ISLET BIOLOGY—APOPTOSISCATEGORY val prior to survey. Cox models were used to compare the 10-year risks of duces proinflammatory lipids that exacerbateβ cell dysfunction and death. developing diabetes across weight progression trajectories based on com- Inhibition of 12/15-LOX thereby provides a potential therapeutic approach binations of body mass index (BMI) category at age 25 and 10 years prior to prevent glycemic deterioration in T1D, but to date no specific inhibitors to survey. 43% of individuals who started out in the normal category (BMI have been tested T1D. Here we assessed the efficacy and mechanism of a of 18.5-24.9) progressed to a higher weight class, with 33% progressing to novel and specific 12/15-LOX inhibitor, ML351. Using culturedβ cells in vitro, overweight (25.0-29.9), 8% progressing to obese I (30.0-34.9) and 2.5% pro- ML351 exhibited no apparent toxicity across a range of concentration and led gressing to obese II (35.0 and above). Among those who started out in the to improved survival in response to pro-inflammatory cytokines (PIC), which overweight and obese categories at age 25, rates of progression to a higher mimic the stress observed in T1D. In isolated mouse islets in vitro, ML351 weight category were 42% and 45%. Compared to individuals who main- inhibited production of the pro-inflammatory lipid mediator 12-hydroxyeicosa- tained a normal weight, those who progressed to a higher weight status tetraenoic acid, and in luciferase assays it dose-dependently increased activ- were at a significantly increased risks of developing diabetes, with hazard ity of the anti-oxidant transcription factor Nrf2. Following oral or intraperito- ratios of 2.49, 4.73 and 5.55 for those progressing to overweight, obese I and neal administration of ML351 in mice penetrance of the drug was observed obese II. Weight stability was associated with a lower risk of diabetes com- in multiple organs, including pancreas, fat, liver and brain. In the low-dose pared to weight gain, however, those who were weight stable in overweight, streptozotocin model of T1D in mice, ML351 administration prevented devel- obese I and obese II had significantly higher risks of developing diabetes opment of diabetes, with coincident enhancement of nuclear Nrf2 in islet than those who were stable normal weight, with hazard ratios of 2.87, 5.67 cells, reduced β oxidative stress, and preservation of β cell mass. In the non- and 9.62. Individuals who lost weight between young adulthood and midlife obese diabetic mouse model of T1D, administration of ML351 in the predia- had lower risks of developing diabetes than those who maintained weight in betic phase prevented glycemic deterioration and reduced invasive insulitis. the higher category; however, they remained at higher risk than those who In conclusion, our data provide the first evidence to date that small mol- were normal weight in both periods. ecules that target 12/15-LOX can prevent progression of β cell dysfunction and glycemic deterioration in models of T1D. Supported By: National Institutes of Health (T32DK064466), (R01DK105588 to ISLET BIOLOGY—APOPTOSIS R.G.M., J.L.N.); Diabetes Research Center (P30DK097512) & 2155‑P Moderated Poster Discussion: Life and Death of the Islet (Posters: 2153-P Physiologic Concentration of IL1β Enhances the Deleterious Effects to 2158-P), see page 17. of Islet Amyloid MAHNAZ MELLATI, DANIEL T. MEIER, THINN THINN KHINE, MEGHAN F. HOGAN, & 2153‑P ANDREW T. TEMPLIN, SAKENEH ZRAIKA, REBECCA L. HULL, STEVEN E. KAHN, Genetic Deficiency of TXNIP Prevents Diabetes Progression in the Seattle, WA, Melbourne, Australia Mouse Model of Wolfram Syndrome Islet amyloid formation by fibrillogenic human islet amyloid polypeptide KIKUKO SHIINOKI, KATSUYA TANABE, MASAYUKI HATANAKA, RISA HARANO, (hIAPP) is associated with β-cell loss in human type 2 diabetes (T2D). Previ- HIROSHI MASUTANI, YUKIO TANIZAWA, Ube, Japan, Kyoto, Japan ous in vitro studies have shown an association between islet amyloid for- Wolfram syndrome (WS), caused by the WFS1 gene mutations, is char- mation and increased IL1β and TNFα levels, and in vivo reduced amyloid acterized by insulin dependent diabetes mellitus. Genetically determined formation when blocking IL1β. As in healthy humans circulating IL1β levels pancreatic β cell loss results from augmented ER and oxidative stresses. are 0.5-12 pg/ml, we determined in vitro whether physiologic IL1β alone TXNIP is induced in response to cellular stresses and is known to be involved exacerbates amyloid deposition and islet inflammation and thereby a vicious in multiple cellular processes. WFS1-deficient islets have a robust increase cycle resulting in greater β-cell loss. in both TXNIP transcription and its protein levels compared to WT islets. We Based on IL1β dose-response studies (0.04-4000 pg/ml), we determined have recently found that WFS1-deficientβ cells revert to endocrine progeni- that 4 pg/ml recombinant mouse IL1β exacerbated amyloid formation in tor like cells expressing Neurogenin3, and that a subset of them takes α cell hIAPP transgenic islets more than 6 fold compared to when IL1β was absent fate. Importantly, such β cell plasticity appears preceding hyperglycemia (amyloid area/islet area: 3.85±0.9 vs. 0.6±0.23%, p=0.02, n=5). When and becomes more apparent along with the progression of hyperglycemia hIAPP transgenic and non-transgenic islets were cultured in the presence or in the setting of insulin resistance. Previously, we demonstrated piogli- or absence of 4 pg/ml IL1β for 48 hours, only when islet amyloid was pres- tazone administration prevented β cell loss and rescued diabetes in WFS1- ent was IL1β treatment associated with reduced insulin but not IAPP gene deficient mice carrying yA /a (WFS1-/-;Ay/a). Interestingly, an increased islets expression, along with increased expression of iNOS and IL6 (Table). TXNIP expression was prevented in the WFS1-/-;Ay/a mice, suggesting exces- In summary, in the presence of islet amyloid, IL1β at a physiological con- sive TXNIP action has implications for the progression of β cell loss in the centration exacerbates islet amyloid formation, decreases insulin gene WFS1-/-;Ay/a mice. To test this hypothesis, WFS1-deficient mice in the pres- expression and enhances expression of pro-inflammatory response genes. ence or the absence of the Ay/a mutation were crossed with the mice lacking Thus, blocking IL1β signaling may prevent amyloid-induced damage and pro- T XNIP. WFS1-/-;Ay/a mice lacking TXNIP did not develop hyperglycemia in gression of T2D. y spite of having more weight gain compared to the nondiabetic obese A /a Table. Expression of Genes (Relative to Cyclophilin) in Non-Transgenic (NT) mice. Further, TXNIP-deficiency completely prevented diabetes progression and hIAPP Transgenic (TG) Islets in Presence or Absence of 4 pg/ml IL1β. in the mice lacking WFS1 (WFS1-/-;a/a) even after 40 weeks. Most antidia- NT + vehicle NT + IL1β TG + vehicle TG + IL1β a b c betic effects of TXNIP-deficiency appear to be due toβ cell preservation without loss of β cell phenotype nor α cells transduction. TXNIP-deficiency Iapp 1.00±0.13 0.95±0.19 0.72±0.17 0.58±0.02 NS NS NS maintains β cell identity by a restoration of MafA expression and suppres- INS2 1.00±0.04 0.85±0.15 1.11±0.17 0.60±0.04 NS NS 0.02 sion of Neurogenin3 emergence. These findings illustrate that inhibition of Nos2 1.00±0.95 1.70±0.53 0.40±0.19 6.80±0.90 NS NS 0.007 TXNIP is a novel therapeutic strategy for diabetes in WS and that TXNIP is a IL6 1.00±0.43 2.05±0.78 1.73±0.82 4.09±0.55 NS NS 0.035 determinant of β cell plasticity in the setting of WFS1-deficiency. Supported By: Japan Society for the Promotion of Science; MSD Life Science IL1β 1.00±0.17 0.61±0.19 0.92±0.17 0.30±0.07 NS NS NS Foundation; Front Runner of Future Diabetes Research; Japan Association for TNF 1.00±0.35 1.51±0.22 1.09±0.09 1.75±0.29 NS NS NS Diabetes Education and Care a = NT + vehicle vs. NT + IL1β; b = NT + vehicle vs. TG + vehicle; c = TG + vehicle vs. TG + IL1β; NS=non-significant; n=4. & 2154‑P Inhibition of 12/15-Lipoxygenase in Mouse Models of Type 1 Dia‑ betes 2156‑P MARIMAR HERNANDEZ-PEREZ, SARAH TERSEY, RYAN M. ANDERSON, CHANELLE & Pregnancy-Induced Proliferation of Human β-Cells Engrafted into BENJAMIN, JERRY L. NADLER, THEODORE R. HOLMAN, DAVID J. MALONEY, Immunodeficient NOD-scid IL2rγnull Mice RAGHAVENDRA G. MIRMIRA, Indianapolis, IN, Norfolk, VA, Santa Cruz, CA, Rockville, AGATA JURCZYK, ROHIT SHARMA, CHAOXING YANG, LEONARD SHULTZ, DALE POSTERS MD GREINER, LAURA C. ALONSO, RITA BORTELL, Worcester, MA, Bar Harbor, ME Islet Biology/ Inflammation and oxidative stress are among the major factors that con- We and others have previously demonstrated that human β-cells in islet- Insulin Secretion tribute to deficiency ofβ cell mass and insulin secretion in type 1 diabetes engrafted immunodeficient mice can be induced to proliferate in response to (T1D). 12/15-lipoxygenase (12/15-LOX) is induced in β cells during T1D, pro- hyperglycemia. The goal of this study was to determine the ability of human

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A565 ISLET BIOLOGY—APOPTOSISCATEGORY

β-cells to proliferate in vivo in pregnant compared with nonpregnant recipi- 2159‑P ent mice. We transplanted 500-2000 human islet equivalents (IEQs) from Development of a High-Throughput Screen to Identify Cell Type- seven individual adult organ donors into normoglycemic female NOD-scid Specific Death in Human Islets IL2rγnull (NSG) mice; the functionality of the human islet grafts was confirmed HEATHER L. HAYES, BENJAMIN M. BUEHRER, Research Triangle Park, NC by detection of human insulin in the serum of the engrafted mice follow- It is a general consensus that loss of beta cell mass results in T1D and ing glucose stimulation. Subsets of engrafted mice were put into mating, contributes to the progression of T2D. Epidemiological studies have sug- and at 13 days of pregnancy, pregnant and nonpregnant mice were given gested that there may be a link between exposure to certain environmen- bromodeoxyuridine (BrdU) in the drinking water for 7 days. The proliferative tal chemicals and T1D onset and T2D onset/progression, but relatively few capacity of human β-cells in the graft was determined by BrdU incorporation studies have assessed environmental contaminants on biological functions of insulin-positive cells. We observed an ~8-fold increase in human β-cell and toxicity of pancreatic cells. Thus, we developed a multiparametric proliferation in donor islets transplanted into pregnant, as compared to high-throughput screen (HTS) with robust Z’ values (0.2 to 0.8) to identify nonpregnant, NSG mice. The proliferative capacity of adult human β-cells cell type-specific death in human islets using only 5000 cells/well. A high- was not dependent on the age (range from 24 to 57 years), gender (3 male content imager and antibodies against insulin and glucagon were used to and 4 female), or body mass index (range from 20.4 to 38.2 kg/m2) of the identify beta and alpha cells, respectively, allowing contaminating exocrine islet donors in this study. The human islet grafts from pregnant mice also cells to be excluded from analyses as they are often more sensitive to cer- had increased levels of insulin (~2-fold) and serotonin (~7-fold) compared tain death stimuli than endocrine cells. Method validation of ZenBio’s HTS to the same donor islets engrafted into nonpregnant mice. Together our involved screening 50 compounds from the ToxCastTM. data suggest that although the endogenous or ‘basal’ rate of human β-cells Phase I Chemical Library. Using human islets from 4 separate donors, Zen- decreases with age, the capacity of human β-cells to proliferate in adapta- Bio’s human islet toxicity screen identified 1 of these 50 compounds to be a tion to an increased need for insulin under a variety of physiologic conditions positive hit as a pancreatic cell toxicant at a 1000-fold lower concentration remains intrinsic throughout much of adult life. than the streptozotocin positive control. This compound, chlorothalonil, is a broad spectrum fungicide commonly used to control fungi in agriculture & 2157‑P crops. Importantly, chlorothalonil poisoning was recently demonstrated to Cathepsin L Is a Downstream Target in the Regulation of Beta-Cell induce diabetic ketoacidosis in a worker exposed to high levels of chlorotha- Adaptive Changes following Antibody Response to Serpin B13 lonil. This positive hit strongly supports our novel screen’s ability to identify CHI WEN LO, YURY KRYVALAP, NADZEYA KRYVALAP, JAN CZYZYK, Rochester, human pancreatic cell toxicants. As there is limited information on environ- NY mental toxicants and their role in islet cell toxicity, any compound identified Successful therapy of type 1 diabetes requires both suppression of auto- using this screen that can induce cell death in human islets will be of inter- immune inflammation and restoration of beta-cell mass. According to our est in understanding the role that environmental toxicants may have in T1D published work exposure to antibodies against serpin B13, a protease inhibi- onset or T2D onset/progression. tor expressed in the exocrine pancreatic ducts, impedes islet autoimmunity Supported By: National Center for Advancing Translational Sciences; National and promotes pancreatic beta-cell proliferation, thereby underscoring the Institutes of Health importance of a functional relationship between the exocrine and endocrine pancreas. 2160‑P We used an in vitro pancreatic islet culture system to examine the mecha- nism by which serpin B13 monoclonal antibody (mAb) causes islet adaptive WITHDRAWN changes. We found that the catalytic activity of cathepsin L (catL), a ser- pin B13 protease target, was augmented in pancreatic cell lysate contain- ing serpin B13 mAb. We also found that addition of serpin B13 mAb to the islets, together with a pancreatic ductal epithelium extract, led to significant upregulation of beta-cell proliferation and expression of Reg genes (impli- cated in islet regeneration). A similar treatment with an extract of nonductal pancreatic cells failed to induce these changes. Moreover, these responses were sensitive to inhibition with E64 pan-protease inhibitor or catL-specific inhibitor thereby, implicating this protease as the downstream molecular target of antibody-mediated neutralization of serpin B13. Consistently with this concept, catL markedly enhanced Reg genes and cell proliferation when directly added to the islet cultures. We conclude that the islet cell proliferation observed with serpin B13 activity is likely caused by catL protease activity and secondary upregula- tion of Reg genes in the islets. In broader terms, our work suggests that crosstalk between exocrine and endocrine pancreatic tissue is dependent on islet sensing signals that are generated in the surrounding tissue and that development of protocols to enhance protease activity within the serpin B13/CatL/Reg pathway may be beneficial in diabetes. Supported By: JDRF

2158‑P WITHDRAWN POSTERS Islet Biology/ Insulin Secretion

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A566 ISLET BIOLOGY—APOPTOSISCATEGORY

2161‑P 2163‑P Lysine Demethylase Inhibition Protects Pancreatic β-Cells from Hyperglycemia Itself Does Not Hamper Quantification of Beta-Cell Apoptosis and Improves β-Cell Function Mass with Indium-Labeled Exendin Probe THOMAS MANDRUP-POULSEN, MARIE B. BACKE, JAN L. ANDERSSON, KARL NAOTAKA FUJITA, HIROYUKI FUJIMOTO, KEITA HAMAMATSU, TAKAAKI BACOS, DAN P. CHRISTENSEN, JAKOB B. HANSEN, JERZY J. DOROSZ, MICHAEL MURAKAMI, HIROYUKI KIMURA, KENTARO TOYODA, HIDEO SAJI, NOBUYA GAJHEDE, TINA DAHLBY, MADHUSUDHAN BYSANI, LINE H. KRISTENSEN, INAGAKI, Kyoto, Japan CHARLOTTE LING, LARS OLSEN, Copenhagen, Denmark, Lund, Sweden Aims: It is reported that beta cell mass (BCM) is decreased in patients Reprogramming of gene transcription definesβ -cell-fate in response to with type 2 diabetes. And also reported that BCM could be non-invasively stress. Post-translational modifications of histone and non-histone tran- quantified with indium-labeled Exendin probe targeting glucagon-like pep- scriptional regulators control gene transcription. Lysine methyltransferases tide-1 (GLP-1) receptors. However, hyperglycemia itself might take influence and demethylases (KDMs) regulate methylation on histone tails, whereby on the accumulation of the probe toward GLP-1 receptors, leading to hamper gene transcription is activated or repressed depending on which lysine quantification of BCM. Therefore, we investigated the influence of hyper- residue is modified. The activating H3K4 mark regulates insulin secretion glycemia on the quantification of BCM with indium-labeled Exendin probe. by controlling transcription of insulin-1/2, MafA and Glut2. The link between Methods: We used indium-labeled Exendin probe, high affinity toward lysine methylation and β-cell-fate in response to stress is unclear. We found beta cells and specific accumulation toward islets have already been con- that the KDMs demethylating H3K4me3/me2 (KDM5B) and H3K27me3/me2 firmed in binding assay and autoradiography with pancreatic sections of (KDM6A/B) were expressed in β-cells and islets. Expression of KDM6B was MIP-GFP mice, respectively. We randomized male db/db mice into 2 groups upregulated by pro-inflammatory cytokines, suggesting a role in inflamma- and gave control diet (Group C) or diet containing Canagliflozin, SGLT2 inhibi- tory β-cell destruction. siRNA mediated knock-down of KDM6B failed to tor, (Group S) for 1 week. After injecting the probe intravenously, we har- affect cytokine mediated β-cell viability. To circumvent possible redundancy vested pancreas and measured its radioactivity. We also calculated BCM between KDM6B and KDM6A/5B we used the KDM5B and 6A/B selective with immuno-histochemical staining method. demethylase inhibitor GSK-J4 which protected insulin-producing cells as Results: Non-fasted blood glucose level and body weight were signifi- well as human and mouse islets from cytokine-induced apoptosis in vitro by cantly lower in Group S than Group C (p < 0.001 and p = 0.02, respectively) blunting NFκB signaling and ER stress response gene expression. KDM5B- after the 1 week intervention, even though there were no significant differ- 6A/B inhibition also improved glucose-stimulated insulin secretion and gene ences between the 2 groups at the start (p = 0.97 and p = 0.37, respectively). expression. Microarray analyses revealed that mRNA clusters regulating On the other hand, there was no significant difference in radioactivity of purinergic and cytokine ligand-receptor interactions were reduced following the harvested pancreas nor calculated BCM between Group S and Group C KDM5B-6A/B inhibition, while protein folding, sorting and degradation, tran- (p = 0.54 and p = 0.40, respectively). Also, there was no significant difference scription, cell replication, repair, growth and death clusters were increased. in radioactivity of the pancreas per calculated BCM between the 2 groups In conclusion, KDM demethylases are important regulators of β-cell dys- (p = 0.94). function and death. Conclusion: Hyperglycemia itself seems not to influence the probe accu- Supported By: SHARE PhD Fellowship; Augustinus Foundation; Arvid Nilsson mulation toward beta cells because there was no significant difference in Foundation; Aase and Ejnar Danielsens Foundation; University of Copenhagen; the radioactivity of the pancreas per calculated BCM between the 2 groups, Danish Cancer Society; Novo Nordisk Foundation; Swedish Research Council; even though exposed blood glucose level was significantly different. Region Skåne; Wallenberg Foundation; Påhlsson Foundation; Swedish Diabetes Foundation; Exodiab; Linné 2164‑P The RNA Decay System Is Regulated in Insulin-Producing Cells 2162‑P and Islets by Cytokines and Contributes to Inflammatory Beta-Cell Toxicity of the Unsaturated Fatty Acid Oleate to Human Beta Cells Damage SIGURD LENZEN, THOMAS PLOTZ, BASTIAN KRUMMEL, ANNA LAPORTE, THOMAS MANDRUP-POULSEN, SEYED M. GHIASI, Copenhagen, Denmark ATTILIO PINGITORE, SHANTA PERSAUD, MATTHIAS ELSNER, ILIR MEHMETI, Inflammatory beta-cell damage requires differential regulation of the Hannover, Germany, London, United Kingdom beta-cell transcriptome. Altered levels of deleterious and protective mRNAs Free fatty acids (FFAs) decrease glucose tolerance and can support the result from a balance between gene transcription and mRNA decay. The reg- development of type 2 diabetes. Pancreatic beta cells play a major role in ulation of the RNA decay systems in beta-cells has not been investigated. this process through toxicity of FFAs. Hence, we analyzed the toxicity pro- By qPCR we found that the key components of both the no-go decay (NGD: file of saturated and unsaturated FFAs in isolated human islets and in the Pelo, Hbs1, ABCE1, Xrn1, DCP2, DCPS, Dis3, Dis3L1, Dis3L2) and nonsense- human EndoC-βH1 beta cell line, especially comparing the toxicity of the mediated decay (NMD: UPF1, 2, 3A and 3B, SMG1, 5, 6 and 7) pathways were physiological FFAs palmitic acid (PA) and oleic acid (OA). For this purpose expressed in INS-1 cells. Pelo, DCP2, UPF2 and SMG1/7 were upregulated by isolated human islets and EndoC-βH1 beta cells were incubated for two days IL-1 (150 pg/ml)+IFNγ (0.1 ng/ml) (CYT) in INS-1 cells under experimental con- with different FFAs and the toxicity was analyzed by caspase-3 assay. Not ditions, where central beta-cell mRNAs were differentially regulated (Ins1/2, only the saturated PA (C16:0) but also the unsaturated OA (C18:1) were toxic Pdx-1, Nkx6.1, Isl-1 down, GLUT2 up). Many of the NGD/NMD component to human beta cells. OA did also not provide protection against the toxicity of expressional regulations were confirmed in human islets at the mRNA level, PA. Fatty acids with a chain length shorter than C16 were not toxic. The FFA and at the protein level in INS-1 cells and rat islets. CYT-induced upregula- linoleic acid (LA) (C18:2) was also toxic, but the poly-unsaturated γ-linolenic tion of Pelo, Xrn1, Dis3L2, UPF2, SMG1/6 were reduced by iNOS inhibition acid (γ-LNA) (C18:3) exhibited no toxicity. The observation that the mono- with NMA, as were ER stress, inhibition of Ins1/2 and accumulated insulin unsaturated OA was as toxic as the saturated PA to human beta cells is an secretion. ROS inhibition by N-acetyl-cysteine, iron chelator or transgenic unexpected new observation. This is in contrast to the situation in rodent DMT-1 knock-out did not affect CYT-induced NGD/NMD component expres- beta cells where only saturated FFAs like PA are toxic. The toxicity profile of sion. siRNA mediated knock-down (KD) of Pelo and Xrn1 ameliorated CYT- the analyzed FFAs in human beta cells is in accordance with the fact that the induced INS-1 cell-death without affecting ER stress, and increased Ins1/2 toxic reactive oxygen species (ROS) are generated in the peroxisomal beta- expression and insulin secretion in both control and CYT exposed Ins-1 cells. oxidation. Interestingly, the mono-unsaturated OA provided no protection We conclude that the RNA decay systems are regulated by inflammatory against the toxicity of the saturated PA in human beta cells. This observation stress in beta-cells in an NO but not ROS dependent manner, either directly has serious consequences for the lipid composition of the daily diet towards or secondary to nitroxidative RNA damage. Knock-down of key NGD compo- protection against lipotoxicity. A high content of OA, which is considered to nents improved beta-cell function and reduced CYT-mediated beta-cell dam- be a desirable element in a cardioprotective diet, would not be a desirable age by preventing clearance of ins1/2 and possibly anti-apoptotic mRNAs. constituent of a beta cell protective diet. While composing cardioprotective Supported By: Augustinus Foundation; Zealand Pharma; Danish Diabetes Acad- diets, it is therefore crucial to avoid components, which exhibit toxic effects emy; Bjarne Jensen Foundation; University of Copenhagen on beta cell function. POSTERS Islet Biology/ Insulin Secretion

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A567 ISLET BIOLOGY—APOPTOSISCATEGORY

2165‑P Figure. Treatment of Hyperglycemia with the SGLT2 Inhibitor Empagliflozin Ameliorates Markers of Islet Endothelial Dysfunction in C57BL/ KsJ.db/db Diabetic Mice REBECCA L. HULL, MEGHAN F. HOGAN, Seattle, WA Islet endothelial cells produce factors that support β-cell function. We have shown that markers of endothelial dysfunction occur in islets from diabetic db/db mice, and induction of islet endothelial dysfunction in vitro results in impaired insulin release. In the present study, we used the SGLT2 inhibitor empagliflozin to define the role of chronic hyperglycemia in the development of islet endothelial dysfunction. Male, 6-week old, C57BL/ KsJ.db/db diabetic mice and littermate controls (db/+;+/+) were treated with empagliflozin (EMPA; 20 mg/kg/day) or vehicle (VEH) administered ad libitum in the diet for 12 weeks (n=5-8). At the end of the study, islet mRNA levels of 2167‑P the cell adhesion molecule E-selectin (Sele), proinflammatory cytokine inter- Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Medi‑ leukin-6 (Il6) and vasoconstrictor endothelin-1 (Edn1) were determined. After ated Beta-Cell Death 12 weeks of treatment, db/db-VEH mice were obese, hyperglycemic, and AMY L. CLARK, FUMIHIKO URANO, DAVID W. PISTON, KOHSUKE KANEKURA, exhibited marked increases in islet Sele, Il6 and Edn1 mRNA levels (Table), CLAY F. SEMENKOVICH, LARRY SPEARS, ZENO LAVAGNINO, St. Louis, MO, Tokyo, consistent with islet endothelial dysfunction. EMPA treatment had no effect Japan in db/+;+/+ mice, whereas in db/db mice it prevented the rise in plasma Pro-inflammatory cytokines are important mediators of islet inflammation glucose, resulted in increased body weight, and significantly reduced islet and β cell death leading to type 1 diabetes. The mechanism of cytokine- mRNA levels of Sele, Il6 and Edn1 (Table). mediated inflammation and pro-apoptotic signaling is known to involve In summary, empagliflozin treatment has beneficial effects on the islet alterations in cellular calcium homeostasis and activation of endoplasmic endothelial cell in db/db diabetic mice. reticulum (ER) stress pathways. In this study, we show that pharmacologic Table. modulation of cellular calcium levels prevents cytokine and ER stress-medi- ated β cell death. INS-1E rat insulinoma cells were pre-treated with known calcium modulating compounds and then exposed to cytokine cocktail (IL1-B and IFN-y) or the ER stress inducing agent thapsigargin. The ryanodine receptor blocker dantrolene and the dipeptidyl peptidase IV inhibitor sita- gliptin both prevented activation of the calcium dependent pro-apoptotic protease calpain and significantly reduced cytokine and ER stress-induced Supported By: National Institutes of Health; National Institute of Diabetes and β cell apoptosis. The drugs were able to improve ER calcium handling by Digestive and Kidney Diseases; Boehringer Ingelheim maintaining functional ER calcium release and sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity during cytokine stress. Real time PCR analy- sis indicated that neither drug had any effect on the expression of canoni- 2166‑P cal ER stress markers such as CHOP and BIP. However, sitagliptin reduced The GLP-1 Receptor as a Homing Beacon for Targeted Delivery expression levels of the ER stress-induced pro-apoptotic protein thioredoxin to Pancreatic β-Cells: Quantitating Tissue Selective Productive interacting protein (TXNIP). Supporting the role of TXNIP as an important Uptake of Antisense Oligonucleotides mediator of cytokine-mediated β cell death, shRNA knock down of TXNIP CARINA AMMALA, INGELA AHLSTEDT, EVA-MARIE ANDERSSON, PATRIK was able to completely prevent cytokine-induced caspase 3/7 activation. ANDERSSON, ERIKA CAVALLIN, WILLIAM DRURY, GENE HUNG, RASMUS Our results demonstrate that targeting maintenance of cellular calcium JANSSON-LÖFMARK, CAMILLA JOHANSSON, LAURENT KNERR, RICHARD LEE, homeostasis presents a novel treatment to prevent cytokine-mediated MARTA PEREZ-ALCAZAR, THAZHA P. PRAKASH, PUNIT SETH, PIA STILLEMARK- β cell death. In addition, we have shown that TXNIP is a crucial mediator BILLTON, LENA S. SVENSOON, LINDA SUNDSTRÖM, ERIC VALEUR, BRETT P. of cytokine-induced β cell apoptosis. These results indicate that targeting MONIA, SHALINI ANDERSSON, Gothenburg, Sweden, Carlsbad, CA cellular calcium homeostasis and suppression of TXNIP present viable thera- Therapies restoring β-cell mass in type 2 diabetes (T2D) remains an unmet peutic strategies for preventing β cell death in type 1 diabetes. medical need. Antisense oligonucleotides (ASO) hold promise as a mean for Supported By: Endocrine Fellows Foundation; National Institutes of Health knock down of genetic targets linked to β-cell loss in T2D. However, islets (K12-HD076224, P30DK020579); JDRF (2-SRA-2016-233-S-N, 2-SRA-2016-225-S-B) are resistant to uptake of ASOs from circulation. Here a GLP-1 receptor (GLP-1R) agonist, conjugated to ASOs (GLP-1-ASO) against MALAT1 or FoxO1, was used to test a hypothesis of β-cell specific 2168‑P ASOs delivery through GLP-1R internalization. Glucagon-Like Peptide-1 Receptor Mediates Delivery of Antisense In vitro, in GLP-1R overexpressing HEK293 cells, GLP-1-ASO treatment Oligonucleotides to Pancreatic β-Cells showed enhanced knock down of the target gene compared to ASO; LINDA SUNDSTRÖM, INGELA AHLSTEDT, EVA-MARIE ANDERSSON, JOHAN with a 40 fold increase in potency for MALAT1 ASO (IC = 870nM vs. BRENGDAHL, JOSEFINE CLAESSON, WILLIAM DRURY, III, GENE HUNG, LAU- 50, ASO RENT KNERR, RICHARD LEE, SVENJA LÜNSE, JOHAN MEULLER, THAZHA P. IC50, GLP-1-ASO = 19nM). Treatment of mouse islets with GLP-1-ASO reduced gene expression by 94% (MALAT1) or 72% (FoxO1) vs. ASO (46% and 11%). PRAKASH, LINDA ROTH, PUNIT SETH, PIA STILLEMARK-BILLTON, ERIC VALEUR, In vivo, single subcutaneous injections of GLP-1-ASOs dose dependently CHARLOTTA WENNBERG-HULT, CARINA ÄMMÄLÄ, BRETT P. MONIA, SHALINI increased ASO exposure and reduced gene expression in mouse islets while ANDERSSON, Mölndal, Sweden, Carlsbad, CA parent ASO was without effect (Figure, FoxO1). Furthermore, three was no Antisense oligonucleotides (ASO) represent a promising drug modality effect on gene expression in liver (Figure) even at doses reaching >70% for β-cell regenerative treatments, since they can be used to knockdown knock down in islets, suggesting that GLP-1 conjugation confers enriched genetic targets linked to β-cell loss in type 2 diabetes. However, pancreatic uptake in islets compared to peripheral tissues. β-cells are resistant to uptake of both generation 2.0 and 2.5 ASOs from We have shown, in vitro and in vivo, that GLP-1R acts as a homing beacon systemic circulation. Cell specific targeted delivery of ASO by conjugation for efficient and selective delivery of ASO cargo to pancreaticβ -cells sparing to a high-affinity ligand for a receptor with enriched expression on the peripheral tissues. target cell, and with ability to internalize, is hypothesized to increase spe- cific uptake. Here, we successfully demonstrate this concept using a GLP-1 receptor (GLP-1 R) peptide agonist to deliver generation 2.5 ASOs to pancre- atic β-cells. Receptor-dependent internalization of the fluorescently labelled peptide was established by confocal imaging with GLP-1 R overexpressing HEK293 cells. Two ASOs targeted against the MALAT1 or FoxO1 genes were

POSTERS conjugated to the peptide. Both ASO conjugates retained GLP-1 R activity Islet Biology/

Insulin Secretion comparable to the peptide agonist, as demonstrated in G protein activation (cAMP and dynamic mass redistribution) and in β-arrestin (β-arrestin and receptor internalization) dependent signaling assays in recombinant cells

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A568 ISLET BIOLOGY—APOPTOSISCATEGORY overexpressing GLP-1 R. Enhanced productive uptake was demonstrated in pathways. MP may prove a promising therapeutic tool in the protection of HEK293 GLP-1 R cells by measuring gene knockdown of MALAT1 or FoxO1. transplanted islets. Following treatment with the GLP-1 conjugates, a 20 to 60 fold reduction in Supported By: Assistance Service Diabète IC50 was achieved compared to the respective unconjugated parent ASOs. Consistent with these results, treatment of primary rodent islet cells with 2171‑P the ASO conjugates significantly increased gene knockdown compared to Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) Activator the parent ASOs. To the best of our knowledge, this study is the first demon- Potentiates Glucose-Stimulated Insulin Secretion and Prevents stration that GLP-1 R, a class B G-protein coupled receptor, can be used to 4-HNE Induced Beta-Cell Apoptosis enhance the productive uptake of ASO cargo and therefore be used for the TIEN JYUN CHANG, MENG WEI LIU, LEE MING CHUANG, Taipei, Taiwan delivery of ASOs to pancreatic β-cells. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde generated by alcohol metabolism and lipid peroxidation. A previous unbi- 2169‑P ased proteomic search identified the activation of ALDH2 correlated with MST1 Inhibition as a Novel Beta-Cell Protective Therapy for Diabetes reduced ischemic heart damage in rodent models. Then a high-throughput KATHRIN MAEDLER, KARTHIKA ANNAMALAI, ALEKSANDRA DOBROWOLSKI, screen yielded a small-molecule activator of ALDH2, named as Alda-1. It has JANINA OETJEN, BLAŽ LUPŠE, LEI DING, SUSHIL AWAL, DELSI ALTENHOFEN, been reported that Alda-1 reduced ischemic damage to the heart through MATTHEW TREMBLAY, AMIN ARDESTANI, Bremen, Germany, La Jolla, CA its inhibitory effect on the formation of cytotoxic aldehydes. However, the Mammalian Sterile 20-like kinase 1 (MST1) is a master regulator of pan- effect of Alda-1 on islet beta cell function and beta cell survival have not creatic beta cell death, a hallmark of the loss of insulin-producing beta-cells been investigated. In this study, we administered different doses of Alda-1 in diabetes. Depletion of MST1 in beta-cells in mice in vivo or in human islets to mouse insulinoma cells (MIN6), and found Alda-1 potentiated glucose- in vitro protects beta-cells from apoptosis and restores beta-cell function stimulated insulin secretion. Alda-1 also increased oxygen consumption rate and normoglycemia. Thus, the identification of MST1 inhibitors represents a in MIN6 cells, which indicated Alda-1 improved mitochondrial function. On promising approach for a beta-cell-protective therapy of diabetes. An MST1 the other hand, Alda-1 also partially rescued 4-HNE induced cell death of inhibition screen across a highly privileged collection of 641 drug-like kinase MIN6 cells. Administration of 4-HNE (50 μM) resulted in increased cleav- inhibitors together with a follow-up strategy for selective, non-cytotoxic age of PARP and caspase 3, and pre-treatment of Alda-1 (10 μM) partially compounds identified Neratinib, initially found as a Her2/EGFR dual kinase decreased the cleavage of these two pro-apoptotic proteins. Administra- inhibitor. It is fairly selective among serine/threonine kinases and currently tion of 4-HNE (50 μM) led to decreased phosphorylation of AKT, Bcl-2/Bad in Phase III trials for breast cancer. Neratinib restored beta-cell survival in ratio, and ALDH2 expression. Pre-treatment of Alda-1 (10 μM) enhanced the vitro under multiple diabetogenic conditions (H2O2, elevated glucose con- expression of the above three proteins. Alda-1 also restored 4-HNE induced centrations, the mixture of 22.2 mM glucose and 0.5 mM palmitate or of the decreased intracellular ATP concentration in MIN6 cells. Taken together, the cytokines IL-1b and IFNg) in human islets and INS-1E cells. While exposure ALDH2 activator, Alda-1, potentiated glucose-stimulated insulin secretion of to the diabetogenic conditions induced MST1 phosphorylation and cleavage beta cells. Alda-1 also rescued 4-HNE induced beta cell apoptosis partially and Caspase 3 cleavage, Neratinib inhibited MST1 activation and apopto- through improving mitochondrial function, suppressed pro-apoptotic pro- sis. In vivo, Neratinib restored normoglycemia, beta-cell function, survival teins and enhanced anti-apoptotic proteins. It may infer that ALDH2 activa- and beta-cell mass in two diabetic mouse models. Diabetes was induced by tor may be a novel drug target of type 2 diabetes. multiple low-dose streptozotocin (MLD-STZ) injections, serving as a type 1 Supported By: Taiwan Ministry of Science and Technology (NSC diabetes mouse model. Obese diabetic Leprdb/db mice served as a mouse 101-2314-B-002-158-MY3) model for type 2 diabetes. In both models, daily intraperitoneal injections of Neratininb over 30 days significantly reduced fasting glucose levels, 2172‑P improved glucose tolerance and the insulin/glucose ratio as well as glucose The Mitochondrial-Derived Peptide MOTS-c Enhances Pancre‑ stimulated insulin secretion during the glucose tolerance test. We show atic Beta-Cell Function and Survival by Ameliorating Endoplasmic that Neratinib is a novel MST1 inhibitor and serves as a potential beta-cell Reticulum Stress protective drug in vitro in human islets and in vivo in two rodent models of SE HEE MIN, HAI YING SHEN, SUN JOON MOON, CHANG HO AHN, YOUNG MIN type 1 and type 2 diabetes. CHO, Seoul, Republic of Korea Supported By: JDRF The role of mitochondrial open reading frame of the 12S rRNA-c (MOTS- c), a newly discovered mitochondrial-derived peptide which ameliorates 2170‑P hyperglycemia and is associated with longevity in humans, in pancreatic Endothelial Microparticles Released by Activated Protein C Protect beta cell function and survival has not been studied. In this study, we inves- Beta Cells through EPCR/PAR1 and Annexin A1/FPR2 Pathways tigated whether MOTS-c improves the survival and function of beta cells GUILLAUME KREUTTER, MOHAMAD KASSEM, ALI EL HABHAB, PHILIPPE BALTZ- under endoplasmic reticulum (ER) stress either alone or in combination with INGER, BLANDINE YVER, FATIHA ZOBAIRI, GENEVIEVE UBEAUD-SEQUIER, a glucagon-like peptide-1 (GLP-1) receptor agonist. LAURENCE KESSLER, FLORENCE TOTI, Strasbourg, France To induce ER stress, we treated cultured mouse insulinoma cells (MIN6) Islet transplantation is associated with early ischemia/reperfusion lead- with thapsigargin or tunicamycin for 12 hr in the presence or absence of ing to localized coagulation events and endothelial damage. Activated Pro- exendin-4 (a GLP-1 receptor agonist) or MOTS-c and both. The cell viability, tein C (aPC) limits thrombin generation and exerts endothelial cytoprotec- determined by the MTT assay, was significantly decreased under ER stress, tion via EPCR/PAR1 pathways. In animal models, islet cytoprotection by aPC which was substantially recovered by exendin-4 or MOTS-c and both. Com- restores islet vascularization and protects graft function. Microparticles pared to exendin-4 or MOTS-c alone, combination treatment of the two (MP) are plasma membrane procoagulant vesicles, surrogates markers of resulted in an additive or synergistic effect on the cell viability under ER stress, and cellular effectors. We measured the cytoprotective effects of stress. Insulin secretion was decreased by thapsigargin treatment com- aPC on endothelial and β-cells, and its role in the autocrine and paracrine pared with control group, which was substantially improved by exendin-4 MP-mediated cell crosstalk during oxidative stress. MP from aPC-treated or MOTS-c and further improved by the combination treatment of the two. endothelial (EC) or β-cells were applied to H2O2-treated β-cells. aPC activ- In addition, exendin-4 or MOTS-c and both of them significantly reduced ER ity was measured by enzymatic assay and ROS species by DihydroEthidium stress markers (e.g., ATF-4 and phosphorylated eIF-2 alpha) and proapoptotic probing. The capture of PKH26-stained MP by β-cells and EPCR expression Bax expression, whereas they increased anti-apoptotic protein Bcl-2 expres- were probed by fluorescence microscopy, apoptosis by flow cytometry. sion. Decreased oxygen consumption rates under ER stress was also sub- aPC treatment enhanced both AnnexinA1 and PAR1 expression in EC and stantially recovered by MOTS-c treatment but further decreased by siRNA to a lesser extent β-cells. MP from aPC-treated EC protected β-cells and targeting MOTS-c. restored insulin secretion whereas MP from β-cells had no effect. After 24 In conclusion, MOTS-c improves survival and insulin secretory function of h, aPC-generated endothelial MP were integrated by 80% of target β-cells pancreatic beta cells under ER stress by alleviating ER stress and apoptosis and induced a 4-fold increase in EPCR expression. These observations were and by improving mitochondrial function. confirmed in rat islets submitted to H2O2, that had increased viability in POSTERS

response to endothelial MP (62% vs. 48% H2O2), reduced apoptosis, and Islet Biology/ preserved insulin secretion by glucose assay (High glucose: insulin 16 vs. Insulin Secretion 5 ng/ml/10 islets H2O2). Endothelial MP delivery to β-cells was concomi- tant with the activation of EPCR/PAR-1 cytoprotective and AnnexinA1/FPR2

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A569 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

2173‑P mice became diabetic (blood glucose ≥ 250mg/dl) by 22 weeks. In contrast, only 20% (2mg/kg) or none (4mg/kg) of the Prl treated mice became diabetic WITHDRAWN by 32 weeks. Shorter treatment (4 weeks) showed reduced insulitis in Prl vs. saline treated mice. To assess therapeutic relevance, recent onset dia- betic (blood glucose ≥ 250mg/dl for 3 days) NOD female mice were treated daily with 4mg/kg Prl or saline, for 4 weeks. One hundred percent of the saline treated mice remained diabetic with average blood glucose reaching >600mg/dl. However, diabetes incidence reversed in 75% of the Prl treated mice, with glucose levels at <250mg/dl (n=10-12 mice/group). The plasma insulin at the end of the treatment was 3-fold higher in the Prl treated mice compared to saline treated controls. Reversal of T1D would suggest that Prl may modulate immunity. We tested this by adoptive transfer of spleno- cytes from prediabetic NOD mice, treated with saline or Prl for 4 weeks, into immunodeficient NOD/SCID mice. The diabetes incidence in NOD/SCID mice which received splenocytes from Prl treated mice was significantly reduced (70%) compared to the mice receiving splenocytes from saline treated con- trols (90%). There was also a delay of 5 weeks to achieve 50% diabetes incidence in Prl vs. saline treated mice (n=25 mice/group). These findings strongly suggest that Prl targets the two critical tissues, the β cell and the immune system, to reverse T1D. Supported By: National Institutes of Health (R01DK072264)

ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND POSTNATAL GROWTH

Moderated Poster Discussion: Islet Function (Posters: 2176-P to 2181-P), 2174‑P see page 19. The Effects of Amyloid Formation on IL-1 Receptor Antagonist Pro‑ duction in Human Islets & 2176‑P QUEENIE HUI, TIMOTHY J. KIEFFER, ZILIANG AO, NOOSHIN SAFIKHAN, ALI Investigating the Impact of the HNF1αG319S Polymorphism on Pan‑ ASADI, MARK MELOCHE, GARTH L. WARNOCK, LUCY MARZBAN, Vancouver, BC, creatic β-Cell Function Canada TIANNA N. FLETT, KRISTIN L. HUNT, MARIO A. FONSECA, CUILAN NIAN, PRA- Growing evidence suggests that islet inflammation plays a key role in SOON AGARWAL, BRANDY A. WICKLOW, ELIZABETH A. SELLERS, VERNON W. pathogenesis of type 2 diabetes (T2D). Recent studies have shown that islet DOLINSKY, FRANCIS C. LYNN, CHRISTINE A. DOUCETTE, Winnipeg, MB, Canada, amyloid, formed by fibrillogenesis of human islet amyloid polypeptide (hIAPP; Vancouver, BC, Canada amylin) promotes interleukin (IL)-1β production and may contribute to islet Manitoba has the highest rate of youth-onset type 2 diabetes (T2D) in inflammation in T2D. Islet levels of IL-1 receptor antagonist (IL-1Ra), a natural Canada, which is amongst the highest in the world. Disproportionately inhibitor of IL-1β, are decreased in T2D, although the mechanism(s) contribut- affected are Indigenous youth with Oji-Cree heritage. Nearly 40% of Oji-Cree ing to reduced IL-1Ra levels in T2D have yet to be identified. In this study, we youth with T2D in Manitoba carry a single nucleotide polymorphism (SNP) in investigated the effects of amyloid formation on islet IL-1Ra production and hepatocyte nuclear factor 1α (HNF1α) where a highly conserved glycine is release during amyloid formation. Isolated human islets (n = 6 donors) were replaced with a serine at position 319 of the protein (“G319S”). Youth with cultured in elevated (11.1 mmol/l) glucose (to potentiate amyloid formation) T2D who harbour this variant have higher HbA1c, lower fasting insulin and for up to 7 days. Islet culture resulted in progressive amyloid formation, as less insulin resistance at diagnosis compared to youth with T2D who carry assessed by quantitative immunolabelling for insulin and oligomers (small the wild type (WT) allele. These observations suggest that T2D in G319S car- aggregates) or thioflavin S (large aggregates). Pre-culture islets had low riers is primarily driven by a β cell defect; however, the mechanistic impact IL-1β levels but amyloid formation markedly increased islet IL-1β production of the G319S variant on β cell function has not yet been directly explored. during 7-day culture. Moreover, in pre-culture islets, IL-1Ra was detected We used CRISPR-Cas9 to knock-in the g>a.955 SNP into MIN6 clonal β cells. mainly in β-cells and to a lesser extent in α-cells. Islet IL-1Ra production and HNF1α protein expression was reduced ~40% in G319S-MIN6 cells com- secretion were progressively reduced during 7-day culture. Interestingly, IL- pared to WT-MIN6 cells. Insulin secretion measured by static incubation in 1Ra levels were higher in oligomer-positive islets than thioflavin S-positive low (2.8mM) and high (16.7mM) glucose showed no impact of the G319S vari- islets, suggesting that IL-1Ra production is reduced during amyloid formation ant on glucose-stimulated insulin secretion (GSIS); however, at low glucose, associated with progressive amyloid-induced β-cell dysfunction and death. insulin secretion was reduced ~5-fold in G319S-MIN6. Exposure of WT- and Amyloid formation did not have any detectable effect on α-cell IL-1Ra levels. G319S-MIN6 cells to palmitate for 24 hrs showed that, unlike WT-MIN6 In summary, these findings show for the first time, that amyloid formation cells where palmitate significantly impaired GSIS, palmitate-treated G319S- disrupts the balance between IL-1β and IL-1Ra in human islets by promoting MIN6 cells maintained significant GSIS. These initial studies suggest that IL-1β production and reducing IL-1Ra production, leading to β-cell dysfunc- the G319S variant may not drive diabetes development via impaired GSIS, tion and death. Targeting imbalance between IL-1β and IL-1Ra may provide but reduces insulin secretion capacity at low (basal) glucose, which could an effective strategy to protect β-cells from amyloid toxicity in T2D. have an important role in controlling endogenous glucose production and Supported By: Canadian Institutes of Health Research triggering hyperglycemia via excessive glucose production. Future studies include ChIP-Seq to examine how the G319S variant affects DNA binding of 2175‑P HNF1α to its target genes in the β cell as well as in vivo rodent studies to Prolactin Prevents and Reverses Recent-Onset Type 1 Diabetes in understand how this gene variant interacts with environmental stressors NOD Mice and contributes to the early onset of T2D in youth. NAGESHA GUTHALU KONDEGOWDA, RAFAEL FENUTRIA, ROSEMARY LI, ROLLIE Supported By: Research Manitoba; Children’s Hospital Research Institute of HAMPTON, RUPANGI VASAVADA, New York, NY Manitoba (OG2014-14); Canadian Institutes of Health Research (ECD-144626) Type 1 diabetes (T1D) is an autoimmune disease which destroys pan- creatic β cells, causing insulin deficiency and hyperglycemia. Prolactin (Prl) enhances β cell function, proliferation, and survival; and has immuno- POSTERS

Islet Biology/ modulatory effects. Therefore, we hypothesized that Prl can improve T1D

Insulin Secretion outcome in NODLtj female mice, an established T1D model. Prediabetic 12-week old NOD female mice were treated daily with saline or Prl (2 or 4mg/kg) for 20 weeks (n=4-7 mice/group). Sixty percent of saline-treated

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A570 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH & 2177‑P that inhibition of glucose metabolism stimulates SOCE leading to enhanced Platelet-Derived Mitochondria Display Embryonic Stem Cell Mark‑ α-cell glucagon secretion. ers and Improve Pancreatic Islet β-Cell Function in Humans Supported By: National Institutes of Health (DK081666, DK097392); Vanderbilt YONG ZHAO, ELIAS DELGADO, ZHAOSHUN JIANG, HENG LI, WEI HU, MARCOS University (P60DK20593 to D.A.J.); Vanderbilt University/National Institutes of PEREZ-BASTERRECHEA, MAO MAO, ZHAOHUI YIN, YE ZHANG, YING LI, JING Health (5T32DK07563) ZHOU, JESUS OTERO, Hackensack, NJ, Oviedo, Spain, Jinan, China, Shijiazhuang, China & 2179‑P Diabetes is a major global health issue. Research to develop a cure must Vascular Endothelial PDK1 Plays Pivotal Roles for Maintenance of overcome multiple immune dysfunctions and the shortage of pancreatic Pancreatic Beta-Cell Function islet β-cells, but these challenges have proven intractable despite inten- ATSUSHI OBATA, TOMOHIKO KIMURA, HIDENORI HIRUKAWA, KENJI KOHARA, sive research over the past decades. In previous clinical trials for type 1 YOSHIYUKI OBATA, SAEKO MORIUCHI, TOMOE KINOSHITA, AKIHITO TANABE, diabetes (T1D), type 2 diabetes (T2D), and other autoimmune diseases we MASASHI SHIMODA, SHINJI KAMEI, SHUHEI NAKANISHI, TOMOATSU MUNE, demonstrated the safety and efficacy of Stem Cell Educator (SCE) therapy KOHEI KAKU, HIDEAKI KANETO, Kurashiki, Japan for re-educating autologous blood mononuclear cells to eliminate autoreac- The phosphoinositide 3-kinase signaling pathway in vascular endothelial tivity. In this study, we found that the percentage of platelets was increased cells is important for systemic angiogenesis and glucose metabolism. We after treatment with SCE therapy. Platelets display immune tolerance-asso- previously reported that vascular endothelial cell-specific PDK1 knockout ciated markers such as the autoimmune regulator (AIRE) and program death mice (VE-PDK1-KO mice) presented the improvement of insulin resistance ligand-1 (PD-L1) that can modulate the function and differentiation of mono- and glucose tolerance fed NC for 6 months or an HFD for 3 months, while cytes/macrophages and other immune cells. Notably, platelets expressed there was no difference in glucose tolerance and insulin sensitivity between embryonic stem (ES) cell- and pancreatic islet β-cell-associated markers VE-PDK1-KO mice and control flox/flox mice fed NC for 3 months (Mol Endo- that are encoded by platelet mitochondrial DNA (mitoDNA). Using freshly crinol. 26, 95-109, 2012). However, we elucidated serum insulin levels ad libi- isolated human pancreatic islets, ex vivo studies established that platelet- tum were significantly decreased in VE-PDK1-KO mice, while blood glucose releasing mitochondria can migrate to pancreatic islets and be taken up by levels ad libitum were comparable. Therefore, we hypothesized that endo- islet β-cells, leading to the proliferation and enhancement of islet β-cell thelial PDK1 might affect pancreatic beta cell function. First, we conducted functions (Figure 1). These findings open up new avenues for the treatment ipGTT at 3 months of age, which resulted in comparable blood glucose levels and prevention of both T1D and T2D in clinics. and significantly reduced insulin levels in VE-PDK1-KO mice. Pancreatic blood flow was significantly decreased in VE-PDK1-KO mice even though there was no difference in blood pressure. We next investigated mRNA expres- sion levels in isolated islets. Intriguingly, mRNA expression levels of ins1, ins2 and their transcription factors such as mafa, pdx-1 and neurod were all significantly decreased in islets of VE-PDK1-KO mice. In addition, mRNA expression levels such as GLP-1R, irs2, glucokinase, glut2 and ccnd1 were all decreased in VE-PDK1-KO mice. The expression levels of antioxidants such as sod2, gpx and catalase were significantly reduced in VE-PDK1-KO mice. In immunohistological analysis, pancreatic beta cell mass and alpha cell mass were both significantly reduced in VE-PDK1-KO mice. Moreover, CD31 positive area in islets was significantly decreased in VE-PDK1-KO mice. Glucose stimulated insulin secretion was significantly impaired in addition to decreased insulin contents in VE-PDK1-KO mice. In conclusion, endothelial PDK1 plays pivotal roles for maintenance of Supported By: American Diabetes Association (1-10-IN-33 to Y.Z.) pancreatic beta cell function.

2178‑P 2180‑P &2+ & Glucose-Dependent Modulation of Store-Operated Ca Entry Con‑ The Swi/Snf Chromatin Remodeling Complex Regulates Mature trols -Cell Glucagon Secretion α β-Cell Function In Vivo MOLLY K. ALTMAN, PRASANNA K. DADI, NICHOLAS C. VIERRA, DAVID A. JASON SPAETH, JIN HUA LIU, MIN GUO, ANIL BHUSHAN, MATTHIAS HEBROK, JACOBSON, Nashville, TN ROLAND W. STEIN, Nashville, TN, Los Angeles, CA, San Francisco, CA In patients with type 2 diabetes glucagon secretion becomes increased Pdx1 is one of the most essential transcription factors (TF) in vertebrates, under elevated glucose conditions exacerbating hyperglycemia. However, as it plays fundamental roles in pancreatogenesis and the development and the mechanisms of glucose inhibition of glucagon secretion remain incom- function of adult islet β-cells. Importantly, inactivation of Pdx1 contributes pletely elucidated. Therefore, we sought to uncover the glucose dependent to diabetic β-cell dysfunction. We are interested in how recruited transcrip- mechanisms that modulate pancreatic α-cell glucagon secretion. We first tional coregulators modulate Pdx1 activity, with our focus here on the Swi/ assessed α-cell glucose uptake with the fluorescent glucose molecule Snf chromatin remodeling complex. Our lab revealed that Swi/Snf was a 2-NBDG. 2-NBDG treatment significantly increasedα -cell fluorescence indi- critical coregulator of Pdx1 controlled gene regulation in β-cell lines. More- cating glucose uptake, which was significantly reduced with 10µ M Dapa- over, Swi/Snf recruitment to Pdx1 increased under high glucose stimulated gliflozin (26.7% ± 6.1%) and 300 μM Phloretin (43.1% ± 1.2%). Thus, glucose conditions in mouse β-cells, and was reduced in the dysfunctional conditions entry into α-cells is mediated via both sodium-glucose transporters as well associated with type 2 diabetes in human β-cells (Mckenna, B. et al, 2015), as glucose transporters. We then determined how glucose metabolism demonstrating the functional interactions between Pdx1 and Swi/Snf are 2+ modulates α-cell Ca homeostasis using the genetically encoded calcium likely dynamic and conserved. indicator GCAMP3 expressed selectively in mouse α-cells. Confocal imag- To evaluate the contribution of Swi/Snf on adult β-cells, we developed a ing of intact whole islets revealed that (46.7% ± 2.3%) of α-cells show an mouse model lacking Swi/Snf activity by removing the two core ATPase sub- 2+ increase in Ca influx following Dapagliflozin inhibition of glucose transport. units of the complex, Brg1 and Brm. This was accomplished by combining the Moreover, in whole islets (26.9% ± 2.4%) of α-cells responded to inhibi- Brm null (Brm-/-) allele with a -cell-specific Brg1 null mutant, with the latter 2+ β tion of metabolism with 2-Deoxyglucose with increased Ca influx. One created by tamoxifen-inducible -cell specific mouse insulin-driven-CreERT 2+ β mechanism that regulates α-cell Ca influx in a glucose dependent man- removal of the floxed Brg1 allele one month after birth. Islet -cell function, 2+ β ner is store-operated Ca entry (SOCE). Therefore, we used an inhibitor of insulin+ numbers, and Pdx1 regulated MafA TF and Glucose transporter 2 SOCE (AnCoA ) to test its role during glucose modulation of -cell Ca2+ entry. 4 α levels were compromised in this adult Swi/Snf β-cell mutant; termed βDKO. In whole islet α-cells inhibition of STIM1/Orai1 interaction with AnCoA4 Strikingly, the -cell enriched TF levels of Pdx1, Mnx1 and Nkx6.1 were 2+ β reduced Ca influx in 1 mM glucose (8.4% ± 1.1%). We went on to test the maintained in islet βDKO cells lacking insulin production, while the normally influence of SOCE on glucose modulation of glucagon secretion, in 1 mM glu- α-cell-specific MafB TF was present in mutant insulin+ β-cells. Interestingly, cose AnCoA4 reduced glucagon secretion in pancreatic islets (53.5% ± 11%). POSTERS

we also found that the newly-identifiedβ -cell dedifferentiation marker, Islet Biology/ However, there was no significant change in glucose inhibition of glucagon aldehyde dehydrogenase 1a3 (Aldh1a3), was produced in a subset of βDKO Insulin Secretion secretion by inhibition of SOCE at 11 mM glucose. Thus, our findings identify insulin+ cells, suggesting the even insulin+Swi/Snf- cells are dysfunctional.

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A571 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

Overall, our studies demonstrate the importance of Swi/Snf in maintaining 2183‑P islet β-cell activity and identity. Analysis of Pathogenic Mechanism by Susceptibility Genes of Supported By: American Diabetes Association (1-16-PDF-109 to J.S.); National T2DM Using Human iPS Cells Institutes of Health (R01DK050203 to R.W.S.) NANAKO SHIMONO, SHUN ICHIRO ASAHARA, YOSHIAKI KIDO, Kobe, Japan Objective: Recently, many susceptibility genes for Japanese type 2 dia- & 2181‑P betes (T2DM), such as Kcnq1 and Eif2ak4, have been identified. However, Nmp4 Is a Novel Regulator of Beta-Cell Mass and Function as they are located in introns, the pathogenic mechanism of T2DM has JOSEPH BIDWELL, RONALD C. WEK, SARAH TERSEY, CARMELLA EVANS- yet to be elucidated well. We have reported that increased expression of MOLINA, Indianapolis, IN p57 in pancreatic islets significantly reduced pancreaticβ cell mass and We have discovered a new regulatory pathway for insulin secretion and induced abnormal glucose tolerance in Kcnq1 mutant mice in whose islets glucose metabolism while investigating cellular secretory machinery dur- the expression of non-coding RNA Kcnq1ot1 was decreased. In addition, we ing bone development. Mice harboring a global loss-of-function mutation have recently clarified that Eif2ak4 knockout mice showed a reduced pan- in the transcription factor Nmp4 are healthy with an unremarkable skeletal creatic β cell mass by a decline in insulin-signaling in pancreatic islets. How- phenotype until challenged with an anabolic stimulus, such as parathyroid ever, these results were obtained from mice. Therefore, to confirm whether hormone, resulting in enhanced bone formation by increasing osteoprogeni- these mechanisms also correspond to human, we aimed to induce the differ- tor number and conversion of the osteoblasts into super-secretors of bone entiation from human iPS cells (hiPSCs) with SNP mutation into pancreatic matrix. Loss of Nmp4 elevates ribosome biogenesis and global mRNA trans- β cells and analyze the effects of each SNP on gene expressions. lation, expanding the processing capacity of the endoplasmic reticulum. Methods: Mutations of Kcnq1 and Eif2ak4 in 11 hiPSC lines including Given the high secretory burden of the pancreatic β-cell, we hypothesized 201B7 were analyzed by sequencing. To induce the differentiation into pan- that β-cell function might be similarly enhanced by Nmp4-/- loss. To test the creatic bud, cells were cultured using reported methods. metabolic phenotype of Nmp4-/- mice, intraperitoneal (IP) glucose tolerance Results: The results of analyzing mutations on 11 hiPSC lines had a Kcnq1 tests were performed. Unexpectedly, 8 wk old Nmp4-/- mice displayed glu- mutation in 8 lines and an Eif2ak4 mutation in 6 lines. From these cell lines, cose intolerance compared to wild type (WT) littermate controls. No differ- we selected 201B7 cell line that had both gene mutations, and then tried ences in fasting glucose levels or peripheral insulin sensitivity, measured by to induce the differentiation into pancreatic β cells. When the cells were an insulin tolerance test, were detected between genotypes. To determine cultured with Activin A and CHIR99021, the expressions of SOX17 and FoxA2 whether Nmp4 influences insulin secretion, we conducted a static glucose- were confirmed by RT-PCR and immunostaining. Moreover, cell clusters stimulated insulin secretion assay on islets isolated from WT and Nmp4-/- were in suspension culture with NOGGIN, KGF and EGF. PDX1 and NKX6.1 mice. Islets from Nmp4-/- mice showed significantly decreased fractional were expressed in the cells, which suggested that hiPSCs differentiated into glucose-stimulated insulin release compared to WT islets. Immunohisto- pancreatic bud. We confirmed that mutations of Kcnq1 and Eif2ak4 didn’t chemical analysis of WT and Nmp4-/- pancreas sections revealed signifi- inhibit the differentiation from hiPSCs into pancreatic bud. cantly smaller islets in the null samples with a significant reduction inβ -cell Conclusion: Human iPS cells with Kcnq1 and Eif2ak4 mutations were mass, consistent with their glucose intolerance. We conclude that Nmp4 is obtained. We confirmed differentiation of these cells into pancreatic bud in required for normal β-cell development and insulin secretion in contrast to which PDX1 and NKX6.1 were expressed. its role as a suppressor of osteogenic cell development and secretion. Supported By: Indiana University School of Medicine 2184‑P Reduced Expression of KPAN2 and Dysfunction of β-Cells in C414A- 2182‑P CRY1 Transgenic Mice SATOSHI OKANO, AKIRA YASUI, SHINICHIRO KANNO, KIYOSHI HAYASAKA, WITHDRAWN MASAHIKO IGARASHI, OSAMU NAKAJIMA, Yamagata, Japan, Sendai, Japan Cryptochrome (CRY) proteins play indispensable roles in the mamma- lian circadian clock. We previously generated transgenic mice ubiquitously expressing mCRY1 with a mutation in cysteine414 (C414A-CRY1). The Tg mice overexpressing C414A-CRY1 showed early onset diabetes mellitus charac- terized by β-cell dysfunction in addition to unusual circadian behaviors. We have already shown that the decrease of insulin secretion from β-cells along with the lowered proliferation of β-cells due to the senescence-like changes in the Tg mice. KPAN2 (Importin α2) is known to be involved in nuclear import of various proteins having a nuclear localization signal. It has been reported that KPAN2 is required for the proper cellular localization of Period proteins that are core clock proteins. In this study, in order to clarify the relation between the β-cell dysfunction and malfunction of circadian clock in the islet, we analyzed KPAN2 in the Tg mice. We found that KPAN2 is highly expressed in the nucleus of pancreatic islet cells in mice by immunohisto- chemical analyses. In wild type mice, the KPAN2 expression was observed in both α and β-cells. In the Tg mice, the expression level of KPAN2 in the islet was markedly reduced at the mature stage (40 weeks of age). These results suggest that KPAN2 plays some important roles in the function and/or the maintenance of pancreatic islet cells, and that the reduction of KPAN2 may be involved in the dysfunction of the β-cell in the Tg mice. Supported By: Japan Society for the Promotion of Science (15K08417); IDAC; Tohoku University

2185‑P Co-culture of Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells Promotes the Proliferation and Differentiation of Human Pancreatic Progenitor Cells into Islet-Like Cell Clusters XING YU LI, SHANG YING WU, PO SING LEUNG, Hong Kong, China The application of clinical islet transplantation is hindered by the inherent lack of human donors; however, directed differentiation of human pancreatic progenitor cells (PPCs) into islet-like cell clusters (ICCs) opens an avenue to POSTERS

Islet Biology/ providing transplantable islets for diabetic patients. Recent reports showed

Insulin Secretion that rat mesenchyme acts on the upstream and downstream of Ngn3 to direct the lineage of pancreatic β-cell fate, while co-transplantation of mouse mesenchymal stem cells (MSCs) enhances islet revascularization

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A572 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH and function. Besides, co-culture with MSCs promotes the proliferation and T1D islets (11±1%), while α cells made up a higher percentage (57%) of endo- differentiation of human cardiac stem cells. However, the effects of MSCs crine cells than in normal islets (39±3%) but lower than found in T1D islets co-culture on the development of human PPCs into ICCs and its functionality (77±6%). On pancreatic analysis, all islets contained β cells and did not meet are largely ambiguous. the consensus definition for insulitis (infiltration of CD45+ cells). By islet peri- We thus co-cultured our two established human cell models of fetal bone fusion, the donor’s β cells responded to cAMP-evoked stimulation but had marrow-derived MSCs and fetal pancreas-derived PPCs/ICCs to examine the impaired insulin secretion in response to glucose and KCl-mediated depolar- effects on the development of the PPCs into ICCs. ization when compared to nondiabetic and T1D islets (relative to islet insulin Results showed that co-culture of MSCs increased the expression of content). When expressed relative to islet glucagon content, α cell function PPC differentiation markers, such as Pdx1, Insulin and Nkx2.2; the resultant was also significantly impaired with loss of response to KCl-mediated depo- ICCs exhibited higher insulin content compared with the cocktail control. larization. Because of these unexpected findings, we sequenced the donor In addition, MSC conditioned medium (CM) stimulated PPCs proliferation DNA for variants associated with monogenic diabetes, revealing a hetero- while reducing cell death. Apoptosis was detected in serum-free condition, zygous mutation in hepatocyte nuclear factor 1 alpha (HNF1A; c.779C>T, which was further confirmed by arrested cell growth in G0/G1 phase and Thr260Met), which is predicted to be pathogenic (previously described in these effects were blocked by the MSC-CM. Moreover, Bcl-2 was upregu- a multigenerational family). These first direct studies of the pancreas and lated whereas BAX was downregulated by the MSC-CM compared with the islets with a HNF1A mutation suggest that the dysfunction in both islet α starvation condition. and β cells contributes to hyperglycemia. Our data indicate that MSCs can promote the proliferation and differen- Supported By: National Institutes of Health; JDRF; U.S. Department of Veterans tiation of human PPCs into ICCs, thus offering an insight into the co-culture Affairs approach to generating functional β-cells for diabetic patients. To this end, we are undertaking ICCs transplantation and microarray to further assess 2188‑P the in vivo functionality and molecular signaling involved. Augmentation of β-Cell Neogenesis and Reversal of Hyperglyce‑ Supported By: Hong Kong Health and Medical Research Fund of Food and mia in Diabetic NOD Mice upon Induction of Mixed Chimerism and Health Bureau (12130611) Administration of Growth Factors MINGFENG ZHANG, QING LIN, YUQING LIU, MICHELLE WON, PERE SANTAMA- 2186‑P RIA, ARTHUR D. RIGGS, DEFU ZENG, Duarte, CA, Calgary, AB, Canada The Modulatory Action of SIRT1 in the Determination of Pancreatic Induction of mixed chimerism has been shown to promote organ transplan- Islet Cell Development tation tolerance in humans (Strober et al, Am J Transplant 2016). We have SHANG YING WU, XING YU LI, PO SING LEUNG, Hong Kong, China reported that induction of mixed chimerism with MHC-mismatched donors Sirtuin1 (SIRT1) is known as a metabolic regulator to affect islet function can reverse autoimmune diseases in murine models of type 1 diabetes (T1D), in adult pancreas; recently, its potential roles in regulating the differentia- systemic lupus, and multiple sclerosis. Here, we show that conditioning tion of various stem cells have been garnered attentions. Whether SIRT1 is with clinically available reagents cyclophosphamide, pentostatin and anti- involved in islet cell development, however, remains to be determined. thymocyte globulin allows the successful induction of MHC-mismatched To address this issue, we sought to characterize the expression profile and mixed chimerism in established diabetic NOD mice. Although induction of examine the effects of SIRT1 in human pancreatic progenitor cells (PPCs)/ mixed chimerism alone or administration of gastrin and epidermal growth islet-like cell clusters (ICCs) system and murine pancreatic rudiment culture factor (EGF) alone could not reverse hyperglycemia in the established dia- system in the present study. betic NOD mice, combination of mixed chimerism plus administration of gas- The expression levels of SIRT1 were significantly increased during the dif- trin and epidermal growth factor restored normoglycemia in 56% (15 of 27) ferentiation of human PPCs into ICCs, while the protein expression of SIRT1 of the diabetic mice. This was associated with increased β-cell neogenesis was observed to be translocated from the nucleus of PPCs to the cytoplasm and replication. Lineage tracing studies showed that approximately 60% of of ICCs. The mRNA expression level of SIRT1 appeared to be opposite in new β-cells arose by neogenesis, with 20-25% came from glucagon+ α cells. the second transition period during the mouse pancreas development. SIRT1 These results indicate that α cells and other progenitors can give rise to inhibitor (EX 527) and lentivirus-mediated knockdown of SIRT1 did not sig- insulin-producing β-cells in diabetic NOD mice after induction of mixed chi- nificantly affect the expression of critical transcriptional markers and insulin merism and growth factor therapy. content of ICCs. However, SIRT1 activator (SRT 1720) and lentivirus-medi- Supported By: Beckman Research Institute of City of Hope ated SIRT1 overexpression significantly enhanced the differentiation and functionality of ICCs. In addition, SRT 1720 not EX 527 exhibited significant 2189‑P increase in the mRNA expression of transcriptional markers in cultured pan- Dynamic Molecular Changes of Porcine Neonatal Pancreatic Cell creatic rudiments. Cluster in Culture and after Transplantation Together, these data provide evidence that SIRT1 is involved in modulat- JYUHN HUARNG JUANG, CHEN YI CHEN, PEI CHUN HUANG, TZU YU KUO, YI TA ing pancreatic islet cell development, and that SIRT1 activation might be an HSIEH, HAO YUAN CHIA, WAN CHUN LI, Taoyuan, Taiwan, Taipei, Taiwan alternative to producing insulin-expressing cells for clinical islet transplanta- Transplantation (Tx) of isolated porcine neonatal pancreatic cell clusters tion in diabetic patients. (NPCCs) in diabetic nude mice needs more than 8 weeks to achieve normo- Supported By: Hong Kong Health and Medical Research Fund of Food and glycemia while ductal epithelium is primarily detected in NPCCs grafts which Health Bureau (12130611) suggests that initial grafts might exhibit progenitor-like phenotype. We used RT-PCR and immunofluorescence staining analysis to further clarify the cel- 2187‑P lular and molecular identity of NPCCs before and after Tx. Soon after NPCCs Analysis of Pancreas and Islets from 33-Year-Old with 16 Years of isolation, pancreatic progenitors, Pdx1+/Insulin- (78.7±1.7% after isolation Type 1 Diabetes (T1D) Reveals MODY3/HNF1A Diabetes vs. 51.9±3.1% in 1-day-old pig pancreas) and Sox9+ (67.1±3.5% after isola- RACHANA HALIYUR, MAY SANYOURA, SAMBRA D. REDICK, SHRISTI SHRES- tion vs. 6.8±0.7% in 1-day-old pig pancreas) dramatically increased. During THA, NRIPESH PRASAD, SHAWN LEVY, RADHIKA ARAMANDLA, GREG POF- 4-day NPCCs culture, insulin- and glucagon-positive endocrine cells gradu- FENBERGER, DIANE C. SAUNDERS, RITA BOTTINO, DAVID M. HARLAN, LOUIS ally increased while amylase- and carboxypeptidase B-positive exocrine H. PHILIPSON, ROLAND W. STEIN, MARCELA BRISSOVA, ALVIN C. POWERS, cells gradually decreased. Moreover, dual-hormonal progenitor-like cells Nashville, TN, Chicago, IL, Boston, MA, Huntsville, AL, Pittsburgh, PA including insulin+/glucagon+, insulin+/somatostatin+ and insulin+/pancreatic Recent observations have highlighted the heterogeneity in the T1D pan- polypeptide+ were observed. Upon NPCCs Tx into nondiabetic nude mice, creas. We describe characteristics of pancreatic tissue and isolated islets both Pdx1+ (50.6, 57.8, 61.5 and 24.0% in 16-, 30-, 60- and 90-day grafts, from a 33-year-old male diagnosed with T1D from age 16 compared with respectively) and Sox9+ (49.7, 9.2, 7.2 and 2.1% in 16-, 30-, 60- and 90-day nondiabetic (n=4; 10-55y of age) and other T1D (n=3; 12-22y of age, 3-7y T1D grafts, respectively) progenitors decreased whereas insulin+ cells increased duration) donors. The donor (BMI 25.8 kg/m2, A1c 8.9%, HLA haplotypes (4.5, 6.9, 9.0 and 9.3% in 16-,30-,60- and 90-day grafts, respectively), in DR4, DQ8) was treated with insulin and had a history of hypertension and accompany with neogenic insulin+ cells budding out from Pdx1+/Sox9+ pro- familial diabetes. His plasma C-peptide was 0.4 ng/mL, slightly greater genitors in NPCCs grafts. Besides, the insulin+ cells in 23-day grafts from POSTERS

than other T1D donors (0.02-0.26 ng/mL; n=3), but all T1D-associated auto- diabetic recipient mice (9.2±1.1%) were more than those from nondiabetic Islet Biology/ antibodies (GAD65, mIAA, ZnT8, ICA512) were negative. FACS analysis of counterparts (6.6±0.5%). Insulin Secretion isolated islets revealed β cells made up 35% of the endocrine population, In summary, our results demonstrated that islet precursors could be acti- which was less compared to nondiabetic islets (53±3%) but greater than in vated during NPCC isolation while NPCCs progenitors maintained their plu-

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A573 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

ripotency in culture. In addition, active NPCCs differentiation occurred after 2192‑P Tx and hyperglycemic environment promoted NPCCs maturation. GLP-1 Induces Neurog3 Re-expression and Beta-Cell Neogenesis Supported By: Taiwan Ministry of Science and Technology (102-2314-B-182A- in Adult Mice 012-MY3) NIDHEESH DADHEECH, YING W. WAYNE, JEAN BUTEAU, Edmonton, AB, Canada Glucagon-Like Peptide-1 potentiates glucose-induced insulin secretion 2190‑P and acts as a growth factor via the promotion of both beta-cell apoptosis Role of BMP9/SMAD1 Signaling during Neonatal Rat Islets Matura‑ and proliferation. However, its potential action on beta-cell neogenesis tion: Regulation of SNAREs Proteins remains elusive. We herein performed lineage tracing experiments in geneti- PRISCILLA M.R. SILVA, JUNIA CAROLINA R. SANTOS-SILVA, JOSEANE MORARI, cally engineered mice to test the hypothesis that GLP-1 induces Neurog3 FERNANDO F. ANHE, GABRIEL F. ANHE, SILVANA BORDIN, Campinas, Brazil, Quebec re-expression in the adult pancreas to stimulate new mature beta-cells for- City, QC, Canada, São Paulo, Brazil mation. Postnatal pancreatic islets continue developing until full function achieve- We used mice expressing a tamoxifen-inducible recombinase under Neu- ment but the mechanisms are not completely understood. Bone Morphoge- rog3 promoter to remove a floxed-stop codon preventing expression of a netic Proteins (BMPs) participate in the body development by activating ZsGreen reporter and permanently label differentiating beta-cells. Neurog3- intracellular proteins called Mothers Against Decapentaplegic Homolog CreER/Rosa26-LSL-ZsGreen mice were challenged with a moderate dose of (SMADs). In a previous work, we have found that SMAD1 is upregulated streptozotocin to induce low levels of beta-cell death and then injected daily during β cell maturation. Moreover, BMP9 treatment increases glucose- with tamoxifen alone or in combination with the long-acting GLP-1 analog stimulated insulin secretion. The aim of study was to identify target genes Exendin4. Pancreata were harvested after 2 or 7 days of treatment. of BMP9-SMAD1 pathway on β cell during the maturation process. Pancre- Control pancreata were devoid of ZsGreen staining, consistent with the atic islets from neonatal rats were isolated by collagenase digestion and fact that beta-cell differentiation does not occur spontaneously in adult cultured during 1 or 3 days with or without recombinant BMP9 (10 ng/mL). mice. Animals treated with Ex-4, showed high frequency of ZsGreen+ cells INS1E cells were transfected with si-RNA targeting SMAD1 (ko-SMAD1). throughout the pancreas, indicating that Ex-4 induces Neurog3 expression in Pancreatic islets and cells were used for expression analysis (PCR array, the adult pancreas. After 2 days, we detected clusters of ZsGreen+ cells that qPCR and Western blot). PCR array from ko-SMAD1 INS1E cells highlighted were polyhormonal, a feature of differentiating endocrine progenitors. Mice the modulation of genes related with granule extrusion process (SNARES treated with Ex-4 for 7 days displayed a high number of insulin/ZsGreen dual proteins; Soluble n-Ethylmaleimide-Sensitive Factor Attachment Protein positive cells, demonstrating that GLP-1 signaling stimulates beta-cell dif- Receptors). Ko-SMAD1 decreased Synaptosomal-Associated Protein 23 ferentiation in adult mice. Interestingly, glucagon cells were negative for and 25 (SNAP23 and 25), decreased Syntaxin 4 (STX4), and increased Syn- ZsGreen after 7 days. We characterized the lineage of ZsGreen+ cells by taxin Binding Protein 1 and 2 (Stxbp1 and 2). Increased protein expression of studying the expression of various progenitor markers. Finally, FACS-sorted Stxbp1 was confirmed by Western blot. Pancreatic islets cultured during 3 ZsGreen+ cells showed important changes in gene expression. days vs. 1 day showed upregulation of SNAP23 and 25 and downregulation Our study shows that GLP-1 stimulates beta-cell neogenesis via a mecha- of Stxbp1 and 2. The presence of BMP9 increased SMAD1 phosphorylation nism that recapitulates embryonic development, thereby unveiling a new and STX4 expression. mode of action for the incretin hormone. In conclusion, we suggest that the modulation of SNARE proteins in post- natal islets induced by BMP9/SMAD1 signaling pathway is likely to contrib- 2193‑P ute to the neonatal pancreatic β cell maturation. BMP6 Stimulates Proliferation of Pancreatic Islet Alpha Cells Supported By: São Paulo Research Foundation; National Council for Scientific LU ZHANG, JONATHAN HALDMAN, JASON GIBSON, LARRY G. MOSS, LISA NOR- and Technological Development QUAY, MATTHEW RANKIN, SEUNGHUN P. LEE, ALESSANDRO POCAI, CHRISTO- PHER B. NEWGARD, SIMON GREGORY, HANS-EWALD HOHMEIER, Durham, 2191‑P NC, Spring House, PA Association between Serum Uric Acid and Bone Mineral Density in Islet β-cell mass expands in response to obesity and pregnancy, and α-cell Type 2 Diabetic Patients mass increases in response to administration of glucagon receptor antago- XU MINGXIN, Shanghai, China nists and other perturbations. Important insights into factors relevant to Introduction: Accumulating evidence has demonstrated that serum uric these responses could come from our recent observations that overexpres- acid (UA), a naturally powerful antioxidant, plays a beneficial role in bone sion of the homeodomain transcription factor Pdx-1 in β-cells stimulates pro- health among general population. But few reports are available about the duction of soluble factors that activate proliferation in islet α- and β-cells. association between serum UA and bone health in patients with type 2 Importantly, overexpression of Pdx-1 in islets from young (8-10 week-old) diabetes mellitus (T2DM). We therefore investigated whether the boon of rats and their co-culture with human islets leads to consistent and robust serum UA for bone health still existed in T2DM patients. stimulation of human α-and β-cell replication. To identify factors mediating Methods: This is a cross-sectional study, including 671 males (older than these responses, we performed RNA-seq analysis on sorted β-cells with and 30 years) and 652 postmenopausal females with T2DM. Serum UA concen- without Pdx-1 overexpression. We found that BMP6, a member of the TGF-β trations and bone mineral density (BMD) measured at lumbar spine (LS), super family, plays an important role in islet α-cell proliferation. Overexpres- femoral neck (FN) and total hip (TH) by dual-energy X-ray absorptiometry sion of Pdx-1 in FACS-sorted rat β-cells caused a 16-fold increase in BMP6 (DXA) were obtained from all subjects. Meanwhile, data on osteoporosis mRNA levels as measured by RT-PCR analysis. Overexpression of BMP6 in prevalence, glucose metabolism, bone turnover markers and other serum rat islets caused a 2.2-fold increase in α-cell proliferation, similar to the 2.0- biochemical indexes were collected. fold increase observed in response to Pdx-1 overexpression or treatment of Results: Positive associations between serum UA concentrations and islets with recombinant BMP6 peptide. The BMP6 pathway inhibitor, DMH1, BMD were found only in overweight T2DM patients (body mass index [BMI] inhibited human α-cell proliferation specifically and completely in the rat- ≥25 kg/m2) after adjustment for potential confounders. The beneficial skel- human islet co-culture setting. Collectively, these studies suggest a novel etal sites were different by genders (males at LS and females at FN). In post- function of BMP6 in islet cell biology, and provide new insight into the poten- menopausal patients, higher tertile of serum UA was associated with sig- tial relevance of α-β cell cross-talk pathways for control of islet cell mass. nificantly lower odds ratios (OR) than those in the lowest tertile (BMI<25 kg/ Supported By: American Diabetes Association (1-16-PDF-136 to L.Z.); Janssen m2: adjusted OR 0.408, 95% confidence interval [CI] 0.175-0.951; BMI≥ 25 Research & Development, LLC kg/m2: adjusted OR 0.238, 95% CI 0.079-0.723). Besides, serum UA levels were inversely correlated with bone turnover markers OC and CTX in males 2194‑P (both p<0.001) while were only negatively related to CTX in postmenopausal The Effects of In Utero High-Fat Diet Exposure on the Endocrine females (p=0.026). Pancreas of the Offspring Conclusions: There is a positive association between serum UA and BMD JOSEPH M. ELSAKR, RAYMOND C. PASEK, DIANA TAKAHASHI, KEVIN L. GROVE, in Chinese overweight T2DM patients. This implies that UA may be a protec- ALVIN C. POWERS, MAUREEN A. GANNON, Nashville, TN, Portland, OR tive factor for bone in these patients. Large intervention studies are needed Recent evidence suggests that nearly one fourth of women in the U.S.

POSTERS to further confirm the outcomes and provide possible explanations. Islet Biology/ are obese at the time of pregnancy. In both humans and animal models,

Insulin Secretion Supported By: Shanghai Municipal Natural Science Foundation (13ZR1432100); maternal obesity leads to a predisposition for obesity, insulin resistance, National High Technology Research and Development Program (2013AA032203); and type 2 diabetes in the offspring. In multiple animal models, offspring National Natural Science Foundation of China (81500650) of high fat diet (HFD)-fed mothers have defects in the mass, function, and

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A574 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH transcriptional profile of their pancreaticα and β cells. Previous work from cose levels (<10mM). 14+ days after STZ injection, combined therapy mice members of our group using a non-human primate (NHP) model has shown showed significantly lower blood glucose levels compared with monothera- that fetuses of HFD-fed mothers have reduced α cell mass, increased β:α pies. Both monotherapies performed as expected, significantly improving cell ratio, and decreased β cell insulin content and expression of Glut2 (the glucose tolerance and insulin sensitivity. Combined therapy was even more key β cell glucose transporter). Additionally, 1-year-old offspring of HFD- effective, as determined by AUC values. Using the two treatments in com- fed mothers maintained on HFD post-weaning (HFD/HFD) have a decrease bination also significantly increased serum insulin and GLP-1 levels while in both β and α cell number, increased β:α cell ratio, and a trend toward decreasing serum glucagon, to a greater extent than monotherapies. Ki67+ decreased Glut2 expression relative to offspring exposed to a control diet and Tunel+ β-cell results were also promising, with both individual therapies (CTR) in utero and weaned onto HFD (CTR/HFD). At 1-year of age, CTR/HFD and combined therapy increasing Ki67+ numbers and decreasing Tunel+ num- animals had elevated β and α cell number relative to CTR/CTR offspring, but bers. Out of the three, combined therapy had the most effective results. Fur- normal β:α cell ratio. The goals of the current study are to characterize the thermore, metabolic studies showed that all therapies significantly reduced longer term effects of in utero HFD exposure and diet switching by analyzing water consumption, with combined therapy again proving superior. β and α cell number and proliferation, β cell ultrastructure, and islet gene In conclusion, GABA/GLP-1 monotherapies delay the onset of MDSD in expression in 3-year-old NHP offspring. At this time point, animals exposed mice, but combined therapy was the most therapeutically effective, presum- in utero to HFD and weaned onto CTR diet (HFD/CTR) did not show changes ably due to enhanced proliferation and decreased apoptosis of β-cells. in β and α cell proliferation or β:α ratio. However, HFD/HFD offspring have Supported By: JDRF; Canadian Diabetes Association a persistently elevated β:α cell ratio; CTR/HFD had normal β:α cell ratio. We conclude that while in utero exposure to HFD alone (HFD/CTR) results in 2197‑P no observable islet phenotype in this model, the negative consequences of The Role of Notch In Human Adult Pancreatic Colony-Forming Pro‑ maternal overnutrition are unmasked when the offspring are weaned onto genitor-Like Cells a HFD (HFD/HFD). These results suggest that in utero exposure to HFD may JANINE C. QUIJANO, LENA WEDEKEN, ISMAIL AL-ABDULLAH, FOUAD KANDEEL, impair to normal islet response to HFD feeding. H. TERESA KU, Duarte, CA Supported By: National Institutes of Health; National Institute of Diabetes and Symptoms of type 1 diabetes are caused by the lack of functional beta Digestive and Kidney Diseases cells that secrete insulin. One potential therapy for type 1 diabetic patients is to implant functional beta-like cells that secrete insulin on demand. We 2195‑P have shown that adult murine pancreatic progenitor-like cells, located in the Glucokinase Overexpression Enhances Beta-Cell Proliferation or exocrine/duct compartment, expand and differentiate into mono-hormonal Beta-Cell Function Depending on Age and Diet endocrine cells in vitro. Our in vitro colony assay allows us to quantify BRIAN LU, KIRAN KURMI, MIGUEL J. MUNOZ-GOMEZ, EGON J. JACOBUS progenitor-like cells and directly analyze their differentiation potential by AMBULUDI, JASON M. TONNE, TARO HITOSUGI, ALEKSEY V. MATVEYENKO, qRT-PCR analysis. In this study, human exocrine tissues remaining from islet YOGISH C. KUDVA, YASUHIRO IKEDA, Rochester, MN isolation were dissociated into single cell suspension and plated into a semi- Type 2 diabetes mellitus (T2DM) is characterized by a combination of solid medium containing methylcellulose, extracellular matrix components, insulin deficiency, beta-cell dysfunction, and beta-cell death. As beta-cells Serum Replacement and various growth factors. We found that the adult largely regenerate from existing beta-cells, enhanced beta-cell prolifera- human pancreas contains colony-forming progenitor cells, which were able tion should increase the beta-cell mass lost in T2DM and improve glycemic to give rise to cystic colonies containing ductal-, acinar- and endocrine-like control. We sought to improve beta-cell proliferation and function by beta- cells. Further characterization of growth factor requirement revealed that cell-restricted glucokinase (GCK) gene delivery. Promoter-restricted adeno- activation of the Notch pathway increases the self-renewing capabilities of associated virus 8 expressing GCK under the mouse insulin 2 promoter was progenitor-like cells. Alternatively, inhibition of the Notch pathway directs delivered into young and aged C57BL/6j mice maintained on normal or high the differentiation of the progenitor-like cells into the endocrine lineages. fat diet (HFD) via a single intraperitoneal injection. Two weeks following This is an unique, non-genetic approach to differentiate adult human pro- injection, we observed a marked increase in beta-cell proliferation com- genitor-like cells in vitro. Our pancreatic colony assay will be valuable for pared to control in young mice fed normal diet (2 fold) without improvements future mechanistic studies to generate large number of beta-like cells for to glucose tolerance tests (GTT). Aged mice fed normal or HFD did not show transplantation from adult human pancreas and paves the way for personal- increased beta-cell proliferation but had improved GTT (p<0.02 by 90 min- ized therapy for type 1 diabetes. utes) with improved insulin stimulation indices in mice fed HFD (control, 1.03; Supported By: JDRF GCK, 1.40). To determine the mechanism of GCK-mediated beta-cell prolif- eration, we overexpressed GCK in the Min6 mouse beta-cell line. Min6 cells 2198‑P overexpressing GCK showed increased proliferation that is dependent on Pancreatic Islet Development Is Affected by Postnatal Antibiotic potassium and calcium channel depolarization. Glucose uptake and glucose Exposure in Pigs utilization were also enhanced in treated Min6 cells, suggesting that GCK KAIYUAN YANG, JANELLE M. FOUHSE, CATHERINE B. CHAN, BENJAMIN P. mediates beta-cell proliferation through the glucose-regulated beta-cell WILLING, Edmonton, AB, Canada proliferation pathway. These data suggest that, following GCK overexpres- Infants exposed to antibiotics during the first months of life are at greater sion, an unidentified pathway controls the switch between beta-cell prolif- risk of childhood obesity and metabolic disorders. Using a piglet model we eration and beta-cell function, likely in an age- and diet-dependent manner. have previously correlated altered microbial acetate production, in response Further study may reveal distinct GCK signaling pathways controlling beta- to antibiotic treatment, with reduced glucose tolerance later in life. Because cell proliferation or functionality. pancreatic islets are crucial in the maintenance of whole body glucose homeostasis, we examined the effects of early-life antibiotics and acetate 2196‑P on pancreatic islets. We hypothesized that early-life antibiotic treatment Combined Therapy of GABA with GLP-1 Prevents the Onset of Dia‑ alters pancreatic islet development, which contributes to metabolic disor- betes by Promoting β-Cell Regeneration in Mice ders later in life. Neonatal piglets received amoxicillin (ANTI, 30mg/kg/day) WENJUAN LIU, HARRY LAU, YINGHUI ZHOU, GERALD PRUDÈHOMME, TIANRU or placebo (CON) (N=6) from post-natal day 0-14. They were assessed at JIN, QINGHUA WANG, Toronto, ON, Canada the end of treatment for islet function (by ex vivo glucose-stimulated insu- Preclinical studies showed both γ-aminobutyric acid (GABA) and gluca- lin secretion (GSIS) from isolated islets) and relevant gene expression (by gon-like peptide-1 (GLP-1) exert β-cell regeneration in diabetic models. To qPCR), and the effect of acetate (10mM) on GSIS. At day 14, pancreatic islets investigate whether combined therapy exerts superior therapeutic effects isolated from ANTI pigs had decreased gene expression in glucose trans- in diabetic animals, we used a multiple low-dose STZ-induced β-cell injury porter 2 (GLUT2), pancreatic and duodenal homeobox 1 (PDX-1), insulin-like model (MDSD) to test our hypothesis. Sitagliptin, a dipeptidyl peptidase-4 growth factor 2 (IGF2), and G protein-coupled receptor 43 (GPR43) compared inhibitor, was used to enhance endogenous GLP-1 action. GABA was admin- to CON pigs, but no differences in INS and GPR41 was observed between the istered alone or with sitagliptin in drinking water. Male C57BL/6J mice were two groups. In vitro acetate treatment inhibited GSIS in islets from ANTI but randomly assigned into 4 groups: non-treated diabetic control, GABA, sita- not CON pigs, although islets from ANTI and CON pigs had similar GSIS and POSTERS

gliptin, or GABA + sitagliptin. Interventions were initiated 1 week before STZ total insulin content. In the present study, we found that early-life antibiotic Islet Biology/ injections and maintained for 7 weeks. 10 days after the first STZ injection, exposure leads to altered expression of genes that are important in islet Insulin Secretion untreated mice developed hyperglycemia (13.9±0.8mM). However, mice on development and function. Acetate affected insulin secretion from islets, either monotherapy or combined therapy had significantly lower blood glu- but only from antibiotic-treated pigs, which had lower expression of GPR43,

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A575 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM

a receptor for short-chain fatty acids. These results suggest a role of gut performed at 25 weeks. βPKD1KO mice under chow diet presented no signifi- microbial metabolites in mediating islet development/function, which will cant difference in glucose tolerance or insulin secretion during OGTTs com- be further explored. pared to controls. Under high-fat diet, βPKD1KO mice were hyperglycemic Supported By: Canadian Institutes of Health Research earlier and had more pronounced glucose intolerance. This was associated with decreased GSIS and C-peptide levels during hyperglycemic clamps. We 2199‑P conclude that beta-cell PKD1 contributes to the compensatory increase in DNA Damage Does Not Cause BrdU Incorporation in Human or insulin secretion in response to metabolic stress. Whether defective PKD1 Mouse Beta Cells function contributes to the pathogenesis of beta-cell failure in type 2 diabe- ROHIT B. SHARMA, YAHUI KONG, RACHEL E. STAMATERIS, BRIAN GABLASKI, tes remains to be determined. LAURA C. ALONSO, Worcester, MA Supported By: Canadian Institutes of Health Research Quantifying beta cell proliferation is important for beta cell mass stud- ies. Incorporation of bromodeoxyuridine (BrdU) into DNA during S-phase is & 2201‑P considered a gold-standard technique for measuring cell cycle entry in vivo Development of Novel Biologic and Nanotechnology-Based Thera‑ and in vitro in other fields. However, in the islet field, the use of BrdU to mea- peutic Agents to Enhance Insulin Secretion and Protect β-Cell sure proliferation has become controversial, due to a perception that human Function in Diabetes beta cells don’t proliferate and since entry into S-phase does not guaran- DONALD E. LEE, ANNA MUNDER, MICHELLE SHIH HUI LEE, JEAN PAUL LEL- tee productive mitosis. Onto this backdrop a belief has emerged, supported LOUCHE, DEREK VALLEJO, LIRON ISRAEL, ABRAHAM LEE, ARIE GRUZMAN, by limited data, that beta cell BrdU labeling may represent DNA damage STEVEN D. CHESSLER, Irvine, CA, Ramat Gan, Israel repair rather than S-phase. The goal of this study was to test whether DNA Despite an ever-increasing understanding of β-cell function, there are damage results in BrdU labeling in primary mouse and human beta cells. only two classes of approved drugs targeting the β cell: sulfonylureas (and Staining for insulin and gH2AX, a DNA damage marker, showed that DNA meglitinides) and GLP-1-related agents. Drugs engaging novel pathways to damage is present at low frequency (1.4±0.1%, mouse; 0.7±0.04%, human) enhance β-cell function and/or prevent loss could have a major impact on in beta cells under control conditions. DNA damaging agents mitomycin C the treatment of types 1 and 2 diabetes. Neuroligins (NLs) and neurexins or UV light increased gH2AX-positive beta cells but strongly decreased the (Nxns) are transmembrane proteins originally identified in the brain. They proportion of BrdU-positive beta cells under glucose-stimulated conditions, are expressed on the surface of neuronal processes and engage in trans- from 10.8±1.2% to 0.1±0.1% (mouse) and from 0.7±0.1% to 0.0% (human). cellular binding interactions essential for synapse formation and function. On the other hand, after overexpression of cyclin D2 33±5% of beta cells Recently, NL-2 and Nxn were found to be present on the β cell surface and, were BrdU-positive, most which were also gH2AX-positive, suggesting beta through similar interactions, to increase insulin secretion and content. The cells forced to enter S-phase by overriding normal controls incur DNA dam- cytoplasmic domain of Nxn binds constituents of the secretory machinery; age. Under more physiologic conditions, of the 10.8% of beta cells that were clustering of Nxn by NL drives assembly of this machinery. NL-Nxn interac- BrdU-positive, 9% co-stained for gH2AX (1% of all cells), whereas 2% of tions underlie, in part, the enhancement of function caused by β cell-to-β BrdU-negative beta cells co-stained for gH2AX. Prolonged (72 h) BrdU expo- cell contact. To test the utility of the NL-2-Nxn interaction as a therapeutic sure increased the gH2AX positive fraction. Thus, S-phase entry, or BrdU target, we designed an NL-2 peptide based on the Nxn binding site and also incorporation itself, induces gH2AX in some cells. We observed many BrdU- produced a recombinant protein incorporating the entire NL-2 extracellular positive doublets, and are working to quantify the frequency of productive domain. These molecules were separately clustered by cross-linking and by cell division following BrdU incorporation. We conclude that actively cycling attachment to nanoparticles and artificial lipid vesicles. Dose-response and beta cells are more likely to stain for gH2AX, but that DNA damage does not time course studies using INS-1E, INS-1 832/13 and mouse islets showed result in BrdU labeling of primary mouse and human beta cells. that these reagents can enhance glucose-stimulated insulin secretion over Supported By: National Institutes of Health (R01DK095140) two-fold while increasing insulin content and resistance to oxidative stress. Interestingly, the candidate agents also increased INS-1 cell proliferation and expression of maturational markers such as PDX-1. ISLET BIOLOGY—BETA CELL— In summary, we have developed candidate therapeutic agents based on a STIMULUS-SECRETION COUPLING AND novel mechanism—the neuroligin-neurexin interaction—and demonstrated METABOLISM the potential utility of this mechanism and these reagents for the develop- ment of new therapies for diabetes. Supported By: National Institutes of Health Moderated Poster Discussion: Insulin Secretion and Signaling (Posters: 2200-P to 2205-P), see page 21. & 2202‑P Using Uniform Reaggregated Pancreatic Islets in a Microfluidic & 2200‑P Perifusion System Enables Studying Insulin Release Dynamics at Protein Kinase D1 in Pancreatic Beta Cells Is Implicated in the Com‑ Single-Islet Level pensatory Increase in Insulin Secretion under High-Fat Feeding in BURCAK YESILDAG, PATRICK M. MISUN, FELIX FORSCHLER, APARNA NEELA­ Mice KANDHAN, ADELINN BIERNATH, ANDREAS HIERLEMANN, OLIVIER FREY, VALERIE BERGERON, JULIEN GHISLAIN, KEVIN VIVOT, NATALIA TAMARINA, Schlieren, Switzerland, Basel, Switzerland LOUIS H. PHILIPSON, JENS FIELITZ, VINCENT POITOUT, Montreal, QC, Canada, Insulin is released from pancreatic beta cells in a biphasic and pulsatile Chicago, IL, Berlin, Germany manner in response to glucose. Loss of first phase, reduction in the second Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is phase and impairment of the oscillatory pattern of insulin secretion are char- markedly amplified by fatty acids, in large part through the G protein-cou- acteristic features of type 2 diabetes. In vitro, dynamic insulin release can pled receptor GPR40. We previously showed that GPR40 signaling involves be observed with islet perifusion assays. However, inherent heterogeneity activation of protein kinase D1 (PKD1) in isolated islets; and that GPR40 is necessitates pooling of multiple islets, which entails a decreased resolution implicated in the compensatory increase in insulin secretion during high-fat due to uncoordinated inter-islet function and inefficient use of donor mate- feeding in mice. The aim of this work was to examine the impact of con- rial. Here, we describe a novel method that enables single-islet perifusion ditional deletion of PKD1 in beta cells in mice under high-fat feeding. We assays for studying alterations in the dynamics of insulin release. Single generated beta-cell specific, inducible PKD1 knock-out mice β( PKD1KO) by reaggreagted islets - uniform in size, cellular composition and function - crossing transgenic mice expressing a tamoxifen-inducible Cre recombinase were loaded into miniature chambers of a newly-engineered microfluidic under the control of the insulin promoter (MIP-CreERT) with mice carrying a system, which facilitates rapid glucose switches and short sampling inter- PKD1 allele flanked by LoxP sites. Experimental animals included MIP-CreERT, vals down to 20 sec. In static culture, reaggregated human islets displayed PKD1flox/flox( βPKD1KO); MIP-CreERT, PKD1+/+ (CRE); PKD1flox/flox (FLOX); and wild highly reproducible and robust glucose dependent insulin secretion across type (WT) littermates. Tamoxifen was injected intraperitoneally 3 times over donors with stable function and viability for more than 28 days. In perifusion 5-days at 9 weeks of age. The efficiency of PKD1 deletion was confirmed by experiments, the step from 2.8 to 16.7 mM glucose induced biphasic insulin POSTERS secretion with a prominent first phase ( 35-fold increase) and a sustained, Islet Biology/ Western blotting. Mice were fed normal chow or a high-fat diet (58% calo- ~ Insulin Secretion ries from fat) from 12 to 25 weeks of age. Food intake, weight gain, glycemia pulsatile second phase (~8-fold increase) potentiated by the GLP-1 agonist and insulinemia were measured every week. Oral glucose tolerance tests Exendin-4. (OGTT) were performed at 12, 20, and 24 weeks. Hyperglycemic clamps were

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A576 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM

Figure. TCF7L2 heterozygous (LKB1f/f:TCF7L2 f/+), or homozygous mice for both genes (LKB1f/f:TCF7L2 f/f). In mice lacking both LKB1 alleles in the β-cell, oral glucose tolerance was unchanged by the deletion of a single TCF7L2 allele (LKB1f/f:TCF7L2 f/+) though glucose-stimulated insulin secretion was marginally enhanced. By contrast, on the LKB1 null background, mice lacking both TCF7L2 alleles (LKB1f/f:TCF7L2 f/f) showed improved oral glucose tolerance (AUC=805±24 vs. 954±63 mmol/L*min, p<0.05), insulin secretion in vivo (AUC=5.2±0.7 vs. 3.1±0.3 ng/mL*min) and in vitro from isolated islets (0.99±0.01 vs. 0.65±0.01 a.u., p<0.01) compared to LKB1f/f:TCF7L2 f/+ mice. Deletion of LKB1 from the β-cell, and the forced activation of mTOR, pro- vides a convenient model of the enhanced growth factor signalling observed in insulin resistance and during pregnancy. Our findings indicate that, under these conditions, TCF7L2 unexpectedly antagonises mTOR action. This may provide a mechanism through which nutrients including glucose, which con- trol mTOR signalling via LKB1 and AMP-activated protein kinase, modulate the impact of TCF7L2 variants on type 2 diabetes risk. Supported By: Medical Research Council, UK; Wellcome Trust UK; Royal Society & 2205‑P Pancreatic Islets in Short-Duration Type 2 Diabetes Have Secretory Dysfunction, Variable Amyloid Accumulation, and Altered Islet- & 2203‑P Enriched Transcription Factor Expression Synaptotagmin-9 Regulates Tomosyn-2 Protein Abundance to JOHN T. WALKER, DIANE SAUNDERS, SHRISTI SHRESTHA, NRIPESH PRASAD, Affect Early Phase of Insulin Secretion SHAWN E. LEVY, RADHIKA ARAMANDLA, GREG POFFENBERGER, RITA BOT- RAJESH GUPTA, SUSHANT BHATNAGAR, Birmingham, AL TINO, ROLAND W. STEIN, MARCELA BRISSOVA, ALVIN C. POWERS, Nashville, Previously we positionally cloned tomosyn-2 gene underlying a diabetes TN, Huntsville, AL, Pittsburgh, PA susceptible locus in an F2 mouse cross. We reported that mice congenic to The progression of pancreatic islet dysfunction in type 2 diabetes (T2D) tomosyn-2 were hyperglycemic, hypoinsulenemic, and have reduced insulin is poorly understood. Efforts to characterize the pathologic and functional secretion from pancreatic islets. Tomosyn-2 binds syntaxin-1A. However, features of T2D islets often do not account for clinical heterogeneity such it is not yet established whether tomosyn-2 binding to syntaxin-1 via its as disease duration or treatment. To address this, we comprehensively C-terminal domain limits the formation of the SNARE complex at the plasma examined pancreatic tissue and isolated islets from the same donors with membrane and exocytosis. Our preliminary data show that in addition to short T2D duration (n=9, age 25-66 years, 2-10 years duration, oral medica- syntaxin-1, tomosyn-2 bind and co-fractionate with synaptotagmin-9 (Syt-9) tions only) and age-matched controls. In islet perifusion assays, T2D islets in sucrose density gradient under conditions of high (15mM) glucose and had reduced baseline insulin secretion and blunted response to glucose, elevated calcium concentrations. Syt-9 protein is a calcium sensor, and is cAMP-evoked stimulation, and KCl-mediated depolarization. Conversely, known to regulate the formation of the SNARE complex. However, its role T2D islets had increased basal glucagon secretion but a blunted response in regulating insulin secretion is not yet well characterized. We observed to cAMP, epinephrine and KCl stimulation. However, T2D and normal islets a significant increase in in vivo insulin secretion at 5 min and 15 min post had similar insulin and glucagon content. In pancreatic sections, there was glucose challenge in 10-week male Syt-9-/- vs. control mice. These mice also no apparent difference in islet cellular composition (T2D vs. control: are more glucose tolerant and show no difference in insulin sensitivity. 61% β, 29% α, and 10% δ cells vs. 63% β, 30% α, and 7% δ cells), although Suggesting that Syt-9 regulates early phase of insulin secretion from beta there was considerable heterogeneity between T2D donors in islets contain- cells. Interestingly, islets of Syt-9-/- vs. control mice have reduced tomo- ing amyloid. Most T2D pancreata had <6% amyloid+ islets, but in 2 donors, syn-2 protein abundance by 50% without altering the levels of other key >50% of islets were amyloid+ without differences in clinical features. In t- or v-SNARE proteins. Moreover, no change in tomosyn-2 mRNA abundance these 2 pancreata, the prevalence of amyloid+ islets varied in pancreatic was observed. Altogether, these results indicate that the reduction in tomo- location with the tail containing the most amyloid+ islets (range: 24.7%- syn-2 protein abundance leads to an increase in insulin secretion observed 87.8%). RNA-sequencing analysis from FACS-purifiedα and β cells showed at early time points in Syt-9-/- mice. The results presented here point to reductions in key transcription factors important for islet cell development an as-yet-undescribed role of Syt-9 in chaperoning or localizing tomosyn-2 and cell identity, including factors such as MafA, MafB, Nkx2.2, Pax6, Isl1 protein from cytosol to the SNARE complex to regulate insulin secretion. and NeuroD1. Together, this data suggests that islets relatively early in the Herein, unpublished data describe a critical role of tomosyn-2 in regulat- course of T2D have impaired insulin and glucagon secretory outputs, het- ing early phase of insulin secretion. Therefore, alternations in tomosyn-2 erogeneous amyloid accumulation, and reduced expression levels of several protein levels will likely lead to elevated insulin secretion and risk potential islet-enriched transcription factors. hypoglycemia. Supported By: National Institutes of Health; JDRF; U.S. Department of Veterans Supported By: National Institute of Diabetes and Digestive and Kidney Dis- Affairs eases/National Institutes of Health (4R00 DK95975-03) 2206‑P & 2204‑P Relationship between Intrapancreatic Fat Content and Beta- and The Type 2 Diabetes Gene TCF7L2 Modulates the Impact of LKB1 Alpha-Cell Mass in Humans With and Without Diabetes Deletion on Beta-Cell Function RIE MURAKAMI, YOSHIFUMI SAISHO, KINSEI KOU, SEIJI SATO, JUN INAISHI, MARIE-SOPHIE NGUYEN-TU, GABRIELA DA SILVA XAVIER, ISABELLE LECLERC, YUUSUKE WATANABE, TAMI TSUCHIYA, MINORU KITAGO, YUKO KITAGAWA, GUY A. RUTTER, London, United Kingdom TAKETO YAMADA, HIROSHI ITOH, Tokyo, Japan Deletion of the tumour suppressor liver kinase B1 (LKB1/STK11) from Background and Aims: It has been reported that ectopic fat deposits in the pancreatic β-cell leads to cellular hyperplasia and enhanced glucose- pancreas induce beta cell apoptosis in rodent studies, called lipotoxicity stimulated insulin secretion by stimulating mammalian Target of Rapamy- hypothesis. Whereas some human studies have shown that pancreatic fat cin (mTOR). Variants in the transcription factor-7-like 2 (TCF7L2/TCF4) gene, content is associated with decreased β-cell function by imaging assess- involved in Wnt signalling, are associated with type 2 diabetes. Since LKB1 ment. However, effect of ectopic fat in pancreas on beta cell mass remains and Wnt signalling interact in primitive organisms, we have used a genetic unclear. The aim of this study was to clarify the effects of intra-pancreatic epistasis approach to explore the possibility that TCF7L2 may be required for fat on β- and α-cell mass in humans with and without diabetes. the effects of LKB1 deletion. Materials and Methods: We analyzed human pancreas at autopsy from POSTERS

C57BL6 mice bearing floxed LKB1 and/or TCF7L2 alleles were bred with 72 Japanese nondiabetic (NDM) adults (NDM-1 group; age 47 ± 11 years, Islet Biology/ 2 mice bearing Cre recombinase knocked in at the Ins1 locus (Ins1.Cre), thus body mass index (BMI) 24.1 ± 5.0 kg/m , HbA1c 5.5 ± 0.6% (mean ± S.D)) Insulin Secretion allowing highly β-cell selective deletion of genes. Breeding pairs produced and pancreas samples from 99 diabetic (DM) and NDM adults who under- wild type mice, LKB1 homozygous null mice (LKB1f/f:TCF7L2+/+), LKB1 null and went pancreatic surgery (NDM-2 group; n = 50, 64 ± 14 years, BMI 22.5 ± 2.7

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A577 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM

kg/m2, HbA1c 5.6 ± 0.5%, DM group; n = 49, 67 ± 9 years, BMI 21.9 ± 3.5 cell processing in these two groups. In addition, the PI:CP ratio in those with kg/m2, HbA1c 7.8 ± 1.6%). Pancreas section was stained with hematoxylin stimulated PI >10pM was greater than those with stimulated PI <10 pM (5.1 and fractional intra-pancreatic fat area to whole pancreas area (PFA) was vs. 3.89, respectively). (PI: CP ratios were calculated as molar ratios with quantified. Associations between PFA and fractionalβ -cell area (BCA), values multiplied by 100 to obtain PI as a % of CP). Our results suggest that α-cell area (ACA), mean islet size, islet density and β-cell turn over markers a PI cut-off of 10pM post MMTT may be biologically relevant, and that a pro- were assessed. portion of individuals with long standing T1D secrete significant amounts of Results: Although the PFA varied among the individuals (median 0.51% PI, despite not having a significant nutrient stimulated change in CP. PI may (0.17-1.27)), there was no difference among three groups (P = 0.37). In all be preferentially secreted following a stimulus, even in long standing T1D. subjects, PFA was significantly correlated with age and BMI (both r = 0.2, Supported By: The Leona M. and Harry B. Helmsley Charitable Trust; Boston P <0.01) but not correlated with HbA1c. PFA was correlated with neither University BCA, ACA, the ratio of ACA to BCA, islet morphology nor β-cell turnover. Conclusions: PFA was positively correlated with age and BMI in humans. 2209‑P There was no significant difference in PFA between subjects with and with- Long-Term Administration of Dulaglutide Does Not Degrade Pan‑ out diabetes. There was no significant association between PFA and BCA, creatic Beta-Cell GLP-1 Receptor Expression in Nondiabetic and ACA or β-cell turnover in subjects with or without diabetes. These findings Diabetic Mice suggest that pancreatic fat deposits have little effects on β-cell mass and TOMOHIKO KIMURA, ATSUSHI OBATA, HIDENORI HIRUKAWA, TOMOE KINO­ development of diabetes in humans. SHITA, KENJI KOHARA, MASASHI SHIMODA, SHUHEI NAKANISHI, TOMOATSU Supported By: Japan Diabetes Foundation; Keio Gijuku Academic Development MUNE, KOHEI KAKU, HIDEAKI KANETO, Kurashiki, Japan, Okayama, Japan Funds; Japan Ministry of Education, Culture, Sports, Science and Technology We have reported the protective effects of GLP-1 receptor (GLP-1R) ago- (15K09399 to Y.S.) nists on pancreatic β-cells. On the other hand, it is well known that chronic exposure to large amount of ligand leads to the down-regulation of its recep- 2207‑P tor. It remains unknown, however, whether GLP-1R agonist down-regulates Cdc42-Pak1 Activation to Evoke Glucose-Stimulated Insulin Secre‑ its receptor. We examined whether GLP-1R expression is reduced after long- tion Is cAMP-Dependent in INS-1 832/13 Cells term exposure to dulaglutide (Dula) in nondiabetic as well as diabetic mice. RAJAKRISHNAN VELUTHAKAL, OLEG G. CHEPURNY, COLIN A. LEECH, FRANK 7-week-old male db/db and db/m mice received Dula (0.6mg/kg×2/week) or SCHWEDE, GEORGE G. HOLZ, DEBBIE C. THURMOND, Duarte, CA, Syracuse, NY, control vehicle (CTL) for 17 weeks. We evaluated various metabolic param- Bremen, Germany eters, glucose-stimulated insulin secretion (GSIS), insulin and TG content in Glucose metabolism stimulate Cdc42-Pak1 activity and initiates F-actin islet after intervention. β-cell-related gene expression was also analyzed cytoskeleton remodeling in pancreatic beta-cells so that cytoplasmic by real-time RT-PCR. In db/m mice, GLP-1R expression in β-cells was not secretory granules can translocate to the plasma membrane where insulin decreased even after long-term administration of Dula. In db/db mice, GLP- exocytosis occurs. Since glucose metabolism also generates cAMP in beta- 1R expression at 24 weeks of age was significantly lower than that at 7 cells, the crosstalk of cAMP signaling with Cdc42-Pak1 activation might be weeks which was presumably due to glucose toxicity. Furthermore, GLP-1R of fundamental importance to glucose-stimulated insulin secretion (GSIS). expression in 24-week-old db/db mice treated with Dula was higher, rather Furthermore, a bicarbonate and calcium regulated soluble adenylyl cyclase than down-regulated, compared to 24-week-old CTL db/db mice, which was (sAC) might serve as a metabolic sensor linking glucose metabolism to cAMP probably due to amelioration of glycemic control. Food intake and blood production and GSIS. Recently, we reported that the novel cAMP antagonist glucose levels in db/db mice treated with Dula significantly decreased until Rp-8-Br-cAMPS-pAB suppresses GSIS from both human and rat islets. Here 24 weeks compared to CTL db/db mice. Expression levels of various β-cell- we use the rat INS-1 832/13 insulin-secreting cell line to demonstrate that related genes, insulin content and GSIS were enhanced after Dula treat- Rp-8-Br-cAMPS-pAB prevents glucose-stimulated Cdc42-Pak1 activation, ment in db/db mice. In contrast, oxidative and endoplasmic reticulum stress, while stabilizing F-actin, and blocking GSIS. Surprisingly, 8-pCPT-2’-0-Me- inflammation, fibrosis and apoptosis were suppressed after Dula treatment. cAMP-AM, a selective activator of Epac proteins, promotes Cdc42-Pak1 Taken together, dulaglutide exerts beneficial effects on glycemic control and activation at 1 mM glucose, and the magnitude of this effect is similar to protective effects on pancreatic β-cells for a long period. GLP-1R expression that evoked by 20 mM glucose alone. However, GSIS from INS-1 832/13 cells level is not reduced at all in nondiabetic as well as diabetic mice even after and human islets is unaffected by the small molecule sAC inhibitor LRE1. long-term exposure to dulaglutide. Collectively, these findings reveal that cAMP acts as a permissive factor, and Supported By: Japan Society for the Promotion of Science; Promotion and possibly as a direct coupling factor, in support of GSIS that is Cdc42 and Pak1 Mutual Aid Corporation of the Private Schools of Japan regulated, but independent of sAC. Supported By: National Institutes of Health (DK067912, DK102233 to D.C.T.); 2210‑P (DK069575 to G.G.H.) Affinity Proteomic Profiling of Plasma Reveals Proteins Asso‑ ciations with Insulin Secretion as Measured with Hyperglycemic 2208‑P Clamps: A DIRECT Study Stimulated Proinsulin in Type 1 Diabetes ELIN BIRGERSSON, MUN GWAN HONG, MARELISE E.M.W. EEKHOFF, SANNA CATHERINE SULLIVAN, JOSE M. CACICEDO, AYSE SAHIN-EFE, DEVIN W. STEEN- BYSTRÖM, CLAUDIA FREDOLINI, DORRET I. BOOMSMA, ECO J.N. DE GEUS, K AMP, Boston, MA MATHIAS UHLEN, SOREN BRUNAK, MARK I. MCCARTHY, HARTMUT RUETTEN, We and others, have recently reported preserved proinsulin (PI) secre- EWAN R. PEARSON, JOCHEN M. SCHWENK, LEEN M. T´HART, DIRECT CON- tion in long standing T1D. There are also reports of a small subset of indi- SORTIUM, Stockholm, Sweden, Amsterdam, Netherlands, Copenhagen, Denmark, viduals who secrete significant amounts of PI, despite negligible C-peptide Oxford, United Kingdom, Frankfurt, Germany, Dundee, United Kingdom, Leiden, (CP), suggesting beta cell ER dysfunction. The role and clinically relevant Netherlands concentrations of PI in this population however are not well defined. We Proteins originating from otherwise inaccessible tissues, like the pancre- performed mixed meal tolerance tests (MMTT) in 10 healthy individuals to atic beta-cells, can be detected in blood plasma and may provide insights on define the normal secretion profiles of CP and PI. Based on our pilot data we beta-cell function. In this study, we investigated whether levels of proteins established that the minimum peak PI concentration following MMTT was in plasma are associated with insulin secretion using samples of a modified 10pM. We applied this cut off to banked sera from 48 T1D subjects enrolled hyperglycemic clamp study in 130 healthy volunteers from the Netherlands in the T1D Exchange Registry (age 42.1 ± 20.7 yrs, median diagnosis 28 yrs Twin Register. We measured glucose, GLP-1 and arginine stimulated insulin and T1D duration 6 yrs), with known CP secretion after MMTT. Samples for secretion to identify proteins enriched for beta-cell function. this analysis were collected within 4 hours of a meal with total PI measured Protein profiles were derived from antibody bead arrays and analyzed via Stellux chemiluminescent assay by ALPCO. All subjects had detectable PI with generalized estimating equations and including age, gender, BMI, insu- (mean 10.92 pM, range 0.33-84.22), with 14 of the 48 demonstrating a non- lin sensitivity and glucose tolerance status as covariates. Among the 741 fasting PI >10 pM (mean 26.19 pM, range 10.16-84.22). These 14 subjects had antibodies from the Human Protein Atlas that were selected by a task force, clinically relevant concentrations of CP (>200pM) but a low ratio of peak vs. we found that SLU7, CCL16, IGFBP2 and TFF3 levels were negatively asso-

POSTERS baseline CP secretion following a MMTT, suggesting presence of residual ciated with glucose stimulated insulin secretion. Levels of MYO3A levels Islet Biology/

Insulin Secretion beta cell function without an expected nutrient response in CP. The 34 sub- associated negatively with GLP-1 and arginine stimulated insulin secretion jects with a PI <10 pM, in contrast, had a twofold larger change in peak vs. and insulin sensitivity (ISI) independent of the covariates (all P<4.0*10-5). A baseline CP after MMTT (ratio of 3.4 vs. 1.4), suggesting differences in beta total of 27 protein profiles were associated with insulin sensitivity. We vali-

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A578 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM dated our most significant findings CCL16, TFF3 and IGFBP2 using immuno- Figure. capture mass spectrometry, epitope mapping and developed dual binder ELISA assays. In this study, we have shown that plasma profiling for pancreatic beta cell enriched proteins identified candidates associated with detailed mea- sures of insulin secretion. Once confirmed in larger and independent studies, levels of these circulating protein fragments may serve as proxies to non- invasively monitor beta-cell function in subjects at risk to develop type 2 diabetes or during clinical trials. Supported By: Innovative Medicines Initiative Joint Undertaking (115317)

2211‑P Role of Proteasome in the Turnover of Insulin and Amylin in Pancre‑ atic Beta Cells DITI CHATTERJEE BHOWMICK, ALEKSANDAR JEREMIC, Washington, DC The major pancreatic hormones, amylin and insulin, are secreted from pancreatic beta cells to maintain glucose homeostasis. Following biosyn- thesis and post-translational modifications, these hormones are stored in secretory vesicles as mature polypeptides, ready to be released by various secretagouges. Studies show that human amylin (hIAPP) misfolds and aggre- gates in pancreatic beta cells of diabetic patients, leading to islet amyloid formation and type 2 diabetes mellitus. Under normal conditions, cellular proteolytic compartments and complexes, such as lysosomes and protea- Supported By: Taiwan National Health Research Institutes (NHRI-EX106- somes, degrade misfolded proteins. Conversely, intracellular accumulation 10524EI) of hIAPP impairs proteasome function indirectly by inhibiting ubiquitinated proteins from entering the 26S proteasome complex. This eventually leads 2213‑P to protein stress, accumulation of toxic amyloid aggregates and ultimately cell death. It is well established that proteasome is involved in the degrada- WITHDRAWN tion of many aggregation-prone proteins. However, the specific involvement of proteasome in proteolytic processing of amylin and insulin, and their turn- over in pancreatic cells is not well known and hence was investigated in our study. Our studies demonstrate a contrasting effect of proteasome on the production of these two hormones in human and rodent pancreatic beta cells. Proteasome inhibition did not significantly deplete the intracellular lev- els of insulin. However, the pharmacological inhibition of proteasome func- tion resulted in a marked reduction in the protein and mRNA levels of hIAPP. Conversely, overexpression of hIAPP upregulated proteasome expression and significantly downregulated the proteolytic activity of proteasomes, thus showing that proteasome regulates hIAPP turnover and vice versa. Because hIAPP secretion was also downregulated in proteasome-impaired beta cells, these findings point to a novel mechanism of hIAPP production and turnover by the proteasome. Supported By: National Institutes of Health

2212‑P Human Pancreatic Neuro-insular Network in Health and Fatty Infiltration SHIUE CHENG TANG, LUC BAEYENS, CHIA NING SHEN, SHIH JUNG PENG, HUNG JEN CHIEN, CHESTER E. CHAMBERLAIN, MICHAEL GERMAN, Hsinchu, Taiwan, San Francisco, CA, Taipei, Taiwan Background: The neuro-insular network has been proposed to enable rapid, synchronized insulin secretion. However, identification of the tissue network is a challenging task due to the lack of 3-D image data. Aims: To examine human pancreatic neuro-insular network in health and fatty infiltration. Methods: We applied tissue clearing to prepare transparent pancreata for 3-D histology. Specimens from subjects with normal BMI (3), high BMI (4), and type 2 diabetes (1) were used to investigate the neuro-insular network. Results: In humans, pancreatic fatty infiltration features adipocytes accu- 2214‑P mulating in the peri-lobular space and scattered in parenchyma. Early (<10% Using Synthetic Analogues to Explore In Vivo Metabolism of CMPF of fat area in specimen), moderate (10-20%), and severe (>30%) fatty infil- and Its In Vitro Effects on Insulin Secretion tration are correlated with subjects with normal BMI, high BMI, and T2D, TIMOTHY B. DURHAM, EDITH NAGY, YING LIU, KACEY J. PRENTICE, KYLE W. respectively. The fatty infiltration, however, is not reflected in mouse obe- SLOOP, PHILLIP E. SANDERS, BATTSETSEG BATCHULUUN, CRAIG D. HAMMOND, sity. Regarding the neuro-insular network, islets and intra-pancreatic gan- MICHAEL B. WHEELER, Indianapolis, IN, Toronto, ON, Canada glia (peri-lobular and intra-parenchymal ganglia) are identified with paired CMPF is a metabolite which circulates at high concentrations in type 2 and insulin/glucagon and PGP9.5/S100B staining to reveal the islet-ganglionic gestational diabetes patients. Data from human clinical studies suggests it association. Significantly, around the infiltrated adipocytes, ganglia increase might have a causal role in these diseases. CMPF has been shown to inhibit in size and projections, suggesting a neurotrophic environment. insulin secretion in mouse and human islets in vitro. Further, CMPF treatment Conclusions: These results provide a new dimension to our understanding impairs insulin secretion in vivo in rodents. However, the metabolic fate of of human pancreas via an integrated approach to examine adipocytes and CMPF in vivo and the relationship of molecular structure to effects on insulin POSTERS

neuro-insular network. secretion in islets have not been significantly studied. We describe the syn- Islet Biology/

thesis of CMPF and analogues of CMPF. These compounds include isotopi- Insulin Secretion cally labeled CMPF molecules designed to probe in vivo metabolism. Study of these labeled materials in mice has led to the first observation of a gluc-

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A579 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM

uronide metabolite of CMPF in plasma. This is notable because CMPF was ities, we studied 86 AA and 86 C normoglycemic subjects, matched for age, believed to be metabolically inert and cleared only by renal filtration. We BMI and parental history of T2D. We used 75 g OGTT, IVGTT and euglycemic have also prepared a wide range of CMPF analogues of varying molecular clamp to derive measures of IS and secretion, and indirect calorimetry and architecture. We characterized these analogues in insulin secretion under DXA for resting energy expenditure (REE) and fat free mass (FFM). Data were high glucose and potassium chloride stimulation conditions in both mouse analyzed by student t- and Chi square tests. Results are shown in Table. and human islets. From these studies, we identified several compounds that Table. showed a range of activities on insulin secretion. Notably, compound 10 dis- played increased potency relative to CMPF. These compounds are promising AA C P-value (n=86) (n=86) chemical probes for further mechanistic studies. Age (years) 37.30 ± 10.28 39.24 ± 11.47 0.2439 Figure. BMI (Kg/m2) 28.07 ± 5.72 26.96 ± 5.19 0.1820 Sex (F/M) 49/37 50/36 1.0000 Parental Diabetes (+/-) 44/42 41/45 0.7605 Fasting plasma glucose (mg/dL) 87.9 ± 6.5 91.0 ± 5.3 0.0007 30 minutes post glucose (mg/dL) 136.3 ± 19.9 146.8 ± 22.5 0.0013 2- hours post glucose (mg/dL) 112.1 ± 18.5 109.8 ± 15.8 0.4058 Area under the curve (AUC) (glucose) 14,538.2 ± 1622.9 15,118 ± 1694.1 0.0231 HOMA-IR 1.06 ± 0.88 1.19 ± 0.95 0.3372 QUICKI 5.43 ± 0.42 5.42 ± 0.42 0.7524 Matsuda index 10.32 ± 6.40 10.66 ± 7.38 0.7457 ISI-clamp (µmol/kg FFM/min/pmol/L) 0.118 ±0.059 0.176 ± 0.081 0.0003 Fasting insulin (µU/mL) 4.79 ± 3.78 5.24 ± 4.02 0.4622 Supported By: Canadian Institutes of Health Research (FDN-143219 to M.B.W.); HOMA-B 69.59 ± 48.19 66.14 ± 47.76 0.6312 Banting and Best Diabetes Centre (to Y.L., B.B.); Canadian Institutes of Health Research (to K.J.P.); National Science Foundation (1257290 to E.N.) Insulinogenic index (OGTT) 1.25 ± 1.01 0.85 ± 0.57 0.0015 Acute insulin response (AIR) [µU/mL] 108.43 ± 79.08 59.69 ± 39.30 0.0004 2215‑P Log AIR 1.94 ± 0.30 1.68 ± 0.29 < 0.0001 Regulation of Sulphonylurea-Induced Insulin Secretion in Beta-Cell Disposition index (DI) (log AIR x ISI) 0.22 ± 0.09 0.29 ± 0.14 0.0095 Lines Resting energy expenditure 28.9 ± 5.9 31.9 ± 6.4 0.0247 DECLAN MCGUIGAN, ANTHONY BJOURSON, PAULA MCCLEAN, CATRIONA (Kcal/kg FFM) KELLY, Derry, United Kingdom Sulphonylureas (SUs) are a front-line therapy for the treatment of type 2 At a lower glycemic burden, AA had lower IS, robust glucose-stimulated diabetes mellitus (T2DM) and act by enhancing insulin secretion from the insulin secretion but lower overall β-cell function (DI) than C. REE also was beta cell. However, given the complex aetiology of T2DM, there is significant lower in AA, which could lead to obesity and worsening IS and β-cell func- variation in response to this drug class with reports suggesting that 50-60% tion, and increased risk of dysglycemia. of those receiving SU therapy fail to achieve glycaemic control. Population Supported By: National Institutes of Health studies suggest that variation in ABCC8, KCNJ11, KCNQ1, HNF1α affect response rates. The aims of this study were (1) to conduct SNP prioritization to identify deleterious mutations in each gene and (2) to gain mechanistic 2217‑P insights into the role that each gene plays in regulating SU-induced insulin Perixiredoxin6 Deletion Impairs Mitochondrial Function and Insulin secretion in beta cell models. Secretion in Pancreatic Beta Cells SIFT and PolyPhen scores identified potentially deleterious SNPs in FRANCESCA PACIFICI, BARBARA CAPUANI, DONATELLA PASTORE, FRANC- ESCA PIERMARINI, ANDREA COPPOLA, ROBERTO ARRIGA, SILVIA REA, GIULIA ABCC8, KCNJ11, KCNQ1, HNF1α. This was cross-referenced with publi- cally available data in the type 2 diabetes Knowledge Portal and the lit- DONADEL, PAOLO SBRACCIA, ALFONSO BELLIA, DAVID DELLA MORTE, DAVIDE erature. Mechanistic studies were conducted in BRIN-BD11 and MIN6 LAURO, Rome, Italy beta cell lines and expression of candidate genes confirmed by qPCR and In pancreatic beta cells, mitochondrial metabolism is essential for ATP production, redox signaling, calcium (Ca2+) handling and glucose stimulated Immunocytochemistry. ABCC8, KCNJ11, KCNQ1, HNF1α were silenced with siRNA (100nm, Qiagen) and the resulting effect on SU-induced secretion in insulin secretion (GSIS). Recent data suggest that pancreatic beta cells response to 20min exposure to SUs (Tolbutamide, Glibenclamide; 200μm) normally contain a dynamic mitochondrial network characterized by a bal- was assessed by ELISA. ance between Fission and Fusion. Furthermore, when mitochondria become SIFT/PolyPhen scores predicted numerous pathogenic (30-50 per candi- largely fused or fragmented, GSIS resulted impaired. In our previous study, date) SNPs in each gene. Several SNPs (ranging from 10% for KCNJ11 to we demonstrated as mice knockout of an antioxidant enzyme, Peroxiredoxin 60% for ABCC8) were further validated in large-scale genomic studies high- 6 (Prdx6), significantly reduced GSIS. Therefore, the main goal of the pres- lighting the number of potential contributors to non-response to therapy. ent study was to evaluate the potential role of Prdx6 in modulating beta SU-induced insulin secretion was significantly reduced (P<0.001) upon cells function through the regulation of mitochondrial homeostasis. Insulin secretion and ATP production were evaluated after glucose stimulation (GS: ABCC8 and KCNJ11 silencing. HNF1α and KCNQ1 did not directly affect SU-induced insulin secretion in these models. Our data suggests that only 30 mM), at different time points, in newly generated murine insulinoma beta cell lines (beta TC6) stably silenced for Prdx6 (Prdx6KD). We observed a ABCC8 and KCNJ11 are directly involved in SU-induced insulin secretion KD from beta cell models. decrease in insulin secretion in Prdx6 cells at 15 min, after GS compared to Supported By: European Union Regional Development Fund (to A.B.); Sustain- control (ctr) cells. At the same time point, we showed a significant reduction of ATP production in association with lower levels of mitochondrial mem- able Competitiveness Programme for Northern Ireland; Northern Ireland Public KD Health Agency; Department for Education, Northern Ireland (to D.M.) brane potential and oxygen consumption, in Prdx6 cells. We, also, demon- strated that reduced ATP production induced a decrease of intracellular Ca2+ levels, which contributed to an impairment in granuli exocytosis machinery. 2216‑P Ultrastructural analysis revealed, in Prdx6KD cells, an irregular mitochondria Glucoregulation and Energy Expenditure in Normoglycemic African shape, sometimes very elongated with abnormal bifurcations, and disorga- Americans and Caucasians nized cristae, typical of alteration in Fission/Fusion ratio. For the first time, EBENEZER A. NYENWE, CHERECHI OGWO, IBIYE OWEI, JIM WAN, SAMUEL we demonstrated as Prdx6 could directly affect mitochondrial structure and DAGOGO-JACK, Memphis, TN

POSTERS function, in pancreatic beta cells. The impairment of mitochondrial Fission Islet Biology/ The etiology of ethnic disparity in type 2 diabetes (T2D) prevalence is and Fusion machinery was, also, responsible for a defect of GSIS. All these Insulin Secretion unclear: T2D incidence in subjects with prediabetes (PD) is ~ 10% in all eth- data highlight a potential novel role of Prdx6, which could open new horizons nic groups. African-Americans (AA) and Caucasians (C) with parental T2D for the treatment of diabetes mellitus. develop PD at an equal rate of 11%. To explore the ontogeny of ethnic dispar-

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A580 ISLET BIOLOGY—BETA CELL—STIMULUSCATEGORY-SECRETION COUPLING AND METABOLISM

2218‑P 2220‑P More than Just Measurable Peptides: Describing the Physiologic Oral Delivery of Probiotics Expressing Angiotensin-(1-7) Improves Importance of Residual C-Peptide through β-Cell Responsivity in Glucose Tolerance in Diabetic Mice Type 1 Diabetes KANG XU, DU TAO, PING ZHU, ZHIBING LIANG, AMRISHA VERMA, TUHINA ASA K. DAVIS, CARMELLA EVANS-MOLINA, CRYSTAL CONNOR, WEI HAO, PRASAD, MANOJ KULKARNI, QIUHONG LI, Gainesville, FL KRISTEN J. NADEAU, MARK A. CLEMENTS, JENNIFER L. SHERR, TAMARA S. Previous studies suggest that Ang-(1-7) improves insulin sensitivity HANNON, RICHARD E. PRATLEY, KELLEE M. MILLER, CARLA J. GREENBAUM, and β-Cell function in vitro and in vivo. In the present study, the probiotic MICHAEL R. RICKELS, Seattle, WA, Indianapolis, IN, Tampa, FL, Aurora, CO, Kansas expressing Ang-(1-7) was generated and orally administered to diabetic mice City, MO, New Haven, CT, Orlando, FL, Philadelphia, PA to investigate its therapeutic potential. Diabetes was induced in mice by Detection of residual C-peptide in those with type 1 diabetes (T1D) for streptozotocin and confirmed by measuring blood glucose. Diabetic mice more than 2yrs is possible; yet, the physiologic significance of very low lev- were orally gavaged daily for eight weeks with 1x109 CFU of Lactobacil- els of C-peptide is unknown. To further characterize how residual C-peptide lus paracasei (LP) secreting GFP (LP-GFP), LP secreting Ang- (1-7) (LP-A), secretion impacts β-cell secretory responses, we performed glucose- and PBS respectively (N=6-8/group). Oral feeding LP-A or LP-GFP reduced potentiated arginine (GPA) studies in adults with T1D classified by the peak the fasted-state blood glucose levels in diabetic mice, however, only LP-A C-peptide achieved during a mixed-meal tolerance test (MMTT) [Low n=14: group showed the significantly lower fasted-state blood glucose compared 0.017-0.200; intermediate n=14: >0.200-0.400; high n=18: >0.400 nmol/L]. with the diabetic mice treated with PBS (p=0.042). Similarly, oral feeding To determine β-cell secretory capacity plasma glucose (PG), C-peptide, and LP-A or LP-GFP improved the intraperitoneal glucose tolerance test (IPGTT) proinsulin were measured while fasting prior to initiation of a hyperglyce- in diabetic mice, and only LP-A group showed the significantly lower area mic clamp (target PG=230 mg/dL) and pre- and post-arginine (5 g IV over 1 under the curve of IPGTT than the PBS-treated diabetic mice (p=0.034). min). Fasting PG was lower (153±37 vs. 149±34 vs. 111±26 mg/dl; P=0.0006) These beneficial effects of LP-A on the blood glucose was associated with with higher fasting C-peptide (0.03±0.02 vs. 0.11±0.03 vs. 0.22±0.13 nmol/L; insulin content in islet. We found higher insulin expression in islets β cells P<0.0001). No difference in proinsulin was seen; thus, the proinsulin-to-C- from LP-A-treated mice compared with LP-GFP group, and no difference in peptide ratio was highest in the low C-peptide group (0.56±0.42 vs. 0.17±0.8 the islets morphology between diabetic mice groups. No significant differ- vs. 0.15±0.17; P<0.0001). In response to hyperglycemia, only the high C-pep- ences were found in fed-state blood glucose, intraperitoneal insulin toler- tide group demonstrated a rise in both C-peptide (P<0.0001) and proinsulin ance test, and proliferation and apoptosis in islets β cells between LP-A or (P=0.01). Conversely, all groups demonstrated responses to GPA stimulation LP-GFP treated and control groups. Taken together, our findings suggest that with incremental rise in C-peptide (0.04±0.03 vs. 0.13±0.04 vs. 0.40 nmol/L; oral administration of a genetically modified probiotics secreting Ang-(1-7) P<0.0001) and proinsulin (0.001±0.001 vs. 0.002±0.001 vs. 0.008±0.007 could improve the blood glucose and glucose tolerance by enhancing insulin nmol/L; P=0.0002); proinsulin secretory ratios were not different across content in islets β cells, thus could be considered a potential therapeutic groups. Secreted proinsulin represents a disproportionate amount of the cir- strategy for diabetes. culating β-cell peptides detectable in those with the lowest residual C-pep- Supported By: American Diabetes Association (1-15-BS-115 to Q.L.); National tide. Our results support that classification of residual C-peptide by peak Institutes of Health (EY021752, EY024564); Bright Focus Foundation MMTT response is consistent with the underlying β-cell secretory capacity derived from GPA. In order to respond to hyperglycemia, peak C-peptide of 2221‑P >0.4 nmol/L was required and may indicate a minimum threshold of physi- Reduced Glucose-Stimulated Insulin Release from Human Type 2 ologic importance. Diabetic Islets Associates with Multiple Proteomic Changes in the Supported By: The Leona M. and Harry B. Helmsley Charitable Trust Secretome KIRSTINE J. BELONGIE, MICHAEL K. HANSEN, MARK WATSON, MARCO BUGLI- 2219‑P ANI, LORELLA MARSELLI, MARA SULEIMAN, LISA NORQUAY, ALESSANDRO Intracellular Effects of Chronic Exenatide Treatment in Islets of POCAI, PATRICIA ANDRADE-GORDON, PIERO MARCHETTI, ELE FERRANNINI, Langerhans of Nonhuman Primates after Partial Pancreatectomy Spring House, PA, Montreal, QC, Canada, Pisa, Italy GIOVANNA FINZI, PAUL B. HIGGINS, FRANCESCA CASIRAGHI, EDWARD J. DICK, β-Cell insulin secretory failure is the hallmark of T2D. Metabolomics has JR., STEFANO LA ROSA, FAUSTO SESSA, FRANCO FOLLI, Varese, Italy, San Antonio, been performed to address the associated molecular changes in the β-cell TX, Lausanne, Switzerland, Milan, Italy but to date no study has looked at the secreted proteome. We evaluated Background: β cell failure results from a loss of β cell mass and from the proteins secreted during a glucose-stimulated insulin release (by the defects in the structure/function of the insulin secretory machinery. We batch sequential incubation method) assay by using a LC/MS platform. undertook an electron microscopy study of insulin secretory granule matu- Human islets from 9 nondiabetic (ND; 5 males/4 females; age: 72±13 yrs; ration, granule insulin content, and cellular ultrastructure in the islets of BMI: 25.5±3.4 kg/m2) and 4 T2D (2 males/2 females; age: 77±3 yrs; BMI: baboons after partial pancreatectomy (ppx) with and without treatment 26.0±2.9 kg/m2) organ donors were incubated at 3.3 and 16.7 mM glucose. with the GLP-1 receptor agonist exenatide. Insulin release (μU.mL-1.islet-1) at 3.3 mM glucose (LG) was 41±19 with ND Methods: Female baboons (Papio sp.) underwent ppx followed by 13 and 58±71 with T2D islets (p=NS), and increased respectively to 230±98 and weeks of continuous exenatide (Ex) at 0.014µg/kg/hr (n=14) or saline treat- 68±87 in response to 16.7 mM glucose (HG) (a stimulation index of 4.1±0.5 ment (n=14). PPx was performed under anesthesia with ~30% of the pan- for ND and 1.5±0.4 for T2D islets, p<0.0001). In the proteomics screen, creas (tail) removed. Baseline and posttreatment specimens were fixed and ND islets secreted higher amounts of insulin (FC=7.99) in response to high embedded in Epon-Araldite. Immuno-gold labelling for insulin and proinsulin glucose than did the diabetic islets (FC=1.86; p<0.015), in good accordance in β cells was also performed. All specimens were examined using a Phillips with the ELISA-based detection. Eighty human proteins were detected in Morgagni electron microscope (FEI, Holland). the incubation media. Six proteins had previous known associations with Results: At baseline, α and β cells were normal in both groups. Ppx with “pancreatic β-cell” (B2M, GCG, INS, REG1A, TTR, UCHL1) and 16 proteins saline treatment resulted in apoptotic features including pycnotic nuclei in had known associations with “type 2 diabetes.” In the comparison of HG vs. β cells and poorly granulated β cells. In contrast, the Ex group had normal LG, 20 proteins were different (p<0.05 and q<0.1), of which 10 have a signal cellular ultrastructure with well-granulated β cells. Ex-treated baboons had peptide and several are intracellular and functionally interact with cell death fewer large, electron clear progranules (27±18 vs. 110±10, p=0.02) and more proteins and stress-response kinases. These proteins are secreted into the electron-dense mature granules (59±12 vs. 13±4, p=0.01) relative to the media along with two nuclear proteins Nucleolin and Prelamin-A/C. The saline group. Immuno-gold labelling showed less proinsulin in the secretory interaction between the HG vs. LG and the disease status identified 11 pro- granules of Ex baboons whereas the number of proinsulin labelled granules teins (2 overlap with the 20 above) (p<0.05). Among them, bile salt-activated was greater in the saline group. Changes consistent with endoplasmic retic- lipase, serpins, zinc-α2-glycoprotein, and complement factor B. This is the ulum and mitochondrial stress present in the saline group were not seen in first time proteomics has been applied to proteins secreted from ND and T2D the Ex group. islets during glucose stimulation. Conclusion: Our findings show that ppx causes islet cellular stress com- bined with features of emerging insulin secretory dysfunction. The islets

of Ex-treated baboons were protected from these effects. Our data provide POSTERS Islet Biology/

evidence that Ex directly affects insulin secretory granule maturation and Insulin Secretion has pro-survival effects. Supported By: National Institutes of Health (R01080148)

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A581 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

2222‑P as well as other published data show that pharmacologic inhibition of PAI-1 Inflammation Reduces Islet TALK-1 K+ Channel Expression Leading prevents weight gain and leads to improved glucose tolerance in fat-fed to Increased Beta-Cell ER Ca2+ Storage and Preserving Glucose- mice. More recently, we have determined that humans with a heterozygous Stimulated Insulin Secretion frameshift mutation in SERPINE-1 (with lower levels of circulating PAI-1) MATTHEW DICKERSON, AVERY BOGART, MOLLY ALTMAN, SARAH MILIAN, have lower rates of obesity and diabetes compared to age-matched con- PRASANNA DADI, DAVID A. JACOBSON, Nashville, TN trols. Taken together, these findings suggest that increased PAI-1 leads to Inflammation during the onset of diabetes leads to an increase of cyto- worsening insulin resistance, either through a role in the adipose tissue and/ kines (TNF-α, IL-1β, and IFN-γ) that negatively impact islet function. Tran- or insulin producing cells. Based on this observation, we hypothesize that scriptome analysis indicates that the K2P channel, TALK-1, which is impor- PAI-1 contributes directly to the development of obesity and diabetes. Here, 2+ we directly assess the effects PAI-1 on the insulin producing pancreatic tant in regulating β-cell membrane potential (Vm), Ca influx, and insulin secretion is downregulated in β-cells exposed to cytokines. Moreover, β cells. We isolated islets from wild type mice and cultured them with recom- cytokine exposure has been shown to disrupt β-cell glucose-stimulated Ca2+ binant PAI-1 as well as with PAI-1 inhibitors. Exogenous PAI-1 increased influx. Therefore, this study sought to uncover the role that TALK-1 channels glucose stimulated insulin secretion in isolated murine islets. Thus, our play in β-cell dysfunction during low-grade inflammatory conditions (i.e., low data suggest that PAI-1 directly affects β-cell function. Future studies are level cytokines). Wild type (WT) and TALK-1 knockout (KO) whole islets and planned to define the role of PAI-1 in islet biology, as well as adipose tissue, isolated β-cells were treated overnight with cytokines (0.1 ng/mL TNF-α, and its fundamental role in insulin regulation and glucose homeostasis. 0.05 ng/mL IL-1β, and 1.0 ng/mL IFN-γ) and the effects on β-cell function Supported By: National Institutes of Health/National Heart, Lung, and Blood investigated. Cytokine treatment decreased islet TALK-1 transcript, TALK-1 Institute (2R01HL051387-18A1, 1P01HL108795-01) protein expression, and reduced TALK-1 channel activity. Wild type β-cells exposed to cytokines behaved similar to TALK-1 KO β-cells with depolar- 2225‑P ization of Vm and increased electrical oscillation frequency. However, β-cell Palmitic Acid-Mediated Nr4a Downregulation Impedes Beta-Cell electrical activity and Ca2+ oscillations were also greatly accelerated in Growth, Survival, and Function TALK-1 KO islets. Basal intracellular Ca2+ and 1st phase glucose-stimulated DANIEL LATHEN, BENJAMIN T. BIKMAN, JEFFERY S. TESSEM, Provo, UT Ca2+ influx were similar in cytokine treated WT and TALK-1 KO islets. How- Studies have shown that beta cells cultured with palmitic acid demon- ever, 2nd phase glucose-stimulated Ca2+ influx and glucose-stimulated insulin strate upregulation of the Nr4a orphan nuclear receptors. However, these secretion were attenuated only in TALK-1 KO islets. As β-cell cytokine expo- studies have two major caveats. First, toxic or near toxic palmitic acid con- sure has been shown to alter ER Ca2+, we further examined whether TALK-1 centrations have been used, and second the beta cells have been exposed to modulates cytokine disruption of ER Ca2+. Interestingly, ER Ca2+ storage was palmitic acid for short periods of two days or less. While elevated free fatty increased in WT but not TALK-1 KO β-cells following cytokine treatment. acid levels are a hallmark of type 2 diabetes (T2D), this occurs due to a grad- These findings suggest that reduction in TALK-1 channel expression may ual increase in circulating levels, rather than an immediate and large change. serve an acute protective role in the ER of β-cells by facilitating increased We sought to model the change in free fatty acid levels in vitro by cultur- ER Ca2+ storage in response to inflammation, which helps to maintain GSIS. ing the 832/13 INS-1 beta cells long-term in the presence of progressively Supported By: American Diabetes Association (1-17-IBS-024 to D.A.J.); Vander- increasing palmitic acid concentrations (0.15mM, 0.3mM and 0.6mM). We bilt University (T32DK101003); National Institutes of Health (DK097392) measured gene expression, beta cell proliferation, cell survival, mitochon- drial respiration and insulin secretion. Interestingly, our data demonstrates 2223‑P that as the beta cells are cultured in progressively higher palmitic acid con- Modeling the Shape of the Glucose Curve during an Oral Glucose centrations Nr4a1 and Nr4a3 expression was significantly decreased. This Tolerance Test as a Risk Factor for Type 2 Diabetes decrease corresponds with a slight decrease in proliferation and a signifi- JOON HA, ARTHUR SHERMAN, ANNE SUMNER, STEPHANIE T. CHUNG, cant increase in thapsigargin induced apoptosis. Interestingly, we observed Bethesda, MD no significant decrease in mitochondrial respiration, while insulin secretion The shapes of the glucose response curve during an oral glucose tolerance was significantly inhibited. Finally, as Nr4a1 and Nr4a3 have been shown to test (OGTT) are classified as mono-, bi-, or tri-phasic. The last two types regulate expression of genes critical for glucose metabolism in the beta cell, are characterized by an early decrease and late increase in glucose concen- we demonstrate that the glycolytic genes AldoA, Pgk1 and Eno1, and the tration during an OGTT. Whether the different shapes are a risk factor for TCA genes Dld and Dlst are significantly downregulated under conditions progression to diabetes is not clear yet. We have updated our mathemati- with elevated palmitic acid levels. Our data suggest that the decreased beta cal model for beta-cell mass and function (Ha et al. Endo 2016) to capture cell survival and function observed as T2D progresses may be due to down- different metabolic abnormalities during an OGTT. We hypothesize that a regulation of the Nr4a’s and decreased expression of the glycolytic genes fast response of insulin granule mobilization to glucose concentration rise driven by these nuclear receptors which are necessary for producing the ATP and discrepancy between hepatic and peripheral insulin resistance under- levels need for proper insulin secretion. lie the bi- and tri-phasic patterns. The former leads to the early increase Supported By: American Diabetes Association (1-17-IBS-101 to J.S.T.); Brigham and subsequent decrease in insulin, leading to both the early decrease and Young University the late rise in glucose. The latter drives the late rise. The model suggests that those with bi- and tri-phasic curves tend to have fast mobilization and a mild degree of hepatic insulin resistance, while those with mono-phasic ISLET BIOLOGY—SIGNAL TRANSDUCTION curves are more likely to have slow mobilization and a severe degree of hepatic insulin resistance. To test the model prediction that fast response 2226‑P of mobilization is required for tri- or bi-phasic, we used data from African HB-EGF Signaling Is Implicated in Glucose-Induced β-Cell Prolif‑ Americans to confirm that the early drop of insulin is strongly correlated eration to the bi-phasic pattern (R2=0.64). Model simulations predict that bi- and HASNA MAACHI, VALENTINE MOULLÉ, JULIEN GHISLAIN, VINCENT POITOUT, mono-phasic patterns are more likely to occur with impaired fasting glucose Montreal, QC, Canada, Paris, France (IFG) and impaired glucose tolerance (IGT), respectively. Model simulations Adult beta-cell proliferation is regulated by a number of nutrients includ- further predict that hypoglycemia at 2 hours compared to baseline is a risk ing glucose; however, the underlying mechanisms are controversial and factor of type 2 diabetes. important species-related differences between rodents and humans have been highlighted. In a rat model of nutrient excess induced by 72 hour- 2224‑P infusion of glucose and lipids, we previously observed that nutrient-induced PAI-1 Directly Regulates Beta-Cell Function β-cell proliferation is prevented by inhibition of either EGF receptor (EGFR) or JOSHUA A. LEVINE, BRIAN T. LAYDEN, DOUGLAS E. VAUGHAN, Chicago, IL mTOR signaling. The aim of this study was to determine the role of the EGFR Plasminogen activator inhibitor 1 (PAI-1) is a member of the serpin family and its ligand HB-EGF in rodent and human beta-cell proliferation. HB-EGF of serine protease inhibitors, and is the major inhibitor of both tissue-type- mRNA levels, as measured by RT-qPCR, were induced in response to a 24-h and urokinase-type-plasminogen activator. Elevated plasma PAI-1 levels exposure of rat islets to 16.7 mM glucose. HB-EGF mRNA levels were also POSTERS

Islet Biology/ have been reported in obese mice and humans, and are associated with increased in human islets from type 2 diabetes donors. Exposure of isolated

Insulin Secretion obesity-related disorders such as metabolic syndrome and type 2 diabetes. rat or human islets to exogenous HB-EGF (100 ng/ml) for 72 h in the presence A direct link between PAI-1 and glucose homeostasis was originally inferred of 2.8 mM glucose potently stimulated β-cell proliferation as assessed by after PAI-1 was find to be a carbohydrate-responsive gene. Data from our lab immunohistochemistry for Ki67 and insulin on islet cryosections. The mag-

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A582 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION nitude of this effect was similar to that observed with 16.7 mM glucose. be significantly reversed by RAGE neutralizing antibody or AGEs cross-link Co-culture in the presence of the EGFR inhibitor AG1478 (300 nM) or the HB- breaker alagebrium chloride (p<0.05 vs. control or AGEs alone, n=5/group/ EGF inhibitor CRM197 (10 ug/ml) completely blocked not only the prolifera- experiment). tive response to HB-EGF but also the response to 16.7 mM glucose, in both Conclusion: These results demonstrate for the first time that AGEs-RAGE rat and human islets. These data demonstrate 1- that HB-EGF is a potent axis is capable of inducing EndoMT and fibrosis in islet ECs. These findings beta-cell mitogen in both rat and human islets; and 2- that the proliferative suggest that AGEs-induced EndoMT may be an important mechanism in the response to glucose requires an intact HB-EGF-EGFR pathway. Our findings pathogenesis of diabetic islet fibrosis. identify a novel player in the complex mechanisms controlling beta-cell pro- Supported By: Taiwan Ministry of Science and Technology liferation in response to nutrients. Supported By: National Institutes of Health; Fonds de recherche du Québec 2229‑P Effects of Obesity and Diabetes on Delta and Pancreatic Polypep‑ 2227‑P tide Cell Mass and the Relative Change among Islet Endocrine Cell Mass in Humans WITHDRAWN YUUSUKE WATANABE, YOSHIFUMI SAISHO, JUN INAISHI, RIE MURAKAMI, TAMI TSUCHIYA, SEIJI SATO, KINSEI KOU, MINORU KITAGO, YUKO KITAGAWA, TAKETO YAMADA, HIROSHI ITOH, Tokyo, Japan Background and Aim: Recent rodent studies have postulated dedifferen- tiation and/or transdifferentiation of beta cell to other endocrine cells as a mechanism of beta cell loss in diabetes. We and others have reported that, by using surgically removed pancreas samples, alpha cell mass did not change in patients with type 2 diabetes (T2D), but changes in other endo- crine cells in the face of obesity and diabetes remains unclear. The aim of this study is to clarify effects of obesity and diabetes on delta and pancre- atic polypeptide (PP) cell mass in these subjects. Materials and Methods: We analyzed human pancreas samples from 98 individuals with (N = 48, 34 men, BMI 21.9 ± 3.5 kg/m2, age 67 ± 9 years (mean ± SD)) or without T2D (N = 50, 26 men, BMI 22.5 ± 2.7 kg/m2, age 64 ± 14 years) who underwent pancreatic surgery between 2000 and 2015. Pancreatic sections were stained for somatostatin or PP, and fractional delta (DCA) or PP cell area (PCA) to the total pancreas area was measured, respec- tively. In addition, the ratios of DCA or PCA to fractional beta (BCA) or alpha cell area (ACA) were assessed. Results: There was no significant difference in DCA or PCA between the subjects with or without T2D (0.080% (IQR 0.048-0.113) vs. 0.090% (0.044- 0.131), p >0.05 and 0.027% (0.013-0.061) vs. 0.032% (0.013-0.087), p >0.05, respectively). Neither DCA nor PCA was significantly correlated with BMI. While the ratio of DCA to BCA was significantly increased in the subjects with T2D compared with those without, it was significantly positively corre- lated with HbA1c (r = 0.311, p = 0.003) and significantly negatively correlated with BMI (r = -0.279, p = 0.018). Conclusion: Although DCA and PCA was not changed with obesity and diabetes, the ratio of DCA to BCA was significantly correlated with obesity and glucose intolerance. The relative change among the islet endocrine cells may be important for the onset and progression of diabetes.

2230‑P Quantitative Modeling and Simulation Analysis of Membrane Excit‑ 2228‑P ability and [Ca2+]i Dynamics under the Control of GLP-1 in Pancre‑ Advanced Glycation End Products Induce Endothelial-to-Mesen‑ atic β-Cells chymal Transition and Cellular Fibrosis in Islet Endothelial Cells YUKARI TAKEDA, GEORGE G. HOLZ, Kyoto, Japan, Syracuse, NY SHING HWA LIU, PEI SHAN TSAI, CHEN YUAN CHIU, CHIH KANG CHIANG, Taipei, Pancreatic β-cells generate bursts of action potentials in response to glu- 2+ Taiwan, Changhua, Taiwan cose, thereby inducing cyclic changes of [Ca ]i that drive pulsatile insulin Background: The intra-islet vascular endothelial cells (ECs) play an impor- release. GLP-1 potentiates this glucose-stimulated insulin secretion by regu- 2+ 2+ tant role in β-cell function. An intra-islet endothelium dysfunction has also lating membrane excitability, [Ca ]i, and Ca -dependent exocytosis. Here been implicated to contribute to the progression of diabetes. Fibrotic islet we perform a quantitative simulation analysis based on published experi- destruction has been suggested to be one of the pathogenic mechanisms mental variables so that a comprehensive mathematical understanding of of impaired β-cell growth and function during diabetic condition. Advanced these processes is achieved. Our analysis reveals how GLP-1 upregulates glycation end-products (AGEs) are implicated to be associated with patho- the oscillatory bursts of action potentials that are important driving fac- genesis of diabetic vascular complications. The experimental evidence sug- tors for insulin secretion. In our model, functional interactions of metabolic gested that endothelial-to-mesenchymal transition (EndoMT) plays a role in coupling factors, second messengers, and ionic events occur at the plasma the pathogenesis of fibrotic diseases. The fibrotic effect of AGEs on islet ECs membrane (PM) and at the endoplasmic reticulum (ER). For PM events, we and the role of EndoMT on islet fibrosis remain unclear. Here, we investi- consider effects of GLP-1 at voltage-gated Ca2+ and K+ channels, ATP-sensi- gated the effects of AGEs on EndoMT and fibrosis in islet ECs. tive K+ channels, and nonselective cation channels (NSCCs). For ER events, Methods: Mouse pancreatic islet endothelial cells (MS1) were cultured we consider effects of GLP-1 at the SERCA Ca2+-ATPase and the inositol and treated with AGEs (10-50 μg/ml) for 48 h. In some experiments, the 1,4,5-trisphosphate receptor (IP3R). Since GLP-1 is a cAMP-elevating hor- streptozotocin-induced diabetic mice or db/db diabetic mice were used. The mone, the model’s novelty is further enhanced by its inclusion of the cAMP protein expressions were determined by Western blot or immunohistochem- sensors Epac2 and PKA as determinants of ion channel and transporter istry. activities. An unexpected prediction based on these simulations is that the Results: We found that the immunoreactivities for AGEs and α-smooth Epac2-mediated stimulatory action of GLP-1 at NSCCs underlies its ability muscle actin (α-SMA) and fibrosis were markedly increased in the diabetic to increase membrane excitability and to reduce the time interval between POSTERS mouse islets as compared with control mice. Non-cytotoxic concentrations bursts. In contrast, a PKA-mediated stimulatory effect of GLP-1 at the IP3R Islet Biology/ of AGEs up-regulated the expressions of receptor for AGEs (RAGE), fibronec- underlies its ability to lengthen the duration of a burst. Thus, these simula- Insulin Secretion tin, connective tissue growth factor, α-SMA, vimentin, and pSmad2/3, and tions indicate complementary Epac2 and PKA mediated actions of GLP-1 to down-regulated E-cadherin and CD31 expressions in MS1 cells, which could promote oscillatory electrical activity and insulin exocytosis. Furthermore,

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A751. A583 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

the mathematical model presented here provides a new explanation for the 2233‑P therapeutically beneficial insulin secretagogue action of GLP-1 in patients GABA Reduces Glucose-Stimulated TxNIP Expression in Pancre‑ with type 2 diabetes. atic Beta Cells Supported By: Japan Society for the Promotion of Science WEIJUAN SHAO, ZHUOLUN SONG, WENJUAN LIU, LILI TIAN, LISA ZHAO, HONG- MEI NING, QINGHUA WANG, TIANRU JIN, Toronto, ON, Canada, Xingxiang, China 2231‑P The destruction of pancreatic beta cells can lead to the development of Identification of Matricellular Protein Fibulin-5 as a Target Mole‑ T1D. Recent studies have shown that GABA administration may serve as cule of Glucokinase-Mediated Calcineurin/NFAT Signaling in Pan‑ a potential treatment for T1D, possibly via increasing alpha cell mediated creatic Islets beta-like cell neogenesis. We reported previously that GABA also pos- TOMOKO OKUYAMA, JUN SHIRAKAWA, HIROMI YANAGISAWA, YASUO TERAU- sesses the protective effect on pancreatic islet beta cells while mechanisms CHI, Yokohama, Japan, Tsukuba, Japan underlying the protection remained largely unknown. Here we show that Glucokinase-mediated glucose signaling induces insulin secretion, prolif- GABA treatment reduced glucose induced expression of TxNIP, an important eration, and apoptosis in pancreatic β-cells. However, the precise molecular mediator of glucotoxicity, in the rodent pancreatic beta cell line INS-1 as mechanisms underlying these processes are not clearly understood. We well as in mouse pancreatic islets. In INS-1 cells as well as in mouse and found that glucokinase activation using a glucokinase activator (GKA) signifi- human pancreatic islets, the repression was also observed at TxNIP mRNA cantly upregulate the expression of Fibulin-5 (Fbln5), a matricellular protein level and GABA treatment attenuated the stimulatory effect of glucose on involved in matrix-cell signaling, in isolated mouse islets. To elucidate the TxNIP promoter expression. As the incretin hormone GLP-1 was also shown roles of Fbln5 in islet function, we investigated how islet Fbln5 is regulated to repress TxNIP expression via protein kinase A (PKA), we determined by glucose or GKA stimulation and the effects of Fbln5 on insulin secretion the stimulatory effect of GABA on PKA and its downstream target cAMP or β-cell proliferation. The islet Fbln5 expression was induced by ambient response element-binding protein (CREB). Curiously, the attenuation effect glucose in a time- and dose-dependent manner and further enhanced by of GABA on TxNIP expression was not observed in GLP-1 receptor knockout high-fat-diet or the deletion of insulin receptor substrate 2 (IRS-2) in islets. mouse islets, suggesting the existence of crosstalk between GABA and the By contrast, the GKA-induced increase in Fbln5 expression was blunted in GLP-1 signaling cascades. Irs-2-deficient islets. Fbln5 protein levels were also increased by the treat- Supported By: JDRF ment with GKA in INS-1 cells, indicating that Fbln5 is expressed in β-cell. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase 2234‑P 2+ inhibitor, KATP channel opener, Ca channel blocker and calcineurin inhibitor, Glucagon Stimulates Insulin Secretion through Both Glucagon and while it was augmented by harmine, a dual-specificity tyrosine phosphoryla- GLP-1 Receptors tion-regulated kinase (DYRK) 1A inhibitor. Although deletion of Fbln5 in mice SARA ENCISCO, BERIT SVENDSEN, DAVID A. D’ALESSIO, JONATHAN CAMPBELL, had no significant effects on the glucose tolerance, glucose-induced insulin Durham, NC, Copenhagen, Denmark secretion (GSIS) or GKA-induced β-cell proliferation, adenovirus-mediated Among the factors regulating islet function, paracrine interactions Fbln5 overexpression increased GSIS in INS-1 cells. Thus, the islet Fbln5 between endocrine cells are increasingly recognized as important. It is well expression is regulated through a glucokinase/KATP channel/calcineurin/ known that β-cell products control α-cell function; however, it is less clear nuclear factor of activated T cells (NFAT) pathway crucial for the mainte- whether α-cell products control β-cell function. β-cells express receptors for nance of β-cells, Fbln5 may be a novel marker of β-cell function. both glucagon (GCGr) and glucagon-like-peptide-1 (GLP-1 R), and we hypoth- esize that proglucagon-derived peptides from α-cells regulate insulin secre- 2232‑P tion. To study this, we developed a mouse with Gcgr specifically deleted Vaspin Improves Islet β-Cell Function via PI3K/Akt and NF-κB Sig‑ in β cells (β-Gcgr KO). In vivo studies comparing WT and β-Gcgr KO mice naling Pathways showed no differences in intraperitoneal or oral glucose tolerance between LIU SHIWEI, LI XIN, DU FANG, DUAN RUIXUE, WU YARU, ZHANG JIAXIN, ZHANG the two groups. To directly measure insulin secretion, islets from WT and QI, Taiyuan, China β-Gcgr KO mice were isolated and studied by perifusion. The response to Vaspin is a newly discovered adipokine that widely participates in dia- 16.7 mM glucose did not differ between the two strains, but 3nM glucagon betes mellitus and other insulin resistance related diseases. However, the enhanced insulin secretion more in WT than β-Gcgr KO mice (AUC: 36.27 ± effects of Vaspin on the function of islet β cells and relevant mechanisms 7.42 vs. 25.18 ± 4.41 ng/mL*min), whereas 0.6nM GLP-1 enhanced insulin are still unclear. Thus, the study was performed to investigate the effects of secretion equally in both groups (AUC: 44.5 ± 6.84 vs. 45.15 ± 5.90). Since Vaspin on the function of pancreatic β cells and the potential mechanisms. the β-Gcgr KO islets retained an insulin response to glucagon, it is plausible INS-1 cells were cultured and divided into six groups: control group, PA that this effect was mediated through the GLP-1 R. Blockade of the GLP-1 treatment group, Vaspin (80, 160, 320ng/ml) treatment groups and vaspin R with exendin-9 (ex-9) in perifused islets reduced the insulin response to (320ng/ml) +ly294002 (inhibitor of PI3K) treatment group. Thirty male hyperglycemia alone in both WT and β-Gcgr KO islets, but did not completely Sprague-Dawley rats (200-220g) were fed in three groups: normal diet suppress the insulin response to 3nM glucagon (AUC: 24.56 ± 3.65 and group, high fat diet group and high fat diet+ Vaspin (320ng/ml) group. Glu- 15.36 ± 1.31). Ex-9 completely blocked insulin secretion in response to 0.6nM cose stimulated insulin secretion (GSIS) assay was conducted in INS-1 cells GLP-1 in both groups. Together, these findings indicate that glucagon has and isolated rat islets. The protien and mRNA of PI3K/Akt and NF-κB singal- insulinotropic actions mediated by both the GCGr and GLP-1 R, while GLP-1 ing pathways were also tested. In addition, glucose tolerance and insulin signaling is mediated only by the GLP-1 R. Glucagon signaling through the sensitivity of rats were assessed with oral glucose tolerance test (OGTT) GLP-1 R may account for the normal glucose tolerance in β-Gcgr KO mice. and insulin tolerance test (ITT). Hyperglycemic clamp test was performed to Inhibition of glucose-stimulated insulin secretion by ex-9 in isolated islets evaluate islet β cell function of these rats. supports a physiologic role for proglucagon peptides to regulate insulin Results showed that secretion function of INS-1 cells and isolated rat secretion. islets treated with 320ng/ml Vaspin were significantly increased compared to control. Furthermore, we found that Vaspin could increase the levels of total IRS-2 protein and Akt (Ser473) phosphorylation while decrease IRS-2 (Ser731) phosphorylation, NF-κB P65 and IκBα (Ser32/36) phosphorylation protein levels and mRNA levels. When INS-1 cells treated with ly294002, the effect of vaspin on insulin secretion function was reduced. The depressed fasting blood glucose and elevated glucose tolerance were found in rats treated with 320ng/ml Vaspin. Furthermore, hyperglycemic clamp test results indicated that Vaspin can improve function of islet β cells in insulin resistant rats. In a word, these results showed that Vaspin can improve islet β cells func- tion via PI3K/Akt and NF-κB signaling Pathways.

POSTERS Supported By: National Natural Science Foundation of China (81200591, Islet Biology/

Insulin Secretion 81471025); Shanxi Scholarship Council of China (2015-109); Shanxi Province Inter- national Technology Cooperation Fund (2015081029); Transformation of Scientific and Technological Achievements in Shanxi Province (201604D131003)

For author disclosure information, see page A751. & Moderated Poster Discussion ADA-Supported Research A584