ISLET BIOLOGY—APOPTOSISCATEGORY

age 44.3 years), comprising 2,078 males (71.7%) and 822 females (28.3%) Multiple regression analysis(STATA 10SE) with leptin, adiponectin, resis- who underwent annual medical check-ups at our center between January tin and ASP as determinants of serum total 25 hydroxy vitamin D used a 2005 and December 2009, were recruited. The subjects were divided into model adjusted for age, gender, adjusted calcium, PTH , BMI, fasting insulin, four groups according to baseline serum triglyceride (TG) level and waist cir- smoking status, season of sampling and statin prescription. hs-CRP was re- cumference (WC): normal WC-normal TG level (NWNT), normal WC-high TG moved due to collinearity with leptin and resistin. level (NWHT), enlarged WC-normal TG level (EWNT) and enlarged WC-high Adiponectin determined circulating vitamin D concentration independent of TG level (EWHT). High serum TG level was defi ned as ≥150 mg/dL and en- other measured adipokines (β 0.39, 95% CI 0.14-0.63, p=0.003). Leptin, resistin larged WC was defi ned as ≥90 cm for men and ≥85cm for women. New cases and ASP did not infl uence vitamin D concentration (r2 of model 0.51, p=0.04). of diabetes were determined according to questionnaires fi lled in by partici- Serum adiponectin infl uences circulating vitamin D concentration inde- pants and the diagnostic criteria of the American Diabetes Association. Cox pendent of other adipokines. Resistin and ASP do not appear to be related to proportional hazards model analysis was used to assess the association of circulating vitamin D in our obese middle aged cohort. HTGW phenotype with the incidence of diabetes. A total of 101 (3.5%) new diabetes cases were diagnosed during the study period. The EWHT group had a higher incidence of diabetes (8.3%) compared with the NWNT group ISLET BIOLOGY—APOPTOSIS (2.2%). The adjusted hazard ratio (aHR) for diabetes for subjects with the EWHT phenotype at baseline was 4.113 (95% confi dence interval [CI] 2.397- 7.059) after adjustment for age, and 2.429 (95% CI 1.370-4.307) after ad- Guided Audio Tour: Islet Biology—Apoptosis (Posters: 2254-P to 2261-P), justment for age, sex, total cholesterol, systolic blood pressure and alcohol see page 15. drinking history. It was attenuated by inclusion of baseline fasting glucose level in the model. Subjects with the HTGW phenotype showed the highest & 2254-P risk of incident diabetes. This tool could be useful for identifying individuals Autophagy Induction Improves Function and Survival of Human at high risk of diabetes. Type 2 Diabetic β-Cells MARCO BUGLIANI, MASINI MATILDE, FAROOQ SYED, SANDRA MOSSUTO, 2252-P MARA SULEIMAN, LORELLA MARSELLI, UGO BOGGI, FRANCO FILIPPONI, DECIO Longevity of the Metabolic Impact of Laparoscopic Sleeve Gastrec- L. EIZIRIK, MIRIAM CNOP, PELLEGRINO MASIELLO, PIERO MARCHETTI, Pisa, Italy, tomy: Results in Morbidly Obese Indian Diabetic Patients at the End Brussels, Belgium of Seven Years Autophagy is the major mechanism involved in degradation and recycling JAYASHREE S. TODKAR, SHASHANK S. SHAH, Pune, India of intracellular components, and its alterations have been proposed to cause Laparoscopic sleeve gastrectomy (LSG) is a standard bariatric operation. β cell dysfunction. In the present study we explored the effects of autophagy It also shows a metabolic impact in terms of improvement in diabetes type 2 modulation in islets prepared from type 2 diabetic (T2D) and non-diabetic (T2DM) in morbidly obese patients. Sparse reports exist about the longivity (ND) human donors, studied under several different conditions. Islets were of its metabolic impact. This is the study to present the results of LSG in Indi- isolated from 5 T2D (age: 77±7yrs; gender: 3M/2F; BMI: 23.9±3.7 Kg/m2) and an obese patients with T2DM at the end of seven years. From 2006 till 2010, 10 non-diabetic (ND; age: 69±19 yrs; gender: 3M/7F; BMI: 23.6±2.9 Kg/m2) 124 patients of Indian origin with morbid obesity and T2DM have undergone organ donors. T2D and/or ND islets were then cultured for 1-5 days with 10 a LSG at our center by a single surgical team. The standard operation of LSG ng/ml rapamycin (autophagy inducer), or 5 mM 3-methyl-adenine (3MA) and and the multidisciplinary care with regular follow ups was provided to all of 1.0 nM concanamycin-A (ConcA) (autophagy blockers), either in the presence them. At the end of seven years we could collect information of 81 patients. or absence of metabolic (0.5 mM palmitate) or chemical (0.1 ug/ml brefeldin N =81. M:F: 29: 62. Age range 22 - 65 yrs. Duration of T2DM: 6 mths to 21 A) endoplasmic reticulum (ER) stressors. Compared to untreated T2D islets, yrs. BMI range: 35 - 68 kg/m2. On OHA only: 56. OHA + Insulin: 25. Average rapamycin exposed (24h) diabetic islets showed improved insulin secretion glycosylated Haemoglobin was 8.5%. At the end of seven years average BMI (glucose-induced insulin stimulation index from 1.6±0.8 to 2.1±1.1, p=0.05), loss was found to be 18 kg/m2. The average glycosylated hemoglobin was reduced amount of β cells with signs of apoptosis (from 6.6±1.7 to 2.0±0.8% 6.8%. The insulin usage (in redced doses) was needed in only 3 patients. by electron microscopy, p<0.05), and better insulin granules, mitochondria 68 patients out of 81 did not need any anti diabetic medication. 10 patients and ER ultrastructure. This was associated with signifi cant reduction of were on a single OHA. 76/81 had other medical comorbidities related to obe- PERK, CHOP and Bip gene expression (qRT-PCR). As expected, in ND islets sity and all showed an improvement even at the end of seven years. 21/81 palmitate exposure (5 days) induced a 4-5 fold increase of β cell apoptosis showed some weight gain (average 5 kg) at the end of 7 years but could re- (from 0.4±0.2 to 2.0±0.8%, p<0.01); this deleterious action was completely tain their metabolic improvements as compared to the baseline. LSG seems prevented by rapamycin (0.3±0.3%) and signifi cantly exacerbated by 3-MA to be a good surgical and metabolic tool to improve the diabetic status in (7.0±1.1%) and ConcA (3.1±1.1%). Substantially similar results were observed morbidly obese Indian T2DM patients even at the end of seven years. with brefeldin treatment (24h). Both palmitate and brefeldin induced PERK, CHOP and Bip gene expression, which was signifi cantly, although partially, 2253-P prevented by rapamycin. This study emphasizes the importance of autophagy Serum Adiponectin Determines Circulating Vitamin D Concentra- in human β cell function and survival, likely in association with ER activity. tion Independently of Other Adipokines Modulation of autophagy could be instrumental to β cell protection. RAM P. NARAYANAN, LUKE D. BOYLE, SARAH NG, AMANDEEP KAHLON, AN- DREW CROSS, CHERLYN DING, CHEN BING, CONOR MAGEE, HASAN Z. MALIK, & 2255-P STEPHEN W. FENWICK, JOHN P.H. WILDING, Warrington, United Kingdom, Liver- Heat Shock Factor 1 Protects Beta Cells against Glucolipotoxicity- pool, United Kingdom induced Endoplasmic Reticulum Stress and Apoptosis Circulating vitamin D concentrations have been linked to adiponectin and INDRI PURWANA, JEAN BUTEAU, Edmonton, AB, Canada leptin, but not studied in the context of other adipokines. Our study aimed Excess exposure to glucose and fatty acid causes endoplasmic reticulum to assess the effects of adipokines on serum vitamin D levels in a predomi- (ER) stress, protein misfolding, and apoptosis, a condition termed “glucolipo- nantly obese middle aged Caucasian cohort in north west England. toxicity”. Heat Shock Factor 1 (HSF1) is a transcription factor that regulates We recruited 53 patients (11 male), the majority (79.2%) of whom were cell response to diverse stressors via the induction of the molecular chaper- due to undergo bariatric surgery. The remainder underwent elective laparo- ones heat shock proteins 70 and 90 (HSP70 and HSP90). Upon stress, HSF1 scopic cholecystectomy. Mean age was 48 years (95% CI 45.3 - 50.9). The binds to the heat shock response element (HSE) to initiate the transcription majority were obese (BMI>30: 88.6%; BMI>40: 77.3%). of HSPs, whose role is to keep proteins from being misfolded thereby al- Fasting serum samples were obtained from patients on the morning of leviating ER stress. the day of surgery. Serum total 25 hydroxyvitamin D (D2+D3), fasting insulin, We herein sought to test the hypothesis that HSF1 would protect beta- calcium, parathyroid hormone (PTH) were measured along with serum leptin, cells against glucolipotoxicity and to identify the posttranslational modifi ca- adiponectin, resistin, ASP and C reactive protein (hs-CRP). Body weight was tions involved in its activation. measured using TANITA scales. Thus, INS1 cells and human islets were treated with or without 25 mM

POSTERS Details of measured proteins expressed as (Mean, SD, 95% CI, units): vi- glucose and 0.4 mM palmitate to induce glucolipotoxicity. Glucolipotoxicity Islet Biology/

Insulin Secretion tamin D (60, 28, 16-149, nmol/L); leptin (21, 8.5, 18.7-23.4, ng/ml); resistin caused ER stress as indicated by a rise in CHOP protein levels. This was (0.2, 0.01, 0.19-0.2, ng/ml); adiponectin (59.6, 61.3, 42-76, ng/ml); ASP (19.2, accompanied by an increase in HSE-luciferase promoter activity and the con- 7, 17.3-21, nm/L). sistent up-regulation of both chaperones hsp70 and hsp90. These effects of

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A574 ISLET BIOLOGY—APOPTOSISCATEGORY glucolipotoxicity were recapitulated by the ER-stress inducer Thapsigargin. treated groups when compared to 1 and 10 mg/kg/bw treated groups, sug- Glucolipotoxicity induced HSF1 acetylation, and our study with point mutants gesting better tolerance in the latter groups. The percentage of non-diabetic identifi ed lysine residue K80 as a main acetylation site. HSF1 overexpres- mice in vehicle, 0.1, 1 or 10 mg/kg/bw BRD3308 treated groups were 30%, sion prevented glucolipotoxicity-induced ER stress and apoptosis. This ef- 30%, 44% and 78%, respectively. These results suggest that selective inhi- fect is abrogated by HSF1-K80R that mimics deacetylation. SiRNA-mediated bition of HDAC3 protects β-cells and could be harnessed to treat T1D. knockdown of SirtT1, a deactylation enzyme, increased HSPs expression, in Supported By: JDRF (17-2013-437) accordance with a positive regulation of HSF1 transcriptional activity by acetylation. We thus identifi ed a novel mechanism by which HSF1 activation & 2258-P via its acetylation promotes beta-cell survival. HSF1 may represent a new Inhibition of Infl ammation-induced Pancreatic Beta-Cell Failure by target to promote the maintenance and expansion of beta-cell mass. Targeting the MAP3 Kinase Tpl2: A Potential New Immunotherapy Supported By: Alberta Diabetes Foundation for Type 2 Diabetes ELODIE VARIN, DANY MULLER, CHRISTOPHE BROCA, MAGALIE RAVIER, FRANCK & 2256-P CEPPO, ERIC RENARD, ANNE WOJTUSCISZYN, JEAN-FRANÇOIS TANTI, STÉPHANE The Islet Amyloid Polypeptide (IAPP) Degradation Product IAPP DALLE, Montpellier, France, Nice, France (16-37) Promotes Aggregation and Toxicity of Full Length IAPP Besides genetic determinants and gluco- and lipo-toxicity, chronic infl am- DANIEL MEIER, LING-HSIEN TU, TANYA SAMARASEKERA, ANDREW T. TEMP- mation is considered a hallmark of type 2 diabetes (T2D), attenuating insulin LIN, MEGHAN F. HOGAN, DANIEL P. RALEIGH, STEVEN E. KAHN, Seattle, WA, action and affecting both beta-cell function and mass. The MAP3 Kinase 8, New York, NY Tumor progression locus 2 (Tpl2), is involved in ERK1/2, JNK and p38 MAPK Amyloid formation by the 37 amino acid peptide human IAPP results in phosphorylation/activation specifi cally in response to infl ammatory stimuli. increased β-cell apoptosis and thus reduced β-cell mass in type 2 diabetes. It is currently unknown whether Tpl2 integrates signals from infl ammatory Upregulation of IAPP-degrading enzymes may have the potential to protect cytokines in beta-cells. We hypothesized that targeting Tpl2 may represent against amyloid-induced damage. We have shown that matrix metallopro- a therapeutic strategy to prevent T2D by limiting cytokine-induced beta-cell teinase 9 (MMP9) cleaves IAPP between residues 15/16 (preferred cleav- death and failure. We report Tpl2 expression in INS-1E beta-cells, mouse age site) and 25/26. Since certain IAPP fragments have been shown to act and human pancreatic islets. We observed increased Tpl2 expression in is- as amyloid inhibitors, we sought to determine whether the MMP9-cleaved lets of diabetic rats, and showed that its pharmacological inhibition in vivo IAPP fragments can modulate IAPP aggregation. Co-incubating full length improved glucose tolerance, decreased fasting blood glucose and serum in- IAPP with up to 10-fold excess of the non-amyloidogenic IAPP fragments sulin levels in diabetic db/db mice, without modifying insulin sensitivity. In 1-15, 1-25 or 26-37 did not alter aggregation kinetics. In contrast, IAPP(16- vitro, Tpl2 was up-regulated and activated by cytokines, required for ERK1/2 37), which still contains the amyloidogenic sequence 20-29 and is itself amy- activation (but not JNK or p38 MAPK) induced by IL-1beta or combined cy- loidogenic, dose dependently accelerated aggregation of 16 µM full length tokines (i.e. IL-1beta + TNFalpha + IFNgamma), and its inhibition protected IAPP (time to 50% maximum: 12.4±0.2 h with vs. 17.8±1.0 h without 16 µM INS-1E cells, mouse or human islets from cytokine-induced apoptosis. Im- IAPP(16-37); mean±sd, n=4, p=0.03). Toxicity studies in INS-1 cells revealed portantly, the insulin secretion in response to glucose was also preserved in that at concentrations ranging from 5 to 60 µM, IAPP(16-37) is actually less mouse and human islets exposed to cytokines and treated with Tpl2 inhibi- cytotoxic than full length IAPP (40 µM for both peptides: 81±24 vs. 28±10% tor. Pharmacological inhibition of Tpl2 in combination with administration of cell viability; n=9, p=0.001). However, since IAPP(16-37) accelerated ag- a glucagon like peptide-1 receptor agonist (i.e. Exendin-4), produced more gregation of full length IAPP, we examined whether addition of IAPP(16-37) powerful synergistic antidiabetic effects on INS-1E cells and human islets also changed the cytotoxic potential of full length IAPP. Incubation of INS-1 survival and function. Neither survival, nor both glucose-induced ERK1/2 cells with a combination of IAPP(16-37) (5 µM, a dose which by itself was phosphorylation and insulin secretion were modifi ed by Tpl2 inhibition. This not toxic) and 20 µM full length IAPP nearly halved cell viability (37±11 vs. preclinical study suggests the use of Tpl2 inhibitors as novel and effective 63±16% compared to full length IAPP alone; n=12, p=0.001). In summary, co- immunotherapeutic strategy to protect beta-cells in T2D. incubation of IAPP(16-37) with full length IAPP enhanced both the aggrega- Supported By: INSERM (France) tion and cytotoxic potential of full length IAPP. Thus, upregulation of MMP9 may aggravate rather than ameliorate islet amyloid deposition and β-cell & 2259-P apoptosis and may therefore not be a viable therapeutic target to protect Wfs1 Defi ciency Causes Beta-Cell Dedifferentiation Associated islets from amyloid burden. with Enhanced ER Stress and Oxidative Stress, Independently of Hyperglycemia & 2257-P KIKUKO SHIINOKI, KATSUYA TANABE, MASAYUKI HATANAKA, YAUSHARU OTA, Selective Inhibition of HDAC3 by a Novel Small Molecule Inhibitor, YUKIO TANIZAWA, Ube, Japan BRD3308, Protects β-Cells from Cytokine-induced Apoptosis and Wolfram syndrome is an autosomal recessive disorder characterized by Protects NOD Mice from Diabetes insulin-dependent diabetes mellitus, and is caused by mutations in the WFS1 ERCUMENT DIRICE, MORTEN LUNDH, FLORENCE WAGNER, RACHAEL MARTI- gene. The Wfs1 defi cient (Wfs1-/-) mice exhibit a selective beta-cell loss and NEZ, EDWARD HOLSON, BRIDGET WAGNER, ROHIT N. KULKARNI, Boston, MA, impaired insulin secretion. This phenotype has been thought to be associat- Cambridge, MA ed with an augmentation of ER stress. However, the underlying mechanisms Inhibition of histone deacetylases (HDACs) appears to be a promising remain unknown. The recent reports have suggested that the loss of beta- strategy to promote β-cell survival by suppressing infl ammatory cytokine- cell in diabetes results from not only apoptosis but also dedifferentiation. induced apoptosis. To test the hypothesis that inhibition of individual HDAC In the present study, we investigated whether loss of beta-cell maturity is isoforms protects β-cells, we tested known HDAC inhibitors in the pres- associated with beta-cell failure in Wfs1-/- mice. Islets of 18 weeks of age ence of infl ammatory cytokines. Transfection of siRNA duplexes specifi c of Wfs1-/- mice, in which blood glucose levels were still maintained, exhib- for HDAC1, 2 or 3 into INS-1E insulinoma cells revealed that knock-down of ited elevated gene expression of ER stress- and oxidative stress-responsive HDAC3 signifi cantly reduced caspase-3 activity in the presence of cytok- genes such as CHOP, Bip, sXBP-1, superoxide dismutase 1 and thioredoxin ines, suggesting that HDAC3 is unique in modulating apoptosis in β-cells. reductase 1, relative to those in islets of WT mice. At the same time, in Based on these fi ndings, we tested the novel HDAC3 inhibitor BRD3308. Wfs1-/- mice, there was a robust reduction of MafA gene expression and a BRD3308 suppressed cytokine-, high glucose- or palmitate-induced apopto- more than three-fold increase in Neurogenin3 gene expression relative to sis in INS-1E cells and rodent and human islets. We next tested the effi cacy WT mice. Immunohistochemistry on pancreatic sections from Wfs1-/- mice of BRD3308 in vivo, using the female NOD mouse model of type 1 diabetes showed that beta cells were severely decreased and that residual beta cells (T1D). Female NOD mice that developed spontaneous diabetes were divided were weakly stained with insulin. Consistently, MafA-expressing cells were into 4 groups (n=18) and injected with vehicle (DMSO), or 0.1, 1 or 10 mg/ markedly reduced. In contrast to that, islet cells with nuclei stained by Neu- kg/bw of BRD3308 intra-peritoneally. All groups received the fi rst injection rogenin3 were appeared in Wfs1-/- mice. These fi ndings were more apparent at age 3 weeks that continued daily for 2 more weeks. At 5 weeks of age, in Islets of Wfs1-/-Ay/a mice in which beta-cell loss was accelerated by the we injected the mice twice a week for an additional 20 weeks. While all addition of insulin resistance. Despite apparent loss of beta-cells, only slight POSTERS

mice gained weight and did not exhibit differences between groups until ~17 increase in dying insulin positive-cells, assessed by TUNEL staining, could Islet Biology/ weeks of age, the group receiving 10 mg/kg/bw showed weight gain during be seen in islets of both Wfs1-/-and Wfs1-/-Ay/a mice relative to WT mice. Insulin Secretion subsequent weeks. Glucose tolerance tests performed at 17 weeks of age Therefore, these results suggested that Wfs1-defi ciency brought about be- demonstrated higher blood glucose excursions in vehicle and 0.1 mg/kg/bw ta-cell dedifferentiation, contributing to loss of functional beta cell.

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A575 ISLET BIOLOGY—APOPTOSISCATEGORY

2262-P & 2260-P Pancreatic Beta-Cell Protection and Reversal of Hyperglycemia in Translation Initiation Factor eIF2A Is Upregulated by Endoplasmic Diabetic Animals by a Small Molecule through Mitigating ER Stress Reticulum Stress and Protects Pancreatic Beta Cells from Apoptosis and Inducing Autophagy EVGENIY PANZHINSKIY, FARNAZ TAGHIZADEH, KWAN-YI CHU, YU HSUAN YU LI, HENDRA SETIADI, DAVID FAN, WEIDONG WANG, Oklahoma City, OK CAROL YANG, ERIC JAN, JAMES D. JOHNSON, Vancouver, BC, Canada, Sydney, Endoplasmic reticulum (ER) stress-mediated pancreatic beta cell dysfunc- Australia tion and death are key elements in the pathogenesis of both type 1 and 2 Endoplasmic reticulum (ER) stress plays important role in beta cell death diabetes. Targeting ER stress-mediated beta cell death could provide a new in diabetes. ER stress leads to activation of the unfolded protein response therapeutic avenue for diabetes. From a high throughput screen, we have (UPR), which inhibits protein translation, activates protein folding and ER- identifi ed a synthetic small molecule RH01687 that protected β cells from associated degradation (ERAD), or if unresolved induces apoptosis. A novel tunicamycin (TM)-induced dysfunction and death. However, the mechanism eukaryotic initiation factor eIF2A has been implicated in the translation of by which RH01687 promotes β cells survival is unknown. In this study, we specifi c mRNAs under stress conditions, such as viral infection, when gen- demonstrated that RH01687 abrogated TM-induced apoptosis as assessed eral translation is suppressed. Therefore, our specifi c hypothesis was that by cleaved caspase-3 and PARP, as well as restored Bcl-2, an anti-apoptosis eIF2A plays a key role in regulating expression of pro-survival genes under protein. Further, RH01687 reduced levels of ER stress markers p-eIF2α and ER stress conditions in beta cells. We showed that mRNA (3.7±0.5-fold) and CHOP. Moreover, autophagy plays a key role in the quality control of unfold- protein (2.3±0.3-fold) expression of eIF2A was up-regulated in MIN6 beta ed proteins and in pathogenesis of diabetes. RH01687 restored the levels of cells treated with ER stress inducer thapsigargin (1 µM), suggesting an as- LC3A/B and Atg7 which were suppressed by TM. These results suggest that sociation with the UPR. We found that expression of eIF2A was maximal at RH01687 inhibits TM-induced apoptosis via induction of autophagy. Impor- 24 hours of thapsigargin treatment. We also showed that another ER stress tantly, blood glucose was reduced and total pancreatic insulin content and inducer palmitate (1.5 mM) increased protein expression of eIF2A by 2.1±0.1- GSIS were enhanced by RH01687 in STZ-induced diabetic mice. Furthermore, fold. The fi ndings were confi rmed using isolated mouse and human islets RH01687 signifi cantly increased insulin expression in TM-triggered INS-1 treated with thapsigargin and palmitate. Fluorescent microscopy showed cells and in the islets of mice with diabetes. Mechanistically, our study re- that eIF2A was localized in the ER under basal and UPR conditions. Using veals that RH01687 protects beta cells against ER stress-mediated dysfunc- annexin and propidium iodide staining we showed 94.3±5.1% reduction in tion and death by mitigating ER stress and inducing autophagy. thapsigargin-induced apoptosis in MIN6 cells overexpressing eIF2A. On the protein level reduced death of beta cells was associated with 90.5±6.1% decrease in protein expression of UPR pro-apoptotic marker CHOP. Knock- 2263-P down of eIF2A with shRNA in MIN6 beta cells had no effect on thapsigargin- The S20G Human Islet Amyloid Polypeptide (hIAPP) Mutant Is More induced apoptosis, but led to 2.6±0.6-fold increase in protein expression of Amyloidogenic and Cytotoxic than Wild-Type hIAPP in Islets translation initiation factor eIF2D, which might compensated for the lack of DANIEL T. MEIER, MEGHAN F. HOGAN, ANDREW T. TEMPLIN, STEVEN E. KAHN, eIF2A. Therefore, we conclude that eIF2A play protective role against apop- Seattle, WA tosis during UPR in ER-stressed beta cells by negatively regulating expres- Human IAPP (hIAPP) is a 37 amino acid peptide that aggregates to form sion of pro-apoptotic CHOP protein. islet amyloid and thus contributes to increased β-cell apoptosis and reduced Supported By: Canadian Diabetes Association β-cell mass in type 2 diabetes. S20G is a naturally occuring hIAPP muta- tion found in Asian populations to be associated with an earlier onset of type 2 diabetes. Physical chemistry studies using synthetic peptides sug- & 2261-P gest that S20G hIAPP aggregates more rapidly than wild-type hIAPP. To RAGE Is a Mediator of β-Cell Toxicity in Type 2 Diabetes determine whether S20G hIAPP has more detrimental effects on β cells JACQUELINE Y. LONIER, ANNETTE PLESNER, ANN MARIE SCHMIDT, ANDISHEH than wild-type hIAPP, we examined islets from mice that express either (a) ABEDINI, New York, NY, Måløv, Denmark non-amyloidogenic mouse IAPP (MM), (b) the S20G hIAPP mutant (GG) or (c) Loss of β-cell mass in type 2 diabetes (T2D) exacerbates hyperglycemia wild-type hIAPP (WW), the latter two knocked into the mouse IAPP locus. and associated cardiovascular disease (CVD). A better understanding of Islets (n=9 studies) were cultured for 144 h in physiological (11.1 mM; low) the mechanisms contributing to β-cell toxicity in T2D is essential for the or elevated (16.7 mM; high) glucose, the latter to induce amyloid formation. development of effective therapies for this disorder. T2D is defi ned by hy- As expected, MM islets did not develop amyloid. The rate of increase in perglycemia, insulin resistance, and signifi cant accumulation of pathologi- amyloid deposition over time was comparable in GG and WW islets, with cal pro-infl ammatory mediators, including the receptor for advanced glyca- culture for 144 h at high glucose inducing similar amounts of islet amyloid tion endproducts (RAGE) ligand families: advanced glycation endproducts deposition in both genotypes (amyloid area/islet area: GG 15.4±5.5%, WW (AGEs), members of the S100/calgranulin family, and toxic oligomers of the 12.1±4.1%; mean±sd, p=0.22). In contrast, at low glucose amyloid deposition misfolded pancreatic hormone, amylin. We thus hypothesized that RAGE is a was signifi cantly increased in GG compared to the negligble amount in WW mediator of β-cell toxicity in T2D, as many of the above pathological factors islets (GG 0.80±0.93%, WW 0.05±0.05%; p=0.003). In high glucose, β-cell associated with hyperglycemia, β-cell dysfunction, and advanced diabetic apoptosis was increased in GG islets (apoptotic β cells: GG: 0.58±0.20%, CVD are ligands for RAGE. Our lab has previously defi ned the properties of WW 0.37±0.22%; p=0.06). This increase in β-cell apoptosis in GG islets was toxic amylin intermediates and demonstrated in cultured β-cells, isolated refl ected in greater β-cell loss (β-cell area/islet area was decreased: GG primary murine pancreatic islets, and mouse models that amylin amyloid 42.4±5.5%, WW 47.2±5.5%; p=0.02). In summary, the S20G mutation may formation and β-cell apoptosis is mediated in a manner dependent on RAGE, render hIAPP amyloidogenic even at physiological glucose levels and, under similar to AGE-induced RAGE activation. Here, we examined human pan- hyperglycemic conditions, may lead to increased amyloid-induced cytotox- creas tissue from T2D and nondiabetic subjects generously provided by the city. Thus we conclude that the enhanced amyloidogenicity and cytotoxicity Network for Pancreatic Organ Donors with Diabetes (nPOD) and show that of the S20G mutation may explain the early onset of type 2 diabetes in pa- amylin-positive amyloid deposits in T2D islets colocalize with loss of insulin tients that carry this variant. immunoreactivity. Islet amyloidosis is accompanied by a signifi cant increase in AGE immunoreactivity and decrease in islet cell numbers, suggesting that RAGE-mediated β-cell toxicity in T2D can be activated in different ways by 2264-P distinct RAGE ligands upregulated in the diabetic milieu. These clinically The Receptor for Advanced Glycation Endproducts (RAGE) Medi- relevant fi ndings provide important insight for identifi cation of new multi- ates Beta-Cell Oxidative Stress in Islet Amyloidosis: Implications targeted therapeutic strategies to preserve pancreatic β-cell mass and im- for Type 2 Diabetes prove glycemic control in T2D. DANIEL J. SARTORI, PING CAO, LING-HSIEN TU, DANIEL P. RALEIGH, ANN MARIE Supported By: Network for Pancreatic Organ Donors with Diabetes; Endocrine SCHMIDT, ANDISHEH ABEDINI, New York, NY, Stony Brook, NY Fellows Foundation Islet amyloidosis by the hormone islet amyloid polypeptide (IAPP) is toxic to pancreatic β-cells, contributing to loss of β-cell mass in type 2 diabetes (T2D). However, the mechanisms underlying IAPP-induced β-cell death re- main poorly understood. Recent studies in our lab have characterized the POSTERS

Islet Biology/ biophysical properties of toxic human IAPP (h-IAPP) oligomers that form dur-

Insulin Secretion ing amyloid assembly, and implicate the multi-ligand receptor for advanced glycation endproducts (RAGE) as a mediator of IAPP-induced β-cell toxicity. We hypothesized that RAGE mediates IAPP-induced oxidative stress, as we

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A576 ISLET BIOLOGY—APOPTOSISCATEGORY have previously shown that toxic h-IAPP oligomers are RAGE ligands and AM gene expression was signifi cantly increased in Wfs1-/- mice islets and that RAGE mediates oxidative stress in various settings. To test this hypoth- Tg treated Min6 cells compared to controls. The expressions of RAMP2, esis, we treated rat insulinoma-1 (INS-1) β-cells with h-IAPP intermediates RAMP3 and CRLR genes were 1.5-3.5 fold increased in Wfs1-/- mice islets and found a dose-dependent reduction in cell viability accompanied by an and Tg treated Min6 cells compared to controls. AM secretion was 2.2 fold increase in production of reactive oxygen species (ROS). We observed simi- increased in Tg treated Min6 cells. Evaluating the apoptosis caused by Tg lar results after β-cells were treated with the prototypic advanced glycation treatment using DNA ladder assay and Western blotting of cleaved cas- endproduct (AGE) ligand of RAGE, carboxymethyllysine (CML), which accu- pase-3 revealed that AM transfected Min6 cells were partly resistant to mulates in diabetes and activates RAGE. CML-induced ROS production was apoptosis. Intracellular cAMP was increased in AM-transfected MIN6 cells accompanied by increased expression of NADPH-oxidase subunits NOX-1 compared to controls. and NOX-4, consistent with prior reports of h-IAPP-mediated oxidative The expressions of AM and AM receptors are up-regulated in β-cells ex- stress. We found that β-cells express mDia1, a requisite intracellular RAGE posed to ER stress. Expressing AM in β-cells prevents apoptosis. The results binding partner, and that expression of RAGE and mDia1 is upregulated by suggest that AM autocrine signaling in β-cells plays an important role on toxic h-IAPP intermediates, further hinting at a RAGE-dependent mechanism attenuating ER stress to protect them from death. Increased intracellular of h-IAPP-induced oxidative stress. These results implicate RAGE in h-IAPP cAMP is likely related to this cytoprotective effect. and CML-induced β-cell oxidative stress and have important implications for the prevention of islet amyloidosis-related β-cell toxicity in T2D. 2267-P Heterogeneity of Autophagy Status in Pancreatic β-Cells Under 2265-P Metabolic Stress Identifying Human Islet miRNAs Associated with β-Cell Loss in a MAI KAMITANI, TAKESHI MIYATSUKA, MASAKI MIURA, TAKESHI OGIHARA, Humanized Mouse Model YOSHIO FUJITANI, HIROTAKA WATADA, Tokyo, Japan REGAN ROAT, JENICA CHRISTOPHERSON, MUNIR HOSSAIN, COLETTE FREE, Autophagy plays an essential role in intracellular quality control through SHALINI JAIN, CLAUDIANE GUAY, ROMANO REGAZZI, ZHIGUANG GUO, Sioux degradation of subcellular damaged organelles and components. We have Falls, SD, Lausanne, Switzerland previously reported that autophagic dysfunction in β-cell specifi c Atg7- Investigating miRNA changes occurring in human islets during β cell de- defi cient mice cause impaired glucose tolerance accompanied by progres- struction in type 1 diabetes (T1D) will help us to identify biomarkers for early sive reduction of β cell mass. In Atg7-defi cient mice and Lprdb/db mice, only disease onset. In this study, we performed adoptive lymphocyte transfer part of damaged β cells exhibited accumulation of p62/SQSTM1, a specifi c (ALT) on a humanized mouse model with transplanted human islets in or- substrate of the autophagy, suggesting that β cells are heterogeneous in au- der to induce engrafted β cell destruction. We then measured the miRNA tophagy status. In addition, Western blotting with isolated islets from db/db profi les of the human islet grafts using a NanoString technology and vali- mouse revealed the elevated expression of microtubule-associated protein dated selected miRNAs using qPCR. Immunodefi cient NOD.scid mice were light chain 3 (LC3) type 2, showing that some islets cells exhibited elevated given streptozotocin to induce endogenous β cell death, and donor human autophagic fl ux. Thus, although autophagic status in whole islets can be al- islets (n = 5 donors; purity 83±7.6%) were subsequently transplanted under tered under metabolic stress, it remains elucidated how different stages of the kidney capsule of diabetic mice. After at least 2 weeks of normoglyce- autophagic fl ux individual β cells have. To address this question, we intro- mia, lymphocytes from diabetic NOD mice were adoptively transferred to duced GFP-LC3 reporter mice, which can visualize autophagic status in each the humanized mice, and blood glucose and body weight were monitored β cell as green-fl uorescent puncta, and cross GFP-LC3 mice with Lprdb/db daily. Plasma and islet graft were collected immediately once blood glucose mice. After 16-hour fasting, less than a third of β cells of GFP-LC3; Lprdb/ reached >200mg/dL. Expression levels of 800 miRNAs in the isolated human db mice exhibited green-fl uorescent puncta, most of which overlapped with islets, the islet graft with control PBS treatment, and the islet graft with ALT p62, whereas few cells of GFP-LC3; Lprdb/+ mice showed green fl uorescence were measured using the NanoString nCounter® miRNA Expression Assay. in their islets. When GFP-LC3; Lprdb/+ mice were treated with S961, which Of the 20 most highly expressed miRNAs in donor islets and PBS-treated is- antagonized insulin signaling without inducing hyperglycemia, part of β cells let grafts, 11 had signifi cantly decreased expression in the ALT-treated islet were positive for GFP, but negative for p62, in contrast with the fi ndings grafts following β cell loss (42.4±15.3% decrease; p < 0.05). qPCR results in GFP-LC3; Lprdb/db mice. These fi ndings suggest that pancreatic β cells validated this decrease in miR-let-7a-5p, miR-let-7b-5p, miR-148a-3p, miR- under metabolic stress were heterogeneous in autophagy status, and au- 200a-3p, miR-23b-3p, miR-27b-3p, and miR-375, as well as in miR-7-5p and tophagic failure in β cells could be induced by hyperglycemia. novel miR-4454 (p < 0.0001). NanoString also revealed several miRNAs with signifi cantly increased expression in the grafts of ALT-treated compared to 2268-P PBS-treated mice (p < 0.01), including miR-142-3p, miR-15a-5p, miR-15b-5p, Attenuation of Streptozotocin-induced Pancreatic Beta-Cell Death miR-150-5p, miR-155-5p, miR-16-5p, miR-191-5p, miR-21-5p, and miR-223- in Transgenic Fat-1 Mice via Autophagy Activation 3p, that are likely related to immunity. Our results suggest that changes in DONG-MEE LIM, JWA-JIN KIM, SEUNG-YUN HAN, KEUN-YOUNG PARK, WON- these miRNAs are associated with the β cell loss and lymphocyte infi ltration MIN HWANG, HEE-KWAN WON, KYU LIM, Daejeon, Republic of Korea characteristic of T1D. Background: Recently, there has been a rapid increase in the number of patients with diabetes, and a change in eating habits has been reported as 2266-P the main cause. In particular, infl ammatory factors, and beta cell dysfunc- Adrenomedullin Has a Cytoprotective Role in Pancreatic β-Cells tion due to a high-fat diet, aggravate chronic diseases and complications. Against ER Stress However, amongst the dietary fats, omega-3 has anti-infl ammatory effects, MASARU AKIYAMA, RISA SUETOMI, YASUHARU OHTA, HIROKO NAKABAYASHI, and the role of autophagy as the etiological cause of diabetes has been re- MANABU KONDO, KATSUYA TANABE, YUKIO TANIZAWA, Ube, Japan cently reported in several studies. In this regard, the authors aim to examine Preventing β-cell loss is a major purpose of T2DM treatment. Recently, ER the protective effects of autophagy on diabetes, using fat-1 transgenic mice stress has been emerged as a major factor that causes β-cell loss. We and with n-3 PUFA self-synthesis capability. others have shown that β-cell loss in Wfs1-/- mice, which are model mice of Methods: To determine whether autophagy machinery was involved in the Wolfram syndrome, is related to apoptosis due to ER stress and β-cells from beta (β) cell protection observed in fat-1 mice, we morphologically detected these mice are susceptible to ER stress. In earlier studies, we showed that the microtubule-associated protein 1A/1B light chain 3(LC3)-immunoreactive Adrenomedullin (AM) is up-regulated in Wfs1-/- mice islets and thapsigargin puncta in β cells. Furthermore, we quantitatively assessed the level of p62, (Tg) treated Min6 cells. In this study, we studied the AM signaling in β-cells which is used as a marker for autophagic clearance, in the pancreas of fat-1 to respond ER stress. mice, and compared this data with that of the control mice. By real-time PCR we analyzed the gene expressions of AM and AM recep- Results: Histological determination using H&E and Masson’s trichrome tor, which is composed of RAMP2, RAMP3 and CRLR, in Wfs1-/- mice isolated staining revealed the protective effects of the fat-1 mutation on cell death islets and Tg treated Min6 cells. We also measured the secretion of AM from and scarring of pancreatic islets after STZ injection. In the β cells of control these cells. In addition, we transfected AM into Min6 cells and studied the mice, while autophagy was rarely detected in basal conditions, autophagy effect of AM expression on apoptosis induced by Tg treatment in these cells. was abruptly activated after STZ-stimulation. However, basal autophagy POSTERS

Apoptosis was evaluated using DNA ladder assay and Western blotting of levels were elevated and this status persisted after STZ-stimulation of β Islet Biology/ cleaved caspase-3. Simultaneously we measured intracellular cAMP using cells of fat-1 mice. Interestingly, quantitative analysis of p62 demonstrated Insulin Secretion ELISA kit. that STZ-induced impairment of autophagic clearance was signifi cantly re- versed in the pancreas of fat-1 mice.

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A577 ISLET BIOLOGY—APOPTOSISCATEGORY

Conclusions: Fat-1 transgenic mice with n-3 PUFA self-synthesis capability Our research focused on the Cdkn1c gene, which codes for a cell cycle inhibitor, have protective effects against STZ-induced β cell death, and n-3 PUFA was found because it is involved in the regulation of pancreatic β-cell mass. The Cdkn1c to have protective effects against diabetes by activating autophagy in β cells. gene is an imprinting gene whose expression is controlled by the non-coding RNA Kcnq1ot1. We found that when the expression of Kcnq1ot1 decreased, 2269-P epigenetic alterations induced an increase in the expression of Cdkn1c and Regenerating Protein (PSP/reg) as a Potential Therapeutic Target a decrease in the pancreatic β-cell mass. We have already reported that the for Beta Cell Endoplasmic Reticulum Stress in Wolfram Syndrome transcription factor C/EBPβ accumulates in the pancreatic β-cells of high fat-fed STEPHEN STONE, BESS MARSHALL, TAMARA HERSHEY, ROLF GRAF, FUMIHIKO mice. It has been reported that a binding motif of C/EBPβ is present in the Cdkn1c URANO, St. Louis, MO, Zürich, Switzerland promoter. On the basis of these fi ndings, we hypothesize that the accumulation The endoplasmic reticulum (ER) is an emerging target for human chronic dis- of C/EBPβ in pancreatic β-cells with reduced expression of Kcnq1ot1 causes fur- eases. Wolfram syndrome, a prototypical human ER disease, is an autosomal ther enhancement of Cdkn1c expression and promotes pancreatic β-cell failure. recessive disorder characterized by juvenile onset diabetes and neurodegen- Methods: Kcnq1ot1-truncated C/EBPβ-overexpressing mice (KT mice) eration. Wolfram syndrome results from the loss of function of the WFS1 gene, were produced by crossbreeding Kcnq1ot1-truncated mice with pancreatic which encodes a transmembrane protein localized to the ER. By identifying the β cell-specifi c C/EBPβ-overexpressing mice. key molecular pathways and biomarkers involved in the ER stress response, Results: The fi ndings showed that the expression of Cdkn1c in MIN6 cells novel therapies may be uncovered. One such molecule, regenerating protein was not infl uenced by the expression level of C/EBPβ, however, it was en- (PSP/reg), was found to be upregulated in islets isolated from WFS1 beta cell hanced by treatment with an inhibitor of epigenetic modifi cation, and was specifi c knockout mice (a mouse model of Wolfram syndrome). PSP/reg is a se- further enhanced by C/EBPβ overexpression. In vivo studies have shown that creted molecule that is abundant in islets regenerating after partial pancreate- compared to C/EBPβ-overexpressing mice and wild-type mice, KT mice show ctomy. In order to establish the role of PSP/reg in Wolfram syndrome, rodent signifi cantly higher blood glucose levels, smaller pancreatic β-cell mass, and beta cells (INS-1 cells) were analyzed by real time PCR, western blot analysis, enhanced expression of Cdkn1c in the pancreatic islets. and the BrdU cell proliferation assay. Immunohistochemistry was performed Conclusions: The fi ndings suggest that the introduction of epigenetic mod- on tissue sections obtained from WFS1 beta cell specifi c knockout mice. Se- ifi cations into MIN6 cells or pancreatic β-cells may lead to enhanced binding rum levels of PSP/reg were also measured in human subjects with Wolfram of C/EBPβ to the Cdkn1c promoter, then an increase in the expression levels syndrome. Our results demonstrated that the expression of PSP/reg was po- of Cdkn1c, and a decrease in pancreatic β-cell mass. tentiated when INS-1 cells underwent knockdown of WFS1 or were treated with the chemical ER stress inducer thapsigargin. PSP/reg and insulin were 2272-P found to colocalize within the islets of the WFS1 beta cell specifi c knockout Targeting Cellular Calcium Homeostasis to Suppress Beta-Cell mice. In human subjects, there was no statistical difference in serum PSP/reg Death in Diabetes levels when comparing subjects with Wolfram syndrome to controls. However AMY L. CLARK, FUMIHIKO URANO, St. Louis, MO there were 3 patients with elevated levels of PSP/reg. These patients tended Beta cell loss is a major component in the development of both type 1 and to be more severely affected. These results demonstrate that PSP/reg may type 2 diabetes. As such, understanding the mechanism behind beta cell death in be a key factor in the beta cell ER stress response. Therefore modifi cation of diabetes could provide novel treatment options. Recently, it has been discovered PSP/reg level or activity may be a possible therapeutic target for patients with that beta cells exposed to diabetes related stressors exhibit altered cytosolic Wolfram syndrome and other ER-associated diseases. and endoplasmic reticulum (ER) calcium levels resulting in apoptosis. Studies Supported By: American Diabetes Association (1-12-CT-61 to F.U.) have also shown that pharmacologic restoration of intracellular calcium levels can protect beta cells from diabetes related cell death. To date studies have fo- 2270-P cused on targeting ER or cytosolic calcium homeostasis alone. Thus, the current Inhibition of Insulin-Degrading Enzyme Increases Islet Amyloid For- study was designed to determine if combination therapy restoring both cytoso- mation In Vitro lic and ER calcium homeostasis can provide enhanced protection of beta cells MEGHAN F. HOGAN, DANIEL T. MEIER, ANDREW T. TEMPLIN, REBECCA L. HULL, against diabetes related beta cell death. In this study INS1E cells and human MALCOLM A. LEISSRING, STEVEN E. KAHN, Seattle, WA, Irvine, CA islets were pre-treated with calcium modulating drugs and drug combinations Islet amyloid formation due to amyloidogenic human islet amyloid polypep- and then exposed to various forms of diabetes related stressors including; cy- tide (hIAPP) is associated with the loss of β cells in type 2 diabetes. Insulin- tokines, free fatty acid (FFA), hyperglycemia, and ER stress. Caspase 3/7 activity degrading enzyme (IDE) is a protease known to degrade insulin, as well as was then quantifi ed as an indicator of cellular apoptosis. In INS1-E cells combi- amyloidogenic peptides including Aβ and synthetic hIAPP. Administration of an nation therapy targeting both ER calcium levels (dantrolene or pioglitazone) and IDE inhibitor to lean and diet-induced obese (DIO) mice (expressing murine IAPP) cytosolic calcium levels (verapamil or nifedipine) provided signifi cantly better increased insulin levels and improved glucose tolerance. However, as the mu- protection against cytokine and ER stress induced cell death compared to drug rine form of IAPP is not amyloidogenic, it is not known whether inhibition of IDE monotherapy (n=3). Additionally, preliminary data in human islets has shown that will have deleterious consequences in islets expressing amyloidogenic hIAPP. treatment with combination therapy provides better protection against cytokine To determine whether inhibition of IDE increases islet amyloid formation induced islet cell death compared to monotherapy (n=1-3). These results indicate and β-cell loss, we cultured isolated islets from amyloid-prone hIAPP trans- that combination therapy targeting restoration of both ER and cytosolic calcium genic mice in 16.7mM glucose for 144 hours in the presence or absence of homeostasis provides protection against diabetes related beta cell death. Thus the non-toxic, selective IDE inhibitor Ii1 (30 µM in DMSO; n=5-6 experiments). presenting a novel treatment for preservation of beta cells in diabetes. After 144 hours, the degree of amyloid deposition was greater in the presence Supported By: American Diabetes Association (1-12-CT-61 to F.U.) of Ii1 (amyloid area/islet area: 4.7±1.0% for 30 µM Ii1 in DMSO vs. 3.3±0.8% for DMSO alone; p=0.004). As expected, this increase in amyloid formation 2273-P was associated with a trend towards the loss of β cells (β cell area/islet area: The Impact of Hexosamine Biosynthetic Pathway in Glucose Ho- 47.9±1.0% for 30 µM Ii1 in DMSO vs. 49.9±1.4% for DMSO alone; p=0.1). meostasis, Analyzing by O-GlcNAc Transferase Knock-out in the In summary, inhibition of IDE in amyloid-prone islets results in increased Multiple Tissues amyloid deposition and the loss of β cells. Thus, while treatment with IDE SHOGO IDA, OSAMU SEKINE, SHINJI KUME, KATSUTARO MORINO, SATOSHI inhibitors may potentially increase insulin levels and improve glucose toler- UGI, KANAKO IWASAKI, NORIO HARADA, NOBUYA INAGAKI, HIROSHI MAE- ance, this benefi cial effect of IDE inhibitors in human type 2 diabetes may be GAWA, Otsu, Japan, Kyoto, Japan negated by them increasing amyloid formation and β-cell loss. O-GlcNAcylation is characterized by the addition of O-linked β-N- Supported By: American Diabetes Association (7-11-MN-28 to S.E.K.) acetylglucosamine to proteins and serves as an intracellular nutrient sensor for modulating cell functions. Although it has been speculated that altera- 2271-P tion of O-GlcNAcylation is associated with metabolic diseases, its exact role Reduction in Pancreatic β-Cell Mass Caused by Enhanced Expression in whole body glucose metabolism has not yet been fully shown. of Cdkn1c via Interaction between C/EBPβ and Epigenetic Control O-GlcNAc transferase (Ogt) is a critical enzyme for O-GlcNAcylation. To SHUN-ICHIRO ASAHARA, YUKA IHARA, HIROYUKI INOUE, KYOKO TERUY- examine the physiological role of O-GlcNAcylation in whole body glucose me- POSTERS tabolism, liver-, skeletal muscle-, adipose tissue- and pancreatic cell-specifi c Islet Biology/ AMA, MIZUKI HARA, MAKI KIMURA, TOMOKAZU MATSUDA, SUSUMU SEINO, β Insulin Secretion YOSHIAKI KIDO, Kobe, Japan Ogt-knockout mice were generated by crossbreeding Ogt-fl ox mice with al- Background: The relationship between pancreatic β-cell mass and the onset bumin-Cre, Mlc1f-Cre, adiponectin-Cre and tamoxifen (TM)-inducible Pdx1-Cre and progression of type 2 diabetes has received a lot of attention in recent years. mouse, respectively, and their metabolic phenotypes were examined.

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A578 ISLET BIOLOGY—APOPTOSISCATEGORY

Liver-, skeletal muscle- and adipose tissue-specifi c Ogt-KO mice showed structure distention by electron microscopy. We also found accumulation of normal growth patterns until 12 weeks of age. Fasting blood glucose (FBG), ubiquitinated proteins in lysates of islets of NOD mice. These observations intraperitoneal glucose and insulin tolerance tests showed little difference show that β-cells of NOD mice display impaired autophagy leading to ac- in glucose metabolism between these tissue-specifi c Ogt KO mice and con- cumulation of ubiquitinated proteins and p62. trol Ogt-fl ox mice. In contrast, TM-inducible β cell-specifi c Ogt KO mice (Ogt- βKO) showed a biphasic pattern of glucose metabolism. At 5 weeks of TM 2276-P injection, Ogt-βKO showed lower FBG levels with hyperinsulinemia and ex- β-Cell Death Occurs Transiently prior to the Development of Obesity- cess weight gain. However, at 10 weeks after TM injection, they developed induced Glucose Intolerance hyperglycemia with impaired insulin secretion and fewer insulin positive ESTHER M. BOLANIS, SARAH TERSEY, MARISA FISHER, FARAH MEAH, THOMAS cells, along with increased TUNEL-positive apoptotic cells in islets. B. FARB, KRISTER BOKVIST, KIEREN J. MATHER, RAGHAVENDRA G. MIRMIRA, Ogt-mediated protein O-GlcNAcylation in insulin sensitive organs affect- Indianapolis, IN ed whole body glucose metabolism minimally, whereas it was essential to In humans with type 1 diabetes (T1D) and animal models of T1D, β cell maintain cell viability. Our results provide new insight into the physiologi- β death is measurable as cell-free, unmethylated preproinsulin (PPI) DNA in cal role of O-GlcNAcylation in glucose metabolism and β cell biology. the circulation. However, the occurrence and timing of β cell death in obesity and type 2 diabetes (T2D) is largely unknown. In this study, we isolated cell- 2274-P free PPI DNA from a cross-sectional cohort of lean or obese/T2D humans, Involvement of Iron Depletion in Palmitate-induced Lipotoxicity to from a longitudinal cohort of C57BL6/J mice subjected to a low fat diet (LFD) Beta Cells or high fat diet (HFD) for 10 weeks, and from diabetic db/db mice. Droplet IKRAK JUNG, SUNG-E CHOI, YUP KANG, Suwon, Republic of Korea digital PCR was used to quantitate the methylation states at the -69 and High level of plasma free fatty acid (FFA) was thought to contribute to the loss of pancreatic beta-cells in type 2 diabetes. In particular, saturated -182 sites, respectively, of the PPI promoter in humans and mice. Compared FFA such as palmitate or stearate is able to induce beta cell apoptosis (li- to lean subjects, subjects with obesity or T2D demonstrated no differences potoxicity). Endoplasmic reticulum (ER) stress was suggested to be a criti- in the levels of either unmethylated or methylated PPI DNA, suggesting that cal mediator for the FFA-induced lipotoxicity. Recently, disorder in respira- in this cross-sectional study, the test groups had either no overt β cell death tory metabolism was also reported to be involved in the lipotoxicity. Since or that β cell death was occurring at a low rate. In mouse feeding studies, iron was critical for mitochondrial respiratory metabolism, our study was mice on a HFD gained more weight and demonstrated elevated fasting blood initiated to determine whether abnormal iron metabolism is involved in glucose levels (105±6 vs. 81±4, P<0.05) and glucose intolerance by GTT (AUC palmitate-induced INS-1 beta cell death. Immunoblotting analysis showed 35033±1054 vs. 21761±402, P<0.05) compared to mice on a LFD at the 6 week that treatment of INS-1 cells with palmitate reduced level of transferrin feeding time point. These differences in glycemia widened over the subse- receptor (TfR), but increased level of heavy chain ferritin (FTH). In accor- quent 4 weeks of feeding. Compared to LFD-fed mice, HFD-fed mice dem- dance with TfR down-regulation and FTH up-regulation, palmitate reduced onstrated transient increases in serum unmethylated PPI DNA at 2 weeks intracellular labile iron pool. Whereas iron depletion through treatment with of feeding (19±4 copies/µl vs. 8.5±2.5 copies/µl, P<0.05) and at 6 weeks iron-chelators such as deferoxamine (DFO) or deferasirox (DS) augmented of feeding (33±19 copies/µl vs. 9.5±5 copies/µl, P<0.05). No differences in palmitate-induced cell death, iron supplementation through treatment with methylated PPI DNA was seen at any time point. Overtly diabetic db/db mice FeCl3, FeSO4, or holo-transferrin signifi cantly protected palmitate-induced exhibited no differences in unmethylated or methylated PPI DNA compared death. Furthermore, overexpression of TfR reduced palmitate-induced death whereas knockdown of TfR augmented the death. In particular, treatment to control db/+ mice. Taken together, these data suggest that markers of β with DFO increased level of ER stress markers such as phospho-eIF2α, cell death may only be detectable in the period before onset of T2D, and may CCAAT/enhancer binding protein homologous protein (CHOP) and phospho- be most readily demonstrated in longitudinal studies of animals or people at c-Jun N-terminal kinase (p-JNK), and, furthermore treatment with chemical risk for progression to T2D. chaperone 4-phenylbutyrate (4-PBA) signifi cantly protected DFO-induced Supported By: American Diabetes Association (7-13-JF-56 to S.T.); Lilly cell death. Iron supplementation also demonstrated protective effect on Research Award (to R.G.M.); Human Islet Research Network (to R.G.M.); American palmitate-induced primary islet cell death. Our data suggest that iron deple- Physiological Society (to E.M.B.) tion plays a role in palmitate-induced beta cell lipotoxicity through induction of ER stress and that attempts to protect iron depletion may be a maneuver 2277-P to prevent beta cell loss in type 2 diabetes. MANF as a Therapeutic Target for Beta-Cell Survival JANA MAHADEVAN, FUMIHIKO URANO, St. Louis, MO 2275-P It has been established that endoplasmic reticulum stress (ER stress) plays Inhibition of Autophagic Turnover May Promote ER Stress and De- a role in beta cell dysfunction and death during the progression of type 1 and crease in Islet β-Cell Mass of Nonobese Diabetic Mouse Model type 2 diabetes. Thus, it would be important to prevent ER stress-mediated SHAKEEL MIR, ZACK GUINN, NORA E. SARVETNICK, Omaha, NE beta cell death for delaying the progression of diabetes. Here we show that Type 1 diabetes (T1D) is a multifactorial disease which is characterized by Mesencephalic Astrocyte derived Neurotropic Factor (MANF) confers pro- diminished insulin production since pancreatic β cells are killed by the im- tection against ER stress-mediated beta cell death and activates beta cell mune system. The clinical onset of T1D occurs when most of the β cells have proliferation. We found that MANF expression was induced by ER stress in been destroyed but the mechanisms leading to β cell destruction are not ful- beta cell lines and primary islets. The treatment of a rat beta cell line, INS-1 ly understood. Because endoplasmic reticulum (ER) stress has been shown 832/13 cells, with recombinant MANF peptide prevented cell death mediated to precede the onset of T1D, we hypothesized that impaired autophagy by ER stress and cytokine treatment. We sought to identify the mechanisms by could delay the appropriate unfolded protein response (UPR) and thus vul- which MANF conferred protection against ER-stress mediated beta cell death, nerability towards ER stress leading to pancreatic β cell death. Prediabetic and discovered that AKT signaling was involved in this process. Recombinant nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID MANF peptide activated AKT phosphorylation in beta cell lines in a dose- mice were utilized for monitoring autophagic fl ux. Haematoxylin and eosin dependent manner. Furthermore, we found that recombinant MANF protein (H&E) staining of islets from 8-week-old NOD-SCID and NOD mice did not stimulated the proliferation of human islets in culture. Our results suggest that reveal any degenerative changes in the islets of NOD mice, however, there MANF is a novel therapeutic target for beta cell survival and proliferation. was a marked increase in the accumulation of LC3B in the NOD mouse pan- Supported By: American Diabetes Association (1-12-CT-61 to F.U.) creatic islets compared to NOD-SCID mice. Furthermore, insulin and LC3B double staining showed greater co-localization in NOD mice compared to 2278-P the control mice. Western blotting analysis of lysates from islets of control Glucagon-Like Peptide-1 Receptor Agonist (GLP-1RA) Protects High NOD-SCID and NOD mice (8 wks) showed a marked increase in autophagy Glucose-induced β-Cell Apoptosis In Vivo and In Vitro through In- when assessed by conversion of LCB-I to LC3B-II in NOD islets. In islets of activation the NADPH Oxidase (Nox2) and the Related Signaling NOD mice, p62 accumulated, likely because it is not being degraded due to Pathway POSTERS defective autophagy. Insulin and p62 double staining showed co-localization MIN DING, YUN-ZHI XING, QIAN YU, CHUN-JUN LI, DE-MIN YU, Tianjin, China Islet Biology/ of insulin-depleted β cells with p62 suggesting that the expression of p62 Aims: Recently, studies have shown that Nox2 is the main source of ROS Insulin Secretion refl ects damaged β cells, and the possibility of impaired insulin secretion. production and plays a critical role in apoptosis of pancreatic β-cells. In this Islets from NOD mice also displayed increase in ER stress as well as ER study, we tested the hypothesis that GLP-1Ra protecting β cells apoptosis

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A579 ISLET BIOLOGY—APOPTOSISCATEGORY

was associated with inhibition the generation of ROS via inactivation of man islet amyloid polypeptide (hIAPP) transgenic and 18 non-transgenic) from 5 Nox2 and related Signaling Pathway. investigators, and observed a broad range of β-cell/islet area (35.8 to 72.2%), Methods: Diabetes was induced in rats by I.V. injection of streptozotocin after amyloid/islet area (0 to 9.4%) and β-cell apoptosis (0.05-0.41%). Within an 8 weeks of high fat diet. The animals were randomly divided into 3 groups: Nor- islet preparation, mean β-cell/islet and amyloid/islet areas did not vary mark- mal groups, and diabetes groups with or without liraglutide treatment. Hyper- edly when more than 10 islets were quantifi ed. However, variability of these glycemic clamp was performed to detect β-cell function. The histomorphology measures declined as up to 30 islets were analyzed, so that with 30 islets the changes of islets andβcells apoptosis were observed with Immunofl uorescent CVs for β-cell area/islet area and amyloid area/islet area were 2.53±0.22% and TUNEL staining respectively. The generation of ROS in Islets was quanti- and 14.9±3.53%, respectively. Increasing the number of islets analyzed from ties by the dichlorofl uorescein diacetate method. Key components of underlying 30 to 90 had a negligible effect on the CV of these measures. In contrast, the signaling pathways including the phosphorylation of AMPK and JNK, caspase-3 CV for β-cell apoptosis was 231±107% when 30 islets were counted. Based on and Nox2 were assayed with Western blot and immunohistochemical. data from these outcomes, when 30 hIAPP transgenic islets within a prepara- Results: βcell secretion and the ratio of α/β were signifi cantly improved tion are examined, 6 separate islet preparations are suffi cient to detect treat- and βcells apoptosis was also signifi cantly decreased compared to diabetic ment effects of 14% on β-cell/islet area, 30% on amyloid/islet area, and 22% groups. Levels of Nox2, ROS generation, and p-JNK, and caspase-3 activity on β-cell apoptosis. When 30 non-transgenic islets within a preparation are were signifi cantly lower and p-AMPK was signifi cantly higher in the lira- examined, 6 islet preparations are suffi cient to detect treatment effects of 9% glutide treatment group compared to diabetic islets. And ROS generation on β-cell/islet area and 45% on β-cell apoptosis. This information is of value was largely reversed by apocynin in vivo. The studies showed that high for the design of studies using isolated islets to compare β-cell area, islet amy- glucose induced JNK activation and AMPK inhibition which resulted in in- loid area and β-cell apoptosis within and between groups. creased Nox2, ROS generation and β cells apoptosis in vitro. Furthermore, Supported By: American Diabetes Association (7-11-MN-28 to S.E.K.) suppression of Nox2 with apocynin or JNK inhibitor could restore high glu- cose-induced ROS generation and βcell apoptosis. 2281-P Conclusions: The GLP-1Ra could increase AMPK activity and reduces the Cold Shipping Study to Improve Human Islet Transport Nox2-ROS-JNK axis in islets, leading to suppression glucotoxicity-induced JOYCE C. NILAND, JAMES CRAVENS, JANICE SOWINSKI, BARBARA OLACK, β cells apoptosis. JOHN KADDIS, GOWRI ARULMOLI, LAURA ZITUR, DAVID SCHARP, LOUIS FER- Supported By: National Natural Science Foundation of China (81300663) NANDEZ, PETER CHLEBECK, BRIAN HAIGHT, Duarte, CA, Irvine, CA, Madison, WI The NIDDK-funded Integrated Islet Distribution Program (IIDP) Coordinating 2279-P Center oversees distribution of human islets for diabetes research. A 2007 IIDP Large Mafs Modulate Autophagic Activity and ER Stress in Diabetic study validated a standardized protocol to ship human islets from isolation Pancreas centers to researchers, used successfully through 2014. Due to shortages of MARIKO TSUCHIYA, RYOICHI MISAKA, KOSAKU NITTA, KEN TSUCHIYA, Tokyo, required shipping supplies and the IIDP’s mission to provide the highest qual- Japan ity islets, a shipping study was conducted to compare the IIDP protocol to a It has been recognized that hyperglycemia damages biological cell functions cold temperature method developed by Prodo Labs Inc., used in shipments such as autophagy or response to ER stress by affecting signal transduction. of 46 million human islets worldwide. The new method requires longer pre- Recently, glucose toxicity has been shown to deteriorate pancreatic cell func- shipment culture time (~48 vs. 12 hrs) for adequate islet healing from isolation tion in which molecular mechanism the expression level of PDX-1 and MafA insults, use of a proprietary media (Prodo Islet Media vs. CMRL), and lower are decreased. We previously observed that other mafs including MafA also temperature to help avoid degradation (6 vs. 15°C). Study endpoints were islet were suppressed in the diabetic pancreas; thus, we speculated that primary quality at receipt and post 24 hrs, convenience of handling, and cost savings. biological cell activities such as autophagy or response to ER stress were af- The experimental design required 2 IIDP islet distribution centers to perform 2 fected according to the change in the expression level of mafs in pancreatic isolations each, divide the yields, culture, and ship 5000 IEQ/isolation/method cells. This was supported by some reports that the ER stress sensors ATF6 to 10 recipients who provided feedback. While individual islet quality rankings and PERK alter the expression level of mafA. Gene profi ling performed after showed no preference, the cold shipping protocol was preferred more often in vivo modulation of mafs expression levels revealed up-regulation of Nupr1 for handling and overall by 8 of the 9 responding laboratories (89%, Table 1). and Ddit3 and downregulation of Hsp8 in mafB siRNA-treated pancreas. Be- Based on these results, the cold shipping protocol has been adopted as a cost- cause MafB locates up stream of MafA in the pancreatic cell lineage, mafs effective replacement for the IIDP. other than mafA are likely to be implicated in the primary biological cell func- Table 1. Comparison of 2007 IIDP (15°C) and experimental cold (6°c) ship- tions. Transfection of mafB siRNA to pancreatic cells (AsPc-1 or BxPC3 cells) ping protocols for 35 shipments*. prominently accelerated autophagy, as assessed by LC3 expression using im- Preference Based on: # Shipments with # Shipments with munostaining, regardless of small changes in cell cycling (BrdU uptake), cell 2007 IIDP Shipping Cold Shipping # Shipments with senescence (beta Gal staining), and apoptosis (Tunnel assay). Regarding cell Protocol Preferred Protocol Preferred No Preference function, although no difference was observed during the steady state, cell N (%) N (%) N (%) proliferation under the stress condition, as assessed by cell scratch assay, was Islet Quality Rankings markedly diminished in mafB-suppressed cells. Addition of Quercetin, a Wnt signaling inhibitor, restored cell migration and proliferation. In addition, the Upon Receipt (total N=35) 2 (5.7%) 9 (25.7%) 24 (68.6%) restration of autophagy was accompanied by suppression of Nupr1 and ATF6 Post 24 Hours (total N=33) 2 (6.1%) 3 (9.1%) 28 (84.8%) by Quercetin treatment in mafB siRNA-treated cells, suggesting that Quece- Handling Convenience 2 (5.7%) 17 (48.6%) 16 (45.7%) tin play a role in regulation of autophagy and response to ER stress. Taken Overall Preference/Laboratory (N=9) 1 (11%) 8(89%) – together, large mafs modulate biological cell activities such as autophagy or response to ER stress under disrupted glucose metabolism. Projected Savings Supported By: Japan Society for the Promotion of Science (C23592021) Projected 2015 Shipping Cost $119,151 $43,062 $76,089 (64%) *5 Recipients were unavailable to receive shipments for fi nal isolation 2280-P Supported By: National Institutes of Health Optimal Sample Size for Quantifi cation of Beta-Cell Area, Amyloid Area, and Beta-Cell Apoptosis in Isolated Islets ANDREW T. TEMPLIN, DANIEL T. MEIER, MEGHAN F. HOGAN, LEON ENTRUP, 2282-P Islet Harvesting in Carbon Monoxide-saturated Mediums Protects THANYA SAMARASEKERA, SAKENEH ZRAIKA, EDWARD J. BOYKO, STEVEN E. Them from Hypoxia-induced Cell Death KAHN, Seattle, WA DO-SUNG KIM, LILI SONG, ZHEN SUN, JINGJING WANG, DAVID B. ADAMS, Cultured islets are often used for histochemical assessments of β-cell area, HONGJUN WANG, Charleston, SC amyloid area and β-cell apoptosis. Knowing the variability of these measures permits determination of the number of islets needed to generate a specifi c Many studies have shown carbon monoxide (CO) functions as a strong data set. We thus determined how the number of islets examined infl uences therapeutic molecule for the treatment of various diseases, due to its anti- infl ammatory and anti-apoptotic properties. We assessed the effi cacy POSTERS mean values of -cell area, amyloid area and -cell apoptosis (using insulin, Islet Biology/ β β and mechanisms of CO-saturated medium-based islet isolation (COMBII)

Insulin Secretion thiofl avin S and propidium iodide positive staining, respectively), the coeffi - cient of variation (CV) thereof, and the sample size required to detect effects in hypoxia-induced mouse and human islet/β cell death. Gaseous CO was on these outcomes. We analyzed 39 individual mouse islet preparations (21 hu- bubbled into enzyme solution and RPMI-1640 medium used for islet isola- tion to generate CO-saturated mediums. Islets were isolated using either

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A580 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH regular mediums (control islets) or CO-saturated mediums (COMBII islets). receptors and transcription factors expression. Strikingly, genetic lineage The potential effects of COMBII on hypoxia (1% O2)-induced apoptosis were tracing revealed that the signaling competent Fltp- β-cell pool compensates assessed in vitro. COMBII methods prevented hypoxia-induced islet/β cell for physiological insulin demand during pregnancy, whereas the metaboli- loss, as evidenced by decrease in LDH release and cytoplasmic DNA frag- cally active Fltp+ β-cell pool accounts for islet hypertrophy after high-fat ments. COMBII islets showed higher insulin secretion in response to glucose diet. Thus, our study reveals a novel unrecognized PCP-mediated heteroge- challenge compare to control islets. Hypoxia-induced elevation of ER stress- neity among β-cells, characterize by Fltp expression. It also illustrates that related protein, CHOP, and autophagy related protein, LC-3II, were abrogated the three-dimensional islet architecture represents a niche that contains in COMBII islets. Moreover islets when exposed to hypoxia showed induc- Fltp- proliferative progenitors and Fltp+ mature β-cells. This is the basis tion of pro-apoptotic proteins (Bim, PARP, Caspase-3) and pro-infl ammatory for future pharmacological targeting of distinct β-cell subpopulations that gene (TNF-α). COMBII islets reversed upregulation of those molecules, to- should allow triggering their distinct biological features, such as maturation, gether with an increase of the anti-apoptotic gene Bcl-2, at protein level, proliferation, and secretion - or even regeneration after chronic insult. as analysed by Western blot. Similar results were observed in both mouse and human islets. Therefore, COMBII preserves survival and function of iso- & 2285-P lated islets from hypoxia-induced islet/β cell death. This simple yet effi cient Insulin Receptor Antagonism Results in Increased Replication of method provides a valuable proof of concept for the potential exploitation of both Endocrine and Non-endocrine Pancreatic Cells islet isolation in clinical settings for the treatment of type 1 diabetes. MATTHEW M. RANKIN, SEUNGHUN P. LEE, FUYONG DU, THOMAS KIRCHNER, Supported By: National Institutes of Health-National Institute of Diabetes and MEGHAN TOWERS, CATHERINE LEE MAY, YIN LIANG, LISA NORQUAY, KEITH DE- Digestive and Kidney Diseases MAREST, ALESSANDRO POCAI, Spring House, PA There has been a high level of attention paid to the use of insulin receptor 2283-P antagonist S961 and its resulting compensatory increase in β-cell replica- Pamidronate Induces Apoptosis in Pancreatic Beta Cells tion. Reports have shown variability in the consistency and magnitude of LU LIU, RUI WANG, CHUNYAN LIU, PENG YANG, XIAOLI ZHANG, YILI LUO, SHEN the effect on β-cell replication, as well as called into question the mecha- QU, HONG LI, Shanghai, China nism which mediates the proliferative response. In an effort to address the Bisphosphonates (BPs) are widely used to treat osteoporosis and are utility of insulin receptor antagonism as a model of β-cell mass expansion highly effective in reducing the risk of fractures. Except for the inhibition of we employed continuous administration of an insulin receptor antagonist bone resorption, recent research indicates that BPs could induce cell death peptide (JNJ-IRa) via subcutaneous osmotic pump for 7 days. Treatment in other cell types including myeloma, breast cancer cells, and prostate of lean C57BL/6 mice with JNJ-IRa resulted in pronounced hyperglycemia cancer cells, among others. As an age related disease, osteoporosis often throughout the course of the experiment that was accompanied by a >25- accompanies T2DM. Therefore, it is essential to clarify the effect of BPs on fold increase in plasma insulin at day 7. A signifi cant increase in cumulative pancreatic beta-cells in patients with T2DM. In this study, pamidronate was food intake was observed in treated vs. control animals, which did not trans- selected to explore the effect on pancreatic INS-1 beta-cells in vitro. INS-1 late into body weight differences between the two groups. As expected, cells were cultured and treated with different concentrations of pamidronate hyperglycemia/hyperinsulinemia was accompanied by a signifi cant increase (0, 10-5 mM, 10-4 mM, 10-3 mM, and 10-2 mM), and then CCK-8 assay was in β-cell replication and expansion of β-cell mass. A 5-fold increase in Ki67/ performed to assess cell viability after incubation. Annexin-V/PI staining and insulin co-positive cells and a 1.6-fold increase in β-cell mass was observed cell cycle analysis were also performed after the cells were treated. The in the treatment group. The effect on β-cell replication in treated mice was CCK-8 assay results indicated that the OD values decreased with increasing not accompanied by altered Betatrophin/ANGPTL8 mRNA in liver nor white concentration of pamidronate, with the values for each day corresponding adipose samples. Increased overall Ki67 staining with highly proliferative to increasing pamidronate concentrations. When treated with 10-3 mM or pockets of cells was observed in pancreas sections of all JNJ-IRa-treated 10-2 mM pamidronate for 4 days, cell death was found in all cells assessed. mice. Upon quantifi cation, continuous JNJ-IRa administration was found to The Annexin-V/PI staining assay(day 3)showed that rates of apoptosis were result in a signifi cant 3.2-fold increase in replication of non-endocrine pan- 1.34%, 7.94%, 12.98%, 23.58%, and 28.42%, respectively corresponding creatic cells. In summary, continuous administration of an insulin receptor with increasing doses of pamidronate. The cell cycle analysis revealed that antagonist consistently increases β-cell replication in mice, however the the proportion of cells in G2-phase was 12.62%, 10.47%, 9.94%, 6.77% and proliferative response is not specifi c to endocrine cells nor dependent on 0%(day 3). This indicated that pamidronate can inhibit proliferation and in- altered expression of Betatrophin/ANGPTL8. duce apoptosis in INS-1 cells in a dose and time dependent manner in vitro. In view of these results, we speculated that the use of pamidronate could & 2286-P exert negative effects on pancreatic beta cells. The function of beta cells In Vivo Hes-1 Knockdown Stimulates Beta-Cell Regeneration in Rat in T2DM patients should be carefully observed when treated with BPs. with Severe Beta-Cell Defi ciency Whether bisphosphonates may exert any infl uence on pancreatic islets in MOHAMAD ALAWIEH, MUNKHZUL GANBOLD, ANISSA ILIAS, DANIELLE BAILBÉ, vivo needs further exploration. BERNARD PORTHA, JAMILEH MOVASSAT, Paris, France Beta cell loss is a root cause for the development of diabetes. Under- standing the mechanisms that govern beta cell regeneration is of major in- ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND terest for cell therapy of diabetes. Notch/Delta signalling pathway is a cru- POSTNATAL GROWTH cial mechanism in the determination of the endocrine versus exocrine fate of pancreatic precursor cells during development. Our aim was to establish whether post-natal down regulation of Hes-1, a pro-exocrine partner of the Guided Audio Tour: Islet Development and Growth (Posters: 2284-P to Notch/Delta pathway, is an effi cient means to promote beta cell regenera- 2290-P), see page 13. tion in two models of severe beta cell defi ciency in rat. We developed an in vivo approach based on local silencing of Hes-1, using antisense morpholino- & 2284-P oligonucleotides in 90% pancreatectomized (90% Px) adult rats, as well as Targeting Beta-Cell Heterogeneity in the Islet of Langerhans in neonatal streptozotocin-induced diabetic rats, and evaluated its impact ERIK BADER, ADRIANA MIGLIORINI, MORITZ GEGG, MARTIN IRMLER, JO- on the regenerative potential of beta cells. Beta-cell mass was evaluated HANNES BECKERS, HELENA CHMELOVA, JULIE A. CHOUINARD, STEPHAN by morphometry. Cell proliferation was assessed by BrdU incorporation SPEIER, HEIKO LICKERT, Garching, Germany, Munich, Germany, Dresden, Germany method. Cell apoptosis was determined by TUNEL assay. The expression of It is long known that pancreatic β-cells differ in size, glucose responsive- Sox9, Ngn3 and PDX1 was evaluated by immunohistochemistry. Insulin se- ness and insulin secretion, but the molecular mechanisms underlying func- cretion in vivo was assessed during an intravenous glucose tolerance test. tional β-cell heterogeneity are unknown. Here, we report a newly identifi ed We showed that Hes-1 knockdown leads to increased functional beta-cell planar cell polarity (PCP) effector gene Flattop (Fltp), displaying heteroge- mass in 90% Px rats by promoting beta-cell proliferation and differentia- neous expression in all islet-endocrine cells. In β-cells, Fltp expression in- tion. Similar results were observed in neonatal streptozotocin-diabetic rats crease from 40% to 80% during post-natal β-cell maturation, suggesting which exhibited enhanced regenerative potential of beta cells, refl ected by POSTERS

that acquisition of 3D architecture and planar polarization correlates with increased beta cell mass, proliferation and neogenesis. Taken together, our Islet Biology/ functional maturation. Whole genome gene chip analysis revealed remark- study reveals that post-natal modulation of the Notch/Delta signalling path- Insulin Secretion able differences between Fltp+ and Fltp- endocrine cells in maturation mark- way through Hes-1 knock down, is a promising approach for the stimulation ers, glucose transporter, mitochondria genes, metabolic enzymes, signaling of beta cell regeneration in the context of cell defi ciency.

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A581 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

Surface availability was assessed by fl ow cytometry of the human beta & 2287-P cell line EndoC-βH1. The density of GPR44 receptor in endocrine (isolated Constant Increase of ER Stress Caused by a Novel Heterozygous human islets and human beta cells) and exocrine tissue was assessed by the Mutation of WFS1 Gene tritiated GPR44 ligand AZD3825. A direct comparison with the putative beta SHUNTARO MORIKAWA, AKIE NAKAMURA, KATSURA ISHIZU, TOSHIHIRO TA- cell marker VMAT2 was performed by radiolabeled [3H]DTBZ. JIMA, Sapporo, Japan, Tokyo, Japan GPR44 was available on the cell surface, and pancreatic RNA levels were Wolfram syndrome (WS) is a disorder characterized by the association of restricted to the islets of Langerhans. [3H]AZD 3825 had nanomolar affi nity early-onset, insulin-dependent diabetes mellitus (DM), diabetes insipidus, for GPR44. The specifi c binding to human islet endocrine preparations was deafness, and progressive optic atrophy. The disease is caused by mutations close to 20 times higher than in exocrine preparations (B). The endocrine-to- of WFS1 located on 4p16 encoding protein that is called WFS1 (wolframin). exocrine binding ratio was approximately 10 times higher for [3H]AZD 3825 This protein is expressed especially in pancreatic islet β-cells and the resi- than for [3H]DTBZ (B). Saturation binding studies showed that the GPR44 dent component of the endoplasmic reticulum (ER) membrane. receptor density (Bmax) in pure human beta cells (C) was >40 times higher When abnormal WFS1 caused by mutation of WFS1 results in ER dysfunc- than in exocrine tissue (D). tion, misfolded and unfolded proteins accumulate in the ER, which leads to In conclusion, GPR44 is a highly beta cell specifi c target, which potentially cell apoptosis (known as ER stress). offers improved beta cell contrast compared to VMAT2. Isotopic radiolabel- We report a Japanese female WS patient with novel heterozygous muta- ing of AZD 3825 for position emission tomography is ongoing. tion in WFS1. She was admitted to our hospital for poor weight gain and DM. Her growth failure was evident at 3 months old and congenital cataract was no- ticed at 7 months old. Her auditory brainstem response (ABR) test revealed severe bilateral hearing loss. Direct Sequence analysis of her WFS1 showed a heterozygous twelve bases deletion in exon 8, resulting in 3 amino acid in- frame deletion (c.973_984del12, p.N325_M328del). As her parents did not have the deletion, the mutation presumably occurred de novo. The functional consequence of the mutant WFS1 identifi ed in this study and previously re- ported (p.H313Y, p.W314R, p.Q194X, p.L543R) were analyzed using ATF6α- luciferase and GPR78-luciferase vector in vitro. As the result, it revealed that the mutant WFS1 in this patient lost the suppression activity of ER stress re- sponse due to the activation of ATF6α, resulting in the constant increase of ER stress. While most WFS1 mutations in WS patients are detected on both alleles and the inheritance of WS is considered to be autosomal recessive, we detected novel heterozygous WFS1 mutation as a cause of WS.

& 2288-P Reprogrammed Gastrointestinal Stem Cells as a Renewable Source of Insulin + Beta Cells QIAO ZHOU, CHAIYABOOT ARIYACHET, ALESSIO TOVAGLIERI, DAVID BREAULT, CATIA VERBEKE, DAVID MOONEY, Cambridge, MA, Boston, MA Supported By: JDRF The adult gastrointestinal (GI) tract contains stem cells that generate a constant supply of new cells in the normal turnover of the GI epithelium. & 2290-P Among the GI epithelial cells are hormone-secreting enteroendocrine cells. Differential Effects of HGF on Infl ammation in Autoimmunity We have devised a strategy to reprogram GI stem cells in vivo and in vitro GINA M. COUDRIET, ANGELA CRISCIMANNA, FARZAD ESNI, JON D. PIGANELLI, with defi ned genetic factors to produce functional insulin+ cells at the ex- Pittsburgh, PA pense of enteroendocrine cells. Using inducible expression of three beta-cell In type 1 diabetes (T1D), recognition of autoantigens leads to pro-infl am- reprogramming factors (Ngn3, Pdx1, and MafA) in mouse genetic models, we matory macrophage differentiation promoting β cell death. However, if the discovered that both gastric and intestinal stem cells can be reprogrammed infl ammation is diminished, endogenous β cell growth can occur. In fact, a to produce insulin+ cells. The induced GI insulin+ cells express key beta cell more controlled infl ammatory response appears to be necessary to induce genes and show robust glucose-responsiveness. Surprisingly, molecular and the regenerative process, characterized by a transition of pro-infl ammatory functional analyses indicate that gastric insulin+ cells more closely resemble macrophages, to an anti-infl ammatory phenotype. Macrophages from auto- endogenous pancreatic beta cells compared with intestinal insulin+ cells. immune-prone mice (NOD) exhibit a differential cytokine and growth factor Gene profi ling studies suggest that this difference likely results from differ- expression profi le compared with immune-competent mice (B6). Our pub- ences in the endogenous endocrine programs of the gastric versus the intes- lished fi ndings demonstrated that macrophage derived regenerative cues, tinal system. The induced GI beta cells can rapidly regenerate from the stems like cytokine switch and growth factor expression, are absent in autoim- cells and are thus capable of maintaining normoglycemia for many months in munity, prolonging infl ammation. Therefore, in this study, we hypothesized diabetic animals even after repeated ablation. To explore therapeutic poten- that the specifi c loss of hepatocyte growth factor (HGF) signaling in autoim- tial of the induced GI beta cells, we created bioengineered stomach tissues munity may to contribute to an exacerbated pro-infl ammatory macrophage using polyglycolic acid (PGA) scaffolds seeded with isolated gastric units or phenotype, and a lack of regenerative capacity by the β cells in the NOD cultured gastric stem cells engineered to express the reprogramming fac- model. Bone marrow derived macrophages (BMφ) from both B6 and NOD tors. Upon transplantation into diabetic animals, the engineered stomach mice were cultured in vitro and stimulated with 100 ng/mL LPS with or with- spheres produced insulin+ cells and suppressed hyperglycemia. These stud- out HGF treatment (10 ng/mL). Protein and gene expression analysis of key ies indicate that engineered GI stem cell could provide a renewable source targets including: TNFα, IL-1β, CCL2, and iNOS, were performed. The modu- of functional beta cells for diabetes studies and treatment. lation of these factors is critical to examine, as they are most responsible for Supported By: Harvard Stem Cell Institute initiating insulitis and β cell destruction. We observed suppression of these pro-infl ammatory genes in the presence of HGF in the B6 BMφ. Alternatively, & 2289-P NOD BMφ demonstrated either an entire loss of suppression, or sub-optimal GPR44 as a Target for Imaging of Beta-Cell Mass suppression of these factors. These results support our hypothesis that BMφ EWA HELLSTRÖM-LINDAHL, ANGELIKA DANIELSSON, FREDRIK PONTÉN, PAUL from the NOD mouse are resistant to the anti-infl ammatory effects of HGF, CZERNICHOW, OLLE KORSGREN, LARS JOHANSSON, OLOF ERIKSSON, Uppsala, likely impeding normal β cell regeneration in vivo. Future studies involve the Sweden, Paris, France use of NOD mice that overexpress HGF specifi cally in the islet in order to To address questions regarding onset and progression of diabetes, surro- determine if HGF is protective to islets upon exposure to pro-infl ammatory

POSTERS gate imaging biomarkers for beta cell mass are urgently needed. GPR44 has cytokines during T1D onset and progression. Islet Biology/ Supported By: JDRF

Insulin Secretion been identifi ed as a beta cell specifi c biomarker through proteomic screening (A). Here, we assessed the potential of GPR44 as a surrogate marker for beta cells, in a direct comparison with clinically used biomarker VMAT2.

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A582 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

2291-P (iPSCs) could serve as a powerful model to interrogate mechanism(s) and CC401 as a Small Molecule Positive Control for Human β-Cell Pro- characterize the pathophysiology of various diseases. Here, we report the liferation Assays generation of iPSCs from skin fi broblasts of a MODY8 patient with diabetes BRIAN RADY, SEUNGHUN P. LEE, LISA NORQUAY, ALESSANDRO POCAI, Spring compared to a family member with the mutation but without diabetes and House, PA a healthy family member. To avoid genomic integration of foreign DNA, we Total or partial β-cell defi cit underlies the pathophysiology of type 1 and applied episomal reprogramming by nucleofecting fi broblasts with a combi- type 2 diabetes. Despite signifi cant efforts to identify a safe approach for nation of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA β-cell mass expansion there has been little progress to date towards a vi- for TP53. The effi ciency of reprograming was higher in fi broblasts from sub- able therapeutic approach. Advancement in this research space has been jects without diabetes compared to the subject with diabetes (subject with hampered by multiple factors including a lack of translatability between ro- diabetes 0.00002±0.00001 vs. subject without diabetes 0.00016±0.00003, dent and human models, poor understanding of the human β-cell cycle, and p=0.001 and vs. healthy individual 0.00036±0.00014, p=0.046, n=8-18). Mul- the inherently low proliferation rate of human β-cells. These issues serve to tiple iPSC clones derived from each subject (n=2-8), were morphologically highlight the importance of primary human islet work. To this end, we have indistinguishable from other reported human iPSCs, and expressed pluripo- formalized an automated, EdU-based, 96-well human β-cell proliferation as- tency markers including OCT4, SOX2, NANOG, SSEA4, and TRA-1-81. We say. Using this assay we have characterized the effect of CC401, a small confi rmed the presence of the single-base deletion (1686delT) in exon 11 molecule JNK inhibitor on human β-cell proliferation. CC401 reproducibly of the CEL gene in the karyotypically normal MODY8-iPSCs. In conclusion, induced proliferation of insulin positive human β-cells an average of 5-fold we report the successful generation of patient-specifi c and integration-free over untreated cells across 4 human islet donors. CC401 was non-specifi c in iPSCs from individuals of a MODY8 family, which provides an invaluable re- its induction of proliferation, as non-insulin staining cells also demonstrated source to model endocrine versus exocrine defects in this form of monogenic signifi cantly increased replication rates. Using this method we have been diabetes. able to rapidly assess the proliferative effect of potential therapeutic tar- gets in human islets. In summary, CC401 is a readily available small molecule 2294-P that we propose as a suitable positive control in human β-cell proliferation Specifi city Protein 1 (SP1) Is Essential for Glucose-mediated β-Cell assays permitting the comparison of islet proliferative potential between Replication: Modulation by Protein Kinase C (PKC) Zeta (ζ) donor samples. CAROLINA ROSSELOT, JAYALAKSHMI LAKSMIPATHI, JUAN CARLOS. ALVAR- EZ-PEREZ, FRANCISCO RAUSELL-PALAMOS, PILI ZHANG, DONALD K. SCOTT, 2292-P ADOLFO GARCIA-OCAÑA, New York, NY SP1 is a transcription factor that binds to GC-rich boxes and regulates WITHDRAWN the expression of genes implicated in cell growth, differentiation, apoptosis and angiogenesis. Recent evidence indicates that PKC ζ phosphorylates SP1 in non-β cells and modulates its transcriptional activity. PKC ζ activation increases β cell replication in young and old mouse islets, and more impor- tantly in human islets. Glucose induces rodent and human β cell replica- tion in vitro and in vivo and stimulates PKC ζ activity. Thus, we wondered whether SP1, downstream of PKC ζ, is involved in glucose-mediated β cell proliferation. Glucose (20mM) signifi cantly and potently increased SP1 phosphoryla- tion and DNA binding activity in INS-1 cells. These effects were eliminated when cells expressed a kinase-dead form of PKC ζ (KD-PKC ζ), indicating that PKC ζ activity is required for glucose-induced SP1 activity in β cells. We have shown that glucose-mediated increase in β cell proliferation is PKC ζ dependent. Since PKC ζ modulates glucose-induced SP1 transcrip- tional activation, we wondered whether impairment of SP1 DNA binding with mithramycin (MTM) or downregulation with siRNA could block glucose- mediated β cell proliferation. MTM or siRNA-mediated SP1 downregulation signifi cantly inhibited INS1 cell replication induced by 20mM glucose. Im- portantly, adenovirus-shRNA-mediated downregulation of SP1 signifi cantly reduced glucose-induced human β cell replication in vitro. Glucose-medi- ated upregulation of Cyclin D2 mRNA was signifi cantly impaired by MTM or siRNA-mediated downregulation of SP1. SP1 binding to GC boxes in the Cyclin D2 promoter was increased by glucose and blocked by KD-PKC ζ, in- dicating that glucose-induced Cyclin D2 mRNA expression in β cells requires the PKC ζ-SP1 pathway. In conclusion, glucose-mediated β cell replication requires SP1 activity. Modulation of the PKC ζ-SP1 pathway specifi cally in β cells can be of thera- peutic value for diabetes. Supported By: National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (R01DK077096)

2295-P Human Bone Marrow Facilitates Human Islets Function and Lon- gevity by Observing Paracrine Effects FENG WU, SOURIYA VANG, JOHN LUO, LUGUANG LUO, Providence, RI 2293-P Islets transplantation holds promise as a long term treatment to type 1 Generation and Characterization of MODY8 Disease-Specifi c iPSCs diabetes. We have previously reported that bone marrow (BM) can support SEVIM KAHRAMAN, ADRIAN K. TEO, NICHOLAS JACKSON, RACHAEL MARTI- human islets function and longevity, but the mechanisms are not well under- NEZ, HELGE RAEDER, ROHIT N. KULKARNI, Boston, MA, Singapore, Singapore, stood. We hypothesize that BM exerts benefi cial paracrine effects on the in- Bergen, Norway jured islet by releasing cytokines and growth factors. Evidence from our pre- MODY8 (maturity onset diabetes of the young, type 8) is a dominantly vious studies supports the hypothesis that BM, through paracrine function, inherited monogenic form of diabetes and exocrine pancreas dysfunction. facilitates human islet vascularization and β-cell regeneration. By exploring POSTERS

This disease is associated with frameshift mutations in the carboxyl ester cytoprotective effect of conditioned medium and screening microRNA from Islet Biology/ lipase (CEL) gene expressed mainly in pancreatic acinar tissue. However, islets, we found that islets/BM contained high levels of growth factors in- Insulin Secretion the mechanism underlying diabetes development in MODY8 patients is cluding VEGF, KGF and PDGF. Interestingly only PDGF remain consistently unknown. Current approaches indicate that induced pluripotent stem cells elevated throughout the entire culture period (210 days) and correlates with

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A583 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

microRNA-24-2 (mir-24-2 0.72 vs. islet only 0.02 and BM only 0.38). Based secretion (miR 342), insulin resistance (miR 143), and obesity (miR 219). To on the fi ndings from our data and others, we proposed a signal transduc- investigate the effects of miR 143 on insulin secretion, we transfected islet tion pathway for BM facilitating islet proliferation by mir-24-2 suppressing cells from control offspring with miR 143 mimic. Transfection of control islet p16INK4a activation. The proposed pathway was supported by identifi cation cells with miR-143 mimic reduced the level of insulin secreted in response to of p16INK4a activation in RT-PCR, western blot and IHC; which show the low glucose, suggesting that miR 143 plays an important role in regulating insulin expression of p16INK4a in co-culture group vs. islet only and consequently in- secretion. Collectively, these data suggest that LP diet during the last week ducing PDX-1 gene expression, a pancreatic β-cell developmental gene. The of gestation is critical and suffi cient to program tissues to affect glucose discovery from current studies revealed BM, through paracrine activation, to homeostasis by inducing permanent changes on specifi c set of miRs. regulate mir-24-2 and PDGF levels in islet synergistically to activate PDX-1 to Supported By: National Institute of Diabetes and Digestive and Kidney Diseases induce β-cell regeneration. BM, mir-24-2, PDGF and PDX-1 pathway may play a critical role in BM repairing and promoting islet longevity. Further studies 2298-P will be required to identify detail of the signal transduction to develop new Distinct Islet Pathology of Pancreatic Islets in Type 2 Diabetes De- protocol for clinical application. veloped in Super Elderly Subjects Supported By: National Institutes of Health (1R01DK097380-01A1) ANN XING, HIROKI MIZUKAMI, WATARU INABA, TARO YOSHIDA, SOROKU YAGI HASHI, Hirosaki, Japan 2296-P There emerges a dramatic increase in elderly patients with type 2 dia- Ontogeny of Ins+Glut2- β-Cell Progenitors in the Human and Mouse betes (T2DM). The basis for the increase is ascribed to decreased organ Pancreas volume, low physical activity or defective insulin secretion. The above fea- CHRISTINE A. BEAMISH, BRENDA J. STRUTT, SUBRATA CHAKRABARTI, SOFIA tures may suggest that different pathogenesis underlies the development MEHTA, MANAMI HARA, PIOTR WITKOWSKI, MICHAEL MILLIS, DAVID J. HILL, of T2DM in elderly patients. It still remains unclear, however, whether there London, ON, Canada, Chicago, IL are distinct pathological changes in the islet in elderly T2DM. In this study, The presence and location of pancreatic β-cell progenitors that could be we attempted to characterize islet changes in elderly T2DM patients. To exploited for the reversal of diabetes is controversial. A rare subpopulation this end, pancreases from 23 young non-diabetic (Y-ND) (mean 25.8 y.o), 21 of Insulin+ Glucose transporter-2- (Ins+Glut2-) cells has been identifi ed as middle-aged non-diabetic (M-ND) (mean 60.2 y.o.), 24 middle-aged diabetic pancreatic progenitors in adult mouse and human islets. We have recently (M-DM) (mean 59.5 y.o., (onset of DM mean 46.1 y.o.)), 29 super-elderly non- shown a signifi cantly higher population of Ins+Glut2- cells in extra-islet diabetic (SE-ND) (mean 89.6 y.o.), and 10 super-elderly diabetic subjects β-cell aggregates (BCA, <5 β-cells) of neonatal mouse pancreas compared (SE-DM) (mean 88.5 y.o., (onset of DM 80.3 y.o.)) were investigated. On the to islets, which can differentiate into multiple lineages. Here, we sought to immunostained sections for insulin and glucagon, β cell and α cell masses identify the presence of these progenitor cells in mouse and human pan- were quantitated. The expressions of insulin transcriptional factors (TFs) creata, and how their abundance and location changes with age. Mouse such as pancreatic and duodenal homeobox 1 (PDX1) or MafA were also ex- pancreata were collected at postnatal (P) days 7, 14, 21, 28, and at 3, 6, 12, amined. Insulin mRNA expression was evaluated by in situ hybridization. As and 18 months of age. Human pancreas samples were examined at 22-30 a result, we found a high frequency of amyloid deposition in SE-DM (70.0%) weeks gestation, infancy (0-1 year), childhood (2-9 yr), adolescence (10-17 compared to M-DM (30.2%) and SE-ND (7.3%). Compared to M-ND, β cell yr), and adulthood (18-40, 41-64, and 65-80 yr). Samples (n ≥ 3/ timepoint) mass was reduced ca 30% in M-DM. In contrast, there was a signifi cant in- were analyzed by immunohistochemistry for the expression of insulin, glut2, crease in α cell mass in M-DM. The β cell mass in SE-DM was dramatically and ki67. In mice, the total number of Ins+Glut2- cells decreased after P7, reduced by 60% compared to SE-ND (p<0.01). The expressions of PDX1 and whilst the abundance of BCA also decreased (16.1 ± 0.8 vs. 2.1 ± 0.7% of MafA were most prominent in Y-ND and well preserved in M-ND. Islets in Ins+ cells, P7 vs. 18 mo, p < 0.005). The Ins+Glut2- cells were consistently M-DM and SE-ND as well as SE-DM all exhibited reduced expressions of TFs more abundant than Ins+GLUT2+ cells in BCA than in islets. A signifi cant to similar extents compared to those in M-ND (p<0.05). These changes of increase in Ins+Glut2- cells occurred in BCA, but not islets, between P7 and TFs were associated with decreased insulin mRNA expression. Collectively, P21 (16.1 ± 2.6 vs. 21.3 ± 2.1%, P7 vs. P21, p <0.05), but decreased thereafter. current results indicate that the pathogenesis of elderly onset T2DM may be The proportion of BCA was higher in human pancreas than in mouse at all distinct from that of middle aged T2DM, characterized by less implication of ages examined, and decreased signifi cantly at adolescence (28.4 ± 2.2% vs. TFs and more robust amyloid deposition. 5.9 ± 0.8%, 0-1yr vs. 10-17yr, p <0.005). Ins+Glut2- cells in human pancreas showed a similar distribution and ontogeny, being more abundant in BCA 2299-P than islets at all ages sampled, and decreasing with age (15.1 ± 3.6 vs. 7.1 ± Senescence-like Phenotype in Pancreatic β-Cells in Diabetic Mu- 1.6%, 2-9 yr vs. 65-80 yr, p <0.05). These data suggest that Ins+Glut2- cells tant Cryptochrome1 Transgenic Mice are preferentially located within BCA throughout life in both mouse and hu- SATOSHI OKANO, AKIRA YASUI, KIYOSHI HAYASAKA, MASAHIKO IGARASHI, man pancreas, and may represent a source of β-cell plasticity. OSAMU NAKAJIMA, Yamagata, Japan, Sendai, Japan, Kaminoyama, Japan Cryptochrome (CRY) proteins play indispensable roles in the mammalian 2297-P circadian clock. We previously generated transgenic (Tg) mice ubiquitously Maternal Low-Protein Diet during the Last Week of Pregnancy Al- expressing mCRY1 with a mutation in cysteine414. The Tg mice showed early ters Specifi c miRNAs Contributing to Insulin Resistance and β-Cell onset insulin-secretory defect of diabetes mellitus characterized by β-cell Dysfunction in Offspring dysfunction. We previously demonstrated that the proliferation of β-cells de- EMILYN ALEJANDRO, MAYA GIANCHANDANI, BRIGID GREGG, SEBASTIAN PAR- creases in Tg mice, which can account for the age-dependent loss of β-cells. In LEE, ORMOND A. MACDOUGALD, ERNESTO BERNAL-MIZRACHI, Ann Arbor, MI order to clarify yet undiscovered molecular pathogenesis of diabetes in which Maternal low-protein diet (LP) throughout gestation (LP0.5) increases the mutant of mCRY1 is involved, in this study we conducted DNA microarray the susceptibility of the offspring to type 2 diabetes by inducing permanent analyses using islets of Tg mice at a young stage. We found that the expres- changes in β-cell mass and function. The present study sought to examine sions of cytokines, chemokines, and CDK inhibitors were promoted in the islet whether LP during the last week of gestation (LP12.5) alone is a critical de- of Tg mice. Tissue remodeling factors were also elevated in Tg mice, which is in velopmental window for insulin resistance and β-cell programming. In con- agreement with the abnormal islet architecture. This expression pattern in the trast to LP0.5, islet morphology revealed normal β-cell fraction in LP12.5 islet of Tg mice is similar to that of the cells showing senescence-associated newborn and normal glucose tolerance at 3-months. By 12-months however, secretory phenotype (SASP), suggesting that β-cells of Tg mice show SASP- LP12.5 offspring displayed glucose intolerance and insulin resistance with like characteristics. We also found that the mRNA level of Sox9 was elevated aging. Glucose-induced insulin secretion assessed in 12-month old LP12.5 in Tg mice already at the young stage. In addition, the expression levels of mice in vivo and in vitro demonstrated an insulin secretory defect. When several factors in the hedgehog as well as in the Wnt signaling pathway were 3-month LP12.5 mice were fed a high-fat diet they too developed glucose also elevated. Immunohistochemical analyses using mature mice showed that intolerance due to increased body weight, insulin resistance, and insuffi - the cells which are Sox9-positive and insulin-negative increased in the islet cient β-cell mass expansion. To uncover the mechanisms underlying insulin of Tg mice. These results suggest that, the dedifferentiation of β-cells, along resistance and β-cell dysfunction induced by LP12.5, we assessed microRNA with the lowered proliferation of β-cells due to the senescence-like changes,

POSTERS (miRs) expression using qPCR-based microarrays in LP12.5 islets and con- are accountable for the age-dependent -cell failure in Tg mice. Furthermore, Islet Biology/ β

Insulin Secretion trols. These studies showed increased in a subset of miRs (i.e. miRs 342, 143, we found that the mRNA level of Rgs4, which is reported to play as a nega- 598, and 219) that might control the alterations observed in LP12.5 offspring. tive regulator of insulin release from pancreatic β-cells via M3 muscarinic Interestingly, previous studies have implicated some of these miRs on insulin acetylcholine receptor, was elevated markedly. This result suggests that the

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A584 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH increased expression of Rgs4 from the early stage is one of the causes of have evidence that α-cells do contribute to new β-cells after mild pancreatic insulin-secretory defect in Tg mice. insult. Supported By: Japan Society for the Promotion of Science (24590473); Tohoku A 50-60% partial pancreatectomy was performed in wild-type and in University GCGR KO mice that exhibit α-cell hyperplasia baseline. Pancreas was har- vested 1 week and 4 weeks after surgery, with the pancreas serially sec- 2300-P tioned, stained and quantifi ed the number of α and β-cells post regenera- tion. Using whole-mount imaging, we visualized and documented any islets exhibiting ductal branching into the islet masses as well as α-cell distribu- WITHDRAWN tion. Alpha-cell fate post regeneration was analyzed using a lineage tracing system Glucagon-cre-CAG tomato. 10% and 18% of alpha cells convert to beta cells after 1 and 4 weeks partial pancreatectomy respectively. Alpha cells express Pdx1 prior to the transdifferentiation process. This conversion was more appreciated with analyzing GCGR KO pancreases. Alpha-cell hyperplasia in GCGR KO mice signifi cantly decreased 4weeks after surgery with a proportional increase in β-cell number. Whole-mount imaging revealed glucagon polarization to- wards the pancreatic ducts. Postnatal expansion of β-cells occurs predominantly through self-replica- tion. Alpha-cells contribute to this expansion after a non-diabetogenic injury model.

2301-P WITHDRAWN

2303-P Differing Perinatal Effects of NF-κB on Pancreatic β-Cell Turnover and Its Role in the Development of Type 1 Diabetes DANIELLE MELLOUL, DROR SEVER, Jerusalem, Israel NF-κB is a well-characterized transcription factor with multiple physi- ological and pathological functions. However, its role in β-cells is still being debated, as it appears to depend on timing and kinetics of its activation. To elucidate NF-κB temporal role in vivo, we have generated two transgenic mouse models, the ToIβ and NOD/ToIβ mice, in which NF-κB activation is specifi cally inhibited in β-cells, in a controlled and reversible manner using the tet-on gene regulated system. In both strains, inhibiting the NF-κB path- way in β-cells during the fetal period led to increased β-cell turnover and re- duced β-cell mass, while its attenuation during the neonatal period caused reverse effects. On the NOD background, these differing effects infl uenced both the degree of insulitis and diabetes incidence. Our data give the fi rst evidence that NF-κB is part of a physiological regulatory circuit that controls the balance of β-cell replication and apoptosis in the early developmental stages of insulin-producing cells and modulates the development of T1D in the young NOD mice. Supported By: JDRF

2304-P Islet-1 and Ldb1 Transcriptional Regulators Interact with Single- stranded DNA Binding Proteins (SSBPs) to Impact Pancreatic Beta- Cell Targets CHAD S. HUNTER, JAMIE GALLOWAY, Birmingham, AL Transcription factors are required to maintain pancreatic beta-cell func- 2302-P tion. However, unlike in other cell types, there is a paucity of interacting Alpha Cell Contributes to New Beta Cells after a Nondiabetogenic coregulators known to impact beta-cell transcription. We showed that the POSTERS Injury to the Pancreas LIM-homeodomain Islet-1 transcription factor interacts with the LIM Domain Islet Biology/ YOUSEF EL-GOHARY, Stony Brook, NY Binding protein 1 coregulator (Ldb1) to promote embryonic islet development. Insulin Secretion It has been demonstrated that after 99% β-cell ablation, α-cells can Sucrose gradient fractionation of beta-cell extracts suggested that Ldb1 and convert to β-cells. Using constitutive lineage-tracing with glucagon-cre, we Islet-1 exist in large protein complexes. Thus, we employed crosslinking-im-

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A585 ISLET BIOLOGY—BETA CELL—DEVELOPMENTCATEGORY AND POSTNATAL GROWTH

munoprecipitation and mass spectrometry to isolate and identify additional CD3 alone. We found no evidence of recruitment of beta-cell progenitors in interacting beta-cell factors. Mass spectrometry datasets indicated that the the cured mice and this improvement in diabetes remission rate in the GLP-1 Single-Stranded DNA Binding Proteins (SSBP2-4) interacted with Ldb1 and/ group was not accompanied by an increase in beta-cell mass in comparison or Islet-1. SSBPs are coregulators known in other tissues to modulate LIM to the prolactin group. However, the two groups treated with growth factors transcriptional complex activity. However, nothing was known of SSBPs in had a higher proportion of insulitis-free islets and we observed the highest beta-cells. We confi rmed that SSBP3, the most abundant SSBP in the data- glucose-stimulated insulin secretion rate and pancreatic insulin content in the sets, interacted with Ldb1 and Islet-1 by co-immunoprecipitation (co-IP). To group that received both prolactin and GLP-1 (in addition to anti-CD3). Hence, it begin understanding the importance of SSBP3 in vivo, we found that Ldb1, appears that GLP-1 can be an effective adjunct in treatment of type 1 diabetes Islet-1 and SSBP3 also interact in purifi ed mouse and human islets. Immuno- by increasing insulin synthesis capacity in the residual beta cells. fl uorescence analysis demonstrated SSBP3 co-expression with Ldb1 and Is- Supported By: Canadian Diabetes Association let-1 in developing and adult pancreas. To test for function, siRNA-mediated SSBP3 knockdown in Min6 cells revealed mRNA reductions similar to those 2307-P found after Ldb1 or Isl1 depletion. Furthermore, SSBP3 occupied Ldb1: Islet-1 Protective Effects of SGLT2 Inhibitor Luseoglifl ozin on Pancreatic target promoters by chromatin immunoprecipitation. Collectively, these data Beta Cells in Obese Type 2 Diabetic db/db Mice suggest that SSBP3 is required for expression of beta-cell Ldb1 and Islet-1 SEIZOU OKAUCHI, MASASHI SIMODA, ATSUSHI OBATA, TOMOHIKO KIMURA, target genes. Future strategies will examine the importance of SSBP3 in HIDENORI HIRUKAWA, KENJI KOHARA, HIDEAKI KANETO, KOHEI KAKU, Kura- vivo. Studies elucidating additional transcriptional complexes will increase shiki, Japan our understanding of fundamental mechanisms governing beta-cell function, Aims: It is well known that SGLT2 inhibitors, new hypoglycemic agents, im- with potential impact on future diabetes therapies. prove glycemic control by increasing urine glucose excretion, but it remained Supported By: National Institutes of Health (DK094842) unclear whether they exert protective effects on pancreatic beta-cells. In this study, we examined the effects of SGLT2 inhibitor luseoglifl ozin on pancreatic 2305-P beta-cell mass and function using obese type 2 diabetic db/db mice. Role of Pancreatic Niche in Delamination and Maturation of Human Methods: 10-week-old male diabetic db/db mice received luseoglifl ozin ES-derived Endocrine Progenitors into Insulin-Producing Beta Cells 0.0025% in chow (Luse 0.0025%), luseoglifl ozin 0.01% (Luse 0.01%) or vehi- DIANE YANG, JOLANTA CHMIELOWIEC, MARISSA ANN SCAVUZZO, MAL- cle (control) for 4 weeks. After such treatment, we measured various param- GORZATA BOROWIAK, Houston, TX eters including body weight, blood glucose levels, serum insulin levels, free Pluripotent stem cells offer a unique opportunity to generate human insu- fatty acids and urinary sugar excretion. In addition, we performed immuno- lin pancreatic beta cells for studies on development, disease, small molecule histochemical study using pancreas tissues and examined gene expression screens or cell replacement leading to new therapeutical strategies for type profi le using isolated islets. 1 and 2 diabetes patients. During development, pancreatic progenitors are Result: Urinary glucose excursion was increased in Luse groups (0.0025% surrounded by mesenchyme, endothelial cells and nerves forming together and 0.01%) compared to mice with vehicle 3 days after the intervention. a pancreatic niche where all the components cross talk with each other Fasting blood glucose levels were signifi cantly lower in mice treated with and infl uence cell fate, migration or survival. Using co-culture of human ES Luse compared to mice with vehicle. Fasting serum insulin concentrations cell derived endocrine progenitors with established here novel embryonic were signifi cantly higher in mice treated with Luse compared to mice with pancreatic mesenchymal and endothelial human fetal pancreatic cells and vehicle. Body weight was increased in Luse groups compared to control hand picked small molecule screen, we studied in details the function of mice. Triglyceride levels tended to be lower in Luse groups compared to con- individual components of pancreatic niche on human beta cell generation trol mice. There was no difference in free fatty acids and adiponectin levels. in vitro. As result we identifi ed the signals provided by components of the In immunohistochemical study using pancreas tissues, pancreatic beta-cell pancreatic niche that facilitate Isl1+endocrine progenitors maturation into mass was larger in Luse groups compared to control group. Furthermore, in insulin producing cells. The identifi ed here signals include agonist of sympa- gene analysis using isolated islets, Ins1, GLUT2, MafA gene expression levels thetic innervation, Wnt family and components of extracellular pancreatic were signifi cantly higher in Luse groups compared to control group. matrix. These effects are manifested by different mechanisms including the Conclusion: SGLT2 inhibitor luseoglifl ozin ameliorates glycemic control and increase in endocrine progenitors delamination and in vitro migration, up- thus exerts protective effects on pancreatic beta-cell mass and function. regulation of beta cell specifi c markers such as NeuroD1, Glut4 and MafA and increased pancreatic cell survival. Using human ES cell pancreatic spe- 2308-P cifi c fl uorescent reporter cell lines and prospective isolation we have charac- The Effects of 3D Culture from Gel and Human Bone Marrow for Islet terized the pooled and single cell of endocrine progenitors and beta cells in β-Cell Function and Survival response to these treatments. The details of the molecular signature along SOURIYA VANG, WU FENG, LUGUANG LUO, Providence, RI with mechanism of induction of islet cells by pancreatic niche and functional Human islet β-cell loss of function is a major reason for Type 1 diabe- evaluation of in vitro derived cells will be presented. All these efforts will tes (T1D) and sustaining β-cell cell function is a signifi cant challenge. We aid to the ongoing efforts to create a robust and adequate source of human hypothesize that 3-D culture condition will create a microenvironment for beta cells from pluripotent stem cells. islet survival and function vs. monolayer condition. In this special report, Supported By: McNair Medical Institute; National Institutes of Health we investigate agarose gel and allogeneic bone marrow (BM) created 3-D conditions to explore the difference between gel scaffolding and BM 3-D 2306-P conditions for islet function and growth. Role of Growth Factors as Adjunct in Treatment of Diabetes in NOD We evaluated insulin release in islet with or without 0.1% gel-scaffolding Mice and islet/BM 3-D conditions and islet function by evaluating islets in 1 hour CLEMENT CHAN, VIPUL SHRISTAVATA, COLIN HYSLOP, CAROL HUANG, Calgary, glucose challenge (20mM) for 14 weeks. Islet morphology and size was moni- AB, Canada tored weekly. After 14 weeks, islets in gel-scaffolding signifi cantly increased Type 1 diabetes mellitus (T1DM) is an autoimmune disease where the de- insulin release, but was less than BM 3-D (866.3µIU/mL no gel, 2785.6µIU/ struction of the beta-cells causes insulin defi ciency and hyperglycemia. Many mL 0.1% gel, and 5733.1µIU/mL BM, P<0.05). Islet function signifi cantly in- immune modulators have effectively prevented the further destruction of beta- creased for gel-scaffolding, but was also less than BM 3-D (157.6µIU/mL cells in animal models of TIDM. However, due to the small beta-cell mass pres- no gel, 409.3µIU/mL 0.1% gel, and 1088.6µIU/mL, P<0.05). Islet in gel-scaf- ent at the time of diabetes, immune modulators (such as anti-CD3) have limited folding also signifi cantly increased growth by morphological analysis at a effi cacy in treating T1DM in humans. We hypothesize that addition of growth 2-D level and was also greater than growth in BM 3-D (70118.8µm2 no gel, factors that can augment beta-cell number (i.e. prolactin) and recruit stem cells 488426.5µm2 0.1% gel, and 184299.7µm2 BM, P<0.05). (i.e. GLP-1) may improve effectiveness of immune modulator in inducing diabe- Our results show that 3-D microenvironment facilitates human islet func- tes remission. Our previous studies have found that prolactin can up regulate tion and survival; however, biological microenvironments (BM created) sig- beta cell mass and function while others have shown that GLP-1 can increase nifi cantly increased function over gel-scaffolding structural microenviron- beta cell proliferation, insulin synthesis, and potentially, recruit stem cells to ments (agarose gel created). Morphological evaluation of islets suggests

POSTERS replete the beta cell mass. Here, we treated diabetic NOD mice with a course that BM 3-D microenvironments may promote better islet growth over 2-D Islet Biology/

Insulin Secretion of anti-CD3 (5 days) ± prolactin (3-weeks) ± GLP-1 (3-weeks) and found that condition. Further investigation for BM 3-D environment facilitation of hu- those treated with anti-CD3+prolactin+GLP-1 had signifi cantly higher diabetes man islet function and survival will need to be addressed. remission rate in comparison to mice treated with anti-CD3+prolactin or anti- Supported By: National Institutes of Health (1R01DK097380-01A1)

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A586 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

2309-P cation of FOXO1 and PDX1 and led to the differentiation of human pancreatic Pancreatic Branching Morphogenesis in Three-Dimensional Ductal ductal cells into β-cells and an increase in insulin synthesis. Concurrently, Cell Cultures Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics TAKATSUGU YAMADA, CLAUDIA R. CAVELTI-WEDER, JENNIFER HOLLISTER-LOCK, analysis identifi ed the Rab43 GTPase-activating protein as a downstream BROOKE SULLIVAN, SUSAN BONNER-WEIR, GORDON C. WEIR, Boston, MA target of Akt. A serial activation of both proteins induced various granular The pancreatic duct cells (PDCs) are a potential cell source for new is- protein syntheses and fi nally enhanced glucose-stimulated insulin secretion. let formation. In previous reports with monolayer culture, however, dense In a pancreatectomized diabetic animal model, exogenous Pref-1 improved cell-to-cell contact may inhibit the potential growth and differentiation of glucose homeostasis by accelerating pancreatic ductal and β-cell regenera- duct cells in vitro. We hypothesized that duct cells in three-dimensional (3D) tion after injury. These data establish a novel role for Pref-1, opening the culture can retain their inherent characteristics such as branching morpho- possibility of applying this molecule to the treatment of diabetes. genesis during pancreatic organogenesis. Primary PDCs isolated from adult DBA/2 mice were cultured in suspension to form spheroids, which were then 2312-P embedded in the collagen gel and cultured three-dimensionally. After seven Early and Late G1/S Cyclins and Cdks Act Complementarily to En- days of 3D culture in collagen gel, PDC spheroids formed 3D structures of hance Authentic Human Beta-Cell Proliferation and Expansion ductal cysts with branching morphogenesis when cultured in DMEM/F12 SHIWANI TIWARI, CHRISTOPHER ROEL, RACHEL WILLS, KAREN TAKANE, NATH- medium supplemented with 10mM nicotinamide and 10% fetal bovine serum ALIE M. FIASCHI-TAESCH, New York, NY, Pittsburgh, PA (FBS), whereas no branch formation was observed in serum-free medium. Beta-cells regeneration is a key goal of diabetes research. Progression Notably, epidermal growth factor (EGF) enhanced the branching morpho- through cell cycle is associated with pRb inactivation via sequential phos- genesis, which was evaluated by total branch length and number of branch phorylation by the “early” cyclins and cdks (D-cyclins, cdk4, cdk6) and the points. Moreover, EGF signifi cantly increased the branching proliferation, “late” cyclins and cdks (cyclin A, cyclin E, cdk1 and cdk2). In beta-cells, ac- when compared to other growth factors such as KGF, VEGF, FGF10 and HGF. tivation of either “early” or “late” G1/S cyclins and/or cdks is an effi cient Immunostaining confi rmed that the branching morphogenesis expressed approach to induce cell cycle entry, but it is unknown whether combined the ductal cell markers, cytokeratin (CK), E-cadherin and Sox 9, suggesting expression of “early” and “late” cyclins and cdks might have synergistic or multipotent progenitor cells. We have developed a particularly promising additive effects. Thus, we explored whether a combination of both “early” approach of a 3D culture system using a collagen gel. This allows branch- and “late” cyclins and cdks might more effectively drive human beta-cells ing morphogenesis to produce newly developed ducts, including progeni- cycle entry than either group alone. We also sought to determine whether tors that could give rise to new islets. This approach may provide important authentic replication with expansion of adult human beta-cells could be insights into the pancreatic ductal differentiation and a potential attractive demonstrated. therapeutic strategy for diabetes. “Late” cyclins and cdks do not traffi c in response to the induction of rep- lication by “early” cyclins and cdks (cdk6 + cyclin D3) in human beta-cells, 2310-P but are capable of nuclear translocation when overexpressed. In contrast The Role of Proinfl ammatory Cytokines on Pancreatic Cell Differ- to the individual cyclins and cdks, combinations of “late” cyclins and cdks, entiation induces a signifi cant increase of BrdU incorporation, with cyclin E and cdk1 IVAN A. VALDEZ, ADRIAN TEO, ERCUMENT DIRICE, ROHIT N. KULKARNI, Boston, being the most effective combination (13.3 ± 1.9%). “Early” (cdk6+cyclin D3) MA, Singapore, Singapore plus “late” (cdk1+cyclin E) cyclins and cdks, acting via pRb phosphorylation Although a pancreatic progenitor cell has been elusive, numerous studies on distinct residues, complementarily induce greater proliferation in human provide evidence of intrapancreatic cellular conversion under diverse states beta-cells (42.8 ± 2.2% BrdU incorporation in beta cells and 54.3 ± 3.1 % of of physiological or non-physiological stress. In this study, we sought to ex- human β-cells positive for Ki67) than either group alone. Importantly, the plore the effect of cellular stress directly induced by infl ammatory cytokines combination of “early” and “late” cyclins and cdks increased human beta- on the differentiation potential of mature pancreatic cells. To this end, the cell numbers in vitro (122.2 ± 5.1%). human ductal cell line, PANC-1, or the human beta cell line, EndoC-bH1, were These fi ndings provide additional insight into human beta-cells expansion. treated either with single or a combination of infl ammatory cytokines (TNFa, They also provide a novel tool for assessing beta-cell expansion in vitro. IFNy, IL-1b; n=3 for all experiments). As a complementary in vivo approach, we Supported By: American Diabetes Association (7-12-BS-046 to N.M.F-T.) performed pancreatic intraductal injection of a cocktail of the three infl amma- tory cytokines in mice (n=3 saline-injected; n=4 cytokine-injected). In addition, we explored the effect of immune cell infi ltration on pancreatic ductal cells ISLET BIOLOGY—BETA CELL— throughout the progression of diabetes in female NOD mice (ages: 4 weeks, 8 STIMULUS-SECRETION COUPLING AND METABOLISM weeks, 12 weeks, 1-2 weeks post-diabetes onset (new onset diabetes) and 5-6 weeks post-diabetes onset (established diabetes); n=4-5 for each time point). Our experiments reveal that infl ammatory cytokines lead to a 2-3-fold upregu- Guided Audio Tour: Stimulus-Secretion Coupling (Posters: 2313-P to lation of stress markers (iNOS and CHOP) and endocrine progenitor markers 2320-P), see page 17. (NGN3, NKX6.1, HNF6, and SOX9) in human ductal and beta cells, in vitro. Moreover, a single injection of the cocktail of infl ammatory cytokines leads & 2313-P to a 2.5-fold increase in ductal cell proliferation (BrdU+ cells) as compared to Very High Adiponectin Levels Are Associated with Impaired Glu- saline-injected controls. Lastly, our studies demonstrate that pancreatic duc- cose Tolerance and Decreased Insulin Secretion tal cells exhibit a 3-4-fold increase in proliferation (BrdU+ cells) during new TIMOTHY E. GRAHAM, JAMES V. POTTALA, STEPHEN A. VARVEL, DAWN L. onset of diabetes when compared to the other time points. In conclusion, our THISELTON, RACHAEL GRIFFITHS, MACIEK SASINOWSKI, JOSEPH P. MCCON- fi ndings suggest that infl ammatory cytokine-induced stress plays a role in the NELL, Salt Lake City, UT, Richmond, VA upregulation of pro-endocrine transcription factors in mature pancreatic cells Adiponectin is an adipocyte-secreted hormone that acts on multiple tis- in vitro and increased ductal cell proliferation in vivo and denote a potential for sues to enhance insulin action, lipid utilization, and endothelial function. endocrine progenitor cell generation. Adiponectin concentrations vary more than 25-fold among healthy individu- Supported By: National Institutes of Health (F31DK098931) als. Although low adiponectin strongly predicts diabetes, cardiovascular disease, and death, little is known about effects of very high adiponectin 2311-P concentrations. 426 patients at risk for diabetes underwent a 75g 2hr OGTT; Preadipocyte Factor 1 Induces Pancreatic Ductal Cell Differentia- blood collected at baseline and during the OGTT was sent to a national ref- tion into Beta Cells and Promotes Insulin Secretion erence laboratory (Health Diagnostic Laboratory, Inc., Richmond, VA) for SEUNG-HWAN LEE, MARIE RHEE, JI-WON KIM, DONG-SIK HAM, HEON-SEOK analysis. Adiponectin groups were defi ned as low (<10 µg/mL; n=198), nor- PARK, JAE-HYOUNG CHO, KUN-HO YOON, Seoul, Republic of Korea mal (10-49 µg/mL; n=210), or high (>49 µg/mL; n=18); the low cut-point was The preadipocyte factor 1 (Pref-1) is involved in the proliferation and dif- reported previously, and the high cut-point was data-derived for best model ferentiation of various precursor cells. However, the intracellular signaling fi t. Ferritin did not differ between the groups. As observed in other studies, POSTERS pathways that control these processes and the role of Pref-1 in the pancreas the low adiponectin group had higher BMI, fasting insulin, C-peptide, CRP, Islet Biology/ remain poorly understood. Here, we showed that Pref-1 induces insulin syn- LDL-P, and triglycerides, lower HDL-C and HDL-P, and increased glucose and Insulin Secretion thesis and secretion via two independent pathways. The overexpression of insulin excursions during OGTT (all P<0.02); the latter were analyzed by re- Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translo- peated measures ANOVA. Remarkably, the high adiponectin group exhibited

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A587 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

enhanced insulin sensitivity but greater glucose excursion and decreased 2316-P insulin response during OGTT as compared to the normal group (P<0.05). Therefore, very high adiponectin levels are unexpectedly associated with impaired glucose tolerance and decreased glucose-stimulated insulin secre- WITHDRAWN tion in some individuals. Because serum adiponectin is a complex mixture of multimeric and post-translationally modifi ed forms, we speculate that very high levels could indicate the presence of structural or post-translational modifi cations that adversely affect insulin secretion. Biochemical character- ization of adiponectin forms in individuals with impaired glucose tolerance and very high circulating levels of adiponectin are underway to assess this possibility.

& 2314-P Molecular Mechanism of GLP-1 Dependent Potentiation of Insulin Granule Exocytosis NEHA SHRESTHA, BINGBING WU, WEIPING HAN, Singapore, Singapore Inadequate insulin secretion is an important facet of type 2 diabetes. Pan- creatic beta cells release insulin in response to signals derived from glucose metabolism. While glucose is the major stimulus for insulin secretion, intra- cellular cAMP signal generated by secretagogues like GLP-1 is crucial in po- tentiation of insulin secretion. GLP-1 modulates exocytosis by coordinating protein kinase A (PKA)-dependent phosphorylation of proteins in exocytosis machinery and PKA-independent activation of cAMP-regulated guanine nu- cleotide exchange factor (GEF/EPAC) signalling. GLP-1 based drugs are being widely used to potentiate insulin secretion and improve metabolic control in diabetics; however, molecular mechanisms underlying GLP-1 potentiation of insulin secretion remain unclear. Here we report that Synaptotagmin7 (Syt7), a principle calcium sensor for insulin secretion in beta cells, is a phosphorylation target of PKA. Phospho- rylation of Syt7 is essential for GLP-1 potentiation of insulin release as phos- phoinactive form of Syt7 fails to elicit an enhancement in insulin secretion in response to exendin-4, a GLP-1 receptor agonist. By performing pull-down assays using phosphomimetic and phosphoinactive forms of GST-Syt7, we further identify that the phosphomimetic form of Syt7 exhibits enhanced binding to Rabphilin3a (Rph3a), a Rab effector protein involved in transport & 2317-P of granules to the plasma membrane. Finally, treatment of insulin-secreting Mice with β-Cell-Specifi c Knockout of Mitochondrial Pyruvate Min6 cells with a cAMP analogue induces in vivo phosphorylation of these Carrier 2 Exhibit Defects in Glucose-Stimulated Insulin Secretion proteins and enhances Rph3a-Syt7 interaction. Our results reveal a potential KYLE S. MCCOMMIS, WESLEY HODGES, WILLIAM G. MCDONALD, JERRY R. mechanism by which GLP-1 modulates the exocytosis process. COLCA, ROLF F. KLETZIEN, MARIA REMEDI, BRIAN N. FINCK, St. Louis, MO, Ka- Supported By: A*STAR Biomedical Research Council lamazoo, MI Glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells is be- & 2315-P lieved to require mitochondrial metabolism of pyruvate and subsequent Evidence of β-Cell Dedifferentiation in Human Type 2 Diabetes effects on ATP-sensitive potassium (KATP) channels. For pyruvate to be FRANCESCA CINTI, RYOTARO BOUCHI, LORELLA MARSELLI, PIERO MARCHETTI, oxidized or carboxylated in the mitochondrial matrix, cytosolic pyruvate DOMENICO ACCILI, New York, NY, Tokyo, Japan, Pisa, Italy must be transported across the inner mitochondrial membrane. Mitochon- Diabetes is associated with a defi cit of insulin-producing β cells. Animal drial pyruvate carrier 1 and 2 (MPC1 and MPC2) proteins are believed to studies show that β cells become dedifferentiated in diabetes, reverting mediate mitochondrial pyruvate import. To investigate the effects of MPC2 to a progenitor-like stage, and partly converting to other endocrine cell defi ciency on GSIS, mice harboring a conditional allele of Mpc2 were gener- types. To determine whether similar processes occur in human type 2 dia- ated, and β-cell-specifi c MPC2 knockout (β-MPC2-/-) mice were derived by betes, we surveyed pancreatic islets from 15 diabetic and 15 non-diabetic crossing MPC2 fl oxed mice with mice expressing tamoxifen-regulated Cre organ donors. We scored dedifferentiation, by immunohistochemistry, using in a β-cell specifi c manner. Knockout was induced by 5 consecutive days markers of endocrine lineage (Synaptophysin and Chromogranin A), β cell- of Tamoxifen injection, and mice underwent metabolic assessment two specifi c transcription factors (NKX6.1, FOXO1, MafA), and a newly identifi ed months later. Isolated islets from β-MPC2-/- mice showed ~50% reduction endocrine progenitor cell marker, aldehyde dehydrogenase 1A3 (ALDH1A3). in MPC2 protein expression compared to littermate controls. Beginning at Dedifferentiated cells, defi ned as Synaptophysin-positive/hormone-nega- one month post induction, β-MPC2-/- mice displayed elevated random fed tive cells, accounted for 31.9% of β cells in type 2 diabetics vs. 8.7% in blood glucose concentrations. β-MPC2-/- mice also displayed elevated blood controls, and for 16.8% vs. 6.5% of all endocrine cells (p<0.001). The number glucose and reduced plasma insulin concentrations during an i.p. glucose of ALDH1A3-positive/hormone-negative cells rose over threefold in diabet- tolerance test (GTT). Isolated islets from β-MPC2-/- mice showed decreased ics compared to controls. We analyzed insulin granule content by electron GSIS during incubation with 16.7 or 23 mM glucose, despite normal cellular microscopy in a subset of 1,290 and 1,377 islet cells, respectively, from non- insulin content. KCl-stimulated insulin secretion was normal in β-MPC2-/- diabetic and type 2 diabetic pancreata. The percentage of cells that didn’t islets, suggesting a specifi c defect in the metabolic pathway upstream of contain any secretory granules rose fourfold in diabetics, from 1.0±0.2 to the KATP channel. Administration of glibenclamide, a sulfonylurea inhibitor 4.0±1.1% (p<0.01). Interestingly, insulin secretion was inversely correlated of KATP channel activity, improved defective GSIS in β-MPC2-/- islets and with the dedifferentiation score (r=0.55, p<0.05). reversed glucose intolerance in β-MPC2-/- mice during a GTT in vivo. These Moreover, β cell-specifi c transcription factors were ectopically found in data are consistent with an important role for the MPC in regulating pancre- glucagon- and somatostatin-producing cells of diabetic subjects. The data atic β-cell GSIS and suggest that augmenting activity of the MPC could be a support the view that pancreatic β cells become dedifferentiated and con- novel treatment for enhancing insulin secretion in diabetes. vert to α- and δ-“like” cells in human type 2 diabetes. We envision dedifferentiation as a mechanism to protect β cells from apoptosis by stealth, preserving them for re-differentiation under more fa-

POSTERS vorable metabolic conditions. The fi ndings should prompt a reassessment of Islet Biology/

Insulin Secretion goals in the prevention and treatment of β cell dysfunction. Supported By: National Institutes of Health (DK64819, DK63608); JPB Founda- tion; Brehm Coalition; JDRF

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& 2318-P & 2320-P Congenital Hyperinsulinism Due to Novel Gain of Function Muta- Nuclear Localization of Glucokinase in Pancreatic Beta Cells Is tions of Glucokinase Mediated by an NLS and Regulated by SUMOylation MING LI, PAN CHEN, KARA E. BOODHANSINGH, ZHIXIN WANG, XINHUA XIAO, INGVILD AUKRUST, MARIE H. SOLHEIM, BENTE B. JOHANSSON, KARIANNE FRANZ M. MATSCHINSKY, CHARLES A. STANLEY, DIVA D. DE LEON, CHANG- FJELD, ANNE DØSKELAND, TORGEIR FLATMARK, PÅL R. NJØLSTAD, LISE BJØRK- HONG LI, Beijing, China, Philadelphia, PA HAUG, Bergen, Norway The importance of glucokinase (GK) in the regulation of insulin secretion Glucokinase (GK) is the predominant hexokinase expressed in hepato- has been highlighted by the phenotype of individuals with gain- and loss- cytes and pancreatic beta-cells. It plays a pivotal role in regulating hepatic of-function mutations in GK. Here we report 6 cases of hyperinsulinism (HI) glucose clearance and glucose-stimulated insulin secretion, illustrated by due to novel gain-of-function mutations of GK, including E67V, K90R, V91L, glucokinase gene mutations causing monogenic diabetes and congenital hy- M197V, Y215C and D217N and characterize the mutant enzyme kinetics. poglycemic hyperinsulinism. While the enzyme is regulated by complex tis- The age at which the fi rst recognized episode of hypoglycemia occurred sue-specifi c mechanisms, little is known about the role of posttranslational in these patients varied from birth to age 20. The severity of the phenotype modifi cations in glucokinase subcellular localization and function. Moreover, was also variable. The patient with the V91L mutation presented at birth the nuclear localization of glucokinase in pancreatic beta-cells represents a with severe hypoglycemia (plasma glucose 1 mmol/L) that did not respond controversial issue. We previously reported that conjugation of Small Ubiq- to diazoxide and required pancreatectomy. The surgical pathology showed uitin-like MOdifyer-1 (SUMO-1) affects the stability and activity of human marked variation in islet size with some extremely large islets. Islet cells pancreatic glucokinase, in vitro and in insulin-secreting model cells. Since were pleomorphic with hyperchromatic and occasionally binucleated nuclei, the level of SUMOylated glucokinase was found increased in the presence suggesting increased β-cell proliferation. The plasma glucose range for the of the nuclear pore SUMO E3 ligase RanBP2, we here aimed to investigate a other cases was 2.4-3.4 mmol/L and 2 of those 5 patients (E67V and Y215C) role of SUMO in the nuclear transport/localization of glucokinase. have poor response to diazoxide. Mutant GK protein were expressed and In this study, we confi rm the nuclear localization of glucokinase in INS-1 purifi ed for determination of enzyme kinetics including, enzyme effi ciency and MIN6 cells by immunofl uorescence, subcellular fractionation and LC- (EE: Vmax/S0.5 ratio) and activity index (AI). MS/MS. We also identify a conserved, seven-residue candidate nuclear All six HI mutations had lower glucose S0.5, 2.8, 2.9, 1.2, 2.5, 2.8, 6.1 vs. localization sequence (NLS) (30LKKVMRR36) in the human enzyme. The sub- WT 7 mM. V91L was the most activating mutation, with EE 7 times higher stitutions K31,32 → A and R35,36 → A in the nuclear localization signal led to a than WT and AI of 33. The other 5 mutations had AI increased; they are loss of glucokinase nuclear localization in transfected cells. Co-transfection 2.6, 5.7, 3.4, 1.1, 1.1. Despite the activation of GK activity, all six gain-of- with SUMO-1 and RanBP2 increased the number of cells demonstrating nu- function mutations still responded to GK activator (Piragliatin) with a 3-7 fold clear accumulation of glucokinase. Moreover, co-localization of glucokinase, increase of enzyme effi ciency. SUMO-1 and Ubc9 (E2) were observed in the nucleus and nuclear membrane. These novel GK gain-of-function mutations are located near previously Thus, glucokinase transport to the nucleus of INS-1 and MIN6 cells is medi- known activation sites, suggesting that only limited specifi c sites of GK can ated through its nuclear localization signal and stimulated by SUMOylation. be associated with enzyme activation. This study also suggests that the The functional implications of glucokinase in the nucleus represent a future possibility of existence of mutation may be stronger than V91L. Examining challenge. enzyme kinetics of the mutant proteins can provide a mechanistic explana- tion for the clinical phenotype. 2321-P Supported By: National Institutes of Health Disruption of Peripheral Clock Gene Nocturnin Leads to Defective Islet Beta-Cell Function and Glucose Intolerance & 2319-P TIEN-JYUN CHANG, CHIH-HAO KUO, SIOW-WEY HEE, LEE-MING CHUANG, Tai- Deletion of O-linked N-acetylglucosamine Transferase Induces pei, Taiwan β-Cell Failure and Type 2 Diabetes in Mice Nocturnin is one of the circadian rhythmic “output gene”, encodes a EMILYN ALEJANDRO, NADEJDA BOZADJIEVA, DOGA KUMUSOGLU, SARAH deadenylase-ribonuclease that specifi cally removes the poly(A) tails from ABDULHAMID, LEENA HAATAJA, PETER ARVAN, ERNESTO BERNAL-MIZRACHI, mRNAs. We investigated the difference of glucose homeostasis, beta cell Ann Arbor, MI function, microRNA and mRNA microarray between wild type (WT) and Noc- O-GlcNAcylation is a dynamic posttranslational modifi cation of nuclear turnin knockout (NOC-/-) mice. and cytosolic proteins solely by O-linked N-acetylglucosamine Transferase Body weight (BW), oral glucose tolerance test, insulin tolerance test, (OGT). This protein modifi cation modulates multiple biological processes in- isolated islet perifusion study, intrapancreatic insulin content, immunohis- cluding transcription, signaling, and metabolism. However, the importance tochemical stain of insulin to measure beta cell mass, micro-array of mRNA of O-GlcNAcylation in the regulation of β-cell mass and function is largely and microRNA were performed and compared between wild type (WT) and unclear. In the present study, we reveal that mice genetically lacking β-cell NOC(-/-) mice. No BW difference between WT and NOC(-/-) mice was found. OGT (βOGT-KO) were normoglycemic until 60 days and developed progres- NOC(-/-) mice are more glucose intolerant but more insulin sensitive than sive hyperglycemia, hypoinsulinemia, and β-cell failure. Despite normal WT mice. Oral glucose-stimulated insulin secretion in 30 min is much less glucose tolerance, 4-week-old βOGT-KO mice also demonstrated reduced in NOC(-/-) mice than in WT mice. During isolated islet perifusion study, the islet insulin mRNA and protein, but increased proinsulin levels in serum, sug- glucose stimulated insulin secretion was much less in NOC(-/-) mice than gesting alteration in function precedes the loss of β-cells. By 10 weeks of in WT mice. No difference of islet beta cell mass and insulin content be- age, βOGT-KO mice have a signifi cant loss in β-cell mass due to enhanced tween WT and NOC(-/-) mice was found. According to microarray study of β-cell apoptosis and decreased proliferation. Markers for ER stress (BiP and mRNA and microRNA of purifi ed islets, the gene expression levels of glucose Chop) and cell death (Caspase 12 and TUNEL) were increased in βOGT-KO transporter 2 (GLUT2), G protein coupled receptor 18 (GPR 18), and calcium mice, suggesting ER stress-mediated cell apoptosis. Interestingly, induc- homeostasis modulator 2 (Calhm2) were much less in NOC(-/-) mice than in ible iβOGT-KO mice exhibited glucose intolerance and revealed a defect on WT mice. We also found the expression of miR142-3p was signifi cantly lower insulin secretion. Moreover, prosurvival Akt phosphorylation (Ser473 and in NOC(-/-) mice compared with WT mice. Thr308) and Pdx-1 protein level were reduced in βOGT-KO islets. Targeted In conclusion, Nocturnin plays a role in regulating glucose homeosta- disruption of Chop gene or Akt overexpression in βOGT-KO mice improved sis. Knock-out Nocturnin gene leads to glucose intolerance with impaired glucose tolerance and hyperglycemia by normalizing β-cell mass. Finally, glucose-stimulated insulin secretion. The differences may attribute to the diabetes in βOGT-KO mice was partly ameliorated with Phloridzin indicating down-regulation of several candidate genes associated with glucose trans- a novel role of OGT in glucotoxicity. These data provide novel insights on the porter, G protein coupled receptor and calcium homeostasis and specifi c role of OGT in ER stress, glucotoxicity, and the maintenance of β-cell mass microRNA. and function. Supported By: National Taiwan University College of Medicine (99C101–601); Supported By: National Institutes of Health National Science Council of Taiwan (102-2314-B-002-022-MY3) POSTERS Islet Biology/ Insulin Secretion

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2322-P increased infl ammation in adipocytes and hepatocytes. Pharmacological Group 2 P21-activated Kinase Phosphorylation Downstream of activation of GPR120 by selective GPR120 agonist appears anti-diabetic GPR40 Potentiates Glucose-Stimulated Insulin Secretion with improved glucose tolerance and enhanced insulin sensitivity. However, VALERIE BERGERON, JULIEN GHISLAIN, STEPHANIE M. YODER, DEBBIE C. whether these anti-diabetic effects of GPR120 ascribe to the regulation of THURMOND, VINCENT POITOUT, Montreal, QC, Canada, Indianapolis, IN pancreatic islet functions remains unclear. To reveal this, we fi rst examined The G protein-coupled receptor GPR40 is highly expressed in pancreatic the mRNA and protein expressions of GPR120 in pancreatic islets. By Taq- beta-cells and plays a key role in the potentiation of glucose-stimulated insu- man real-time RT-PCR analysis of FACS purifi ed different islet cell popula- lin secretion (GSIS) by fatty acids. We previously showed that protein kinase tions and immunohistochemistry of rodent and human pancreatic sections, D1 (PKD1) acts downstream of GPR40 to control cortical actin remodeling and we found that GPR120 is expressed in both beta cell and non-beta cells, indi- the potentiation of GSIS by fatty acids. In this study we investigated whether cating its possible roles in islets. Then, we performed in vitro islet hormone P21 activated kinases (PAK), known effectors of PKD1, are involved in GPR40- release assays in mouse and human islets treated with GPR120 agonist. We dependent potentiation of GSIS. To investigate the activation of PAKs in beta- found that activation of GPR120 potently enhanced glucose stimulated-insu- cells in response to fatty acids we used group 1 or 2 PAK phospho-specifi c lin secretion from mouse and human islets but not from GPR120 KO mouse antibodies in Western blotting. In MIN6 cells, oleate (0.5 mM) increased the islets. Meanwhile, treatment with GPR120 agonists inhibited islet soma- phosphorylation of the group 2 PAK, PAK4, whereas the group 1 PAK, PAK1, tostatin release. Furthermore, the stimulatory effect on insulin secretion was unaffected. Glucose increased the phosphorylation of both PAK1, as has and the inhibitory effect on somatostatin release by GPR120 agonist were been shown previously, and PAK4. We then asked whether PAK4 phosphoryla- blocked by pertussis toxin, an inhibitor of Gi/Go signaling. Thus, our data tion in response to oleate is dependent on GPR40. Treatment of islets from WT suggest that, in addition to improving insulin sensitivity, GPR120 may exert mice resulted in an oleate-dependent PAK4 phosphorylation that was absent critical regulatory functions on pancreatic islets, and that GPR120 agonist in GPR40 knock-out islets. To investigate a functional role of group 2 PAKs in enhances beta cell insulin secretion directly or indirectly through inhibition the potentiation of GSIS by fatty acids we performed knock-down experiments of local somatostatin release, which in part underlies the anti-diabetic ef- in the INS832-13 cells using siRNAs specifi c to the group 2 PAKs, PAK4, 6 and fects of GPR120 agonist. 7. RT-qPCR analysis 48 h after electroporation confi rmed effi cient (70-80%) and specifi c knock-down by each PAK siRNA. We then performed 1-hour static 2325-P insulin secretion assays in response to 1 and 10 mM glucose with or without The Missense Mutation of R583G in HNF-1a Increases Insulin Se- 0.5 mM oleate. Whereas PAK7 knockdown impacted GSIS, knock-down of cretion in the Presence of Sulfonylureas and Incretin either PAK4 or 6 signifi cantly decreased oleate potentiation of GSIS without YANYAN LIU, SHIN-ICHI HARASHIMA, YU WANG, DAISUKE TANAKA, NOBUYA affecting the glucose response. In summary, we show, for the fi rst time, that INAGAKI, Kyoto, Japan both PAK4 and 6 are involved in the potentiation of GSIS by fatty acids and that Combination therapy with sulfonylureas (SUs) and DPP-4 inhibitors is PAK4 is phosphorylated in a GPR40-dependent manner. These results suggest more effective for glycemic control in type 2 diabetes in Japanese than it is that GPR40-dependent actin remodeling in insulin secretion may involve group in Caucasian because of lower insulin secretion capacity. We have treated a 2 PAK4 and 6. three-generation family with diabetes mellitus. Two members of the family Supported By: Canadian Institutes of Health Research (proband, 79 years old, female; her eldest daughter, 45 years old) were treat- ed with basal insulin and SUs. However, HbA1c levels of the two patients 2323-P were over 9.0%. Therefore, both patients’ treatment was changed from SUs Integrated Analysis of the Human Pancreatic Islet Phenotype, Me- and basal insulin to SUs and sitagliptin. HbA1c levels of both patients were tabolome, and Transcriptome dramatically decreased to less than 6.5%. Glucagon loading tests showed PETER SPÉGEL, JIANGMING SUN, LOTTA ANDERSSON, PETTER STORM, ISABEL the C-peptide index of the proband and her eldest daughter to be improved GOEHRING, HINDRIK MULDER, Malmö, Sweden from 1.09 to 1.48 and from 1.67 to 1.96, respectively. There is preponderance for genes involved in β-cell function among the Genomic DNA was then extracted from peripheral blood samples of all >80 variants associated with future risk of type 2 diabetes (T2D). β-cell func- members the family. PCR-RELP revealed that an R583G mutation was pres- tion is controlled by metabolism of glucose, yielding signals that trigger and ent in proband, her eldest daughter, and her grandson. To determine whether potentiate secretion of insulin. Hence, β-cell failure is likely associated with this R583G mutation affected insulin secretion, human HNF-1α-R583G and perturbed β-cell metabolism. normal HNF-1α were synthesized by insert-PCR method and subcloned into Here, we investigated glucose metabolism in islets from T2D (n=7) and an adenoviral expression vector. Seventy-two hours after transfection of non-diabetic (control, n=31) human donors. Metabolites were profi led by gas both genes into INS-1D cells, insulin secretion was examined in the pres- chromatography/mass spectrometry after a 1 h incubation in 2.8 mM or 16.7 ence of 10 mM glimepiride and/or 10 nM exendin-4 under low or high glucose mM glucose. Gene expression in islets from the same donors was investi- conditions. In HNF-1α-R583G-infected cells, insulin secretion for 30 min was gated by RNAseq and microarray. about 1.3 fold increased in the presence of glimepiride or glimepiride and We found glycolytic metabolism to be glucose-responsive in both T2D and exendin-4 with low glucose and about 1.5 fold increased in the presence of control islets, whereas metabolites in Krebs cycle, mitochondrial shuttles and glimepiride and exendin-4 with high glucose, compared to those in normal shunts were glucose responsive only in control islets. This blunted response in HNF-1α-infected cells. T2D islets was largely accounted for by increased basal metabolite levels. Recently it has been reported that not only SUs but also DPP-4 inhibi- Next, we linked metabolite levels to clinical data. Levels of fatty acids and tors are useful for MODY3 treatment. Although the full details are unknown, amino acids correlated with basal and fold-change of insulin secretion, where- our results from human and in vitro study suggested that R583G mutation as glycolytic and Krebs cycle intermediates correlated with HbA1c and BMI. in HNF-1α is involved in insulin secretion by activating both triggering and Finally, we correlated metabolite levels to gene expression. We found amplifying pathways. 6423 gene metabolite pairs (nominal, p<0.05), using RNAseq replicated by microarray (correlation for individual donors 0.8). Hydroxyproline, alanine 2326-P and malate were correlated to a large number of genes; these genes were CMPF Induces Persistent β-Cell Metabolic Infl exibility and Im- enriched in KEGG pathways of T2D, MODY, and insulin secretion. paired Insulin Secretion In conclusion, we found that mitochondrial metabolism was perturbed in hu- KACEY J. PRENTICE, YING LIU, EMMA M. ALLISTER, JUDITH A. EVERSLEY, XINYE man islets from T2D donors. Furthermore, we propose that association of me- S. WANG, JAKOB B. HANSEN, MICHAEL B. WHEELER, Toronto, ON, Canada tabolite levels with gene expression data is a powerful tool to identify genes Recently, CMPF was shown to be elevated in plasma of diabetic and pre- implicated in β-cell metabolism, perturbations of which are hallmarks of T2D. diabetic patients. Acute CMPF treatment induces glucose intolerance, re- duces glucose utilization, and impairs glucose-stimulated insulin secretion 2324-P (GSIS). Here we examine if CMPF induces metabolic infl exibility, an impaired The Roles of GPR120 in Rodent and Human Islet Function ability to switch from fatty acid to carbohydrate substrates, which may be NINA XIAOYAN LI, YUE FENG, TARO AKIYAMA, LIANGSU WANG, Kenilworth, NJ responsible for persistent impairment of β cell function and recovery in dia- G-protein coupled receptor 120 (GPR120) is activated by unsaturated long- betes.

POSTERS Measurements of O consumption showed that acute treatment of mouse Islet Biology/ chain free fatty acids and has been shown to mediate various physiological 2

Insulin Secretion processes, including infl ammation, adipogenesis, and regulation of appetite or human islets with CMPF reduced glucose, but enhanced palmitate utiliza- and food preference. In high fat diet fed animals, lack of GPR120 leads to tion with no change in oxidative capacity. To determine if this preferential obesity and glucose intolerance accompanied by impaired lipogenesis and fat utilization persists as a state of metabolic infl exibility, we treated mice

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A590 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM with CMPF, vehicle, or oleate for 7d and followed them for 16wks. CMPF was vitro. Pancreatic islets may therefore represent a new target tissue for in- absent from plasma and tissues within 24hrs after the fi nal injection. Inter- fl ammation and glucolipotoxicity to activate the TKP. Since infl ammation is estingly, despite the absence of CMPF in circulation, CMPF mice were sig- now recognized as a crucial mechanism in the development of the metabolic nifi cantly more glucose intolerant than controls, with dramatically impaired syndrome and more specifi cally at the islet level, it is needed to evaluate the GSIS both in vivo and ex vivo. Impaired GSIS corresponded to decreased islet potential induction of the TKP in the endocrine pancreas during obesity and/ insulin content, but no difference in number or size. Consistent with acute or diabetes and its relationship to the islet cell functional alterations. studies, islets from CMPF mice had impaired response to glucose in terms Supported By: Metabrain Research of MMP, O2 consumption, and ATP production, but an enhanced response to fat compared to controls. This corresponded to a signifi cant increase in 2329-P fat-stimulated insulin secretion. Mechanistically, CMPF treatment was as- PERK eIF2a Kinase Regulates ER Chaperones to Control the Bal- sociated with lower expression genes required for glucose metabolism, and ance between Proinsulin Secretion and Degradation in Pancreatic increased expression of β-oxidation genes, implicating induction of a persis- β-Cells tent fasting-like state. CARRIE R. LEWIS, JINGJIE HU, RONG WANG, SARAH C. BEVILACQUA, BARBARA Overall, our data suggests that CMPF impairs β cell function by induc- C. MCGRATH, DOUGLAS R. CAVENER, State College, PA ing preferential fat oxidation and a state of metabolic infl exibility, which The most severe cellular dysfunction seen in PERK-defi cient pancreatic persists in the absence of CMPF. CMPF may therefore be responsible for β-cells is an aberrant accumulation of proinsulin in the endoplasmic reticu- the failure of β cell function and recovery, and the high rate of progression lum (ER) as an insoluble, high molecular weight (HMW) aggregate, which from GDM to T2D. we refer to as Impacted-ER. This is in contrast to normal β-cells in which proinsulin is localized to the Golgi. Insofar as the literature has focused on 2327-P the regulatory role of PERK in the unfolded protein response, it was gen- Chronic Effects of Neuroendocrine Regulatory Peptide on Insulin erally accepted that the Impacted-ER phenotype seen in PERK-defi cient Secretion and Gene Expression in Pancreatic Beta Cells β-cells was caused by derepression of protein synthesis. However, we have JI-WON KIM, HAE KYUNG YANG, JAE-HYUNG CHO, SEUNG-HYUN KO, YU-BAE largely disproven this claim by showing that reducing protein synthesis fails AHN, KUN-HO YOON, Seoul, Republic of Korea to block the accumulation of proinsulin in PERK-inhibited β-Cells. Instead, Neuroendocrine regulatory peptides (NERP-1 and -2) are novel amidated we propose that PERK regulates proinsulin quality and quantity control in peptides derived from VGF, a polypeptide secreted from neurons and endo- the ER by regulating the expression of key ER chaperones, including GRP78 crine cells through a regulated pathway. Dr. Nakazato Masamitsu reported and ERp72, which control the balance between anterograde traffi cking and that NERP-1 and -2 may have a local modulator function on the human endo- ER-associated degradation (ERAD). To test this hypothesis, we have manipu- crine system, and clearly showed expression of NERP-1 and -2 in human pan- lated the expression levels of ER chaperones in conjunction with PERK abla- creas islets. Based on these data, we investigated the alteration of insulin tion to assess how this determines the fate of proinsulin along the secretory secretion, insulin granule-related protein, and pancreas-specifi c transcrip- pathway. We found that siRNA-mediated knockdown of GRP78 or ERp72 in tion factors in response to NERPs expression. We confi rmed the expression INS1 832/13 cells partially alleviates the Impacted-ER phenotype in PERK- of NERP-1 and -2 in the pancreas of a human diabetes patient, in addition to ablated β-cells. More profoundly, overexpression of adenovirally transduced diabetic animal models. When INS1 cells and primary rat islets were incu- GRP78 causes an Impacted-ER phenotype that is indistinguishable from bated with 10 nM NERPs for 3 days, glucose-stimulated insulin secretion lev- what is seen in PERK-ablated β-cells. In addition, GRP78 was found in in- els were blunted by NERP-1 and -2. The number of insulin granules released soluble HMW proinsulin aggregates in PERK-inhibited β-cells and is highly from the readily releasable pool, which is associated with the fi rst phase of colocalized with proinsulin in Impacted-ER cells. We speculate that PERK glucose-stimulated insulin release, was decreased by NERP-1 and -2. Insulin functions to modulate proinsulin quantity and quality control in response to granule-related proteins and mRNAs were down-regulated by NERP-2 treat- metabolic demand for insulin, which is communicated to β-cells by glucose ment. In beta-cells, transcription factors, translocated FOXO1 and decreased and other insulin secretagogues. Pdx-1, BETA2/NeuroD and insulin levels were shown by NERP-2 treatment. Supported By: National Institutes of Health (DK088140) In normal/diabetic human and mice models, NERP-2 levels were dramati- cally increased in diabetic pancreas. In conclusion, NERP-2 may play an im- 2330-P portant role in insulin secretion through the regulation of insulin secretory The Oxygen Sensing Protein, Prolyl Hydrolase (PHD), Plays a Role in granules and beta-cell transcription factors. In addition, NERP-2 expression Regulating Insulin Secretion from Pancreatic β-Cells is increased in diabetic conditions. Therefore, we suggest that NERPs may SABINA PAGLIALUNGA, MEI HUANG, MONICA HOANG, SARA PULLACK-MC- be potent endogenous suppressors of glucose-dependent insulin secretion. CORMICK, JAMIE W. JOSEPH, Waterloo, ON, Canada Type 2 diabetes is associated with impaired nutrient-regulated anaple- 2328-P rosis and insulin secretion in pancreatic β-cells. One key anaplerotic sub- Expression of the Kynurenine Pathway Enzymes in the Pancreatic strate that may be involved in regulating insulin release is α-ketoglutarate Islet Cells—Activation by Cytokines and Glucolipotoxicity (αKG). αKG has been suggested to regulate the K-ATP channel-independent JUNJUN LIU, SOPHIE RAYNAL, DANIELLE BAILBÉ, BLANDINE GAUSSERÈS, pathway involved in insulin release from islets. Since PHDs can metabolize CHRISTINE CARBONNE, VALÉ RIE AUTIER, JAMILEH MOVASSAT, MICHELINE αKG, we sought to explore the role of this enzyme in the regulation of β-cell KERGOAT, BERNARD PORTHA, Paris, France, Chilly-Mazarin, France function. The oxygen sensing PHD proteins regulate the stability of hypoxia Cytokines activated tryptophan/kynurenine pathway (TKP) is now at the inducible factor 1α (HIF1α) as well as other proline containing proteins by center of stage in virtually all major neurodegenerative and psychiatric disor- catalyzing the hydroxylation of proline residues. This reaction is dependent ders. Experimental and clinical data have clearly established pancreatic islet on the presences of suffi cient levels of oxygen, iron and αKG. In the present tissue itself is a site of infl ammation during type 2 diabetes. Since pancreatic study, we utilized both pharmacological and genetic approaches to assess β-cells share a large number of similarities with neuronal cells, cytokines may the impact of PHD activity on β-cell function. We demonstrated that a non- induce islet TKP in a way resembling that seen in the brain. Using cultured selective iron chelator (ethyl-3, 4-dihydroxybenzoate; EDHB) can both inhibit normal rat islets and INS1 cells, we have demonstrated (by qPCR, western PHDs and signifi cantly diminish insulin secretion from 832/13 clonal cells as blotting, immunocytochemistry, islet cell sorting, islet perifusion) for the fi rst well as reduce rat islet glucose-stimulated insulin secretion (GSIS). While, time that: 1/only some TKP genes are constitutively expressed, in β-cells as EDHB treatment had no negative effect on cell viability, citric acid cycle well as non β-cells; 2/the rate-limiting enzyme indoleamine 2, 3-dioxyge- metabolites such as pyruvate, citrate and malate were all downregulated nase (IDO1) is not constitutively expressed, at variance with TDO2; 3/IDO1 suggesting the possible involvement of reduced mitochondrial function and/ and kynurenine 3-monoxygenase (KMO) expression are potently activated or mitochondrial αKG. Therefore, to directly test the effects on PHD activ- by proinfl ammatory cytokines (2500U/ml IFNγ or 500U/ml IL1β exposure for ity, we also treated cells with dimethyloxalylglycine (DMOG, a specifi c PHD 48h) and glucolipotoxicity (20mM glucose+0.4mM palmitate exposure for inhibitor) to show an -80% (p<0.05) reduction in GSIS. Moreover, genetic 48h) respectively, rather in β-cells than in non β-cells; 4/islet kynurenine/ knock-down of the individual PHDs by siRNA transfection reduced GSIS by kynurenic acid production ratio is enhanced following IFNγ and glucolipo- -41%, -34%, and -53% (p<0.05) for PHD1, PHD2, and PHD3 in cells, respec- POSTERS

toxicity; 5/ oxidative stress (50µM H2O2 exposure for 0.5h+47.5h normal tively. Taken together, the current results demonstrate an important role for Islet Biology/ culture) and glucorticoid (10µM corticosterone exposure for 48h) modulate PHD as mediators of islet insulin secretion, highlighting a novel mechanism Insulin Secretion TKP genes only marginally; 6/some TKP metabolites such as kynurenine or in which cytosolic α-ketoglutarate can regulate pancreatic β-cell function. kynurenic acid acutely potentiate the glucose-induced insulin secretion in

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A591 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

2331-P 2333-P Mitochondrial Network Fragmentation in Response to Excess Nu- Expression and Action of Activin and Other TGFbeta Ligands in Hu- trient Environment Represents a Compensatory Adaptation that man Islets Maintains Beta-Cell Function MELISSA BROWN, NATHAN UNGERLEIDER, LARA BONOMI, DANIELLE ANDRZE- KYLE M. TRUDEAU, SAM SEREDA, NATHANIEL MILLER, PATRICK MACDONALD, JEWSKI, AMY BURNSIDE, ALAN SCHNEYER, Amherst, MA, Springfi eld, MA ORIAN SHIRIHAI, Boston, MA, Edmonton, AB, Canada Diabetes results from a loss of β-cell function that leads to inadequate We have previously reported that high glucose and fatty acids, termed insulin secretion to control blood glucose. Understanding the role of intra- glucolipotoxicity (GLT), induces mitochondrial fragmentation and is concur- islet signaling molecules in regulating β-cell function might lead to novel rent with beta-cell dysfunction in cultured insulinoma (INS1) cells and mouse strategies for therapeutic development. Members of the TGFβ superfamily, islets. The goal of this work was to determine if fragmentation plays a com- including activins A and B, TGFβ, and BMPs and their signaling receptors pensatory or pathological role during GLT-induced beta-cell dysfunction, and and second messengers have been identifi ed in islets of various species, but to reveal if this mechanism is also observed in human islet. To address if GLT expression in human islets has not been systematically analyzed. alters mitochondrial shape in human beta-cells, islets were cultured for 4 We determined TGFβ superfamily expression in human islets and po- days in media supplemented with low glucose (control) as compared to the tential roles for activin in regulating β-cell function. Human islets (33 combination of high glucose, oleate and palmitate (GLT). Assessing mito- shipments) were divided into: 1) normal functional (SI > 1); 2) normal non- chondrial morphology and membrane potential (MMP) by confocal imaging functional (SI

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A592 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

2335-P 2337-P The Selective Reduction of Gluco- or Lipo-toxicity Alters β-Cell SERCA2b Plays a Critical Role in the Maintenance of Pancreatic Gene Expression Profi les in db/db Mice Beta-Cell Function and Mass in Response to Diet-induced Obesity NAOKI SHIMO, TAKAAKI MATSUOKA, TAKESHI MIYATSUKA, DAN KAWAMORI, XIN TONG, TATSUYOSHI KONO, CARMELLA EVANS-MOLINA, Indianapolis, IN IICHIRO SHIMOMURA, Suita, Japan The Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) 2b pump is a It has been reported that reduction of chronic hyperglycemia or/and hyper- P-type ATPase that transports 2 Ca2+ molecules into the ER lumen at the lipidemia improves β-cell function under diabetic conditions. However, the expense of one ATP. SERCA2b activity is responsible for maintaining a steep underlying molecular mechanisms involved in improvement of β-cell failure Ca2+ gradient between the cytosol and ER compartments, and our previous still remain to be uncovered. Here, we investigated the effects of selective data demonstrate signifi cantly reduced ex vivo and in vitro β cell SERCA2b alleviation of glucotoxicity or lipotoxicity on β-cell function and the underly- expression in human and rodent models of type 1 and type 2 diabetes mel- ing molecular mechanisms. We treated 7-week-old db/db mice (U0), a model litus, leading to altered insulin secretion, activation of ER stress pathways, of obese type 2 diabetes, for 1 week with or without the following reagents; and β cell death. However, the effect of SERCA2 insuffi ciency on whole ani- empaglifl ozin (E; SGLT2 inhibitor), bezafi brate (B), combination of E and B mal glucose homeostasis in response to diet-induced obesity has not been (D), no reagents (U). Age-matched non-diabetic db/m mice (N) were used previously tested. To this end, mice with a total body heterozygous deletion as controls. Fed blood glucose levels were lower in N, E and D than U and of SERCA2 (S2HET) and wild-type littermate controls (WT) were placed on B, whereas plasma triglyceride and free fatty acid levels were lower in N, B high fat diet (HFD) containing 45% of kilocalories from fat for 16 wks. Body and D than U and E, which clarifi ed that 1-week treatment of empaglifl ozin composition, glucose metabolism, β cell mass and function, and changes or/and bezafi brate could reduce glucotoxicity or/and lipotoxicity, respec- in islet gene expression were analyzed. Compared to WT controls, S2HET tively. On these conditions, real-time PCR analysis showed that the expres- mice showed no signifi cant difference in body weight or fat content. At the sion levels of transcriptional factors Pdx1, Mafa, and their potential target end of HFD treatment, S2HET mice demonstrated signifi cantly impaired glu- genes Insulin1, Slc2a2, Glp1r in the islets were higher in E and D than U, and cose tolerance (AUC 23258 vs. 41869; p<0.001), lower random and glucose- Insulin2 and Mki67 were higher in D than U. Moreover, the expression levels stimulated serum insulin levels (p<0.05), and a trend towards higher random of Mafa, Insulin1, and Slc2a2 in E and D were greater than U0. Consistent re- and fasting blood glucose. Insulin sensitivity and muscle, adipose, and liver sults were obtained on immunohistochemical analysis with Mafa, Glut2, and insulin signaling were not different between groups. Analysis of pancreatic Ki67 antibodies. These results indicated that suppressed gene expression sections revealed signifi cantly reduced β cell mass in S2HET mice (5.37mg of β-cell factors under diabetic conditions could be restored by reduction vs. 2.66mg; p<0.01) and reduced β cell proliferation. Meanwhile, qRT-PCR of glucotoxicity and lipotoxicity. On the other hand, these molecular-based results showed increased expression of the ER stress markers BIP and CHOP improvements made no difference in glucose-stimulated insulin secretion in islets isolated from S2HET mice. In aggregate, these data demonstrate a between each group of db/db mice. These fi ndings suggested that 1-week critical role for SERCA2b in the β cell compensatory response to diet-induced treatment for glucotoxicity and lipotoxicity improves expression profi les of obesity and suggest involvement of β cell ER Ca2+ dyshomeostasis during β-cell-specifi c genes, although not enough to better insulin secretion. the transition from normal to impaired glucose tolerance and diabetes. Supported By: National Institutes of Health (R01DK093954); U.S. Department of 2336-P Veterans Affairs (101BX001733) GPR120 Selective Agonism Stimulates Insulin Secretion in Rodent but Not in Human Islets 2338-P LISA NORQUAY, BRIAN RADY, S. PAUL LEE, TONYA MARTIN, JUNE XU, KIRSTINE The Individual Roles of Inositol Triphosphate and Diacylglycerol, JUHL, MATTHEW RANKIN, YUANPING WANG, JENSON QI, JAMES LEONARD, Downstream Messengers of FFAR1/GPR40 Produced by Fasiglifam MICHAEL P. WINTERS, MARK J. WALL, MARK MACIELAG, ALESSANDRO POCAI, on the Glucose-Dependent Insulinotropic Effect Spring House, PA KENSUKE SAKUMA, CHIORI YABUKI, MINORU MARUYAMA, AKIKO ABIRU, GPR120 is a G-protein-coupled receptor activated by long chain fatty acids HIDETOSHI KOMATSU, NOBUYUKI NEGORO, YOSHIYUKI TSUJIHATA, KOJI whose chronic activation has been shown to improve glucose tolerance in TAKEUCHI, YUGO HABATA, MASAAKI MORI, Fujisawa, Japan insulin resistant mice via stimulation of peripheral glucose uptake. Free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875) Expression of GPR120 has been demonstrated in β-cell lines, mouse islets improves glycemic control in patients with type 2 diabetes (T2DM) with and human islets. Acute GPR120 activation with non-selective synthetic ag- minimum risk of hypoglycemia. Fasiglifam potentiates glucose stimulated onists and endogenous ligands suggest a potential role of GPR120 on insulin insulin secretion (GSIS) from pancreatic β-cells in a glucose-dependent man- secretion in rodents. A recent report demonstrated that GPR120 mediates ner, but the precise mechanism remains unknown. We previously reported the enhancement of glucose uptake by palmitic-acid-9-hydroxy-stearic acid that fasiglifam acts as an ago-allosteric modulator for FFAR1, which is sup- (9-PAHSA), a member of a new class of mammalian lipids with anti-diabetic ported by the X-ray co-crystal structure demonstrating the unique binding effects whose levels are reduced in insulin-resistant humans. PAHSAs di- mode of fasiglifam. In this study, we investigated key cross-talks between rectly bind to and activate GPR120 in a cell-based GPCR activity assay and insulin secretory pathway and FFAR1 signaling activated by fasiglifam using incubation of PAHSA enhances glucose-stimulated insulin secretion (GSIS) mouse insulinoma MIN6 cells. Insulinotropic effect of fasiglifam in the pres- in human islets. ence of high glucose was cancelled by KATP channel opener and conversely These data raise the possibility that GPR120 is involved in fatty acid-in- potentiated by exogenous high K+ stimulation indicating that membrane duced insulin secretion in humans and that GPR120 mediates the enhance- depolarization is essential for the effect. FFAR1 mainly couples with Gαq ment of GSIS in human islets. protein, leading to the two signaling branches, inositol 1, 4, 5-triphosphate 2+ Here, a GPR120 selective synthetic agonist (cpd A) was evaluated for its (IP3)/Ca release from endoplasmic reticulum (ER) and diacylglycerol (DAG)/ insulinotropic properties in vivo and in isolated mouse and human islets. Cpd protein kinases activation. We demonstrated that fasiglifam as well as an A activates mouse and human GPR120 receptors with EC50 values of 21 nM IP3 mimicker, D-myo-inositol 1, 3, 5-trisphosphate hexakisacetoxymethyl es- 2+ and 16 nM, respectively. ter, 2, 4, 6-tri-O-butyryl-(Bt3(1, 3, 5) IP3/AM) increased amplitudes of Ca 2+ GPR120 agonism dose-dependently enhanced glucose-stimulated insulin oscillation and potentiated GSIS, suggesting that IP3/Ca release from ER secretion (GSIS) in mouse islets but not in islets obtained from GPR120 KO activated by fasiglifam has a pivotal role in the insulinotropic effect. A DAG mice. However, cpd A failed to stimulate GSIS in human islets. mimicker, 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GSIS with little In conclusion, selective GPR120 agonism enhanced GSIS in mouse islets effects on Ca2+ mobilization and oscillation, suggesting that fasiglifam may but not in human islets. These data support the notion that GPR120 plays a also enhance insulin secretion via protein kinases activation independently role in fatty acid-induced insulin secretion in mice but not in humans and that of Ca2+ dynamics. These results give us mechanistic insights into the insu- the GSIS effect of PAHSAs observed in human islets may not be mediated by linotropic effect via FFAR1 which help better understanding of its agonist direct GPR120 activation. therapy for the treatment of T2DM. POSTERS Islet Biology/ Insulin Secretion

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A593 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

2339-P 2341-P Peroxiredoxin6 as Modulator in Pancreatic Beta Cells Function and The Role of Prolyl Isomerase Pin1 in Beta-Cell Functions Survival: A New Mechanism for Exendin 4 YUSUKE NAKATSU, KEIICHI MORI, YASUKA MATSUNAGA, TOSHIAKI FUKU- FRANCESCA PACIFICI, BARBARA CAPUANI, FABIOLA CICCOSANTI, ROBERTO SHIMA, HIDEAKI KAMATA, SUGURU YAMAGUCHI, HISAMITSU ISHIHARA, TO- ARRIGA, DONATELLA PASTORE, ANDREA COPPOLA, VALERIO SANTOLINI, MI- MOICHIRO ASANO, Hiroshima, Japan, Tokyo, Japan CHELA LUISA COLARELLI, GIULIA DONADEL, SARA CARATELLI, FRANCESCA Pin1 binds to the pSer-Pro or pThr-Pro motif and isomerizes the peptide FERRELLI, ANGELICA GALLI, MARIA ROMANO, ALFONSO BELLIA, PAOLO SBRAC- bonds of target proteins, thereby regulating their functions. We previously CIA, GIUSEPPE SCONOCCHIA, GIAN MARIA FIMIA, DAVID DELLA-MORTE, DA- reported that Pin1 expression is markedly upregulated in various tissues by VIDE LAURO, Rome, Italy high-fat diet (HFD) feeding, and that Pin1 regulates glucose and lipid metabo- Oxidative stress is associated with impairment in insulin secretion contrib- lism through binding with IRS-1 or CRTC2. uting to onset of diabetes mellitus. Pancreatic β-cells are very sensitive to Interestingly, Pin1 expression in pancreatic islets was found to be mark- oxidative stress for their low expression of common antioxidant enzymes but edly increased by HFD, an observation similar to those in muscle, liver and with high expression of peroxiredoxins (Prdxs). In our previous study we dem- adipose tissues. In this study, to elucidate the roles of Pin1 in pancreatic beta onstrated a reduced insulin secretion after glucose stimulation in Prdx6-/- cells, we generated beta cell specifi c Pin1 KO mice (βKO mice) employing mice, suggesting a pivotal role of this enzyme in glucose homeostasis. In Ins-Cre Tg mice. Successful deletion of Pin1 limited to beta cells was con- the present study we aimed to further clarify the molecular mechanisms un- fi rmed by western blotting and immunohistochemistry. Glucose tolerance derlay the correlation between Prdx6, glucose-mediated insulin secretion, testing revealed higher serum glucose and lower serum insulin concentra- β-cells function and survival. We stably infected pancreatic cell line βTC-6 tions, after glucose administration, in βKO mice than in controls. In contrast, with Lentiviral vectors containing shRNA Prdx6, confi rming the altered insu- insulin tolerance testing showed no signifi cant difference in insulin-induced lin secretion in Prdx6 knockdown cells. However, we found similar intracel- hypoglycemic effects between the βKO and control mice. The beta cell mass lular insulin levels compared to control, suggesting that the defect in insulin was smaller in the βKO than in the control pancreas. Consistent with this release linked with lacking in Prdx6 could be associated to impairment in result, the rate of Ki67 positive cells was decreased in the pancreatic beta insulin secretion’s mechanism. We, also, evaluated the effect of TNFα- cells of βKO mice, while the proportion of TUNEL positive cells did not differ induced cellular apoptosis by demonstrating that cells knockdown for Prdx6 between the wild type and βKO mice. after TNFα treatment increased apoptosis by 2 folds compared with control In addition, elevated AMPK and reduced p70S6K phosphorylations were cells, confi rming the protective role of Prdx6. In order to further confi rm that observed in the beta cells of βKO mice. protection against apoptosis was mediated by Prdx6, cells were pre-treated Taken together, these observations suggest that Pin1 contributes to in- with Exendin-4 (ex4), which it is known to protect β-cells against apoptosis. creasing the beta cell mass andthereby to greater insulin release, at least We shown that ex4 reduced apoptosis even in Prdx6 knockdown cells, sug- partially via suppression of the AMPK pathway. gesting that protection induced by Prdx6 and ex4 is mediated by different mechanisms. We also demonstrated that ex4 treatment increased Prdx6 2342-P expression while didn’t modify the expression of other Prdxs. In the present Insulin Release (IR) in Humans: Lessons from 399 Nondiabetic (ND) study, we demonstrated an association between Prdx6 and β-cells function and 69 Type 2 Diabetic (T2D) Isolated Islet (HI) Preparations and survival. Moreover, for the fi rst time, we showed a link between Prdx6 MARA SULEIMAN, MARCO BUGLIANI, FAROOQ SYED, FRANCESCO OLIMPICO, and ex4 suggesting a novel mechanism of action of ex4 in inducing insulin LUCIANA MARIOTTI, SILVIA FORNACIARI, FABRIZIO SCATENA, UGO BOGGI, secretion which deserves further exploration. FRANCO FILIPPONI, LORELLA MARSELLI, PIERO MARCHETTII, Pisa, Italy HI are used for research and clinical studies. However, little information 2340-P is available on the functional properties of large series of ND and T2D HI, The Effects of SGLT2 Inhibitor on Pancreatic β-Cell Regeneration and the variables affecting IR ex-vivo. We prepared HI from 399 ND [age, and Glucose Homeostasis in Type 1 Diabetes 61±17 yrs; M/F, 51.5/48.5%; BMI, 25.2±3.8 kg/m2; ICU stay: 3.2±3.3 days; SAM TSZ WAI CHENG, STEPHEN YU TING LI, ERIC MAYOUX, PO SING LEUNG, plasma glucose during ICU (PG): 154±46 mg/dl; pancreas cold ischemia time Hong Kong, China, Ingelheim, Germany (PIT): 15±6 h] and 69 T2D [age, 71±8 yrs (p<0.01 vs. ND); M/F, 59.4/40.6%; Type 1 diabetes mellitus (T1DM) accounts for about 10% of all adults BMI, 26.8±4.0 kg/m2 (p<0.01); ICU stay: 2.6±3.3 days; PG: 217±72 mg/dl with diabetes, in which pancreatic β-cell loss occurs as a consequence of (p<0.01); diabetes duration: 9.2±6.9 yrs; PIT: 15±8 h] organ donors. Islet pu- immune-mediated destruction. Importantly, a recent study has reported rity (IP, estimated by DTZ staining) was 64±23% in ND and 46±25% in T2D that a novel SGLT2 inhibitor empaglifl ozin can improve glycaemic control (p<0.01). In ND, IR (µU/10 islet/min, assessed 4.0±2.5 days after isolation) in an insulin-independent manner via an increase in urinary glucose output was 0.36±0.15 at 3.3 mM glucose (3.3G) and increased to 1.03±0.76 at 16.7 in streptozotocin-induced T1DM rats. Moreover, a study in a patient with mM glucose (16.7G), 1.02±0.7 with 100 µM glyburide (gly) and 0.85±0.57 recent onset T1DM has shown an enhancement in β-cell replication. Based with 20 mM arginine (arg) (all p<0.01 vs. 3.3G). IR values in T2D (assessed on these fi ndings, it is unclear whether empaglifl ozin also has an insulin- after 3.9±2.7 days) were: 0.31±0.10 at 3.3G, 0.50±0.30 at 16.7G, 0.58±0.35 dependent effect. The present study was thus designed to investigate the with gly and 0.56±0.32 with arg (p<0.01 vs. ND for all stimulated IR). Ac- role of empaglifl ozin in pancreatic β-cell regeneration and glucose homeo- cordingly, insulin stimulation index (S-Ind) was 43 (G), 35 (gly) and 25% (arg) stasis in STZ-induced T1DM. Results showed that glucose tolerance was lower in T2D (all p<0.01). In ND, IR was not correlated with any of the studied signifi cantly improved in mice treated with empaglifl ozin, as evidenced by donor characteristics; however, glucose S-Ind was inversely correlated with intraperitoneal glucose tolerance tests. Real-time PCR also showed that days from isolation to IR assessment (p<0.01). In T2D, IR was lower (p<0.01) increase in insulin mRNA expression was observed in the pancreas after at increasing anti-diabetic therapy (from diet to insulin). IR was positively empaglifl ozin administration. Results with an ultra-sensitive insulin ELISA correlated with islet purity (p<0.01) in both ND and T2D. Correlations were kit assay further showed that a higher serum insulin level was detected af- confi rmed by multiple regression analysis. In conclusion, this large series of ter empaglifl ozin treatment. In addition, immunohistochemistry proved that HI preparations unequivocably show reduced IR from T2D islets; islet purity both the insulin to glucagon ratio and the expression of cell proliferation and time from isolation to the day of IR experiments are major factors af- marker Ki67 (co-stained with insulin) were enhanced by empaglifl ozin treat- fecting IR from ND and/or T2D HI; in T2D, IR is lower at increased intensity ment. Furthermore, BrdU cell proliferation assay showed that empaglifl ozin of pharmacological therapy. This should be taken into account when HI are signifi cantly promoted INS-1E cell proliferation under high glucose condi- studied in the molecular or clinical setting. tions. Taken together, this study indicates that empaglifl ozin might have an insulin-dependent effect on improving blood glucose homeostasis in T1DM, 2343-P probably via induction of pancreatic β-cell proliferation. A Potential Role for Altered SOCE and ER Ca2+ Dysfunction in the Supported By: Boehringer Ingelheim Pharma GmbH & Co. KG Pathogenesis of Type 2 Diabetes TATSUYOSHI M. KONO, XIN TONG, WATARU YAMAMOTO, CARMELLA EVANS- MOLINA, Indianapolis, IN Store-operated Ca2+ entry (SOCE) is activated in response to endoplasmic

POSTERS 2+ 2+

Islet Biology/ reticulum (ER) Ca depletion and involves infl ux of Ca from the extracellular

Insulin Secretion space to facilitate refi lling of ER Ca2+ stores. In recent years, studies have identifi ed key molecular components of the SOCE complex, including stromal interaction molecule (STIM), which functions as an ER Ca2+ sensor, and the

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A594 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM plasma membrane Ca2+ channel Orai1. While previous work has identifi ed improved in animals receiving SCHADKO islets compared with those receiv- loss of cell ER Ca2+ homeostasis during the development of type 2 diabetes ing normal islets. These data suggest that the hypoglycemic phenotype of (T2D), the function of SOCE in the cell has been controversial and its role in SCHAD-CHI is islet cell autonomous. the development of T2D has not been well explored. To this end, expression of STIM1 and Orai1 were fi rst confi rmed in rat INS-1 832/13 cells and mouse and human islets. Next, Ca2+ imaging experiments were performed in INS- 1 cells using the Ca2+ indicators Fura-2AM or calcium 6. INS-1 cells were incubated in calcium free buffer and then treated with thapsigargin (Tg) to empty ER Ca2+ stores. Activation of SOCE was detected as an elevation of cytosolic Ca2+ following extracellular Ca2+ supplementation, and SOCE was found to be signifi cantly increased in INS-1 cells transfected with Orai1 and decreased by the STIM1 inhibitor ML9. To determine if SOCE was altered under diabetic conditions, INS-1 cells were treated with 5 ng/ml of the pro- infl ammatory cytokine IL-1. IL-1 led to a signifi cant decrease in Tg-elicited SOCE as well as a ~74% reduction in STIM1 mRNA expression, while Orai1 expression was unchanged. Finally, STIM1 and Orai1 expression were tested in human cadaveric islets. Similar to results obtained in IL-1-treated INS-1 cells, STIM1 expression was signifi cantly reduced in islets isolated from do- nors with T2D; Orai1 mRNA levels were not different. These results confi rm activation of SOCE in the pancreatic β cell in response to ER Ca2+ depletion and suggest a role for altered STIM1 expression and loss of SOCE in ER Ca2+ dyshomeostasis during the progression of T2D.

2344-P DPP-4 Suppresses Glucose-stimulated Insulin Secretion in Cultured Supported By: Novo Nordisk Foundation; Helse Vest Norwegian Regional Pancreatic Beta Cell Line that Is Partially Recovered by Vildagliptin Health Authority or Mannose-6-phosphate DAE HO LEE, DONG-SUNG LEE, EUN-SOL LEE, Iksan, Republic of Korea, Incheon, 2346-P Republic of Korea Ethnic Differences in Insulin Secretion Tone May Require Different DPP4-inhibitors that prolong the insulinotropic effect of glucagon-like Diagnostic Indicators peptide-1 (GLP-1) are now in active clinical use for the management of type 2 JOON HA, KYONGYEUN JUNG, SOO LIM, SUNGHEE CHOI, HAKCHUL JANG, AR- diabetes. Studies have shown that DPP-4 inhibitors have pleiotropic effects THUR SHERMAN, Bethesda, MD, Seongnam, Republic of Korea beyond the glycemic control. And, soluble DPP-4 has been considered as an Insulin exocytosis is a major component of the insulin secretion machin- adipokine of which actions need to be further characterized. In the present ery, which may be distinct in different ethnic groups. For example, a sub- study, we investigated the direct effects of soluble DPP-4 on glucose-stimu- group of Korean exhibits relatively high glucose 30 minutes into an oral lated insulin secretion in cultured pancreatic beta cell line, RINm5F cells. We glucose tolerance test (OGTT) but normal fasting plasma glucose (FPG) and used two types of DPP-4 inhibition, alone and in combination; vildagliptin, as two-hour glucose (2HG). Despite not satisfying the standard criteria for type an its enzymatic inhibitor, and mannose 6-phosphate (M6P) as a competi- 2 diabetes (T2D), this group has elevated risk. On the other hand, African- tive inhibitor of binding of soluble DPP-4 to its insulin-like growth factor II/ Americans have relatively low glucose in the early portion of an OGTT to- M6P (IGF-II/M6P) receptor. Soluble DPP-4 suppressed glucose-stimulated gether with normal FPG and 2HG, and also have elevated risk for T2D. We insulin secretion in a concentration-dependent manner in RINm5F cells. have developed a comprehensive model of the pathogenesis of T2D, which The suppressed insulin secretion was partially recovered by vildagliptin or includes a subsystem for exocytosis, and have applied it to simulate OGTTs. M6P pre-treatment before the addition of soluble DPP-4. And, the combined The model suggests that the Korean OGTT pattern results from a defect pre-treatment with vildagliptin and M6P was more effective than the pre- in fi rst-phase insulin secretion and a delay in second-phase secretion. Both treatment with single agent alone. Because M6P was not fully effective in data and model simulations support that this defect contributes to rapid blocking the binding of soluble DPP-4 to cell surface receptor, its protective progression to diabetes. In contrast, African Americans typically exhibit effect on insulin secretion was weaker when compared to vildagliptin. And, stronger fi rst-phase insulin secretion than Whites but second-phase insulin soluble DPP-4 suppressed the expression of PDX-1 protein in glucose-stim- secretion similar to that of Whites. The simulations for this case suggest ulated RINm5F cells that was recovered by vildagliptin pre-treatment. Our that the strong fi rst phase results from a large readily releasable pool of fi ndings suggest that soluble DPP-4 has a direct inhibitory effect on glucose- insulin granules but that the diabetes risk results from traffi cking of reserve stimulated insulin secretion in pancreatic beta cells. Further studies are re- vesicles to the plasma membrane that is more susceptible to deterioration quired to determine the role of soluble DPP-4 in beta cell functions. under the stress of insulin resistance. In summary, different temporal insulin Supported By: National Research Foundation of Korea (2008-0062484) secretory patterns among ethnic groups during the OGTT suggest different diagnostic indicators that are more informative than the current standard. 2345-P The Hypoglycemic Phenotype of Short-Chain Hydroxyacyl-CoA De- 2347-P hydrogenase (SCHAD) Defi cient Mice Is Islet Cell Autonomous miRNA374-Common Regulator of PGC-1a in Hepatocytes and Beta ANDERS MOLVEN, JENNIFER HOLLISTER-LOCK, JIANG HU, RACHAEL MAR- Cells TINEZ, PÅL R. NJØLSTAD, CHONG WEE LIEW, GORDON C. WEIR, ROHIT N. JI-WON KIM, YU-BAE AHN, KUN-HO YOON, Seoul, Republic of Korea KULKARNI, Bergen, Norway, Boston, MA, Chicago, IL Glucotoxicity plays a major role in the progressive deterioration of β-cell Persistent hyperinsulinemic hypoglycemia of infancy, also termed con- function and development of type 2 diabetes mellitus. MicroRNAs (miRNAs) genital hyperinsulinism (CHI), can be caused by inactivating mutations in the represent small noncoding RNAs that play a role in many diseases, including gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), an diabetes. This study was to search the common gene mechanism between ubiquitously expressed enzyme of fatty acid oxidation. The hypersecretion liver and pancreas in diabetic condition such as glucotoxicity and to search of insulin may be explained by a lost interaction between SCHAD and glu- the miRNAs related to glucotoxicity-induced β-cell dysfunction and glucose tamate dehydrogenase in the pancreatic beta-cells. However, in affected metabolism dysfunction in liver. To study the role of miRNAs in glucotoxicity- individuals there is also a general accumulation of metabolites specifi c for induced β-cell dysfunction and glucose metabolism dysfunction, we ana- the enzymatic defect, and it remains to be explored whether the hypoglyce- lyzed the miRNA expression patterns of primary rat islets and hepatocytes mic phenotype of SCHAD-CHI can be uncoupled from the systemic effect on for three days after exposure to glucotoxicity and confi rmed up-regulated fatty acid oxidation. We therefore transplanted islets from global SCHAD and down-regulated miRNAs. POSTERS

knock-out (SCHADKO) mice into mice with streptozotocin-induced diabetes. Among the down-regulated miRNAs, we demonstrate that miR-374 is Islet Biology/

Mice receiving SCHADKO islets exhibited signifi cantly lower random and a key player in glucotoxicity-induced β-cell dysfunction and glucose me- Insulin Secretion fasting blood glucose compared with mice receiving normal islets and non- tabolism dysfunction. miRNA expression is decreased by glucotoxicity, and diabetic, non-transplanted controls. Intraperitoneal glucose tolerance was extrinsic inhibition of miR-374 by antisense miRNA induced β-cell dysfunc-

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tion. Overexpression of miR-374 suppressed PGC-1α and FOXO1 expression that is superior to treatment with either as a monotherapy. We synthesized in glucotoxicity. MiR-374 was found to target the 3’-untranslated region recombinant GLP-1 and GIP Fc fusion proteins to investigate these combina- (3’UTR) of PGC-1α through miR-374 binding sites and to downregulated PGC- tions in rodents. In lean C57Bl/6 mice, acute doses of both Fc-GIP and Fc-GLP-1 1α protein abundance at the post-transcriptional level by luciferase activity resulted in dose-dependent reductions in glycemic excursions following in- assay combined with mutational analysis. traperitoneal glucose tolerance tests. Acute doses of Fc-GLP-1 also produced Here we demonstrate that miR-374 mediated suppression of PGC-1α gene dose-dependent reductions in food intake. These acute dose-response studies expression is an important initiation step of glucotoxicity induced β-cell dys- guided the selection of low and high doses for longer term combination stud- function and glucose metabolism dysfunction in liver and appropriate new ies. Diet-induced obese (DIO) or diabetic db/db mice were treated every other target for early treatment. day for 2 or 3 weeks, respectively, with vehicle, Fc-GIP (high dose), Fc-GLP-1 (high dose) or a combination of either low doses or high doses of Fc-GIP and 2348-P Fc-GLP-1. The weight loss observed in DIO mice and the HbA1C reductions ob- Alterations in Fatty Acid Metabolism Associated with Glucolipo- served in db/db mice were signifi cant relative to vehicle, but similar between toxicity in β-Cells the Fc-GLP-1 (high) monotherapy arm and the high dose combination arm. In- MAHMOUD ELAZZOUNY, ROBERT T. KENNEDY, CHARLES F. BURANT, Ann Arbor, MI terestingly, the low dose combination reduced HbA1C as effectively in db/db Acute fatty acid (FA) exposure can augment insulin secretion by the mice as high dose Fc-GLP-1 monotherapy. In summary, our data indicates that generation of important signaling molecules through esterifi cation with high dose combinations of GIP and GLP in DIO and db/db mice produce com- glucose-derived glycerol-3-phosphate. Chronic increase in glucose and fatty parable weight loss and glucose-lowering, respectively, to high dose GLP-1 acid is known to causes “glucolipotoxicity” of β-cells leading to increased monotherapy. In diabetic db/db mice we observe that low dose combinations basal insulin and reduction in glucose stimulated insulin secretion. We used of GLP-1 and GIP interact to produce greater improvement in glucose control. INS-1 (832/13) cell line to monitor the metabolomics changes that are as- Thus, addition of GIP activity may reduce the GLP-1 doses required to achieve sociated with glucolipotoxicity. Two days of incubation of β-cells with 25 good glucose control in type 2 diabetic subjects. mM glucose and 1 mM of fatty acid mixture (2:1 Oleate: palmitate) was used to probe these changes. Using LC-MS and metabolomics approaches we 2351-P were able to monitor changes in several pathways involved in glucose and The Roles of Cellular Adhesion Molecule 1 (CADM1) in the Develop- fatty acid oxidation as well as esterifi cation. After the two days of vehicle ment and Maintenance of the Insulin Secretory Machinery or glucose+fatty acids, cells were exposed to 250 µM fully labeled U-13C TOM A. CALDWELL, CHARLES ZHANG, REZA MIRBOLOOKI, STEVEN D. CHESSLER, Oleic acid to monitor fatty acid fl ux. Glucolipotoxic cells showed higher fatty Irvine, CA acid oxidation as evidenced by a 3-fold increase in absolute amount and Recent advances in neurobiology have yielded important insights into how percentage labeling of the M+2 isotopomer of citrate and 5-fold increase in transcellular interactions drive neuronal synaptogenesis. Our prior results the +18 labeled oleoyl carnitine (all p<0.01). This increase in apparent fatty revealed that this new knowledge is applicable to the beta-cell insulin se- acid oxidation was accompanied by a 2-fold reduction in the generation of cretory machinery: Many of the proteins_such as neurexins, neuroligins, and M+18 labeled DAG (34:1) (P=0.01). The increase in fatty acid oxidation was CASK_that are involved in the development of the pre-synaptic terminals of confi rmed using a SeaHorse apparatus by the fi nding of a 50 % increase in neurons have parallel roles in the assembly of the insulin secretory machin- the basal oxygen consumption rate (OCR) under low glucose conditions and ery. We asked whether CADM1, a neural transmembrane protein also pres- a two fold reduction of glucose stimulated increase in OCR (P<0.05). These ent in beta-cells, is important for the maturation of the secretory machinery. data suggest that reduction in the formation of signaling glycerolipids play We have previously shown that CADM1 inhibits insulin secretion, which was a role in glucolipotoxicity. revealed by an increase in the stimulation index of INS1 cells treated with Supported By: National Institutes of Health (DK097153) CADM1-targetting siRNA. Here we show that CADM1 transcellular contact is suffi cient to induce assembly of the insulin secretory machinery. This was 2349-P indicated by a correlation between syntaxin-1 punctateness in INS1 cells The Type 2 Diabetes-associated Channel TALK-1 Is a Key Determi- and CADM1 expression levels in cocultured, transfected COS7 cells. CADM1 nant of β-Cell Excitability and Insulin Secretion directly interacts with proteins in the insulin secretory machinery as shown NICHOLAS C. VIERRA, PRASANNA K. DADI, IMJU JEONG, FARAH LADAK, DAVID by coimmunoprecipitation of CASK and syntaxin-1 from INS1 cell lysates. POWELL, DAVID A. JACOBSON, Nashville, TN, Norfolk, VA, The Woodlands, TX The effect on insulin secretion of CADM1 transcellular interactions differed Two-pore-domain potassium (K2P)channels play an important role tuning between INS1 cells (enhancement of secretion) and rat islets (repression β-cell glucose-stimulated insulin secretion (GSIS). The K2P channel TALK-1 of secretion). CADM1 is known to infl uence actin polymerization. Here we is linked to type 2 diabetes risk through a coding sequence polymorphism test the hypothesis that transcellular CADM1 contact enhances actin po- (rs1535500), however, its physiological function remains unclear. Here, we lymerization and thereby inhibits secretion in rat islets. Treatment of rat show that functional TALK-1 channels are expressed in mouse and human islets cocultured with CADM1-expressing COS7 cells in 5 µM Latrunculin- β-cells, where they serve as key regulators of electrical excitability and GSIS. B signifi cantly enhanced insulin secretion, corroborating prior fi ndings that Genetic ablation of TALK-1 results in β-cell membrane potential (Vm) depolar- fi lamentous actin acts as a barrier to insulin secretion. The data presented ization, increased islet Ca2+ oscillation frequency, and enhanced 2nd-phase here strongly suggest that CADM1 is a functional participant in the insulin GSIS. Moreover, we fi nd that the rs1535500 polymorphism, which results in secretory machinery and that it may exert its effect on insulin secretion in an alanine to glutamate substitution in the C-terminus (Ct) of human TALK-1, part through cytoskeletal remodeling. increases channel activity. Interestingly, TALK-1 channels are also activated Supported By: National Institute of Diabetes and Digestive and Kidney Diseases by membrane phospholipids through a charge-dependent interaction with the Ct. These fi ndings reveal TALK-1 channels as important modulators of 2352-P 2nd-phase insulin secretion, and suggest a clinically relevant mechanism for Effect of Human Myotube Conditioned Media on Glucose-stimulat- rs1535500, which may increase diabetes risk by limiting GSIS. ed Insulin Secretion from Mice Islets Supported By: National Institutes of Health (DK081666, DK097392, DK20593) MARIA L. MIZGIER, KATIE LOUCHE, GLORIA GARRIDO, RODRIGO CATALDO, JOSE L. SANTOS, MARIANA CASAS, PAOLA LLANOS, ARIEL CONTRERAS, CEDRIC 2350-P MORO, JOSE E. GALGANI, Santiago, Chile, Toulouse, France The Effects of Combinations of GIP and GLP-1 Fc-Fusion Proteins in Fasting-to-postprandial transition requires tight adjustment of insulin se- Obese and Diabetic Mice cretion to its demand, so tissue glucose supply is assured while hypo-/hyper- ARTHUR T. SUCKOW, DAVID HORNIGOLD, JACQUELINE NAYLOR, HAMID SAL- glycemia is prevented. A putative skeletal muscle-pancreas crosstalk might ARI, ISABELLE SERMADIRAS, PETER RAVN, SAFINA ALI, NICHOLAS BHAGROO, participate considering that muscle is pivotal for insulin-mediated glucose ANDIE COLLINSON, JOE GRIMSBY, JAMES TREVASKIS, ANISH KONKAR, Gaith- disposal and muscle-derived factors play an endocrine role. We studied the ersburg, MD, Cambridge, United Kingdom myokine profi le of conditioned media (CM) from insulin-stimulated human GIP and GLP-1 are gut peptides secreted from enteroendocrine K- and L- myotubes, and the effect of the CM on Glucose-Stimulated Insulin Secretion cells, respectively, following meal consumption. These peptides act directly on (GSIS) from mice islets. POSTERS Human satellite cells were isolated from vastus lateralis biopsies (4 Islet Biology/ pancreatic beta-cells to promote glucose-dependent insulin secretion via the Insulin Secretion GIP- and GLP-1-receptors. GLP-1 also acts via neuronal GLP-1 receptors to in- healthy men, 23±1 kg/m2, 24±1 y). After differentiation, myotubes were in- hibit food intake and promote weight loss. In principle, the combination of GIPR cubated with/without 100 nmol/L insulin for 24 h and CM was collected. and GLP-1R agonists might result in glucose-lowering and body weight loss Glycogen content and synthesis, glucose oxidation and extracellular lac-

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A596 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM tate concentration were measured. Myokines were characterized using an age in genetic defects of long-chain L-3-hydroxyacyl-CoA dehydrogenase or antibody-based array. Pancreatic islets from adult male C57BL6/J mice were the mitochondrial trifunctional protein accumulate in islets under GLT: 3) litera- incubated for 24 h with CM and GSIS was assessed. ture (Tonin et al., 2010) suggesting that LC3HFAs behave as strong uncouplers Insulin increased myotube glycogen content and synthesis by 1.6±0.3 fold of oxidative phosphorylation in concentration that might occur in islet tissue. (p<0.01) and 2.3±0.9 fold (p<0.01) while glucose oxidation and lactate con- We proposed that bioenergetics failure is due to partial uncoupling/inhibition centration remained similar when compared with control myotubes. Over 20 of oxidative phosphorylation by a novel mechanism of GLT. myokines were found in control CM, with MCP1, GRO, ENA78 and IL8 hav- Supported By: American Diabetes Association (7-11-BS-34 to N.M.D.) ing the highest relative content. Moreover, eotaxin2, GCP2, IGFBP2 and IL8 were modifi ed by insulin. Islets incubated with control CM vs. unconditioned 2355-P media showed similar basal insulin secretion (p=0.99) while increased GSIS Dynamic Changes of Blood Betatrophin, Glucose, and Insulin during (p<0.05). Such difference did not remain in islets incubated with CM from STZ Induction of Diabetes in Rhesus Monkeys insulin-treated myotubes. YONGQIANG LIU, BINGDI WANG, GUOFENG SUN, XUNHUA DING, XIAOLI WANG, Non-insulin-stimulated human myotubes secrete several factors that may YI-XIN (JIM) WANG, YONG-FU XIAO, Jiangsu, China increase in vitro GSIS. Such effect was not observed in response to CM from Betatrophin can proliferate β-cells and enhance insulin secretion in sever- insulin-stimulated myotubes. Eotaxin2, GCP2, IGFBP2 and IL8 may play a role al animal studies. However, its effect on human β-cells has not been clearly on GSIS considering that their content differed in CM from insulin- and non- evidenced. This study was to investigate betatrophin and β-cell prolifi cation insulin-stimulated myotubes. in nonhuman primates (NHPs, rhesus monkeys) with normal blood glucose Supported By: National Fund for Scientifi c and Technological Research of Chile (<80 mg/dL) and then treated with streptozocin (STZ). In STZ-treated NHPs (1130217 to J.E.G.); University of Chile (PUCNUT1403 to J.E.G.); National Research (n=7), both blood glucose and betatrophin increased dramatically (Fig. 1, Pan- Agency (ANR-12-JSV1-0010-01 to C.M.); Société Francophone du Diabète (to C.M.) el A). Blood insulin initially increased on day 1 after STZ and then decreased. Two NHPs with blood glucose back to ~80 mg/mL on day 7 after STZ injec- 2353-P tion showed the recovery of insulin secretion. In another 2 STZ-treated NHPs Secreted Frizzled Related Protein 4 Does Not Regulate Glucose Ho- with blood glucose >250 mg/dL, proliferation of both β-cell and non-β-cell meostasis or Body Weight in Mice was observed in their islets (Fig. 1, Panel B). The rest 2 NHPs with more JASON MASTAITIS, MARK ECKERSDORFF, SOO MIN, YURONG XIN, KATIE CAV- completed β-cell destrotion after STZ treatment showed much higher blood INO, JOHNPAUL AGLIONE, HARUKA OKAMOTO, ERQIAN NA, TREVOR N. STITT, glucose (>300 mg/dL) with no obvious β-cell regeneration and ivGTT showed MELISSA G. DOMINGUEZ, JENNIFER P. SCHMAHL, CALVIN LIN, NICHOLAS W. almost no insulin secretion (Fig. 1, Panel C). Our data demonstrate that blood GALE, DAVID M. VALENZUELA, ANDREW J. MURPHY, GEORGE D. YANCOPOU- betatrophin rised parallelly with glucose elevation during STZ induction of LOS, JESPER GROMADA, Tarrytown, NY diabetes in NHPs. Such increase in blood betatrophin could lead to β-cell Secreted frizzled related proteins constitute a family of fi ve circulating regeneration in some animals, 5 out of 7, but not in those NHPs with severely modulators (SFRP1-5) of wingless-type MMTV integration site family (WNT) destroyed islets, 2 out of 7. Our data may explain, at least partially, why cir- signaling. Members of the SFRP family of proteins have been implicated in culating betatrophin level is elevated in patients with type 2 diabetes. metabolic control. Here we report on the metabolic phenotype of Sfrp4 defi - cient (Sfrp4-/-) mice. Sfrp4-/- mice (75% C57BL/6NTac and 25% 129/SvEvTac background) were generated by replacement of a 8.9 kb segment containing all six Sfrp4 exons with LacZ fused in frame just after Sfrp4’s ATG start site. Sfrp4-/- mice on a chow diet had normal glucose tolerance, beta cell mass and body weight when compared to their wildtype littermates. Energy expenditure and food intake showed no genotypic differences. Sfrp4-/- mice on a high-fat diet gained weight and developed insulin resistance and hy- perinsulinemia similar to wildtype mice. In a separate series of studies we found that overexpression of SFRP4 in livers of mice using hydrodynamic DNA delivery resulted in high circulating levels of biologically active SFRP4 protein but did not alter glycemic control. Messenger RNA expression and LacZ reporter studies in mice detected Sfrp4 in several metabolic tissues, including hypothalamus, white adipose tissue, intestine and muscle but not in pancreatic islets. In conclusion, these data do not support a role for SFRP4 in the control of body weight, food intake or glycemic control.

2354-P Long-Chain Beta-Hydroxylated Fatty Acids as Mediators of Pancre- atic Beta-Cell Bioenergetics Failure NICOLAI M. DOLIBA, QIN LIU, CHANGHONG LI, PAN CHEN, JIE CHEN, CHENG- YANG LIU, ALI NAJI, MICHAEL BENNET, FRANZ M. MATSCHINSKY, Philadelphia, PA 2356-P Hyperglycemia of type 2 diabetes mellitus develops when pancreatic beta- G-protein Coupled Receptor 40 Enhances Insulin Secretion through cells damaged by chronic exposure to elevated blood glucose and lipids (gluco- Two Classes of Protein Kinase C in INS-1 cells lipotoxicity (GLT)) fail to synthesize and secrete suffi cient quantities of insulin TAKUYA HASHIMOTO, HIDEO MOGAMI, TETSU MORIOKA, DAISUKE TSURIYA, for maintaining plasma glucose level. To duplicate GLT in vitro, islets were HIROSHI MORITA, SHIGEKAZU SASAKI, TATSURO KUMADA, YUKO SUZUKI, TET- cultured for 3-5 days with 0.5 mM palmitic acid (PA) or a mixture of PA and SUMEI URANO, YUTAKA OKI, Hamamatsu, Japan oleic acid (OA) (2:1 ratio) at 1% albumin and different glucose concentrations. The mechanism by which G-protein coupled receptor 40 agonist, GW9508 Glucose and acetylcholine (Ach) were used as stimulus of insulin secretion (IS) (GW) amplifi es insulin secretion by activating protein kinase C (PKC) remains providing a test unmatched in sensitivity. Inhibition of IS by PA was seen in to be determined. We focus on two classes of PKC, conventional PKC (cPKC) islets cultured 3 days at 10 mM glucose and the effects became more severe and novel PKC (nPKC). The former is activated by diacylglycerol (DAG) and with increasing glucose during culture. Ach potentiation of IS was reduced Ca2+, the latter by DAG. We have shown that GW activates myristoylated even more drastically in PA/OA exposed islets. The FFA-induced IS defect was alanine-rich C kinase substrate (MARCKS), irrespective of changes in intra- absent when glucose during culture was replaced with glutamine plus leucine cellular Ca2+ concentration ([Ca2+]i) at a substimulatory concentration of and also when islets were cultured with high glucose and FFAs but tested with glucose (G), 3mM G. This indicates that GW predominantly activates nPKC an amino acid mixture indicating absolute glucose requirement to induce and at 3 mM G. Here, we examined whether two distinct PKC isoforms, PKCα demonstrate the lipotoxicity. Our results point towards bioenergetics failure of cPKC and PKCε of nPKC, which are dominantly expressed in INS-1 cells, as a primary cause of beta-cell damage as based on the following fi ndings: enhance insulin secretion in response to GW at 3mM G and a stimulatory POSTERS

1) a marked reduction of the oxygen consumption rate below normal control, concentration of glucose, 20 mM G. Islet Biology/ and a reduced ability to increase islet ATP with high glucose following 5 days Using epifl uorescence microscopy, we monitored the relative change in Insulin Secretion of exposure to GLT: 2) new evidence showing that long-chain 3-OH-fatty acids cytosolic fl uorescence intensity (cF) of Fura2-AM as a marker of changes (LC3HFA) (considered to be the causal factor for widespread severe cell dam- in [Ca2+]i and MARCKS-GFP as a marker of PKC activation in response to

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A597 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

GW at 3 mM or 20 mM G in INS-1 cells, rat insulin-producing cells. To as- in interstitial volume using EDTA to improve penetration. We measured the sess activation of the two PKC isoforms, the relative change in membrane media exchange rate through islets and determined a suitable range of oper- fl uorescence intensity (mF) of PKCα-GFP and PKCε-GFP were measured using ating fl ow-rates. Using similar treatments, we achieved highly effi cient ad- total internal refl ection fl uorescence microscopy. enovirus transduction and deeper penetration into intact islets as reported GW induced the sustained increase in cF of MARCKS-GFP at 3 mM G in by fl uorescent protein expression. We will further measure islet metabolic spite of Ca2+ spikes whereas the transient increase on the sustained one responses (NAD(P)H and calcium) to confi rm normal beta-cell function and took place at 20 mM G following a transient increase of [Ca2+]i. The increase coupling as well as determine whether smaller AAV viruses can be used to in area under the curves of cF of MARCKS-GFP was plotted against that of achieve transduction in fully intact islets. [Ca2+]i. There was a stronger correlation between them at 20 mM G than at 3 mM G. GW increased the repetitive spikes of mF of PKCα-GFP and the sus- tained one of PKCε-GFP at 3 mM G. GW didn’t enhance insulin secretion at 3 ISLET BIOLOGY—SIGNAL TRANSDUCTION mM G but did at 20 mM G (1.97±0.30 fold increase; p < 0.05). These results indicate that GW activates both PKCs not at 3mM G but 20mM G thereby enhancing insulin secretion in INS-1 cells. Guided Audio Tour: Signal Transduction (Posters: 2359-P to 2366-P), see page 17. 2357-P A Red-shifted Photoswitchable Sulfonylurea for the Remote Control & 2359-P of Pancreatic Beta-Cell Function Insulin Secretory Defect in a Mouse Model of Chronic Kidney JOHANNES BROICHHAGEN, JAMES A. FRANK, NATALIE R. JOHNSTON, RYAN Disease K. MITCHELL, KATJA ŠMID, PIERO MARCHETTI, MARCO BUGLIANI, GUY A. LAETITIA KOPPE, ELSA NYAM, VALENTINE MOULLE, VINCENT POITOUT, Mon- RUTTER, DIRK TRAUNER, DAVID J. HODSON, Munich, Germany, London, United treal, QC, Canada Kingdom, Pisa, Italy Disorders of glucose homeostasis are common in chronic kidney disease Photopharmacology harnesses the power of light to fi nely regulate drug (CKD). Whereas insulin resistance in CKD is well characterized, the presence activity and modify biological processes accordingly. Thus, using a sulfony- and the mechanisms of impaired insulin secretion remain unclear. The pur- lurea-based synthetic photoswitch to confer light-sensitivity on endogenous pose of this study was 1-to examine whether insulin secretion is altered in a + ATP-sensitive K (KATP) channels, we have recently demonstrated the optical mouse model of CKD (5/6 nephrectomy); 2-to ascertain whether urea has a control of beta cell function and insulin release. However, a barrier to the direct negative impact on insulin secretion, 3-to assess the role of oxidative use of such compounds is their UV-blue absorption spectra, increasing pho- stress and O-GlcNacylation in both models. At 3 weeks post-surgery, β-cell totoxicity and limiting tissue penetration due to photon scattering. mass, insulin content and secretion were measured. Insulin secretion was To address this issue, we have devised a novel approach for the synthe- assessed by hyperglycemic clamps. Isolated mouse or human islets were sis of wavelength-tuned sulfonylureas with red-shifted activation profi les. exposed to pathological concentrations of urea (20 mM, 24h) and insulin Using glimepiride as a template, this was achieved by extending the sul- secretion was measured in 1-h static incubations. Total O-GlcNacylated fonylurea aromatic core to a heterocylic azobenzene, rather than electron- proteins levels were evaluated by Western blot. The effect of an antioxi- donating halogen or amine moieties. The resulting azosulfonylurea, termed dant (N-acetylcysteine, NAC) on insulin secretion was assessed after treat- JB558, undergoes trans- to cis- isomerization in response to green-yellow ment of CKD mice or in urea-treated islets. Hyperglycemic clamp revealed (520-560 nm) light, and this can be readily reversed following rapid (ms) a decrease in both glucose- and arginine-induced insulin secretion in CKD thermal relaxation in the dark. The accompanying alterations to molecule mice. Insulin levels during the steady-state phase of the clamp were 1.2±0.1 motion/shape unusually allow photoactivation of Epac2A, a critical mediator vs. 2.2±0.3 ng/ml in sham mice (n=7, p<0.05). Isolated islets from CKD mice of sulfonylurea action. As a result, multicellular Ca2+ fl uxes can be reversibly showed reduced insulin secretion in response to 16.8 mM glucose (-41% manipulated in both rodent (n = 10 recordings) and human (n = 9 recordings) n=5-7, p<0.05) and 35 mM KCl (-49%, n=5, p<0.05). β-cell mass and islet pancreatic islets using green (520 nm), but not violet-blue (400-490 nm) light, insulin content were not altered. A similar reduction in insulin secretion was yielding remote control over insulin secretion (n = 6 animals). No cytotoxic observed after exposure of normal mouse islets (-42% vs. islets exposed effects of JB558 could be observed, as assessed using propidium iodide/ to 20 mM mannitol, n=11, p<0.05) or human islets (-47%, n=4, p=0.08) to calcein incorporation (n = 6 animals). Moreover, JB558 was resistant to E. 20 mM urea. We observed an increase in total O-GlcNAcylated proteins in Coli azoreductase, (n = 3 assays), expected to limit oral bioavailability due to CKD and urea-treated islets. NAC decreased O-GlcNAcylation levels and re- diazene cleavage in the intestine. stored insulin secretion in both models. In summary, CKD is associated with In summary, JB558 displays properties that are well-suited to the in vivo an insulin secretory defect which likely lies at the level of exocytosis. This manipulation of pancreatic beta cell function with light. defect is recapitulated by exposure of islets to urea, and is associated with Supported By: Diabetes UK; European Research Council (UK); UK Medical Re- an increase in protein O-GlcNAcylation and oxidative stress. search Council; UK Wellcome Trust; European Association for the Study of Diabetes & 2360-P 2358-P Glucocorticoids Regulate Beta-arrestin-1 Expression and Impact Towards a Microfl uidic Device for Highly Effi cient Adenoviral on the GLP-1 Receptor Signalling in Pancreatic Beta Cells Transduction of Pancreatic Islets MORGANE ROUSSEL, JULIA MATHIEU, FABIENNE CHARRIER-SAVOURNIN, ERIC PAMUDITHA N. SILVA, ZAID ATTO, UILKI TUFA, YIH YANG CHEN, ALLEN VOLCHUK, TRINQUET, GYSLAINE BERTRAND, STÉPHANE DALLE, Montpellier, France, Cod- DAWN M. KILKENNY, JONATHAN V. ROCHELEAU, Toronto, ON, Canada olet, France Beta-cells act as a functional unit within pancreatic islets through multi- Strategies based on activating GLP-1 receptor (GLP-1R) in pancreatic beta- ple cell-cell interactions resulting in a strict threshold of glucose-stimulated cells are extensively developed for type 2 diabetes treatment. Deciphering insulin secretion. To study beta-cell metabolism, viral transduction is com- the behavior of the signaling pathways linked to activated GLP-1R within the monly used on ex vivo islets to genetically manipulate the tissue. However, beta-cells is of great importance. The nonvisual arrestin, beta-arrestin-1, is a poor penetration of adenovirus and adeno-associated virus (AAV) to the multifunctional scaffold protein which has been reported to play key roles in center of the tissue requires dispersion to single cells to achieve suffi cient beta-cell GLP-1R signaling. On binding of activated GLP-1Rs, beta-arrestin-1 transduction effi ciency, but ultimately results in loss of beta-cell connectiv- mediates GLP-1 signaling to cAMP production and insulin secretion. Using ity. To improve transduction effi ciency in intact islets, we now aim to fl ow vi- INS-1E beta-cell line and isolated mouse pancreatic islets, we found that a ral particles deeper into the tissue using a custom-made microfl uidic device. glucocorticoid exposure (dexamethasone, 100 nM, 24 h) increased beta-ar- We created a device featuring hydrodynamic traps along a spiral-shaped restin-1 protein expression. This up-regulation of beta-arrestin-1 expression channel to hold and transduce islets. This device was engineered to provide was associated with a two-fold enhancement of cAMP production elicited similar resistances and pressure drops along individual traps, allowing for by GLP-1 (20 nM, 20 min) measured by HTRF technology. These effects were similar media exchange in each islet. Viral particle penetration was physi- mediated through the nuclear glucocorticoid receptor (GR) determined using cally modeled using fl uorescently labeled 25 and 100 nm gold nanoparticles, a GR antagonist (RU486, 1 µM). In contrast, the cAMP production induced POSTERS

Islet Biology/ diameters of similar size to AAV and adenovirus, respectively. Even with sig- by an adenylyl cyclase activator (forskolin, 100 nM, 20 min), or by glucagon

Insulin Secretion nifi cant media exchange to the center of islets in our device, we observed (20 nM, 20 min), were not modifi ed by the glucocorticoid exposure. This spe- exclusion of the 100 nm particles from the tissue. Consistently, exclusion of cifi c enhancement of cAMP production by GLP-1 was not due to inactivation these larger nanoparticles and adenovirus necessitated a transient increase of phosphodiesterases shown using a phosphodiesterase inhibitor (IBMX,

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A598 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

1 mM), or increased GLP-1R expression measured by western blot. While & 2363-P 24 h-exposure of beta-cells to glucocorticoid decreased glucose (8.3 mM)- Pro-infl ammatory Cytokines Disrupt Islet Gap Junctions Early in the induced insulin secretion, it did not modify the insulin secretion potentiated Development of Type 1 Diabetes in NOD Mice via Pkcd- Phosphory- by GLP-1. This study reports that glucocorticoids regulate beta-arrestin-1 lation of Connexin36 expression, and have the capacity to modulate the cAMP levels produced NIKKI L. FARNSWORTH, ALIREZA HEMMATI, RICHARD K. BENNINGER, Aurora, CO by activated GLP-1R in beta-cells. This study further identifi es a previously Pro-infl ammatory cytokines have been implicated as mediators of the de- undescribed cross-talk between nuclear and plasma membrane receptors cline in islet mass and function in type 1 diabetes. Connexin36 (Cx36) gap in beta-cells whereby glucocorticoids may impact on the clinical effects of junctions regulate β-cell calcium (Ca2+) signaling and insulin secretion across GLP-1R-based drugs. the islet. Altered Ca2+ signaling occurs upon treatment with low levels of Supported By: INSERM (France) cytokines and is consistent with disrupted Cx36 gap junction coupling. The goal of this study is to understand the role of altered Cx36 gap junction cou- & 2361-P pling in islet dysfunction in early type 1 diabetes and how pro-infl ammatory Interleukin-1β Regulates ChREBP and TXNIP Expression in Pancre- cytokines may mediate disruption. atic β-Cells Ca2+ dynamics, insulin secretion and Cx36 gap junction coupling were KYUNGHEE HONG, GUANLAN XU, ANATH SHALEV, Birmingham, AL monitored in islets from C57BL/6NHsd mice and human islets treated for 24 The cytokine interleukin (IL)-1β, has been implicated in diabetes, but its or 1h with a cocktail of Th1 cytokines (10ng/mL TNF-α, 5ng.mL IL-1β,100ng/ effects especially on pancreatic β-cells are still not fully understood. In the mL IFN-γ), as well as NOD and NOD/RAG mice at 6, 9, and 12 weeks of age. present study, we surprisingly found that in INS-1 β-cells as well as in pri- Glucose tolerance tests were administered at the specifi ed time points in mary islets IL-1β down-regulated the expression of thioredoxin-interacting NOD and NOD/RAG mice. protein (TXNIP), a key regulator of β-cell apoptosis and function. In addi- Low levels of cytokines disrupted Ca2+ synchronization and decreased tion, luciferase studies revealed that IL-1β caused a signifi cant reduction in Cx36 gap junction coupling in both mouse and human islets, through nitric 2+ human TXNIP promoter activity. Interestingly, mutation of the E-box motif, oxide mediated phosphorylation of Cx36 by activated PKCδ. Altered Ca which acts as the carbohydrate responsive element binding protein (ChREBP) and decreased coupling was observed as early as 6 weeks of age in NOD binding site, blunted this effect, indicating that IL-1β may inhibit TXNIP ex- mice, while glucose tolerance was not signifi cantly different from controls pression through ChREBP. Indeed, IL-1β treatment resulted in a decrease in until 12 weeks of age. Gap junction coupling further declined with increasing ChREBP nuclear localization as demonstrated by nuclear fractionation and age, where at 12 weeks of age, prior to the onset of diabetes, coupling was led to reduced ChREBP occupancy of the TXNIP promoter as assessed by decreased to similar levels as in isolated islets treated with high levels of chromatin immuneprecipitation assays. Moreover, IL-1β also inhibited ex- pro-infl ammatory cytokines. pression of liver pyruvate kinase (LPK), another bona fi de ChREBP target In conclusion, gap junctions are disrupted early in the development of gene, indicating that the effects are not restricted to TXNIP. In summary, type 1 diabetes, through cytokine mediated phosphorylation of Cx36 by our data suggest that IL-1β modulates ChREBP signaling and thereby reveal nitric oxide activated PKCδ. As gap junction coupling has been shown to a novel aspect of IL-1β function in the pancreatic β-cell. protect islets from cytokine-induced apoptosis, modulating Cx36 via these Supported By: American Diabetes Association (7-12-BS-167 to A.S.); National mechanisms of disruption may reduce the decline of β-cell mass and delay Institutes of Health (IHR01DK078752); JDRF (JNJSI40-2011-1) or prevent the onset of type 1 diabetes. Supported By: National Institutes of Health (F32DK102276-01A1, R00DK085145); & 2362-P JDRF (5-CDA-2014-198-A-N) Coordinate Regulation of Chrebpa and ChREBpβ Gene Expression by T3 and Glucose, and Subsequent β-Cell Proliferation & 2364-P LIORA S. KATZ, LUCY LI, DONALD K. SCOTT, New York, NY GCN2, a Type 2 Diabetes Mellitus Susceptibility Gene, Is Associ- Carbohydrate response element binding protein (ChREBP) is a glucose- ated with the Regulation of Pancreatic β-Cell Mass responsive transcription factor required for glucose mediated β cell prolif- AYUMI KANNO, KATSUHISA MASUDA, SHUN-ICHIRO ASAHARA, MAKI KIMU- eration. In β cells, ChREBP regulates liver-type pyruvate kinase (Pklr), a gly- RA, TOMOKAZU MATSUDA, MASATO KASUGA, WATARU OGAWA, SUSUMU colytic enzyme, as well as thioredoxin-interacting protein (Txnip) - involved SEINO, YOSHIAKI KIDO, Kobe, Japan, Tokyo, Japan in oxidative stress and regulation of β cell mass. ChREBP has two splice Objective: Single-nucleotide polymorphism (SNP) analysis of Japanese isoforms - α and β, derived from different promoters. The expression of diabetes patients has revealed a signifi cant correlation between gen- ChREBPβ (but not ChREBPα) is induced in response to glucose and leads eral control nonderepressible 2 (GCN2) SNPs and type 2 diabetes mellitus to increased β cell proliferation. Type 2 diabetes is closely associated with (T2DM). GCN2 is a molecule activated by amino acid defi ciency. A compari- both hypo- and hyper-thyroidism, and thyroid hormone (T3) is required for is- son of GCN2 expression in different mouse tissues showed that GCN2 was let cell development, maturation and function. Since T3 regulates ChREBP in prominently expressed in pancreatic islets. We analyzed the role of GCN2 in the liver, we hypothesized that T3 and glucose coordinately regulate ChREBP pancreatic β-cells and its involvement in the onset of T2DM. expression and β cell mass. Methods: We generated generalized GCN2 knockout mice (GCN2-/- mice) We identifi ed conserved thyroid response element (TRE) binding sites and analyzed their metabolic parameters and histology. We also investigat- on both promoters of ChREBPα and β. To study glucose and T3 effects on ed changes in insulin signaling by using islets from GCN2-/- mice. ChREBP target gene expression, we used rat Ins1 cells, mouse and human Results: Although GCN2-/- mice fed a normal-chow diet (NCD) did not islets. In all models, ChREBPα and Txnip expression was dose dependent exhibit any changes in glucose tolerance or pancreatic β-cell mass up to on T3 in high (20 mM) but not in low (2 mM) glucose. By contrast, ChREBPβ 24 weeks of age, those fed a high-fat diet (HFD) exhibited signifi cant aggra- and Pklr expression was dose dependent on glucose both in the presence vation in glucose tolerance and reduction in pancreatic β-cell mass. Islets (10 nM) and absence of T3, which was amplifi ed in the presence of T3 (all, isolated from GCN2-/- mice showed signifi cant increase in mTORC1 activity n=3, p < 0.05). Together, our data suggest a model where glucose activates and decrease in insulin signaling. To clarify the mechanism underlying this ChREBPα, driving expression of ChREBPβ, and the TREs of both isoforms phenomenon, we investigated the activity of GCN2 and found that GCN2 enhance the feed-forward loop, increasing target gene expression and β cell was markedly activated in islets from mice fed a HFD. Because it seemed mass. Next, we assessed the effect of T3 and glucose on β cell prolifera- plausible that phosphorylation of GCN2 increases when translation of insu- tion by co-staining for insulin and ki67. Surprisingly, while glucose promoted lin is enhanced, we measured the concentration of amino acids and found proliferation as expected, the highest percentage of cells double-positive that this concentration is reduced in islets from mice fed a HFD. for insulin and ki67 was obtained in low glucose and high T3 (n=3, p < 0.05). Conclusion: Our results showed that chronic activation of mTORC1 signal This suggests that fi ne-tuning of glucose and T3 levels is crucial for ChREBP is one of the causes of reduction in pancreatic β-cell mass in GCN2-/- mice. expression, β cell proliferation, and regulation of β cell mass. In islets from mice fed a HFD, the activation of GCN2, caused by the en- hancement of translation of insulin, might contribute to maintenance of pan- creatic β-cell mass. We suggest that GCN2-/- mice are one of the models of the pathogenesis of T2DM in patients who have a SNP in GCN2. POSTERS Islet Biology/ Insulin Secretion

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A599 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

& 2365-P 2367-P Receptor Binding, Biased Receptor Signaling and Glucose Stimulated Palmitate Reversibly Inhibits Inositol Trisphosphate-mediated Ca2+ Insulin Secretion by Partial and Full GPR40 Agonists Release and Induces Mitochondrial Fragmentation in Pancreatic SEUNGHUN P. LEE, JUNE XU, JENSON QI, SHUYUAN ZHAO, IVONA BAKAJ, Beta Cells JOSEPH GUNNET, RHYS SALTER, HUI HUANG, SANATH MEEGALLA, MARK COLIN LEECH, HEATHER A. NELSON, JAMAL ALAFIFI, RICHARD F. KOPP, FORREST PLAYER, ALESSANDRO POCAI, Spring House, PA A. WRIGHT, RICHARD J.H. WOJCIKIEWICZ, MICHAEL W. ROE, Syracuse, NY GPR40 is a G-protein coupled receptor that mediates fatty acid-induced The molecular mechanisms underlying the detrimental effects of lipo- glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells and incre- toxicity on pancreatic beta cells remain incompletely understood. Here, we tin release from enteroendocrine cells. Numerous GPR40 ligands have been report a novel mechanism of palmitate action in MIN6 cells that involves in- 2+ investigated for their anti-diabetic actions. Using two potent and selective hibition of Ca mobilization from intracellular stores mediated by Gq protein- GPR40 agonists, JNJ-GPR40-A and JNJ-GPR40-B, we have previously dem- coupled receptors (GPCR) for carbachol and ATP. The effects of palmitate onstrated that there are at least 2 distinct ligand binding sites with differing on endoplasmic reticulum (ER) Ca2+ release were dose-dependent, evident ligand specifi cities and degrees of intrinsic activity. We demonstrated that within 3 h, and after a 24-h exposure, were reversible within 3-6 h. Neither the endogenous ligand, α-linoleic acid, fully competed the binding of 3H-JNJ- oleate nor control media affected intracellular Ca2+ signals generated by GPR40-A with a Ki value of 3.5 µM, but did not inhibit binding of 3H-JNJ- GPCR activation. Inhibition of ER Ca2+ mobilization did not result from an in- GPR40-B. Importantly, the two compounds did not compete with each other. hibition of inositol 1,4,5-trisphosphate (IP3) production downstream of GPCR Furthermore, JNJ-GPR40-B and α-linoleic acid were determined to be full activation of phospholipase C. Using D1ER, an ER Ca2+ indicator, we found 2+ agonists whereas JNJ-GPR40-A was a partial agonist as assessed by an that palmitate did not signifi cantly reduce [Ca ]ER. In addition, expression of inositol phosphate (IP-1) assay. Recent evidence suggests that GPR40 couples genes encoding type 1, 2, and 3 IP3 receptors and IP3R protein levels were to either Gq or to Gq and Gs depending on the ligand. Here, we examined the not affected by lipotoxicity. Taken together, these fi ndings suggest that IP3R ability of a full and partial GPR40 agonist to induce cAMP production in a activation was inhibited by palmitate. Palmitate also reversibly fragmented recombinant human GPR40 low–expressing cell line. These compounds were MIN6 cell mitochondria. The time courses for the palmitate-induced effects also tested for their effi cacy in inducing GSIS in rat and human islets. on mitochondrial morphodynamics and GPCR-stimulated Ca2+ signaling were The full agonist was determined to promote both IP-1 and cAMP whereas similar. Using ddGFP, a genetically encoded biosensor of mitochondrial-as- the partial agonist only induced IP-1 accumulation. Interestingly, the partial sociated ER membrane (MAM) integrity in living intact cells, we found that agonist was found to have small enhancements in GSIS whereas the full palmitate rapidly disrupted MAM contact sites. Our data suggest that pro- 2+ agonist had robust GSIS in isolated rat islets and human islets. These data longed exposure of beta cells to palmitate inhibits IP3-mediated Ca tran- suggest that full and partial GPR40 agonists may be defi ned by their receptor sients by causing mitochondrial fragmentation and loss of MAM integrity. binding, biased signaling and insulin secretory characteristics. These differ- The effect of lipotoxicity on beta cell MAM integrity could contribute to the ences at the GPR40 receptor may lead to diverse therapeutic opportunities development of diabetes in obese subjects by reducing islet sensitivity to for the treatment of diabetes. neuronal or paracrine signaling through Gq-coupled receptors. Supported By: American Diabetes Association (1-12-BS-109 to C.L.); National & 2366-P Institutes of Health (R01DK092616) Human Islet Amyloid Polypeptide Inhibits Thioredoxin-Interacting Protein Expression through pAMPK/ChREBP Signaling Pathway 2368-P GU JING, CLARA WESTWELL-ROPER, JUNQIN CHEN, GUANLAN XU, BRUCE Nuclear Export of FoxO1 Is Associated with ERK Signaling in β-Cells VERCHERE, ANATH SHALEV, Birmingham, AL, Vancouver, BC, Canada Lacking Insulin Receptors Pancreatic islets in patients with type 2 diabetes are characterized by loss TERESA MEZZA, JUN SHIRAKAWA, RACHAEL MARTINEZ, JIANG HU, ROHIT N. of functional beta-cell mass and formation of amyloid deposits. The latter KULKARNI, Boston, MA is derived from human islet amyloid polypeptide (hIAPP), a 37-amino acid Insulin signaling has been reported to play critical roles in the regulation polypeptide co-expressed and co-secreted with insulin by pancreatic beta- of β cell biology. To investigate how insulin regulates downstream signal- cells. We previously identifi ed the thioredoxin-interacting protein (TXNIP)/ ing proteins in vivo, we designed two experimental approaches. First, we miR-124a/Forkhead boxA2/IAPP signaling cascade linking the critical beta- examined the effects of a physiological stimulus by performing either a glu- cell signaling pathways of TXNIP and IAPP. Using INS-1 beta-cells, primary cose (GG) (1 gm/kg) or saline gavage (SG) separately in β-cell specifi c insulin mouse and human islets, and hIAPP-transgenic mice, we now demonstrate receptor knockout (βIRKO) or C57/B6 (control) mice (n=6, total 4 groups). that hIAPP inhibits beta-cell TXNIP expression both in vitro and in vivo. Inter- Second, to explore the potential endocrine effects of circulating insulin we estingly, rat IAPP and pramlintide which in contrast to hIAPP lack the ability performed either a 15-min saline infusion (sham clamp-SC) or a hyperinsu- to aggregate into insoluble fi bers and therefore are non-toxic to beta-cells linemic clamp (HC) (150 mUI/kg priming followed by 2.5 mU·kg-1·min-1 insulin show no effect on TXNIP. This suggested inhibition of pro-apoptotic TXNIP infusion rate) (n=6, total 4 groups). Pancreas sections were co-immunos- by hIAPP might represent a compensatory survival attempt by the cell in tained for pAKT, p70S6K, pERK or FoxO1 with insulin. In the control group, response to hIAPP-induced beta-cell toxicity. TXNIP induces mitochondrial we observed a signifi cant increase in pAKT, p70S6K and pERK+ β-cells in apoptosis and recent fi ndings have shown that mitochondrial membrane GG (p<0.05 for each stain vs. controls) and HC group (p<0.01 for each stain disruption contributes to the toxic effects of hIAPP. In fact, we found that vs. controls). In contrast, the % of nuclear FoxO1+ β cells were signifi cantly staurosporine (STS), a well-known stimulus of mitochondrial damage and decreased in the GG and HC group (p<0.01 vs. respective controls). In βIRKO apoptosis, could mimic the effects of hIAPP on TXNIP. Real-time PCR, West- mice, we observed no signifi cant changes in pAKT or p70S6K+ β-cells in ei- ern blotting and chromatin immunoprecipitation further revealed that hIAPP ther experiment, while pERK+ β-cells were signifi cantly increased in GG and downregulates TXNIP expression by activating AMP-activated protein ki- HC groups (p<0.01, vs. respective controls). Consistently, the % of nuclear nase (AMPK) and decreasing nuclear carbohydrate response element-bind- FoxO1+ β cells was decreased in GG and HC group, (p<0.01 vs. controls). ing protein (ChREBP) localization and function. Moreover, we determined These data were validated in β-cells derived from wild-type (WT) or βIRKO that p-AMPK/ChREBP signaling pathway also contributes to the regulation mice in vitro. Thus, a signifi cantly decreased % of nuclear FoxO1+ β cells of TXNIP by STS. Thus, our fi ndings have identifi ed a novel compensatory was observed in both WT and βIRKO β cells after treatment with 22.2 mM cellular response to hIAPP toxicity that helps block expression of pro-apop- glucose+10 nM insulin. However, blocking MAPK signaling with a Mek1/2 totic TXNIP. They thereby shed new light on the role of TXNIP and IAPP in inhibitor had no effect on FoxO1 nuclear export in WT β cells, in contrast to beta-cell biology. attenuated FoxO1-nuclear export in βIRKOs (n=5, p <0.05). These data sug- Supported By: American Diabetes Association (7-12-BS-167 to A.S.); National gest that insulin and glucose minimally activate the Akt pathway, while ERK Institutes of Health (R01DK078752) phosphorylation and FoxO1 nuclear export occur independently of insulin signaling in βIRKO β-cells. POSTERS Islet Biology/ Insulin Secretion

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A600 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

2369-P 2371-P Combined Stimulation of IL-6 and Dexamethasone Activated Human Triiodothyronine Acts as Protective Factor against Hyperglycemia REG Ia and REG Iβ Gene Transcription in Human Pancreatic β Cells Mainly via Thyroid Hormone Receptor α via the JAK/STAT Signaling Pathway YOHSUKE OHKUBO, TAKASHI SEKIDO, KEIKO SEKIDO, SHIN-ICHI NISIO, SATORU AKIYO YAMAUCHI, ASAKO ITAYA-HIRONAKA, SUMIYO SAKURAMOTO-TSUCHI- SUZUKI, MITSUHISA KOMATSU, Matsumoto, Japan, Fukushima, Japan DA, MAIKO TAKEDA, KIYOMI YOSHIMOTO, TOMOKO MIYAOKA, TAKANORI Triiodothyronine (T3) has diverse effects on cellular function including FUJIMURA, HIROKI TSUJINAKA, CHIKATSUGU TSUCHIDA, HIROYO OTA, SHIN glucose homeostasis. Herein, we demonstrate T3 directly affects insulin TAKASAWA, Kashihara, Japan secretion. Using Q-PCR that the treatment of INS-1 insulinoma cells with T3 Reg (Regenerating gene) gene was originally isolated from rat regenerat- increased insulin mRNA levels. Transient transfection assays of HIT T15 and ing islets and its encoding protein was revealed as an autocrine/paracrine INS-1 cells revealed that thyroid hormone receptor (TR) α and TRβ enhanced growth factor for β cells. The Reg and Reg-related genes were isolated and transcriptional activity of rat insulin II promoter in a ligand-dependent man- revealed to constitute a multigene family, the Reg family, which consists of ner. Deletion analyses of the insulin promoter showed that the region of four subtypes (types I, II, III, and IV). The rat type I Reg gene, Reg I, was the positive thyroid hormone responsiveness within the rat insulin II promoter fi rst isolated Reg family gene, and is activated in infl ammatory conditions for was localized at nucleotides from -238 to -188bp relative to the transcription β cell regeneration. In human, fi ve functional REG family genes (REG Iα and start site. Mutations in the DNA binding domain of TRβ1 (G345R) abolished REG Iβ as type I, REG III and HIP/PAP as type III, and REG IV as type IV) were the activation. The activation was mediated by BETA2/E47 binding sites, but isolated. However, their expressions in β cells under infl ammatory condi- not by a TRE-like sequence in the promoter. Expression plasmids for Gal4 tions remained unclear because of their restrict supply of human β cells. DBD fusions of the transcription factors that bind E47/BETA2, the active re- In this study, we found that combined addition of IL-6 and dexamethasone gion on the insulin promoter were created and we co-transfected with TRβ (Dx) induced REG Iα, REG Iβ, and HIP/PAP transcription in human 1.1B4 β expression vector and UAS reporter plasmid. TRβ repressed the activity of cells. The mRNA levels of REG Iα and REG Iβ were increased in response BETA2 in a T3-dependent manner in HIT-T15 cells. Next we performed intra- to the combined stimulation of IL-6 and Dx. Promoter assay revealed that a peritoneal glucose tolerance tests (1.5 mg/g BW) in wild mice, TRαΚΟ mice signal transducer and activator of transcription (STAT)-binding site in each and TRβΚΟ mice. Fasting plasma glucose concentration (FPG) in wild mice promoter of REG Iα (TGCCGGGAA, the -142~-134 region) and REG Iβ (TGCCA- and TRβΚΟ mice were similar (49+/-17 vs. 68+/-18 mg/dL, respectively). In GGAA, the -151~-143 region) was essential for the IL-6+Dx-induced promoter contrast, FPG was higher in TRαΚΟ mice (111+/-17 mg/dL; p<0.05 vs. wild activation. A Janus kinase (JAK)2 inhibitor, AG490, signifi cantly inhibited the and TRβ ΚΟ mice). The blood glucose concentration at 60 (PG60) and 120 IL-6+Dx-induced REG Iα and REG Iβ gene activation. Electrophoretic mobility (PG120) min after glucose load of TRαΚΟ were signifi cantly higher (PG60 shift assay and chromatin immunoprecipitation revealed that IL-6+Dx stimu- 232+/-45 mg/dL; PG120 161+/-30 mg/dL) than those of wild (PG60 136+/- lation increased STAT3 binding to the REG Iα promoter. Furthermore, small 28 mg/dL, PG120 99+/-23 mg/dL) and TRβΚΟ mice (PG60 107+/-28 mg/dL, interfering RNA-mediated targeting of STAT3 blocked the IL-6+Dx-induced PG120 99.2+/-23 mg/dL). These results imply that T3 could act as protective expression of REG Iα and REG Iβ. These results indicate that, among the factor against hyperglycemia mainly via TRα by increasing insulin secretory human REG family genes, the expression of type I REG genes should be up- capacity. regulated in human β cells under infl ammatory conditions through the JAK/ STAT signaling pathway. 2372-P Supported By: Japan Society for the Promotion of Science In-Depth Proteomics Characterization of Directed Differentiation of Human-induced Pluripotent Stem Cells 2370-P HEIDRUN VETHE, ADRIAN TEO, NICHOLAS JACKSON, JOAO A. PAULO, YN- System Biology Informed by Islet Transcriptome Analyses Identifi es GVILD BJØRLYKKE, MARC VAUDEL, HARALD BARSNES, STEVEN P. GYGI, HELGE a GSK3-beta Regulatory Node that Reciprocally Controls Estrogen RÆDER, ROHIT KULKARNI, Boston, MA, Bergen, Norway Receptor and TCF7L2 Activities Human induced Pluripotent Stem Cells (hiPSCs) represent a unique re- ELLEN DOVER, WAYNE GRAHAM, PETER ANTINOZZI, Winston-Salem, NC source used to derive diverse mature cell types including hormone-produc- To identify potential interactions of key regulatory nodes and transcription ing cells. The alterations in the hiPSC proteome during the differentiation factors, we implemented the following tiered approach. RNA-Seq transcrip- process that simulate normal human pancreatic development is not fully ex- tome analysis was performed on isolated human islet samples to determine plored. Here we present a quantitative mass spectrometry-based study with islet expressed genes and prioritized into two gene lists categorized as: 1) in-depth time course characterization of proteomic changes during directed well-described regulatory nodes or 2) transcription factors. differentiation of hiPSCs towards pancreatic progenitors. We undertook Using a system biology approach, these gene lists were analyzed to con- in vitro simulation of early human pancreatic development by modulating struct an interactome of regulatory relationships. Identifi ed interactions multiple signaling pathways, including Wnt, activin, hedgehog, EGF, retinoic included GSK3B (glycogen synthase kinase beta) regulation of both ESR1 acid, and TGF-β signaling and performed a comprehensive characterization (estrogen receptor alpha) and TCF7L2 transcription activity. of the proteome to defi ne the changes at each of the differentiation steps. The impact of GSK3B activity on TCF7L2- or ESR1-transcription was evalu- Time course studies during differentiation of hiPSCs revealed that the pluri- ated in the INS1E beta-cell model with 7 unique TCF7L2 splice variants and 3 potency factors OCT4 and SOX2 decreased rapidly from day 0 to day 5, and ESR1 splice variants. Transcriptional activity of TCF7L2 and ESR1 splice iso- that the hiPSCs go through a mesendoderm transition with high protein lev- forms were assessed by ERE- or TRE-specifi c luciferase reporters, respec- els of EOMES at day 3, followed by a shift to defi nitive endoderm, marked tively, under conditions of GSK3B activation or inhibition. As anticipated, by CXCR4 and GATA6. Further differentiation towards foregut endoderm and inhibition of GSK3B activity with the kinase specifi c inhibitor SB216763, po- pancreatic progenitors by day 12 and day 17, respectively, were marked by tentiated TCF7L2 activity. GSK3B inhibition had a consistent potentiation ef- increased levels of GATA4 and ISL1, and increased levels of PBX1, a complex fect amongst each of the TCF7L2 splice isoforms. Overexpression of GSK3B partner of PDX1. Comparative proteomics analysis allowed for the discov- inhibited TCF7L2 transcriptional activity and, again, with no distinguishing ery of a novel set of protein markers for each of the developmental stages isoform preference. Reciprocal to its effects on TCF7L2, GSK3B inhibition towards pancreatic progenitor cells. Our quantitative dataset represents a reduced ESR1 transcriptional activity by 40%, where GSK3B overexpression unique characterization of hiPSC-derived cells and presents a reliable and increased ESR1 activity two-fold. GSK3B effects were specifi c to the 66kD accurate method for quantitative proteomics analysis of differentiating hu- isoform of ESR1, where no changes in activity were observed with the 36 man induced pluripotent stem cells along the pancreatic lineage. and 46kD ESR1 isoforms. In conclusion, this experimentally validated system biology approach 2373-P identifi ed a novel ESR1 splice isoform specifi c interaction with GSK3B. This Identifi cation and Functional Implication of Sodium/Myo-inositol result highlights the complex infl uence of isoform expression which can po- Co-transporter 1 in Pancreatic Beta Cells and Type 2 Diabetes tentially impact effi cacy of pharmacological treatments for diabetes. Mellitus Supported By: National Institutes of Health (DK-080151) STEPHEN YU TING LI, SAM TSZ WAI CHENG, PO SING LEUNG, Hong Kong, China Myo-inositol (MI), the precursor of the second messenger phosphati- POSTERS

dylinositol (PI), is known to mediate multiple cellular events, including osteo- Islet Biology/

genesis and the development of peripheral nerves through the involvement Insulin Secretion of multiple pathways such as Akt/PKB and PLC/PKC signaling cascades. Meanwhile, rat islets have been known to actively transport MI. Despite

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A601 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

this, the underlying transport mechanism remains ambiguous. In this regard, we identifi ed, for the fi rst time, the expression of SLC5A3 gene and its en- coded protein, sodium/myo-inositol co-transporter 1 (SMIT1) in rat pancre- atic islets and INS-1E β-cells using real-time PCR and Western blot analyses. We also showed that INS-1E β-cells had an intact biological system for the conversion of MI into PI, implying a potential role of extracellular MI in regu- lating β-cell function. In addition, functional characterization of SMIT1 by pharmacological inhibition in INS-1E β-cells revealed a signifi cantly impaired glucose-stimulated insulin secretion (GSIS), probably due to the defi ciency of intracellular PI which, in turn, led to a down-regulation of PI signaling. Furthermore, we found that the expression of SMIT1 was signifi cantly re- duced, consistent with the defects in GSIS, as evidenced by the high-glucose treated INS-1E β-cells. Taken together, our data indicate that SMIT1, being a transporter for MI, is required to maintain a stable PI pool in the pancreatic β-cells so as to sustain the rapid turnover of PI signaling cascades for insulin secretion. Given that hyperglycemia suppresses SMIT1 expression in pan- creatic β-cells, the defect in myo-inositol transport may provide a clinical im- plication for the pancreatic β-cell dysfunction and type 2 diabetes mellitus. Supported By: Research Grants Council of Hong Kong (470413) POSTERS Islet Biology/ Insulin Secretion

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A602