis induced by palmitate (PA), and the isotope dilution technique using an intravenous primed-constant infusion of potential mechanisms related with mitochondrial permeability transition ex-vivo labeled [1-14C]VLDL-TG in combination with frequent blood sampling (mPTP). Hepatocytes L02 were incubated for 72 h in the medium containing and sampling of expired air. different glucose concentrations with or without PA (0.125mmmol/L): control VLDL secretion rate was unchanged during the basal and hyperglycemic (NG, 5.5 mmol/L), SHG (33.3 mmol/L) and IHG (5.5 mmol/L and 33.3 mmol/L period (35.1 ±26.8 vs. 35.3 ±19.7 µmol/min; ns). A modest, non-signifi cant

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A163 DIABETIC DYSLIPIDEMIA

rise in plasma VLDL-TG concentration (0.25 ±0.22 vs. 0.30 ±0.21 mmol/l) was observed while total plasma TG levels were unchanged. VLDL clearance rate was not altered signifi cantly (155 ±41.1 to 144 ±77.8 ml/min) in response to hyperglycaemia. Average VLDL-TG fatty acid oxidation rate increased from 18.17 ±10.30 µmol/min during the basal period to 25.15 ±14.8 µmol/min during hyperglycemia, but the increase was insignifi cant. FFA levels were not altered during hyperglycemia. We conclude that 4 hours of acute hyperglycaemia (16 mmol/l) without POSTERS

Complications concomitant insulin increase does not alter VLDL-TG kinetics in type 1

Acute and Chronic diabetic patients.

648-P Safety of Statin Therapy in Patients With Prediabetes or T2DM and NASH: A Long-Term Prospective Study FERNANDO BRIL, ROMINA LOMONACO, BEVERLY ORSAK, JOAN FINCH, CARO- LINA ORTIZ-LOPEZ, CELIA DARLAND, AMY WEBB, KENNETH CUSI, Gainesville, FL, San Antonio, TX Patients with prediabetes or type 2 diabetes mellitus (T2DM) have a high cardiovascular risk but are often denied statins due to elevated liver enzymes (LFTs) in the setting of nonalcoholic steatohepatitis (NASH). We performed the fi rst prospective analysis on the safety of statins in biopsy-proven NASH patients with LFTs ≤2.5 fold ULN (n=86, age: 52 ± 1 yr, BMI: 34.4 ± 0.5 kg/ m2, A1c: 6.4 ± 0.1%, T2DM: 51%, AST/ALT: 45/64 IU/L). They were part of an 18-month randomized controlled trial of pioglitazone (PIO) vs. placebo in patients with prediabetes or T2DM and NASH. This was followed by an open- label extension of PIO treatment for up to 36 months. We measured LFTs every 1-2 months and performed a liver biopsy at 0, 18 and 36 months. There 650-P were no clinical, biochemical (LFT levels) or histological differences between Elevated Lipoprotein Lipase (LPL) Does Not Account for the Asso- statin and non-statin users at baseline. At study entry, all had an indication ciation between Increased Adiponectin (APN) and High HDL in Type for statins but due to elevated LFTs they were prescribed in only 37%. This 1 Diabetes (TID) increased to 67% after the run-in and to 90% at 6 months (simvastatin used SYLVIA DIAZ, ROSANNA CALDERON, ANGELA SZETO, ARMANDO MENDEZ, in 67%; median dose = 40 mg/d). Plasma LDL-C was reduced by 24% (p=0.02). THOMAS A. HUGHES, RONALD B. GOLDBERG, Miami, FL, Memphis, TN In patients on placebo (n = 42), plasma ALT decreased at 18 months (60 ± 6 We have shown that APN levels correlate strongly with HDL-C in T1D to 39 ± 3 U/L; p<0.001) although BMI and histology were unchanged. Even suggesting a link between the elevated APN and the increased HDL-C found in those with elevated LFTs at baseline, statin therapy was well tolerated in T1D. APN levels have been shown to be associated with LPL, and elevated without any further LFT increase. Two patients on statins (1 on placebo, LPL could contribute to the high HDL-C in T1D. Here we investigated if the 1 on PIO) had a 2-fold increase in LFTs during follow-up but returned to association between APN and HDL-C in T1D is accounted for by elevated normal within 2-3 months without statin discontinuation or dose-titration. In LPL. We measured APN, HDL-C, apo A-I, LPL and the cholesterol content (Ch) another 2 patients, the increase in LFTs predated statin therapy initiation. In of light (HDL-L), medium (HDL-M) and dense (HDL-D) HDL subfractions using patients on PIO (n = 44), statin therapy was also well tolerated, with plasma density gradient ultracentrifugation in 69 T1D (39 women) and 42 healthy ALT decreasing at 18 months (68 ± 6 to 28 ± 2 U/L; p<0.001) and with an controls (CON, 23 women). APN, LPL, Apo A-I, HDL-C, and HDL-LCh levels improvement in histology that was likely related to PIO therapy. were signifi cantly higher in T1D than controls (p<0.002) with differences by Conclusion: statin therapy is safe for patients with prediabetes or T2DM sex (table). APN correlated with HDL-C and HDL-LCh in CON (p<0.01) and T1D and NASH under adequate monitoring. Given their high CV risk, statin use (p<0.05) women, and with HDL-C in T1D men (p<0.01) LPL was not related to should not be withheld in this population. APN or HDL-C overall in T1D or CON, but among T1D women LPL correlated Supported by: Burroughs Wellcome Fund with HDL-C (r=0.501), apo A-I (r=0.475), and HDL-LCh (r=0.522); p<0.002, and 649-P in T1D men with apo A-I only (r=0.435; p<0.02) but not in CON women or Ezscan as a Screening Tool for Oxidative Stress Parameters Based men. In regression analyses in T1D women, the APN association with HDL-C on Sudomotor Assessment was not accounted for by LPL (r=0.389 p<0.02). While LPL is elevated in T1D JEAN-HENRI CALVET, ISABELLE DUFAURE, PETER SCHWARZ, Paris, France, Dres- women and LPL and APN are both correlated with similar HDL measures, LPL den, Germany does not account for the relationship between elevated APN and increased Type 2 diabetes is associated with high cardiovascular morbidity and HDL in men or women, suggesting that this linkage is independent of mortality. Oxidized LDL (Ox-LDL) and anti-oxLDL antibody levels (anti-ox-LDL) elevated LPL in T1D. can be used as markers of atherogenesis, which often precedes diabetes onset. Sweat glands are innervated by small C-fi bers that can be damaged in All data are mean+/-SD; a=p<0.05 vs CON; b=p<0.001 vs CON the early stages of dysglycemia. Ezscan was developed to be a non invasive, APN LPL umol Apo A-I HDL-C HDL-LCh HDL-MCh HDL-LCh quick, simple and quantitative measurement of sweat function. This study (ug/ml) FFA/ml/hr (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) aimed to determine the ability of this method to detect disturbances in T1D-M 11.2±6.2a 3.7 ± 3.0 151±24a 56±14b 20.1±9.1b 25.8± 4.9 6.5 ± 1.8b oxidative stress parameters in German subjects with a family history of type T1D-W 16.6±7.3b 5.6± 3.9b 182±34a 73±19b 32.9±16.6a 29.5± 6.5 6.2 ± 2.1b 2 diabetes, obesity or dyslipoproteinemia. CON-M 8.9 ± 3.0 3.7 ± 1.8 138± 19 46± 10 12.1 ± 4.8 29.5± 6.5 8.3 ± 1.6 Plasma levels of Ox-LDL and anti-ox-LDL were measured by enzyme immunoassay and by ELISA, respectively. Small C-fi ber status using Ezscan CON-W 11.4± 4.2 2.7 ± 1.6 164± 28 61± 16 21.1± 12.0 31.0± 6.5 8.4 ± 2.1 was assessed by measurement of hand and foot sweat function and calculation of a risk score. Among the 82 subjects involved in the study, 38 had impaired glucose 651-P tolerance and 6 had newly diagnosed diabetes. Correlation between Ezscan Meta Analysis: Intensifi ed LDL-c Target of Statin Therapy and Risk risk score and ox-LDL/anti-ox-LDL levels and between ox-LDL and anti-ox- of Incident Diabetes LDL levels were 0.32, p=0.004 and 0.42 p<0.0001 respectively. Receiver RONGRONG CAI, YI ZHOU, YUE YANG, WENQING XIA, YAN HUANG, PIN WANG, Operating Characteristics (ROC) curve for detection by the risk score of ox- SHAOHUA WANG, Nanjing, China LDL level higher than 77 U/L and/or anti-ox-LDL level lower than 212 U/L Meta analysis: Intensifi ed LDL-c target of statin therapy and risk of incident is displayed in Figure. For a value of the risk score of 50% sensitivity and diabetes Evidence based medicine prompted that intensifi ed low-density specifi city were 73% and 67% respectively. lipoprotein cholesterol (LDL-c) goal may provide even greater cardiovascular Ezscan could be used as a screening tool for oxidative stress risk in daily benefi ts. However, there were fi ndings suggesting that statin therapy may practice. increase the development of new-onset diabetes. We done a meta-analysis

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A164 DIABETIC DYSLIPIDEMIA among large randomized controlled trials(RCT)to identify whether there drugs). Lipids were obtained at baseline (after 4 weeks off anti-lipid drugs or is relationship between statin use and development of incident diabetes 1st treatment drug) and after each drug. among patients with intensifi ed LDL-c target goal. Twenty (55% male) T1DM and 27 (44% male) T2DM completed the study. A total of 60287 non-diabetic participants with intensifi ed target goal T2DM were older (65 ± 10 vs 49 ± 13 years, p < 0.01), had higher BMI (28 ± 2 level of LDL-c in statin group (LDL-c target of <2.59 mmol/L (100 mg/dl), vs 26 ± 3 kg/m2, p = 0.01), and lower HbA1C levels (6.4 ± 0.8 vs 7.2 ± 0.0, p < or relative LDL-c reduction at least 30% of baseline) from 8 randomized, 0.01) than T1DM. There were no signifi cant differences between mean lipids placebo-controlled, double-blind trials were included in this meta-analysis. at 1st and 2nd baseline (after washout) within each group. The results (Table) We used the I² statistic to measure heterogeneity between the 8 trials and remained signifi cant after adjusting for age, gender, and baseline lipids. POSTERS calculated an overall OR with a random-effects model meta-analysis to The study suggests that statins are not as effective in T1DM as in T2DM, Complications identify potential effects of statin therapy on incident diabetes. and ezetimibe should be considered for treatment of hyperlipidemia among Acute and Chronic Among 60287 participants, 2994 patients (1613 assigned to statins and patients with T1DM. Studies are needed to confi rm these fi ndings and assess 1381 assigned to placebo) developed diabetes during a mean of 4 years. CVD protection conferred by ezetimibe among subjects with T1DM. Intensifi ed LDL-c target of statin therapy was associated with an 18% increased risk for diabetes (odds ratio [OR] 1.18; 95% CI 1.10-1.28), with little Simvastatin Ezetimibe p value heterogeneity (I²=0.0%) between trials. There was one additional case of (% change ± SD) (% change ± SD) diabetes per 130 patients taking statin therapy for 4 years. Fail-safe number TYPE 1 DM was 53 (P=0.05) in this meta-analysis. Total Cholesterol -15 ± 25 -19 ± 12† 0.97 Intensifi ed LDL-c target of statin therapy slightly increase risk of development HDL-Cholesterol 5 ± 22 -2 ± 25 0.85 of new-onset diabetes, but the risk is low compared to the decrease in coronary heart disease. Since there is low risk of development of incident diabetes in LDL-Cholesterol -9 ± 68 -28 ± 16† 0.80 intensifi ed LDL-c target of statin therapy, clinical practice with general statin Triglycerides -10 ± 30 -6 ± 30 0.71 therapy shouldn’t be changed in patients with cardiovascular risk or existing TYPE 2 DM cardiovascular disease before better drugs appear. Total Cholesterol -24 ± 18 -2 ± 23 0.0001 Supported by: Zhongda Hospital of Southeast University HDL-Cholesterol 24 ± 39 3 ± 18 0.002 652-P LDL-Cholesterol -38 ± 26 -4 ± 36 <0.0001 Association of Medication Adherence Levels and Achievement of Triglycerides -9 ± 31 12 ± 33 0.04 Lipid Goals † p< 0.05 in the effect of ezetimibe between T1DM and T2DM JONATHAN H. WATANABE, MARK BOUNTHAVONG, TIMOTHY CHEN, Pomona, CA, San Diego, CA Supported by: NCATS (8UL1TR000055) We evaluated the relationship between different categories of the medication adherence metric medication possession ratio (MPR) and 654-P achievement of American Diabetes Association goal for levels of Low Different Proteins Expression in Liver of Insulin Receptor Knock- Density Lipoproteins (LDL) as well as a common goal for non-High Density out Mice: Role of HMGB1 as an Alarmin in Diabetic and Steatotic Lipoproteins (non-HDL). Conditions We performed a retrospective cohort study of 4 018 new statin users from BARBARA CAPUANI, SARA CARATELLI, ANDREA COPPOLA, DONATELLA PAS- the Department of Veteran Affairs (VA). Subjects were required to be eligible TORE, FRANCESCA PACIFICI, ROBERTO ARRIGA, FRANCESCA FERRELLI, MARIA for VA medical and pharmacy services for the 1 year study period from index PAOLA CAPUTO, ELISA DE CARLI, ANNA NERI, ALFONSO BELLIA, MASSIMO FED- date and to have complete data for exposure, outcome, and adjustment ERICI, GIULIA DONADEL, GIUSEPPE SCONOCCHIA, DAVIDE LAURO, Rome, Italy variables. MPR was defi ned as days supplied of medication divided by days Different conditions are important in the pathogenesis of non-alcoholic of observation. Fatty Liver Disease (NAFLD) as insulin resistance, diabetes mellitus (DM) Multiple logistic regression adjustment variables were age, gender, race, and metabolic syndrome. The availability of murine model with DM and copayment status, income category, statin used, and baseline: lipid levels, steatosis, induced by deletion of one or two allele of insulin receptor (IR- body mass index, medication count, and presence of comorbidities. MPR /-, IR+/-) gene, allowed us to investigate and identify new molecules which exposure categories were 0 to 0.39 (reference), 0.4 to 0.69, 0.7 to 0.79, 0.8 to defi ne the switch between severe hepatic insulin resistance and hepatic 0.89, 0.9 to 1.0. Main outcome measure was achievement of 1 year follow- pathological lipid accumulation in diabetic conditions. To reach this purpose up levels of < 100 mg/dl and < 130 mg/dl for LDL and non-HDL respectively. we compared by different proteomic approaches, hepatic proteins from mice Analyses performed via SAS 9.3 (Cary, NC) with α = 0.05. wild type, heterozygous and null in insulin receptor (IR+/+, IR+/-, IR-/-). The Odds ratios (ORs) for both outcomes reached a maximum for the 0.9 to complete list of identifi ed proteins was loaded into IPA (Ingenuity Pathways 1.0 MPR category with ORs of 7.30 (95% Confi dence Interval (CI), 5.61, 9.50) Analysis), to fi nd new possible networks. for the LDL outcome and 7.67 (95% CI, 5.90, 9.97) for the non-HDL outcome. First, we determined hepatic steatosis severity in all phenotypes For both outcomes, The ORs for the 0.9 to 1.0 MPR category estimates observing an higher steatosis grade in IR-/- than IR +/- and IR+/+. Then, we exceeded those for the 0.8 to 0.89 MPR category. Direct comparison of the identifi ed 28 proteins differentially expressed in the three phenotypes by 0.9 to 1.0 MPR category versus the 0.8 to 0.89 MPR category demonstrated 2D-GE, and 25 proteins in nLS MS/MS spectrometry. It’s very interesting to increase in odds of achieving lipid goals. This analysis was performed in a VA outline that immune system process, glycolysis, gluconeogenesis, transport population. Findings may be modifi ed in other populations. and lipid metabolism were regulated from the same set of proteins. IPA study We conclude that improvements in lipoprotein goal attainment are realized determined two networks strictly associated to energy production and lipid as MPR increases. This progression continues beyond the common threshold metabolism functions while a third network showed molecular relationships of 0.8 MPR. Our fi ndings suggest additional lipid reduction may be possible between IR (Insulin Receptor) and some transcription regulators such as with elevated MPR adherence thresholds than commonly targeted. HMGB1 known to be involve in NAFLD onset. For this reason we tested HMGB1 presence in diabetic sera patients with different steatosis grade, 653-P using ELISA assay, immunoprecipitation and western blot. We showed Statin Effi cacy in Type 1 and Type 2 Diabetes Mellitus how HMGB1 had reached maximum sera levels, in diabetic patients with KEVIN CIRIACKS, SHANTHI KRISHNASWAMI, SHAILENDRA PATEL, SRIVIDYA higher hepatic steatosis score. In conclusion we found by proteomic analysis KIDAMBI, Milwaukee, WI interesting molecules whose function it’s not well known in DM and NAFLD; Statins lower total and LDL cholesterol (TC and LDLC) levels among particularly HMGB1 because its secretion in serum correlates with NAFLD patients with type 1 and 2 diabetes (T1DM and T2DM). Studies suggest severity. patients with T1DM have high gut cholesterol absorption; hence cholesterol Supported by: MEDIGENE absorption inhibitors such as ezetimibe may be as effective. We compare the effects of statins and ezetimibe among T1DM and T2DM in this study. Subjects with T1DM and T2DM with hemoglobin A1C (HbA1C) ≤ 8.5%, BMI ≤ 31 kg/m2, and no cardiovascular disease (CVD) were recruited. Subjects were assigned to alternating therapy with 40 mg of simvastatin (statin) or 10 mg of ezetimibe therapy for 6 weeks (with 4 week washout period between

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A165 DIABETIC DYSLIPIDEMIA

655-P SS, interleukin (IL)-6 and IL-8 production, and cleavage of α-fodrin to produce A Critical Role of Ceamide-Mediated ATF3 on Hepatic Steatosis in 120-kDa fragments are increased, which is followed by apoptosis in salivary Metabolic Disease Models By Changes of Caveolin-1/CD36 Compo- gland epithelial cells (SGECs). We previously reported that palmitate induces sition IL-6 secretion in human SGECs. In this study, we investigated whether JI YEON KIM, KEON JAE PARK, WON HO KIM, Chungbuk, Republic of Korea high levels of FFAs, especially saturated fatty acids, promote severity of In the onset and progression of ASH and NASH, ceramide is signifi cantly primary SS. Palmitate treatment induced IL-6 and IL-8 production, α-fodrin increased by alterations of sphingolipid. However, the regulatory mechanisms degradation, and apoptosis in human SGECs, but not in human squamous carcinoma cells and keratinocytes. Unsaturated fatty acids failed to induce

POSTERS by which ceramide drives hepatic steatosis remain poorly understood.

Complications Here, we examined the role of ATF3 on ceramide-mediated liver steatosis α-fodrin degradation in human SGECs. Furthermore, when model mice of Acute and Chronic in ethanol-fed mice or HFD-fed rats. Hepatic steatosis increased in both primary SS were fed with high-fat diet to elevate the serum level of FFAs, models are correlated with the increase of ceramide levels in their serum their salivary glands and lacrimal glands exhibited infl ammation signifi cantly or liver tissues, accompanied with the expression of caveolin-1, CD36/FAT, more advanced than those observed in model mice fed with normal diet. and sphingomyelin synthase-2, which was determined by TNF-α production Taken together, these results suggest that higher levels of FFAs, presumably and TNFR1-dependent pathway. Concomitantly, a stress-inducible ATF3 was saturated fatty acids, may promote the severity of primary SS. signifi cantly increased in these models and it plays as a potent regulator for de novo ceramide synthesis and ceramide-mediated lipid accumulation. 658-P Also, ATF3 directly regulates caveolin-1 and CD36/FAT expression, thereby Changes in BMIz Infl uence the Lipid Profi le in Youth With Type 1 increases ceramide-mediated lipogenesis and inhibits insulin receptor Diabetes: The SEARCH for Diabetes in Youth Study signaling, which were abolished by ATF3 siRNA. As well, macrophage AMY S. SHAH, LAWRENCE M. DOLAN, DANA DABELEA, JEANETTE S. AN- infi ltration into hepatocytes is dependent on the induction of ATF3. ATF3- DREWS, RALPH B. D’AGOSTINO, JR., ELIZABETH J. MAYER-DAVIS, SANTICA mediated hepatic steatosis and macrophage infi ltration in the liver of HFD- M. MARCOVINA, GIUSEPPINA IMPERATORE, R. PAUL WADWA, STEPHEN R. fed rats or ethanol-fed mice are completely abolished by delivery of ATF3 DANIELS, KRISTI REYNOLDS, RICHARD F. HAMMAN, DAVID M. MAAHS, Cincin- siRNA using in vivo-jetPEI system. As well, resveratrol-induced AMPK/ nati, OH, Aurora, CO, Winston-Salem, NC, Chapel Hill, NC, Seattle, WA, Atlanta, GA, SIRT1 signaling pathway has a negative role for ATF3-mediated hepatic Pasadena, CA fi brosis and hepatic apoptosis. Taken together, our studies suggest that Dyslipidemia contributes to the increased risk of cardiovascular disease in ceramide-mediated ATF3 may play as a critical regulatory pathway in persons with type 1 diabetes (T1D). Weight control is commonly recommended dynamic regulation of hepatic steatosis via induction of caveolin-1 or CD36/ as a treatment for dyslipidemia. However, the effect of changes in weight FAT. Also, ATF3 may play as a potent activator of macrophage infi ltration on the lipid profi le in youth with T1D is not known. Therefore, we tested into hepatocytes and thus promote the progression of type-2 diabetes by the hypothesis that changes in BMI-z score (BMIz) are associated with the induction of the impaired glucose metabolism and insulin insensitivity concomitant changes in the lipid profi le in youth with T1D. and could be considered as a potential therapeutic target in treating Type-2 We studied 1142 youth with incident T1D who had at least 2 lipid diabetes. measurements over 2 years (initial visit mean: age=10.8±3.9 years, Supported by: Korea National Institute of Health BMIz=0.55±0.97, T1D duration =10.7±7.6 months; 47.5% female, 77.9% non- Hispanic white) in the SEARCH study. Longitudinal mixed models were used 656-P to examine the relationships between change in BMIz and change in total, LDL Receptor Knock-Out Mice Impaired Spatial Cognition With Hip- LDL, HDL, non-HDL cholesterol, and log triglycerides (TG) adjusted for initial pocampal Vulnerability to Apoptosis and Synapse Defi cits age, sex, race, clinical site, season of study visit, T1D duration and A1c. YAN HUANG, WEN-QING XIA, YI ZHOU, PIN WANG, PIN WANG, YUE YANG, We found that 1) all lipid levels except LDL increased signifi cantly over 2 RONG-RONG CAI, SHAO-HUA WANG, Nanjing, China years (data not shown), p<0.05; and 2) changes in BMIz were associated with Evidences from clinical studies support that abnormal cholesterol changes in HDL and TG only and the magnitude of these changes depended metabolism in the brain lead to the progress of cognitive dysfunction. LDL on the initial BMIz value (interaction p<0.05) (Table). receptor (LDLR) is well known for its role in regulating brain cholesterol These data suggest that decreases in BMIz over time appear to have a homeostasis. Whether LDLR plays role in cognitive impairement and benefi cial impact on TG and HDL resulting in less TG increase and greater potential mechanisms were unknown. Twelve-month-old Ldlr-/- mice (n =10) HDL increase. Thus, weight loss may be an effective, though limited, and wild-type littermates (n =14) were subjected to morris water maze test. therapeutic approach for dyslipidemia in youth with T1D. All animals were killed for synapse and apoptosis study uses 1 week after the Table: Examples demonstrating the association of change in BMIz with completion of the behavioral test. The Ldlr-/- mice were confi rmed to have change in TG and HDL higher plasma cholesterol concentrations than wild-type mice (t(4) = 4.076, Initial BMIz Change in BMIz Change in TG Change in HDL P = 0.015). The results of behavioral test revealed that Ldlr-/- mice displayed impaired spatial memory with decreased expression levels of synaptophysin 0.5 (normal weight) ↑ by 0.5 ↑16.6% (13.2-20.1) ↑1.63mg/dl (0.90-2.36) and number of synaptophysin-immunoreactive presynaptic bouton in the 0.5 (normal weight) No change ↑14.2% (11.4-17.1) ↑2.14mg/dl (1.54-2.75) hippocampal CA1 and dentate gyrus areas (all P<0.05). Furthermore, electron 0.5 (normal weight) ↓ by 0.5 ↑11.9% (8.5-15.3) ↑2.66mg/dl (1.91-3.41) microscopy observation and TUNNEL staining indicated that apoptosis 1.25 (overweight) ↓ by 0.5 ↑11.1 % (7.7-14.6) ↑2.80mg/dl (2.03-3.57) occurred in the hippocampus of Ldlr-/- mice with the elevated Bax/Bcl-2 ratio for gene and protein expression (t=4.369, P=0.012 ; t= 6.163, P=0.004, 1.75 (obese) ↓ by 0.5 ↑10.6% (7.2-14.2) ↑2.89mg/dl (2.10-3.68) respectively), and activated-caspase3. In conclusion, LDLR defi cient induced Data are % or mg/dl (95% confi dence interval) deterioration of cholesterol homeostasis contributing to impaired spatial Supported by: CDC cognition, probably via its negative effects on hippocampal vulnerability to apoptosis and synapse defi cits. 659-P 657-P Discriminant Ratio and Biometrical Equivalence of Measured vs. High Levels of Free Fatty Acids may Advance the Severity of Primary Calculated Apolipoprotein B100 in Patients With T2DM Sjögren’s Syndrome MICHEL P. HERMANS, SYLVIE A. AHN, MICHEL F. ROUSSEAU, Brussels, Belgium YOSUKE SHIKAMA, NAOZUMI ISHIMARU, YASUSEI KUDO, YUKIKO BANDO, Background: Apolipoprotein B100 (ApoB100) determination is superior NANAKO AKI, YOSHIO HAYASHI, MAKOTO FUNAKI, Tokushima, Japan to low-density lipoprotein cholesterol (LDL-C) to establish cardiovascular Obesity and type 2 diabetes (T2D) are characterized by decreased insulin (CV) risk, and does not require prior fasting. ApoB100 is rarely measured sensitivity and higher concentrations of free fatty acids (FFAs) in the serum. alongside standard lipids, which precludes comprehensive assessment of Among FFAs, saturated fatty acids (SFAs), such as palmitate, have been dyslipidemia. reported to play a role in obesity-associated infl ammation. Primary Sj gren’s Objectives: To evaluate two simple algorithms for apoB100 as regards syndrome (SS) is an autoimmune disease characterized by infi ltration of their performance, equivalence and discrimination with reference apoB100 infl ammatory mononuclear cells and destruction of epithelial cells in lacrimal laboratory measurement. and salivary glands. Although epidemiological studies have suggested a Methods: Two apoB100-predicting equations were compared in 87 type 2 link between primary SS and dyslipidemia or T2D, little is known about the diabetes mellitus (T2DM) patients using the Discriminant Ratio (DR). Equation clinical signifi cance of elevated serum level of FFAs in primary SS. In primary 1: apoB100 = 0.65 x non-high-density lipoprotein cholesterol + 6.3; and

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A166 DIABETIC DYSLIPIDEMIA

Equation 2: apoB100 = -33.12 + 0.675 x LDL-C + 11.95 x ln[triglycerides]. The 661-P underlying between-subject standard deviation (SDu) was defi ned as SDu = Effect of Pitavastatin on Glucose Metabolism in Patients With Type SQRT (SD²b - SD²w/2); the within-subject variance (Vw) was calculated for m 2 Diabetes: Randomized Controlled Study of Pitavastatin versus (2) repeat tests as (Vw) = S(xj -xi )² /(m-1)), the within-subject SD (SDw) being Pravastatin its square root; the DR being the ratio SDu / SDw. TSUGUHITO OTA, SHUICHI KANEKO, TOSHINARI TAKAMURA, Kanazawa, Japan Results: All SDu, SDw and DR’s values were nearly similar, and the observed Several recent meta-analyses suggest that treatment with HMGCoA differences in discriminatory power between all three determinations, reductase inhibitors (statins), with the possible exception of pravastatin, i.e. measured and calculated apoB100 levels, did not reach statistical

can not only increase the incidence of new-onset diabetes, but also impair POSTERS signifi cance. Measured Pearson’s product-moment correlation coeffi cients glucose metabolism. Pitavastatin is a new statin that has LDL-C-lowering Complications between all apoB100 determinations were very high, respectively at 0.94 effects in the range of 35-45%; its effect on glucose metabolism remains Acute and Chronic (measured vs. equation 1); 0.92 (measured vs. equation 2) ; and 0.97 (equation unknown. In the present study, we compared the effects of pitavastatin and 1 vs. equation 2), each measurement reaching unity after adjustment for pravastatin on glucose metabolism in Japanese patients with either IGT attenuation. or mild type 2 diabetes mellitus (T2DM). The 38 patients (61.6±10.8 years, Conclusion: Both apoB100 algorithms showed biometrical equivalence, HbA1c 6.4±0.5%, LDL-C 149.3±30.4 mg/dL) were randomiozed to 10 mg and were as effective in estimating apoB100 from routine lipids. Their use pravastatin (M, n = 12), 2 mg pitavastatin (LP, n = 14), and 4 mg pitavastatin should contribute to better characterize residual cardiometabolic risk linked (HP, n = 14). All patients were tested with a 75 g OGTT before as well as after to the number of atherogenic particles, when direct apoB100 determination 6 months of treatment. Six months after treatment, LDL-C decreased by 21% is not available. in M, 38% in LP, and 45% in HP (P < 0.001). HbA1c increased by 0.09±0.21% in M, 0.13±0.33% in LP, and 0.06±0.26% in HP. However, these changes in 660-P HbA1c from baseline were not signifi cant in any of the groups. HOMA-IR A Novel Total Lipoprotein Electronegativity Index for Predicting increased by 0.52±1.28 in M but decreased by 0.30±1.72 in LP and increased Cardiometabolic Risk by 0.05±1.51 in HP, whereas HOMA-β increased by 51.1±24.3 in M but JING-FANG HSU, TZU-CHIEH CHOU, JUYI HSIEH, JONATHAN LU, SHU-HUA decreased by 2.8±18.9 in LP and by 1.3±42.4 in HP, with no signifi cant inter- CHEN, STEVEN SHAYANI, CHU-HUANG CHEN, Taichung, Taiwan, Houston, TX, group differences. We next compared the change in incremental area under New York, NY the OGTT curves (ΔiAUC) for glucose from baseline to 6 months of treatment. The most electronegative subfractions of chromatographically resolved ΔiAUC was +31.6±63.7% for M, +9.5±23.8% for LP, and -5.9±32.3% for HP, low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL), L5 and with no signifi cant differences. In conclusion, treatment of IGT or mild T2DM V5, are highly atherogenic. High-density lipoprotein (HDL) can similarly be with 2 or 4 mg pitavastatin resulted in greater reductions in LDL-C levels resolved into subfractions; H5 exhibits reduced cholesterol effl ux capacity than 10 mg pravastatin. Pitavastatin, however, had no signifi cant effect on and appears dysfunctional. Therefore, we examined the clinical implications glucose tolerance, insulin sensitivity, or insulin secretion, regardless of dose of lipoprotein electronegativity by analyzing plasma H5, L5, and V5 levels compared to pravastatin. The present study demonstrates that pitavastatin in 33 asymptomatic subjects with cardiometabolic risk factors. H5, L5, may be a useful and potent LDL-C lowering agent in patients with T2DM. and V5 concentrations were 18.4±10.6, 19.6±18.9, and 10.5±7.6 mg/dL, respectively. The Jonckheere’s trend test revealed that the total lipoprotein 662-P electronegativity index (H5+L5+V5) increased with the number of metabolic Comparison of Lipoprotein-Derived Insulin Resistance Score and syndrome criteria (P<0.001). When total cholesterol (TC) and age were also Standard Measures of Insulin Sensitivity included in the analyses, the index was signifi cantly (P<0.05) associated CATHERINE R. MIKUS, MAHESH J. PATEL, CRIS A. SLENTZ, LORI A. BATEMAN, with age (Spearman’s rho, 0.41), waist circumference (0.44), systolic blood LESLIE H. WILLIS, WILLIAM E. KRAUS, Durham, NC pressure (0.40), and levels of fasting glucose (0.49), TC (0.52), and triglyceride Insulin resistance (IR) is associated with distinct lipoprotein subclass (0.49). Stepwise regression analysis revealed that fasting glucose (FG) size and concentration profi les characterized by larger very low density and TC levels contributed to 40% of index variance; thus, we derived the lipoprotein particles (VLDL-P) concurrent with smaller LDL-P and high density following formula for predicting plasma total lipoprotein electronegativity: lipoprotein (HDL-P). Based on these observations, a Lipoprotein IR Score (LP- ([0.36 × FG] + [0.23 × TC] - 34). Large-scale clinical trials are warranted to IR; LipoScience Inc.) derived from NMR measures of LDL-P, VLDL-P, and HDL-P test the reliability of this formula and the clinical importance of the total sizes and concentrations has been developed as a surrogate marker of IR. The lipoprotein electronegativity index. purpose of this study was to compare the LP-IR score to standard measures of IR [(Si) from intravenous glucose tolerance tests and the homeostatic model assessment of IR (HOMA-IR)] in two distinct cohorts. Cohort1: N=220; 49% female; 82% Caucasian; 52±1 y; 88±1 kg; LP-IR 60±2; HOMA-IR 2.36±0.11; Si 3.45±0.18 and Cohort2: N=290; 52% female; 85% Caucasian; 49±1 y; 89±1 kg; LP-IR 57±2; HOMA-IR 2.26±0.01; Si 4.65±0.40. In both cohorts, there was a signifi cant inverse correlation between LP-IR and Si (r=-0.52, P<0.0001 and r=-0.47, P<0.0001, respectively) and a signifi cant positive correlation between LP-IR and log HOMA-IR (r=0.38, P<0.0001 and r=0.41, P<0.0001), demonstrating reproducibility across cohorts. In comparison, associations between Si and log HOMA-IR were r=-0.62 (P<0.0001) and r=-0.41 (P<0.0001) for cohorts 1 and 2, respectively. Incomplete concordance between the three indices of IR is not surprising, as they each refl ect distinct facets of metabolic control. Additional studies are needed to determine whether they may contribute complementary information. In summary, the strength of association between LP-IR, a novel lipoprotein-derived IR score, and Si, an established measure of IR, was similar to that observed between Si and HOMA-IR, two established indices of IR. Supported by: NIH (HL112575 to C.R.M.), (HL57354 to W.E.K.)

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A167 DIABETIC DYSLIPIDEMIA

663-P 665-P Altered HDL and LDL Subfraction Profi les of Patients With Mono- The Effect of Metformin on Mean Platelet Volume in Diabetic genic and Autoimmune Diabetes Patients WOJCIECH FENDLER, MACIEJ BOROWIEC, MANFREDI RIZZO, BEATA MALA- ILHAN DOLASIK, SELCUK YUSUF SENER, UGUR KORKMAZ, ZEKI AYDIN, KORAY CHOWSKA, KAROLINA ANTOSIK, AGNIESZKA SZADKOWSKA, MACIEJ BA- CELEB, ZEYNEP CANTURK, BERRIN CETINARSLAN, Kocaeli, Turkey NACH, MAGDALENA SZOPA, MACIEJ MALECKI, WOJCIECH MLYNARSKI, Lodz, Diabetes Mellitus (DM) is an independent risk factor for cardiovascular Poland, Palermo, Italy, Kraków, Poland diseases. Metformin, the most commonly used antidiabetic, also have an

POSTERS Patients with monogenic diabetes exhibit an altered risk of cardiovascular antiatherojenic effect. Mean platelet volume (MPV) is increased in patients

Complications complications which may be linked to a specifi c lipid profi le, To investigate at high thrombogenic activation, also risk for atherosclerosis. The purpose Acute and Chronic this, we performed detailed cholesterol subfraction profi ling in young adult of this study was to examine the effects of metformin on MPV values in patients and healthy controls. The study group consisted of 15 controls, 18 newly diagnosed type 2 DM patients on metformin monotherapy. In this patients with GCK-MODY, 22 with type 1 diabetes (T1DM), 5 with HNF1B- study, 60 newly diagnosed type 2 DM patients (45 female, 15 male) who had MODY and 16 with HNF1A-MODY. Median age was 25.7 years (25-75% 22- applied to Kocaeli University School of Medicine Endocrinology outpatient 44 years), mean BMI was 23.1+3.2 and neither differed between the groups. clinic, and 47 healthy individuals (35 female, 12 male) were included. Two LDL and HDL were assessed by non-denaturing, linear polyacrylamide gel groups have similarity for age, sex and body mass index. The patients with electrophoresis. LDL were distinguished in 7 subfractions: large (LDL-1;- additional disease, nephropathy, smoking and using drugs that may effect 2) and small (LDL-3 to -7), while HDL are distinguished in 10 subfractions: the MPV was excluded. At baseline and 6 months after metformin treatment, large (HDL-1 to -3), intermediate (HDL-4 to -7) and small HDL (HDL-8 to -10). patient demographics and laboratory values were compared. MPV was Individuals with GCK-MODY had higher VLDL levels than controls (21.0+5.2% higher among type 2 DM patients than the control group (p<0.001). After vs 14.3+3.3%, p=0.0006), higher IDL-C than controls and T1DM patients 6 months metformin treatment, MPV values were signifi cantly decreased (11.0+3.3%, 6.0+1.2% p=0.0001 and 6.7+2% p=0.0001) and lower LDL1 (p<0.001). HbA1c and mean platelet mass were also signifi cantly decreased levels than controls and T1DM groups (12.2+6.7%, 10.3+5.0% p=0.0003 (p=0.022; p=0.001, respectively). There was no correlation between MPV and 16.7+5.3% p=0.048). The group of patients with GCK-MODY also had a and HbA1c values (r=-0.13, p=0.926). Metformin; which has been shown substantially altered HDL profi le with signifi cantly higher HDL4 (10.9+2.0% to exhibit antiatherogenic effect through positive effects on cholesterol vs 8.8+0.5 p=0.001) and lower HDL7 (3.9+2.3% vs 6.2+0.8% p=0.002), HDL8 levels, infl ammatory markers, and vascular adhesion molecules; decreased (2.8+1.9% vs 4.6+0.8% p=0.02), HDL9 (2.1+1.6% vs 3.4+/-0.6% p=0.047) and MPV values that appear to play a crucial role in the at the beginning of HDL10 (2.6+2.4% vs 5.6+1.6% p=0.01) levels than individuals with HNF1A- atherosclerosis development. We conclude that our result may conribute to MODY. Patients with HNF1A-MODY had higher IDL-C levels (8.9+1.9% vs the explanation for anti-atherogenic effect of metformin. 6.0+1.2%), lower LDL1 (14.4+3.5% vs 20.7+3.6%) and LDL2 (5.8+2.2% vs 10.3+5.0%) than controls. HNF1A-MODY and GCK-MODY groups showed 666-P considerable within-group similarities in hierarchical clustering. In conclusion HDL Remodeling by Over Expression of β Chain of Cell Surface F0F1 lipid profi les were dependent on the genetic background of diabetes, with ATPase GCK-MODY patients showing lower levels of atherogenic LDL particles than KEXIU SONG, XIAOYU PAN, SHANGYU CHAI, YUEYE HUANG, XIAOWAN JIANG, T1DM patients. Moreover, distinct HDL profi les of GCK- and HNF1A-MODY FEI LI, HONG LI, SHEN QU, Shanghai, China may have diagnostic and prognostic consequences. HDL cholesterol as well as apolipoprotein A-I (apo A-I) are known to Supported by: Foundation for Polish Science be inversely correlated with cardiovascular disease due to theirs diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL’s 664-P cholesteryl esters. Knock-down of SR-BI diminishes but does not completely Bone Microstructure Characteristics in High Fat Diet Induced Fatty block the transport of HDL, other receptors may be involved. Ectopic β-chain Liver Disease in Mice of F0F1 ATPase in hepatocytes has been previously characterized as an RAN CUI, YUNZI LIU, YI ZHANG, CHUNLEI JIANG, SHEN QU, Shanghai, China apoA-I receptor, triggering HDL internalization. This study was undertaken It has been proved that non-alcoholic fatty liver disease (NAFLD) had to identify the overexpression of ectopic β-chain of F0F1 ATPase on DIL- detrimental effect on bone mineral density (BMD) in both adults and HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in adolescents. However, most of the evidence was from clinical cross section SR-BI -knockout mice. Human ATPase β-chain cDNA was delivered to the study, which cannot ensure the adverse effect. Thus, we performed an mouse liver by adenovirus , and GFP adenovirus as control. The adenovirus- animal experiment to certify this phenomenon.46 C57 male mice, 8-10 mediated over-expression of β-chain was identifi ed at both mRNA and weeks old, average body weight 15-20g at the beginning of the experiment, protein levels on mice liver, and validated by its increasing of DiL-HDL uptake were enrolled and divided into two groups, feeding for 12 weeks either in primary hepatocytes. In response to hepatic overexpression of β- chain, standard chow (control; N = 21) or high-fat diet (2% cholesterol, 7% lard, plasma HDL-c levels and cholesterol were reduced in SR-BI knockout mice, 8.3% yolk, 16.7% sucrose, and 66% base forage, HFD; N = 25). Femur, Tibia compared with the control (P<0.05). In SR-BI knockout mice hepatocytes, and liver samples were collected after sacrifi ce. The bone samples were HDL uptake increase about 24%, while the WT increase less than 22%. scanned using a micro-CT system (mCT-80; Scanco Medical AG) in a high- Maybe SR-BI contribute to the uptake in WT mice. In addition, increased resolution scanning mode (a voxel size and slice thickness of 20 mm). In HFD ATPase β- chain may have no interaction with SR-B1(the known pathway group, 20 mice were proved with fatty liver by liver biopsy. Tissue mineral for HDL metabolism) in reducing plasm HDL levels. The present data suggest density (TMD), total bone volume fraction (BV/TV), trabecular thickness (Tb. that ATPase β-chain can serve as the endocytic receptor of HDL, and its Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), connectivity over- expression can reduce plasma HDL-c. We are conducting the study density (Conn.D) and structure model index (SMI) were compared between to further explore its advantage or disadvantage in the management of control and NAFLD group. Compared to control group, fatty liver group had dyslipidemia. a lower level of TMD (59.741±23.038 vs. 112.196±54.940, P=0.028), BV/TV Supported by: NSFC (81070238) (0.017±0.003 vs. 0.068±0.044, P=0.007), SMI (3.918±0.568 vs. 2.860±0.579, P=0.006) and Tb.Th (0.050±0.006 vs. 0.063±0.006, P<0.001). For tibia, fatty liver also had a lower level in TMD (25.078±19.822 vs. 85.462±34.011, P=0.021), BV/TV (0.023±0.012 vs. 0.068±0.017, P<0.001), Conn.D (0.359±1.688 vs. 8.522±6.458, P=0.023) SMI (3.428±.412 vs. 2.529±.454, P=0.002), Tb.Th (0.048±0.007 vs. 0.061±0.007, P=0.004) and Tb.Sp (0.636±0.158 vs. 0.441±0.145, P=0.022). Our results convinced the adverse effect on bone microstructure of high fat diet induced fatty liver mice. However, further study was needed to investigate the mechanism.

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A168 FOOT CARE—LOWER EXTREMITIES

FOOT CARE—LOWER EXTREMITIES BMC numbers 7 days after implantation decreased to 28% of that in non- diabetic mice. Blood fl ow volume of ischemic limbs measured using Doppler method in diabetic mice 14 days after BMCs administration reduced to 44% Guided Audio Tour: Walking Around on the Diabetic Foot (Posters: 667-P to of that in non-diabetic mice. ICS co-implantation with BMCs signifi cantly 674-P), see page 17. recovered the reduced cell numbers and blood fl ow of the ischemic limbs in diabetic mice. In conclusion, we demonstrated the usefulness of ICS as & 667-P an enhancer for cell-based therapeutic angiogenesis even in diabetic limb ischemia. Self-Reported Quality of Life and Diabetic Foot Infections POSTERS Supported by: Japan Science and Technology Agency (AS2314097F to S.F.) Complications DANE K. WUKICH, KATHERINE M. RASPOVIC, Pittsburgh, PA Acute and Chronic Diabetic foot infections (DFI) are serious complications that can result in hospitalization, need for amputation and premature mortality. The aim & 669-P of this study was to assess self-reported quality of life (QOL) in patients The Attitude Towards Feet in Diabetic Patients With and Without hospitalized with DFI and to compare the QOL with diabetic patients without Diabetic Foot Ulcers foot pathology. QOL assessment was performed using a region specifi c CECIL THOMAS, E.K. NADERALI, G. GILL, Liverpool, United Kingdom lower extremity assessment tool. The Foot and Ankle Ability Measure Introduction: Foot problems are the commonest cause of hospital (FAAM) has been demonstrated to valid, reliable and reproducible in patients admissions amongst diabetic patients in the United Kingdom. The foot ulcers with diabetes, and is comprised of a general/ADL section and sports section. usually take considerable time to heal ultimately putting marked fi nancial Patients hospitalized with DFI were asked to participate in this prospective strain on health care system. There is little or no information on impact of study. The control was obtained from patients with diabetes who attended diabetic foot ulcers on quality of life from patients prospective. Therefore, routine foot screenings and had no foot complaints. Results: see Table this study was designed to evaluate psychological impact of foot ulcers on diabetic patients. Diabetic Infection Control P value Methods & Results: One hundred patients (50 cases, 50 matched controls) (N=43) (N=43) were enrolled to evaluate their attitude towards their own feet in the Age in years ± SD 53.5 ± 9.8 56.5± 12.3 P =0.204 presence or absence of diabetic foot ulcers using a specifi cally designed questionnaire developed by Carrington et al in 1996. The questionnaire Gender (Male/Females) 35/8 33/10 P = 0.791 consisted of 12 pairs of opposites, rated on a 7 point scale using a semantic Diabetes Type (Type 1/Type 2) 5/38 3/40 P =0.713 differential methodology with maximum and minimum achievable scores of Duration of DM years± SD 15.0 ± 9.6 9.2 ± 8.3 P =0.004 84 and 7, respectively. Higher score indicate a more positive attitude while Insulin Use (N,%) 34(79%) 15(35%) P <0.0001 a lower score indicative of negative attitude. Patients with foot/feet ulcer had signifi cantly (p<0.0001) higher mean score of 55.20 + 11.44, while the Neuropathy (N,%) 43(100%) 16(37%) P < 0.0001 mean score for controlled groups was 74.56 + 9.21. The Bivariate analysis Foot Ankle Ability Measure failed to show any signifi cant correlation between attitude towards feet and FAAM General/ADL 31.1± 20.8 68.5 ± 15.9 P <0.0001 duration of diabetes, age, degree of glycaemic control, BMI and the duration Maximum score 84 of foot ulcer. % (FAAM General-ADL/84 times 100) 37.0 ± 24.7 81.6 ± 18.9 P <0.0001 Conclusion: Our study indicates that the presence of foot ulcer per se Sports Maximum score 32 4.1 ± 5.6 19.7± 9.4 P <0.0001 exerts marked psychological strain in diabetic patients. Therefore, our fi ndings highlight important implications for the evaluation, planning and % (FAAM Sports Score/32 times 100) 12.6 ± 17.6 63.0 ± 30.0 P <0.0001 management of patient care in diabetic foot disease. Patients hospitalized with DFI had signifi cantly reduced QOL when com- pared to diabetic patients without foot complaints. In fact, patients with DFI & 670-P had a score that was more than two standard deviations lower on the general/ Assessing the Cost of Lower Extremity Amputation in U.S. Veterans ADL scale and 1.5 standard deviations lower on the sports subscale. Patients With Diabetes hospitalized with DFI had longer duration of disease, more likely to use insu- HEATHER REBECCA-CRAIG FRANKLIN, MANGALA RAJAN, CHIN-LIN TSENG, lin and more likely to have neuropathy. Self-reported measurement of function LEONARD POGACH, ANUSHUA SINHA, East Orange, NJ and QOL are important assessments in following patients with DFI. Region Diabetes-related lower extremity amputations (LEA) are a prevention specifi c measurements which track foot and ankle function can be utilized in quality indicator by the Agency for Healthcare Research and Quality and are conjunction with a general measure of health such as the SF-36 to provide refl ective of the quality of foot care delivered within a health care system. important outcome assessments. Therefore, the aim of this study was to estimate individual level health care costs associated with LEA within the Veterans Health System (VHA). & 668-P This was a cross-sectional study of 3,429 VHA clinic users identifi ed as The Effectiveness of Injectable Cell Scaffold for Enhancing Cell- having diabetes, non-traumatic LEA in fi scal year (FY) 2004 (10/1/2003- Based Therapeutic Angiogenesis in Diabetic Limb Ischemia 9/30/2004). LEA expenditures related to inpatient medical surgical & KOJI TAKEDA, SHINYA FUKUMOTO, YOHEI MIMA, KOKA MOTOYAMA, TOMOAKI outpatient care were evaluated using VHA Health Economics Resource MORIOKA, KATSUHITO MORI, TETSUO SHOJI, MASANORI EMOTO, TSUTOMU Center average cost fi les. Pharmaceutical costs incurred up to 7 days post- FURUZONO, MASAAKI INABA, Osaka, Japan, Wakayama, Japan amputation surgery were obtained from the 2004 VHA Decision Support The clinical success of cell-based therapeutic angiogenesis in patients System national extract. with critical limb ischemia is still limited, especially in diabetic patients. We The median cost associated with care for diabetes related LEA per patient reported a biodegradable and injectable cell scaffold (ICS), which is a nano- in the VHA healthcare system in FY2004, adjusting for infl ation to United scaled hydroxyapatite (HAp)-coated polymer microsphere, as an enhancer State dollar in 2012, was $33,530; the infl ation adjusted total cost was $169 of therapeutic angiogenesis. In this study, we examined the effectiveness million dollars. The median of the overall cost (inpatient, outpatient, and of this ICS in diabetic limb ischemia. Bone-marrow mononuclear cells pharmacy) for individual patients by level of amputation was $24,352 in toe (BMCs) were intramuscularly injected, with or without ICS, or with uncoated amputations and increased to $40,610 in above knee amputations. For those polymer microsphere (PM) as a control into the ischemic hind limbs of with one amputation, the total median cost was $31,117 and on the upper mice. Kaplan-Meier analysis demonstrated the marked benefi cial effects end, those with four amputations, had a median cost of $131,464. Patients of ICS + BMC (vs. BMC alone, vs. PM + BMC; p < 0.05) for limb necrosis with more severe and/or multiple amputations had higher costs for inpatient after the ischemic operation. The number of BMCs in ischemic tissues surgery and pharmacy. The median cost of an amputation in the VHA is was estimated by determining the tissue GFP expression in BMCs derived comparable to the cost reported in a study in an HMO in Michigan in 2003 from EGFP-transgenic mice. Seven days after the implantation, the number ($37,600; CI: $23,300-$62,200). This study is signifi cant in that it assesses of BMCs that were injected with ICSs in ischemic tissues was 5 times the expenditures of LEA, a less frequent but debilitating complication of greater than that for BMCs injected alone or with PM. Intramuscular levels diabetes in a large managed care setting like the VHA. This information of proangiogenic cytokines, VEGF, and FGF2, were signifi cantly elevated in is useful for comparative effectiveness studies that assess the impact of ischemic tissues treated with ICSs + BMCs than those treated with BMCs different treatments on diabetes complications for veterans with diabetes. alone or with PM. Increased collateral blood fl ow was confi rmed using 3D computed tomography angiography. In streptozotocin-induced diabetic mice,

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A169 FOOT CARE—LOWER EXTREMITIES

& 671-P & 673-P Retrospective Study of Non-Healing Neuropathic Forefoot Ulcers The Performance of Serum Infl ammatory Markers for the Diagnosis Treated With Gastrocnemius Recession and Follow-Up of Patients With Osteomyelitis STEPHANIE JAMESON, RODNEY STUCK, Chicago, IL NIKOLAOS TENTOLOURIS, EDWARD JUDE, CHRISTOS LIASKOS, MARIOS MI- Institutional Review Board approval was obtained at Edward Hines CHAIL, SPYROS KARAMAGKIOLIS, Athens, Greece, Ashton-under-Lyne, United Veterans Affair (VA) and Loyola University Medical Center (LUMC) and a Kingdom retrospective study was conducted using patients from both hospitals. Serum infl ammatory markers like C-reactive protein (CRP), erythrocyte POSTERS

Complications Patients were included if they underwent a gastrocnemius recession for sedimentation rate (ESR), white blood cell count (WBC), and procalcitonin (PCT)

Acute and Chronic the history of a non- healing neuropathic plantar forefoot ulcerations from have been used for the diagnosis of patients with diabetic foot infections. In January 2010 to July 2012. Patients undergoing the procedure for another the present prospective study we examined the performance of these markers diagnosis were excluded in this study. Twenty patient charts were reviewed, in the diagnosis and follow-up of patients with osteomyelitis. A total of 61 which included nineteen patients were from the VA, and one patient from patients (age 63.1 ± 7.0 years, 45 men and 16 women, 7 with type 1 and 54 with LUMC. All surgeries were performed by a single surgeon (R.S). Pre-operative, type 2 diabetes) with untreated foot infection (34 with soft tissue infection peri, and post operative data was collected from all patients who had the and 27 with osteomyelitis) were recruited. Diagnosis of osteomyelitis gastrocnemius recession procedure, CPT code 26787. The charts of sixteen was based on clinical examination and was confi rmed by imaging studies diabetic male patients (20 feet) with an average age of 62 years old, were (X-ray, scientigraphy, MRI). Determination of the infl ammatory markers was reviewed in total from Hines VA (19 patients) and Loyola University Medical performed at baseline, after 1 and 3 weeks and after 3 months of treatment. Center (1 patient). Eighteen out of the twenty (90%) ulcerations healed, and At baseline, the values of CRP, ESR, WBC and PCT were signifi cantly higher only two ulcers re-ulcerated in the same location of previous ulceration. The in patients with osteomyelitis than in those with soft tissue infections. The average time of wound closure was 29 days post gastrocnemius recession. sensitivity and specifi city for the diagnosis of osteomyelitis of CRP (cut-off There were no transfer ulcerations. Two patients died of unrelated events value >14 mg/l) were 0.85 and 0.83, of ESR (cut-off value>67 mm/h) 0.84 and following resolution of their ulcerations. The average length of time the 0.75, of WBC (cut-off value >14.000/ml) 0.74 and 0.82, and of PCT (cut-off value patient had the ulceration pre-operatively was 12.35 years and the average >0.30 ng/ml) 0.81 and 0.71. All values declined after initiation of treatment followup was 17.4 months. A tendon lengthening should be a treatment with antibiotics; the WBC, CRP and PCT values returned to near-normal levels option once any underlying infection has been appropriately treated and by day 21 in both groups, while the values of ESR remained high until month the vascular status has been shown to be appropriate for wound healing. A 3 only in patients with bone infection. In conclusion, all infl ammatory markers gastrocnemius recession should be considered in neuropathic patients who have adequate performance for the diagnosis of osteomyelitis; ESR is the best have failed to respond to conservative wound care, including application marker for the follow-up of patients with osteomyelitis. of serial human derived dermal substitute grafts when appropriate for the treatment of plantar forefoot ulcerations. & 674-P Serum Levels of TNF-α In-Peripheral Neuropathy Patients and its & 672-P Correlation With Nerve Conduction Velocity in Type 2 Diabetes Mel- A Silent, Painless Emergency? The Burden of Diabetic Foot Infec- litus tions and Emergency Department Admissions in the U.S. GAUHAR HUSSAIN, S. AIJAZ ABBAS RIZVI, SANGEETA SINGHAL, JAMAL AHMAD, GRANT H. SKREPNEK, JOSEPH L. MILLS, DAVID G. ARMSTRONG, Tucson, AZ Aligarh, India Objectives: To evaluate the magnitude and impact of diabetic foot Objective: To compare serum levels of TNF-α in patients of peripheral infections (DFIs) in emergency department (ED) settings from 2006-2010 in neuropathy and patients without neuropathy in type 2 diabetes mellitus. the U.S. Material and Methods: This cross sectional study was conducted in Methods: We used Agency for Healthcare Research and Quality diagnosed type 2 diabetes mellitus patients. They were divided in groups, Healthcare Cost and Utilization Project discharge records of ED visits among Group I ( n= 37) with clinically detectable diabetic peripheral neuropathy persons ≥18 years with any-listed diagnoses of DFIs. Beyond descriptive of shorter duration and Group II ( n= 27) with clinically detectable diabetic statistics, a predictive logistic regression reported odds ratios (OR) for peripheral neuropathy of longer duration. They were compared with inpatient admissions based upon patient demographics, payer, hospital patients without clinical neuropathy (n= 22), Clinical diagnosis was based characteristics, and comorbidities. on neuropathy symptom score (NSS) and Neuropathy disability score (NDS) Results: Overall, 1.0 million cases of DFI presented to EDs in the US for signs. Blood samples were collected for baseline investigations and from 2006-2010, constituting 1.9% of the 54.2 million total diabetes cases. estimation of serum TNF-α. Nerve conduction velocity was measured in Patients averaged 62.5 years of age with 59.4% being male. In the ED alone, both upper and lower limbs. Median, Ulnar, Common Peroneal and Posterior the national bill was $1.2 billion (USD, 2012), increasing to $41.5 billion ($8.3 Tibial nerves were selected for motor nerve conduction study and Median billion/annum) when including inpatient charges for the 81.2% of cases and Sural nerves were selected for sensory nerve conduction study. that were admitted. Clinically, mortality occurred in 2.0%, sepsis in 9.6% Results: The comparisons were done between various clinical and of cases, surgical complications in 8.0%, and amputation in 10.5% (Hi-Low biochemical parameters in clinically detectable and undetectable peripheral amputation ratio of 0.46). The top comorbidities in these patients included neuropathy groups of type 2 diabetes mellitus. The study showed raised chronic or acute renal failure, hypertension, fl uid/electrolyte disorders, heart serum levels of TNF-α in peripheral neuropathy patients and signifi cant disease, infective arthritis and osteomyelitis, heart failure, and neurologic correlation with nerve conduction velocity. Conclusion: High level of TNF-α disorders. The logistic regression found 28 of 30 Elixhauser comorbidities to in serum of T2DM patients with neuropathy shows possible contribution in be signifi cantly (p<0.05) associated with odds of inpatient admission, ranging development of neuropathy. Due to its independent association this cytokine from renal failure (OR=1.82) and uncomplicated hypertension (OR=1.69) to might be used as biomarker for diabetic peripheral neuropathy. anemia (OR=12.27) and wasting syndrome (OR=12.64). Conclusion: DFIs exact a substantial toll on resource utilization as a percentage of overall diabetes care in the ED. Development of better 675-P systems of comprehensive outpatient diabetic foot care to provide earlier Can the Mobility of the Ankle Joint Predict which Foot Is at Higher coordinated care for this frequently silent condition, should be a major point Risk of Ulcer in Patients With Diabetes? of healthcare emphasis as it has the potential to reduce costs of emergency PIERGIORGIO FRANCIA, ALESSANDRA DE BELLIS, ANNA TEDESCHI, MASSIMO care and improve outcomes. GULISANO, ROBERTO ANICHINI, ARIANNA BERNINI, Firenze, Italy, Pistoia, Italy Limited joint mobility (LJM), particularly of the ankle, is common in diabetes and is known to play a role in the development of foot ulceration. The aim of this study was to evaluate the relationship between LJM and foot ulceration in diabetic patients. In 2006 we measured the range of motion (ROM) in plantar and dorsal fl exion of the ankles in 71 patients with diabetes (Group D), mean age 61.11 ± 9.74 years, duration of diabetes 17.28 ± 11.36 years, (40/31 male/female; 16/55 Type 1/2) and in 30 healthy subjects (Group C), mean age 58.42 ± 5.97 years, (11/19 male/female).

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A170 FOOT CARE—LOWER EXTREMITIES

Group D was then subdivided into two groups, the Ulcer Group (DU), study of 170 Japanese outpatients(mean [± SD] age: 61 ± 14 years, men: represented by 19 patients, who had one foot ulcer or more (14 detected 72.4%). The endpoint was the incidence of LEA. Hazard ratio for reaching before evaluation, 5 detected by the 6th year of evaluation), mean age the endpoint was calculated using Cox proportional hazard model analysis. 64.58 ± 7.62 years, duration of diabetes 16.58 ± 12.28 years, (14/5 male/ During the mean follow-up period of 9.9 ± 12.4 months, 64 reached the female; 1/18 Type 1/2), and No Ulcer Group (DNU), comprised of the other 52 endpoint. The univariate Cox proportional hazard analysis revealed the patients, mean age 59.85 ± 10.18 years, duration of diabetes 17.64 ± 11.12 signifi cant association between HDL cholesterol levels and the endpoint years, (26/26 male/female; 37/15 Type 1/2). [hazard ratio 0.961, 95% confi dence interval (CI) 0.944 - 0.978, p< 0.001]. Ankle ROM in plantar and dorsal fl exion was measured in the different Meanwhile, neither logarithmically transformed triglycerides (hazard ratio POSTERS groups by inclinometer and gave the following results: Group C 133.72° ± 1.648,95% CI 0.460 - 5.911, p= 0.443) nor LDL cholesterol levels (hazard ratio Complications

18.43°, Group D 101.93° ± 27.62°, Group DNU 108.53 ± 27.46 and Group DU 0.996,95% CI 0.987 - 1.006, p= 0.437) were associated with the endpoint. In Acute and Chronic 83.86° ± 18.97°. the multivariate Cox proportional hazard analysis using a stepwise variable- ROM value of the ankle in plantar and dorsal fl exion in Group D was selection procedure, HDL cholesterol levels (hazard ratio 0.972, 95% CI signifi cantly lower than in Group C: 31.27° (s1= 18.43°, s2= 27.62°, P< 0,001). 0.950 - 0.995, p= 0.019) in addition to the presence of peripheral arterial Furthermore, within Group D, ROM value of the ankle in Group DU was disease (ankle brachial index ≤ 0.9) and serum albumin levels were selected signifi cantly lower than in Group DNU: 24.27° (s1= 18.97°, s2= 27.46°, P< as independent predictors for the endpoint. Age, sex, decreased kidney 0,001). function, C-reactive protein, logarithmically transformed triglycerides and In Group DU ankle joint mobility of the fi rst foot presenting ulceration was LDL cholesterol levels were excluded from the model. signifi cantly lower than in the foot without ulceration: 6.90° ± 7.42°, P<0.01. Lower HDL cholesterol level may be a useful clinical predictor for the In 15 out of 19 cases (78.9%) the fi rst ulceration was detected in the incidence of LEA in patients with diabetic foot ulcer/gangrene. corresponding foot with lower ankle joint mobility: 9.46° ± 5.91°, P<0,01. The identifi cation of decreased ROM of the ankle joint may be an early 678-P indicator of higher risk ulceration in diabetic patients and also indicate the A Role for Wnt3a in Recruiting Host-Derived Stem Cells for Wound foot at higher risk. Repair Therapies DANIEL A. PETERSON, FARHEEN WAHID, EMILY REISENBIGLER, SARAH SCHUCK, 676-P STANLEY BAZAREK, SAMUEL LAKE, STEPHANIE C. WU, Chicago, IL Depression and Diabetic Foot Ulcer Healing: The Association Diabetes is a major risk factor for developing chronic non-healing wounds. Revisited Frequently, healing of these wounds is resistant to traditional therapeutic LORETTA VILEIKYTE, BIING-JIUN SHEN, RYAN T. CREWS, ANDREW J.M. BOUL- approaches. Using a mouse model of type 2 diabetes (db/db), we previously TON, MARK F. PEYROT, Manchester, United Kingdom, Athens, OH, Chicago, IL, showed that engraftment of human mesenchymal stem cells (hMSCs) do not Baltimore, MD survive in the wound bed, but recruit host-derived stem cells to the wound Recently conducted studies, though limited by their methodology, bed resulting in enhanced repair and wound closure. We hypothesized that indicate that depression impedes healing of diabetic foot ulcers (DFU). wound repair in impaired subjects could be initiated by signals that recruit However, failure to account for potential confounders such as adherence endogenous stem cells. Identifi cation of such signals could provide an to offl oading undermines their validity. Moreover, the potential endocrine- alternative to costly cell-mediated therapies. Surgical debridement samples immunologic mechanisms by which depression affects DFU healing have from wounds of healing and non-healing diabetic patients were assessed not been explored. One hundred ten type 2 DM patients (84% male; mean for gene expression. Angiogenesis PCR array analysis showed elevation of age 57yrs) with plantar neuropathic DFU (UT Classifi cation: 69% grade 1A; both angiogenic factors and proteases and inhibitors in non-healing diabetic 11% 1B; 16% 2A; and 4% 2B, Ankle Brachial Index: 1.2±0.3) completed the patients. Targeted qPCR for selected factors and cytokines found that chronic Hospital Anxiety and Depression scale, (HADS-D: 16.1±1.9). DFU healing was wound tissue was more infl ammatory than surrounding healthy tissue. To defi ned as wound area reduction from baseline (253.6±349.6) to 6 weeks address stem cell-related signals, an MSC-related gene array revealed (143.1±402.7), WAR-6. Subjects were provided a removable offl oading device elevation of many stemness factors and genes, but reduction in others for their DFU. Adherence to offl oading (AO) was assessed using a validated in non-healing diabetic patients. Targeted qPCR was also performed on dual activity monitor method. Activity data were uploaded to a centralized samples from diabetic mice. Diabetic mice wounds have reduced expression server via internet (AO: 59±23%) Systemic biomarkers (IL6: 18,7±25.8pg/ of Wnt3a, a signaling molecule important for activation and recruitment of ml and IL1beta: 7.3±1.6pg/ml) were measured from patient serum by ELISA. stem cells. In vitro evaluation of Wnt3a found that Wnt3a increased MSC DFU (tissue)-specifi c biomarkers (IL6: 468.6±449.8; IL1beta: 2.3±5.5; MMP2: proliferation in a dose-dependent fashion. Administration of the antagonist 4.3±8.9; MMP9: 7.3±28.4; and TGFbeta1: 719.2±762.6) were determined DKK-1 produced a loss of function blockade of Wnt3a without altering basal by quantifi cation of immunohistochemical tissue localization and/or MSC proliferation, suggesting that Wnt3a is necessary for MSC expansion, normalized biopsy gene expression. Signifi cant bivariate associations were but not normal proliferation. An in vitro wound closure assay found that found between WAR-6 and baseline: wound size (β=.62; p < .001); HADS-D Wnt3a increased MSC migration/invasiveness. Paralleling our previous (β=.28; p=.002); systemic IL6 (β=30; p<.01); TGFbeta1 (β=-.33; p<.05). In report, daily delivery of Wnt3a to wound beds of wild-type and diabetic mice multivariate regression analyses controlling for study site, demographic, accelerates wound closure in diabetic mice in the absence of MSC grafts. disease characteristics and AO, less WAR-6 was independently predicted Supported by: Rosalind Franklin University (to D.A.P. and S.C.W.) by larger baseline DFU size (β=.49; p<.001), more severe HADS-D (β=.23; p=.028) and higher levels of IL6 (β=.30; p=.005). The model explained 51% of the variance in WAR-6. These data confi rm that depression is a risk factor 679-P for DFU chronicity but not that IL6 accounts for the relationship between 25-Hydroxyvitamiv D [25(OH)D] Levels and Diabetic Foot Ulcer: Is depression and impaired DFU healing. There any Relationship? Supported by: 5R01DK07106605 MOHAMMAD ZUBAIR, ABIDA MALIK, DILNASHEEN MEERZA, JAMAL AHMAD, Aligarh, India 677-P In recent years, there has been an effort to understand possible roles Lower HDL Cholesterol Levels are Associated With the Incidence of 25(OH)D, including its role in the immune system particularly on T cell of Lower-Extremity Amputation in Patients With Diabetic Ulcer/ medicated immunity, pancreatic insulin secretion and insulin action. Gangrene 25(OH) D stimulates the cell differentiation and reduces cell proliferation, KAZUKI IKURA, TAKAMICHI SHINJYO, KOU HANAI, YASUKO UCHIGATA, Tokyo, which is essential for cell growth and wound healing. However, data on Japan the association between low level of plasma 25(OH) D and diabetic foot Dyslipidemia has been shown to be a risk factor for both micro- and syndrome are scarce. Circulating plasma 25(OH) D levels were measured in macroangiopathy in diabetic patients; however, the association of diabetic patients with ulcer (n=162) and without ulcer (n=162) in a case control dyslipidemia with lower-extremity amputation (LEA) remains unclear. A study. Of these patients, 85.1% had type 2 diabetes. Subjects with diabetic recent cross-sectional study reported that lower triglycerides levels were foot ulcer showed lower median plasma level of 25(OH)D [6.3(4.2-11.1) vs. paradoxically associated with LEA in patients with diabetic foot ulcer. 28.0(21.4- 37.0)]ng/ml after adjusting the age and BMI. Regardless of the low Therefore, we longitudinally examined whether serum lipid profi les are levels of 25(OH) D in cases and controls, it was associated with neuropathy, associated with the incidence of LEA in patients with diabetic foot ulcer/ sex (female), duration of ulcer healing, and smoking status and independent gangrene.This was a single-center observational longitudinal cohort of confounding factors, including BMI (kg/m2), A1c (%), hypertension,

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A171 DIABETESCATEGORY EDUCATION

nephropathy, foot ulcer, retinopathy, CAD, PAD, HDL-C (mg/dl) and LDL-C college (3.4), Hispanic (0.47), and white race (1.72), all p < 0.0001], diabetes (mg/dl). The factors which predict the risk of developing ulcer independent (OR = 0.82) was no longer a predictor of internet usage (p = 0.12). Both of 25(OH) D status were A1c (>6.9%) [OR 4.37; RR 1.77], HDL-C(<40mg/dl) [OR crude and adjusted health-related internet use were comparable between 1.16; RR 1.07], LDL-C (>100mg/dl) [OR 1.07; RR 1.03], triglycerides(>200mg/dl) diabetes and no diabetes for seeking information on medical problems (OR = [OR 1.40; RR 1.19], neuropathy [OR 6.88; RR 3.12], retinopathy [OR 3.34; RR 1.06), treatments (0.88) and health care providers (1.07), all p > 0.4. For all 1.91], hypertension [OR 1.64; RR 1.28], nephropathy [OR 3.12; RR 1.87] & respondents mean ± SD internet/email use from home [rated 1 (never) - 7 smoking [OR 4.53; RR 2.99] using odds and risk ratios. In conclusion, it is not (several times/day)] was 5.4±0.03, but 0.3 units lower in persons with clear whether the suppression of delayed wound healing seen during 25(OH) diabetes (p = 0.007). Internet/email usage from home was more frequent D defi ciency is due to the secondary effect or is a direct action of vitamin D than at work (5.4 vs. 3.8, p < 0.001). Effect modifi cation analysis indicated on certain components of the immune system. Long-term randomized trials that higher socioeconomic and education status increased internet use more are needed to see the impact of vitamin D supplementation on the outcome for those with diabetes. Thus, age, education, income, race and ethnicity of diabetic foot patients. were important mediators of internet use among patients with diabetes. These factors need to be considered when implementing internet-based diabetes management and education programs. DIABETES EDUCATION & 682-P

POSTERS Launching a Lifestyle Intervention Program for Diabetes Prevention Guided Audio Tour: Improving Diabetes Prevention and Treatment through at the Worksite Education (Posters: 680-P to 687-P), see page 15. M. KAYE KRAMER, KARL K. VANDERWOOD, VINCENT C. ARENA, RACHEL G. MILLER, GERALD L. SCHAFER, ELIZABETH M. VENDITTI, ANDREA M. KRISKA,

Behavioral Medicine, Clinical & 680-P Pittsburgh, PA Nutrition, Education, and Exercise Integration and Utilization of Peer Leaders for Long-Term Self-Man- The Group Lifestyle Balance (GLB) program, adapted from the Diabetes agement Support: Results from Project SEED (Support, Education, Prevention Program’s lifestyle intervention, has been shown to reduce risk and Evaluation in Diabetes) factors for diabetes and cardiovascular disease (CVD) in several settings. The GRETCHEN PIATT, SHARLENE EMERSON, PATRICIA JOHNSON, DEBRA TILVES, current project evaluated the effectiveness of GLB program implementation JANICE C. ZGIBOR, Ann Arbor, MI, Pittsburgh, PA at a large corporate worksite. Study investigators collaborated with worksite Long-term self-management support (SMS) is critical; however, effective, representatives to develop and implement GLB program recruitment and sustainable models for SMS are scarce. We determined whether a peer leader intervention strategies. A progressive screening approach using telephone (PL) SMS model was as effective in achieving and maintaining improvements and in-person methods was employed. Adults with a BMI≥ 24 kg/m2 and in glycemic control following a DSME and SMS intervention compared pre-diabetes and/or the metabolic syndrome were eligible to participate. A to a traditional DSME support model in 6 rural primary care practices in randomized controlled-delay study design was utilized with two-thirds of Pennsylvania. Practices and eligible participants (n=221; mean age: 63.0 subjects randomly assigned to begin the intervention immediately (NOW), years, 63.8% female, 96.8% Caucasian, 28.5% at or below poverty level, and one-third delayed for 6 months (DELAYED). Weight loss and physical 32.5% using insulin, A1c ≥ 7%: 54.2%) were randomized to the intervention activity were assessed at 6 and 12 months from baseline, as were changes (DSME+PL SMS; n=119) or usual care (UC) group (DSME+traditional DSME in risk factors for diabetes and CVD. A total of 89 individuals enrolled in support with no PL; n=102). Each intervention practice had one PL to support the study (N=60 NOW, 29 DELAYED). At 6 months the NOW group (N=55) participants. Data were collected at baseline, 6 weeks, 6, and 12 months. demonstrated signifi cantly greater mean weight loss compared to the Marked decreases in A1c were observed in both groups following DSME DELAYED group (N=29) (-5.3% v. -1%, p<0.001), as well as signifi cantly (intervention: -0.24%, p<0.0001; UC: -0.31%, p=0.01); however, as SMS greater improvements in HbA1c (-0.1% v. -.004%, p=0.01) and other diabetes continued over time, intervention participants’ average A1c levels continued and CVD risk factors. Further, a signifi cantly higher proportion of the NOW to signifi cantly decrease at 6 months (-0.32%, p=0.04) and at 12 months group achieved 5% weight loss than the DELAYED group (46% v. 7%), with (-0.37%, p=0.02) in comparison to UC which had increases in average A1c a signifi cantly higher proportion of the DELAYED group demonstrating during the support period (6 months: +0.08%, p=0.09; 12 months: +0.02%, weight gain than the NOW group (7% v. 45%). Most importantly, regardless p=0.13). In those who achieved A1c <7% following DSME, 94% and 100% of of when they started the intervention, both NOW and DELAYED groups participants sustained glycemic control at 6 and 12 months, respectively, in demonstrated signifi cant weight loss (~5%) and improvements in diabetes the intervention group. Similarly, in those who achieved A1c<8% following and CVD risk factors after 6 and 12 months of intervention, as well as a DSME, 98% and 96% of participants sustained glycemic control at 6 and 12 signifi cant increase in physical activity levels. These results suggest that the months, respectively, in the intervention group. Results were similar for UC. GBL lifestyle intervention can be effectively delivered in a worksite setting These data demonstrate that PL SMS is as effective as traditional DSME and can serve as a potential model for other worksite programs. support in helping participants to maintain glycemic control in the long- Supported by: NIH term. With the growing prevalence of diabetes and shortage of diabetes educators, it is important to integrate and utilize low-cost interventions in & 683-P high-risk communities that build on available resources. Virtual Diabetes Education Improves Resident Physician Knowl- Supported by: IDF edge and Performance: A Cluster-Randomized Trial JOANN SPERL-HILLEN, PATRICK J. O’CONNOR, HEIDI EKSTROM, STEVE ASCHE, DEEPA APPANA, PAUL E. JOHNSON, Minneapolis, MN & 681-P We conducted a cluster-randomized trial to evaluate the impact of Socioeconomic and Demographic Predictors of Internet and Social an online virtual diabetes educational experience on resident physician Media Use in Persons With Diabetes knowledge and ability to care for patients with diabetes mellitus. MARCIA A. TESTA, DONALD C. SIMONSON, Boston, MA Nineteen primary care residency programs with 341 consented residents Internet and social media tools to access and disseminate health were randomly assigned to receive (n=177) or not receive (n=164) the information have increased dramatically; however, there are limited data on intervention. The intervention included 18 virtual learning cases, 3 of which their use in persons with diabetes. To help design internet-based technologies were performed each month for 6 months. Each unique learning case required to monitor diabetes patients and enhance health messaging, we analyzed about 15 minutes to complete using a Web-based interactive electronic health two (2008 & 2010) Pew Research Center cross-sectional telephone surveys. record-like interface that challenged providers to take clinical actions to bring Logistic regression was used to assess the impact of clinical diagnoses, patients to evidence-based care goals within 6 months of simulated time. demographic and socioeconomic factors on internet usage. A total of 5,254 Physiologic modeling simulated realistic outcomes of provider actions, and individuals participated [age 50±19 yrs, 44% male, 50% married, 42% HS providers received learning feedback designed to critique and guide them grad or lower, 73% Caucasian, 41% full-time employed, 25% retired, median between simulated encounters in each learning case. After the intervention income $35,000]. 678 individuals (13%) reported having diabetes with period, all residents were assigned a 10-question knowledge test and 4 comorbidities of hypertension in 67%, heart disease (24%), and pulmonary virtual assessment cases. Generalized linear mixed models were used to disease (20%). Internet usage was reported by 73% of those with no test for study-arm differences in knowledge scores and the proportion of diabetes and 47% with diabetes (p < 0.001). After adjustment [(Odds Ratio) residents bringing each assessment case to composite goals for glucose, age in yrs (OR = 0.94), male gender (0.64), income / $10,000 (1.55), some blood pressure, and lipids.

For author disclosure information, see page 829. & Guided Audio Tour poster ADA-Funded Research

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