ISLET BIOLOGY—APOPTOSISCATEGORY subjects completing 56 weeks of treatment, NB was generally well tolerated comparable levels with fresh islets. Improved proIAPP processing in exenatide- and associated with dose-dependent sustained weight loss and numerous treated islets was associated with reduced hIAPP proto(fi brils) detected by improvements in weight-related risk factors, including lipids and glycemic electron microscopy and TUNEL-positive β-cells (human: -Ex: 15.2±2.4% vs +Ex: parameters, as well as improved quality of life and control of eating. 7.6±1.7%; mouse: -Ex: 12.3±2.7 vs +Ex: 5.8±2.3, P<0.05). These data suggest Supported by: Orexigen Therapeutics that treatment with GLP-1 analogues such as exenatide may enhance proIAPP processing and reduce amyloid formation in T2D patients and pre-transplant ISLET BIOLOGY—APOPTOSIS culture of human islets.

[See also: Presidents Posters 469-PP to 470-PP, page A130.] & 1931-P Mammalian Sterile 20-Like Kinase 1 (MST1) Mediates Pancreatic β-Cell Apoptosis Guided Audio Tour: Apoptosis (Posters 1929-P to 1937-P), see page 11. AMIN ARDESTANI, FEDERICO PARONI, JULIE KERR-CONTE, KATHRIN MAEDLER, Bremen, Germany, Lille, France & 1929-P Apoptotic cell death is the hallmark of the loss of insulin producing b-cells Impact of Vildagliptin on Glucose Tolerance, and beta Cell Mass in and a fundamental cause of diabetes. Loss of function, of survival signals Insulin Receptor Substrate-2-Knockout Mice and activation of pro-apoptotic mediators are characteristic for diabetic KOICHIRO SATO, AKINOBU NAKAMURA, JUN SHIRAKAWA, TOMONORI MURA- β-cells. In search for a common pro-apoptotic pathway, we investigated OKA, YU TOGASHI, KAZUKI ORIME, YASUO TERAUCHI, Yokohama, Japan whether the serine/threonine kinase Mammalian sterile 20-like kinase 1 Loss of beta-cell mass underlies the development of diabetes and insulin (MST1), triggers β-cell death in diabetes. receptor substrate-2 (Irs2) is critically required for beta cell growth and Exposure of isolated human islets and INS1-cells to a diabetic milieu survival. GLP-1 reportedly increased islet proliferation and reduced apoptosis (cytokines IL-1β/INF-γ, high glucose or thapsigargin) signifi cantly increased of beta-cells in rodents. In this study, we explored the chronic effect of MST1 activity (phosphorylation & cleavage) and Akt phosphorylation was dipeptidyl peptidase-4 (DPP-4) inhibitor vildagliptin on glucose tolerance acutely increased, but dramatically decreased after 48h and 72h. Increased and beta-cell mass in Irs2-knockout (Irs2-/-) mice fed a high-fat diet for 20 MST1 activity and its downstream effectors p38 and JNK correlated with weeks. Wild type (WT) mice and Irs2-/- mice of the same genetic background increased b-cell apoptosis demonstrated by increased caspase-3- and PARP- were fed standard-chow until 8 weeks of age, and then given free access cleavage. Blocking MST1 by overexpression of dominant negative protein to high-fat diet for 20 weeks. Half of the animals in each genotype were (DN-MST1) inhibited b-cell apoptosis and potentiated insulin-induced AKT given vildagliptin in the drinking water (0.3 mg/ml) and others were given phosphorylation, whereas MST1 overexpression was suffi cient to induce tap water without vildagliptin. Whereas there were no differences in body and potentiate b-apoptosis and abolished AKT phosphorylation. Activation of weight or food intake, vildagliptin signifi cantly reduced fasting blood glucose the PI3K-Akt pathway through exogenously added insulin or GLP1 abrogated in Irs2-/- mice. In both genotypes of mice, vildagliptin signifi cantly decreased glucose-, cytokine- and thapsigargin-induced MST1 activation and β-cell apoptosis, whereas suppression of PI3-K/Akt signaling (by LY294002, AIP-2 or AUC0-120min blood glucose and signifi cantly increased the insulin response to glucose during oral glucose tolerance test (OGTT). To evaluate the chronic siRNA to Akt), induced MST1 activity and b-cell apoptosis. Our results indicate effect rather than the direct effect, we also performed OGTT one day after that MST1 and Akt negatively regulate each other and constitute a stress- sensitive survival pathway. Under acute stress conditions, Akt promoted discontinuation of vildagliptin administration. AUC0-120min blood glucose was still signifi cantly decreased and the insulin response to glucose during OGTT cell survival by inhibiting MST1, but prolonged stress decreased Akt, which was signifi cantly increased in Irs2-/- mice treated with vildagliptin compared allowed pro-apoptotic MST1 signaling. MST1 overexpression caused PDX1 with those without vildagliptin. Histochemical analysis of pancreatic islets shuttling from the nucleus to the cytosol and its subsequent degradation, revealed that treatment with vildagliptin signifi cantly increased beta-cell which occurred by direct MST1-induced phosphorylation of PDX1. Importantly, mass (p<0.05) and proportion of beta-cells to islet cells (p<0.01) in Irs2-/- mice. MST-1 was highly activated in islets isolated from patients with T2DM, from Vildagliptin decreased the proportion of TUNEL-positive beta-cells (p<0.05), diabetic db/db mice and high fat/high sucrose fed mice. but failed to increase the BrdU incorporation in Irs2-/- mice. These results Our results show that MST1 plays an important role in triggering b-cell suggest that vildagliptin improved glucose tolerance and increased beta-cell death via suppressing survival signals and PDX1 functionality. Its inhibition mass by reducing apoptotic beta-cells in Irs2-/- mice and that vildagliptin is provides a new strategy to restore b-cell survival. able to reduce apoptotic beta-cells in Irs2-independent manner. & 1932-P & 1930-P Identifi cation of microRNA Expression Profi le Induced by Palmitate Exenatide Treatment Improves Processing of Pro-Islet Amyloid in Human Pancreatic Islets Poly peptide in Human and Mouse Islets during Culture YUNXIA ZHU, HONGDONG WANG, XIAO HAN, Nanjing, China HAO CHEN, TIMOTHY J. KIEFFER, C. BRUCE VERCHERE, ZILIANG AO, MADELEINE Among all the pancreaticβ-cell dysfunction related factors in T2DM, FFA SPECK, DAVID THOMPSON, GARTH L. WARNOCK, LUCY MARZBAN, Vancouver, seems to play a key role in inadequate insulin secretion. Several proteins BC, Canada were involved in the pathology of FFA-induced β-cells impairment, such Islet amyloid is a pathologic characteristic of type 2 diabetes (T2D) that also as pdx1, neurod1, sur2, kir6.1, oncut2, vamp2, et al. Since the discovery forms in islets during culture and post-transplant. Islet amyloid is comprised of microRNAs (miRNAs), an entirely new line of thoughts has emerged as mainly of human islet amyloid polypeptide (hIAPP; amylin), a β-cell peptide regard to the relationship between miRNAs and disease proteins. Moreover, that is produced by cleavage at the C- and N-termini of its precursor proIAPP. a particular link seems present between miRNA and palmitate-induced Elevated synthesis, fi brillogenic sequence, and impaired processing of proIAPP β-cells failure. To investigate the possible involvement of miRNAs in due to β-cell dysfunction have been implicated in hIAPP aggregation. Exenatide palmitate-induced human islets impairment, the isolated adult human islets is a long acting analogue of glucagon-like peptide-1 (GLP-1) that is used for were picked up and incubated for 48 hours in the presence of 0.5 mmol/l treatment of T2D and has been shown to enhance β-cell mass and function. palmitate. The GeneChip® miRNA Array (Affi metrix) was used to identify the We tested the effect of exenatide on restoring impaired proIAPP processing expression profi le and 289 miRNAs were detected. 11 miRNAs were highly in islet β-cells. Freshly isolated human or hIAPP-expressing transgenic mouse expressed in untreated human islets. Especially, 21 miRNAs up to two fold islets were cultured in CMRL or Hams-F10 (5 or 10 mmol/l glucose), respectively, altered were determined in palmitate treated group. Among those miRNAs, without or with exenatide (10 nM, 7 d). (Pro)IAPP forms were detected in 9 miRNAs were up-regulated and 12 miRNAs were down-regulated. For islet lysates by Western blot followed by immunoblot using an antibody that further functional analysis, we used miRNA target prediction algorithm recognizes all (pro)IAPP forms or antibodies that we generated specifi c for N- named miRanda and Targetscan to search possible regulation target of and C-termini of human proIAPP. Mature IAPP was the major form detected in these miRNAs. Connected with pancreatic β-cell proliferation and insulin fresh human and mouse islets with low levels of immature proIAPP also present. secretion associate proteins, miR-377 may involve in β-cell failure via down- Islet culture caused impaired proIAPP processing manifest as elevated proIAPP regulating the protein level of puma, bcl-xL, kir 6.1, sur2, et al. The reported and its intermediates and reduced mature IAPP compared to fresh islets. The miR-146a was also signifi cant increased in treated group, demonstrating major intermediate form present in cultured human and hIAPP-expressing mouse POSTERS

that miR-146a played a main role in human islets induced by palmitate as Islet Biology/ islets was N-terminally unprocessed proIAPP, suggesting that mainly cleavage at well. Our results may provide other insights into the causes of β-cell failure Insulin Secretion N-terminus was impaired. Exenatide-treated islets had markedly lower proIAPP implicating the development of type 2 diabetes. and its intermediates and higher mature IAPP than untreated cultured islets and Supported by: National Basic Research Program of China (2011CB504003)

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A521 ISLET BIOLOGY—APOPTOSISCATEGORY

& 1933-P & 1935-P Glucose-Dependent Survival of Beta-Cells Is Achieved by Maintenance Endosomal TLR3 Activation Initiates Virus-Induced β-Cell Death of Optimal CREB Expression Resulting in Suppression of Apoptosis FEDERICO PARONI, ERNA DOMSGEN, JULIE KERR-CONTE, ANDREAS DOTZAUER, SAFIA COSTES, ELODIE VARIN, CHRISTOPHE BROCA, NATHALIE LINCK, ANNE KATHRIN MAEDLER, Bremen, Germany, Lille, France WOJTUSCISZYN, STÉPHANE DALLE, Montpellier, France Group B coxsackievirus (CVB) infection of β-cells is associated with the Pancreatic beta-cell survival is both positively and negatively regulated development of diabetes. Intra-islet viral particles were detected in patients in response to the glucose conditions to which beta-cells are exposed. with T1DM and T2DM, but the mechanisms of a correlation between virus The transcription factor CREB (cAMP Response Element Binding protein) infection and diabetes progression are poorly understood. In this study we plays a key role in beta-cell survival. Using MIN6 beta-cells, rat and human asked the question which intracellular signals are involved and whether pancreatic islets, we observed that in vitro exposure of beta-cells to low virus-induced CXCL10 correlates with β-cell destruction? Human islets and a glucose concentrations (≤5 mM) favors the emergence of a cleaved caspase- human β-cell line were infected with two different serotypes of coxsakievirus 3-mediated apoptotic program, chromatin condensation and fragmentation, (CVB3 and 4). Immunoprecipitation-RT-PCR coupled assays showed that and a series of morphological changes corresponding to distinct (early and extracellular viral components bind to transmembrane-anchored, endosomal late) phases of apoptotic process detected by electron microscopy. These members of the Toll-like receptor family (TLR3) and transduce a signal via observations reveal that the beta-cells are not synchronized for their cytoplasmic adaptor proteins. In a parallel pathway, cytoplasmic receptors sensitivity to the apoptotic condition induced by glucose limitation and such as Protein kinase R (PKR) mediated the induction of response genes that glucose-dependent survival of beta-cells is achieved by suppression from intracellular pathogen-produced substrates. In contrast, retinoic-acid of apoptosis. This caspase-3-mediated apoptotic program induced by inducible gene I (RIG-I) and Melanoma differentiation associated protein 5 low glucose exposure is found to be associated with CREB protein down- (MDA-5) were not involved in the recognition of CVB’s dsRNA, despite the regulation, while CREB mRNA levels remained unaffected. This indicates upregulation of RIG-I and MDA-5 mRNA in islets. CXCL10, IFNβ, IL-1β, TNFα, that the decreased CREB protein expression occurs most probably at a IL-6, MCP1 and IL-8 mRNA levels were increased in the infected islets. Among post-transcriptional level. Down-regulation of CREB is further found to them, CXCL10 was the highest induced factor (∼35-fold increase in secretion, be associated with loss of bcl-2, IRS-2 (Insulin receptor substrate 2) and 15-fold increase in mRNA, p<0.001), compared to uninfected islets. This was PDX-1 (pancreatic duodenal homeobox-1) protein contents. Notably, we TLR3 and not IFNγ-dependent. TUNEL staining revealed that β-cell apoptosis observed that the emergence of apoptosis in beta-cells following glucose was 7-fold increased in CVB infected islets. Blocking of CXCL10 had no effect limitation is mainly due to the lack of CREB, since transfection of wild-type on β-cell apoptosis in virus-infected islets, indicating that during the early CREB blocked by 70% the emergence of apoptosis induced by low glucose phase of virus infection, β-cell apoptosis occurs independently of CXCL10. exposure. Expression of wild-type CREB in beta-cells exposed to low Analysis of survival and apoptotic pathways showed that the protein Akt glucose concentration prevented bcl-2, IRS-2, and PDX-1 protein expression was acutely activated during CVB infection and lasted for several hours, decreases. Together, these observations indicate that the glucose- but switched to pro-apoptotic signaling at 24h p.i. with Akt downregulation, dependent survival of beta-cells is achieved through the suppression of activation of pJNK and Caspase-3 and the appearance of the viral protein apoptosis by maintaining optimal expression of CREB which, in turn, controls VP1. In summary, we show that CBV infections have a direct deleterious the expression of crucial genes, such as bcl-2, IRS-2, and PDX-1 that are effect on β-cell survival, through activation of TLR3, which will be of required for beta-cell survival. particular interest in order to develop new therapies to rescue the β-cell.

& 1934-P & 1936-P Islet Amyloid-Induced Upregulation of Fas Receptor in Human Islets Is Pancreatic β-Cells Depend on Basal Expression of ATF6α-p50 Potentiated by Elevated Glucose and Prevented by Amyloid Inhibitors for Cell Viability as Acutely Reducing ATF6α-p50 Levels Induces YOO JIN PARK, NOOSHIN SAFIKHAN, TIMOTHY J. KIEFFER, ROBERT BAKER, Apoptosis Via JNK and p38 Pathways in an ER-Stress Independent ZILIANG AO, ALI ASADI, GARTH L. WARNOCK, LUCY MARZBAN, Vancouver, BC, Manner Canada TRACY TEODORO, ALLEN VOLCHUK, Toronto, ON, Canada Type 2 diabetes (T2D) is characterized by reduced β-cell mass and islet Pancreatic β-cells produce large amounts of proinsulin in the endoplasmic amyloid formation. Islet amyloid, formed by aggregation of human islet reticulum (ER) and rely on the unfolded protein response to maintain ER amyloid polypeptide (hIAPP), is associated with β-cell death. Islet amyloid homeostasis and cell survival during times of ER stress. ATF6α is one of also forms in cultured and transplanted human islets. The mechanisms three principle ER stress sensing proteins that make up the unfolded protein underlying β-cell death by endogenous hIAPP are unclear. We previously response and becomes activated when ER homeostasis is perturbed. This showed that formation of hIAPP (proto)fi brils in islets is associated with involves its translocation from the ER to the Golgi where it is proteolyzed to upregulation of the cell death Fas receptor. In this study, we used cultured release a transcription factor ATF6α-p50. ATF6α-p50 functions to increase human islets as an ex vivo model of amyloid formation to: 1. Examine if ER capacity by stimulating transcription of ER-resident chaperone genes inhibition of the interaction between hIAPP (proto)fi brils and β-cells can including the principle Hsp70 chaperone GRP78. Using an antibody that prevent Fas expression and protect β-cells; 2. Test the effect of elevated recognizes active ATF6α-p50 we found that ATF6α-p50 was detectable in glucose on amyloid-induced Fas expression. Freshly isolated human islets insulinoma cells and rodent islets even under basal non-stress conditions (n=3 donors) were cultured (5.5, 11.1, 16.7 mmol/l glucose, 7 d) with or and the levels were further increased by ER stress-inducing compounds without the amyloid binding dye Congo red (CR) or Fas antagonist (FA). or chronic high glucose treatment. To examine the function of ATF6α we Human islets in which amyloid formation was suppressed by adeno-siRNA- depleted endogenous ATF6α protein levels using siRNA in INS-1 insulinoma hIAPP were used as control for amyloid-independent Fas expression. cells. Knock-down of endogenous ATF6α-p50 protein levels by ∼60% Thiofl avin S (amyloid)-positive islets were absent in fresh islets but were resulted in a reduction in the steady-state levels of GRP78 mRNA and protein present after culture (16.7: 16±4%; 11.1: 9±3%; 5.5: 4±1%). Fas was not levels in non-stressed cells. Furthermore, ATF6α knock-down resulted in an detectable in fresh islets by RT-PCR, Western blot, or immunostaining but apoptotic phenotype. We hypothesized that removal of the ATF6 branch of was upregulated in cultured islet β-cells with maximal expression at 16.7 the UPR would be suffi cient to induce ER stress. However, neither the PERK mmol/l. The proportion of Fas, active Casp-3, and apoptotic β-cells were nor the IRE1 branches of the UPR were activated in ATF6α knock-down cells. markedly higher in thiofl avin S-positive than negative islets at all glucose Interestingly, phosphorylation of JNK and p38 stress kinases was increased levels. Inhibition of the interaction between hIAPP (proto)fi brils and β-cells in ATF6α knock-down cells and inhibitors of JNK or p38 activation prevented by CR reduced Fas expression by 50% (11.1) and 60% (16.7). CR had no effect ATF6α knock-down-induced apoptosis. These results suggest that basal on Fas level in islets in which hIAPP was suppressed. Finally, FA markedly expression of ATF6α-p50 in pancreatic β-cells is essential for cell survival reduced hIAPP-induced β-cell apoptosis (Cont (16.7): 18.9±3.4% vs +FA: even in the absence of ER stress, and that cells depleted of ATF6α initiate 12.4±2.6%, P<0.05). In summary, these data suggest that: 1. hIAPP-induced apoptosis via an ER stress-independent mechanism which involves the β-cell death in human islets is mediated, at least partially, by Fas pathway; stress kinases JNK and p38. 2. interaction between endogenously formed hIAPP (proto)fi brils and Fas is required for this process; 3. high glucose and hIAPP proto(fi brils) upregulate POSTERS Islet Biology/ Fas in an additive manner. Insulin Secretion

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A522 ISLET BIOLOGY—APOPTOSISCATEGORY

1939-P & 1937-P Bim, a Pro-Apoptotic Protein, and Caspase-3 Activity Are Asso- A Novel Small-Molecule Suppressor of Cytokine-Induced Beta-Cell ciated with Apoptosis InducedWITHDRAWN by Lentivius shRNA Grb10 in the Apoptosis Pancreas In Vivo DANNY HUNG-CHIEH CHOU, PATRICK FALOON, ERIN FORBECK, DEEPIKA WALPI- BRUNO DOIRON, LUKE NORTON, RALPH A. DEFRONZO, San Antonio, TX TA, JEREMY DUVALL, SIVARAMAN DANDAPANI, VLADO DANCIK, MONICA Grb10 binds with a family of cell survival-related proteins and has been SCHENONE, EAMON COMER, CLAIRE REDDY, CHRISTIE CIARLO, JACOB JAFFE, implicated in apoptosis. Lentiviral vector constructed shRNA targeting Grb10 PAUL CLEMONS, LISA MARCAURELLE, BENITO MUNOZ, MICHELLE PALMER, was injected in vivo via the intraductal pancreatic route to target pancreatic STUART SCHREIBER, BRIDGET WAGNER, Cambridge, MA tissues in adult mice who were studied 2 weeks post-injection. Control mice Infl ammatory beta-cell apoptosis involves a set of signaling cascades were injected with lentivirus scramble. 14-days post-injection, the pancreas initiated by interleukin-1β, interferon-γ, and tumor necrosis factor-α. We used was removed for immunohistologic examination. In mice injected with shRNA high-throughput screening to identify small-molecule suppressors with little Grb10, we did not observe any increase in cell proliferation with antibodies effect on other cell types. Using the rat INS-1E beta-cell line, we developed against BrdU or Ki-67. In contrast, using TUNEL assay, we demonstrated for a cell-based assay for ATP levels and screened >300,000 compounds for the the fi rst time that in vivo injection of lentivirus shRNA Grb10 directly into ability to suppress the negative effects of cytokines on ATP levels after two the adult mouse pancreas induced apoptosis in both exocrine and endocrine days. Three secondary assays provided complementary readouts of beta- (alpha and beta cells) tissues. In the present study, expression of the cell biology and apoptosis: caspase-3 activity, cellular nitrite secretion, and pro-apoptotic protein Bim in islets was higher in Lentivirus shRNA Grb10 glucose-stimulated insulin secretion (GSIS). compared to Lentivirus shRNA scramble mice. In the apoptotic pathway, We identifi ed one in-house compound as a particularly good screening the protein Bim initiates apoptosis signaling by binding to the Bcl-2-like pro- hit. To determine the structural requirements for activity, we synthesized survival proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1), thereby releasing Bax 27 analogs for testing and identifi ed BRD0476, which was fairly potent and/or Bak which cause a decrease in the mitochondrial outer membrane in both the ATP (EC50 3.5μM) and caspase (EC50 2.2μM) assays. BRD0476 potential, release of cytochrome c, and activation of the caspase cascade. was not active in other models of beta-cell apoptosis, and was not toxic to We propose that, when complexed with Grb10, Bim is inactive. Upon cellular other cell types tested. This compound was also active in human islet cells, activation by stress signaling, or in the present study following injection of both decreasing caspase activity and increasing GSIS in the presence of Lentivirus shRNA Grb10 into the pancreas, Bim becomes unbound from Grb10 cytokines after six days. and causes activation of Bax-Bak, thereby inducing apoptosis by activation We then sought to determine the mechanism of action of BRD0476. We the caspase cascade. Indeed, the activity of caspase-3 in islets was higher performed gene-expression profi ling of INS-1E cells in the presence or absence in the experimental group compared to the control group. The apoptosis of cytokines and compound; used mass spectrometry to fi nd proteins pulled induced by shRNA Grb10 resulted in a decrease in fasting plasma glucagon down by immobilized BRD0476; and developed a computational method to concentration and improved IP glucose tolerance despite a decrease in identify compounds with similar activity within our screening collection. Our glucose-stimulated insulin secretion. Fasting plasma insulin was similar in data suggest that BRD0476 acts on-mechanism, interfering with cytokine both groups. Our data provide evidence that Grb10 is critically involved in signaling pathways to inhibit a transcriptional response to cytokines. Further alpha-cell survival and, as a result, plays an important role in regulating work to determine the precise intracellular target(s) and to optimize the basal glucagon secretion and glucose tolerance in adult mice. pharmacokinetic properties of BRD0476 is ongoing. These results indicate that small molecules identifi ed by this screening approach may protect beta cells from autoimmune attack, and may be good candidates for therapeutic 1940-P intervention in type 1 diabetes. Cytokine Induced β-Cell Cytokine Production Is Regulated by HDACs Supported by: NIDDK Type 1 Diabetes Pathfi nder Award MATTIAS S. DAHLLÖF, DAN P. CHRISTENSEN, PAOLO MASCAGNI, CHARLES DINARELLO, LARS G. GRUNNET, THOMAS MANDRUP-POULSEN, Copenhagen, Denmark, Milano, Italy, Denver, CO 1938-P Destruction of the pancreatic β-cells occurs through an infl ammatory Autophagy Is Protective in a Cellular Model of HNF1A-MODY process in which pro-infl ammatory cytokines, in particular interleukin (IL)- SEÁN M. KILBRIDE, ANGELA M. FARRELLY, MARIA M. BYRNE, JOCHEN H.M. 1β, but also interferon (IFN)γ and interferon gamma-induced protein 10 kDa PREHN, Dublin, Ireland (IP-10/CXCL-10), have been implicated. Clinical proof of concept has been Heterozygous inactivating mutations in the hepatocyte nuclear factor 1A provided that IL-1β is involved in β-cell dysfunction in Type 2 Diabetes (HNF1A) gene have been shown to result in the pathogenesis of Maturity onset mellitus (T2DM), and clinical trials of IL-1 blockade are underway in patients diabetes of the young type 3, (HNF1A-MODY). We suppressed endogenous with recent-onset Type 1 (T1) DM. β-cell produced IL-1β is suffi cient to HNF1A function by overexpressing a dominant negative mutant (DN-HNF1A) in destroy β-cells in glucose and leptin-exposed islets. We have previously INS-1 cells. Induction of DN-HNF1A expression led to the transcriptional down- shown that inhibition of histone deacetylaces (HDACs) protects β-cells from regulation of several genes required for glucose metabolism, and decreased cytokine-mediated decay. The pathways through which cytokines mediate the intracellular ATP level. This resulted in increased phosphorylated AMP- β-cell death are not fully understood, but induction of nitric oxide production activated protein kinase (AMPK) levels, indicating increased AMPK activity. is a major player. AMPK negatively regulates mTOR kinase activity, and signalling through mTOR By means of quantitative (q)PCR and nitric oxide (NO) assay we here was indeed suppressed, as indicated by ribosomal s6 kinase and Akt-Ser473 show 1) that IL-1β induces IL-1β (p< 0.001, n=3), and CXCL-10 (p<0.001, dephosphorylation. Both of the latter two serine/threonine kinases have n=3) transcription as well as NO production in insulin producing cells, 2) important roles in cell survival, and prolonged overexpression of DN-HNF1A that IFNγ+IL-1β induce IL-1β (p<0.001, n=6) and CXCL-10 (p<0.001, n=6) led to activation of the mitochondrial apoptosis pathway. transcription and NO production (n=6) and furthermore these data suggest Increased AMPK signalling and decreased mTOR activity have also been that IFN-γ potentiates IL-1β induced IL-β, 3) that IFNγ itself induces CXCL- shown to activate macroautophagy (‘autophagy’), the process whereby a 10 but not IL-1β transcription or NO production (n=4), 4) that CXCL-10 does cell recycles its components. The role of autophagy in cell death signalling not induce IL-1β transcription nor NO production (n=3) and 5) that the novel is controversial, with reports showing that autophagy can both protect HDAC inhibitor givinostat diminishes both IL-β induced IL-1β (p <0.01, n=3) and mediate cell death. We therefore investigated whether the DN- and CXCL-10 (p<0.05, n=3) transcription and NO production and IL-1β+IFNγ HNF1A-induced cell stress led to activation of autophagy, and whether induced IL-1β transcription (p<0.001, n=6) and NO production, as well as autophagy contributed to cell death. In cells transiently transfected with IFNγ induced CXCL-10 transcription. a vector expressing LC3-GFP, overexpression of DN-HNF1A increased the Taken together these data suggest that feed-forward IL-1β induction of IL- percentage of the population which displayed punctae, indicating activation 1β transcription contributes to cytokine induced β-cell death, that the role of of autophagy. We next used gene silencing technology to knock down Atg5, IFNγ in this death is primarily enhancing IL-1β caused cell death, that CXCL-10 a protein which is essential in the autophagic process. Interestingly, knock is not pivotal in cytokine induced β-cell cytokine production and that HDACs down of Atg5 resulted in a potentiation of DN-HNF1A-induced apoptosis. regulate cytokine induced β-cell cytokine production. Givinostat is already This suggests that autophagy is activated in response to dominant negative in several phase II trials and has been granted European Union orphan drug suppression of HNF1A function, and plays a protective role against DN- designation, and is thus a promising anti-diabetic drug candidate. POSTERS HNF1A-induced cell death, potentially by providing energy substrates for Islet Biology/

Supported by: JDRF# 26-2008-893 & Novo Nordisk, Inc. Insulin Secretion metabolically deprived cells. Supported by: Health Research Board RP/2008/14 and Science Foundation Ireland 08/IN1/1949

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A523 ISLET BIOLOGY—APOPTOSISCATEGORY

1941-P 1943-P Direct Role for IL-12 in Islet Beta Cells Implications of the RAS in Beta-Cells DAVID A. TAYLOR-FISHWICK, JESSICA WEAVER, WOJCIECH GRZESIK, SWARUP NADINE S. SAUTER, YULIYA YEUDAKIMOVICH, MARC Y. DONATH, Basel, Switzer- CHAKRABARTI, JERRY L. NADLER, Norfolk, VA land, Zurich, Switzerland Increasing evidence suggests that infl ammation leads to islet β-cell Recent clinical trials have suggested that blockade of the renin- damage in type 1 and type 2 diabetes. Following an acute incubation with angiotensin system (RAS) reduces the incidence of new-onset diabetes. pro-infl ammatory cytokines (TNFα, IL-1β, IFNγ), human islets upregulate The RAS is classically known as a systemic hormonal system, however the gene expression for IL-12 and IFNγ. Both IL-12 p35 and p40 ligand chains are presence of local tissue RAS has been recognized and all the components upregulated, with p40 expression being infl uential. of a functional RAS are observed in the endocrine pancreas. RAS blockade We have investigated if IL-12 signaling components exist in β-cells. The in animal models of type 2 diabetes results in the improvement of islet receptor chains for IL-12 (R1, R2) are expressed in islets and their genes are structure and function and glucose tolerance. In β-cells, Angiotensin II upregulated following stimulation; either 5.2 and 4.5 fold (R1 and R2 resp.) (AngII) regulates insulin secretion, pro-insulin synthesis and expression of after treatment with pro-infl ammatory cytokines (PICs) or 2.3 and 2.5 fold monocyte chemoattractant protein-1. The aim of this study is to explore the (R1 and R2 resp.) after addition of IL-12. Immunochemistry detected IL-12 consequences of chronically elevated AngII and its role in the deterioration receptor (R1-chain) staining in non-diabetic human islet sections (nPOD of islet function during the development of diabetes. obtained) and this staining co-localized with insulin-positive cells, indicating INS-1E β-cells and isolated human islets were cultured under control β-cells are able to respond to IL-12. Evidence of IL-12 signaling in human conditions (11.1 resp. 5.5 mM glucose), in elevated glucose (22.2 resp. 11.1 islets was refl ected by a 3 fold increase in STAT4 expression following PIC mM) or in the presence of AngII (1 μM) with or without the addition of stimulation, and an increase in IFNγ gene expression following stimulation Interleukin-1Receptor Antagonist (IL-1Ra, 1 µg/ml) or an IKK-2 inhibitor (10 with either PICs or IL-12. Further, a role for IL-12 in mediating PIC effects was µM) for 4 days. confi rmed using a blocking IL-12 antibody that reduced, but did not eliminate, Our studies revealed that elevated glucose levels regulate mRNA and upregulation of IFNγ. To determine whether IL-12 directly regulated β-cells, protein levels of most of the members of the RAS family in islets and in homogeneous β-cell lines (INS-1 and βTC-3) were studied. In INS-1 cells, β-cells. Most importantly, glucose up-regulates the precursor angio- expression of IL-12 receptor chains (R1,R2) and IL-12 ligand chains (p35, p40) tensinogen as well as the angiotensin II receptor type 1, necessary factors were increased following stimulation with murine PICs (14.1, 6.9, 1.8 and 3.9 whose expression results in active signaling. The main component of the fold for R1,R2,p35 and p40 resp.). Increased gene expression of candidate RAS family, AngII, in turn up-regulates cytokines such as Interleukin-1β signaling pathways (PCR array and confi rmed by RT-PCR) following IL-12 and Interleukin-6 in isolated human islets and chemokine KC in INS-1E stimulation of β-cell lines support a direct effect of IL-12 on β-cells. These cells. In parallel, AngII treatment results in impaired glucose-stimulated pathways are also upregulated in islets from human diabetic donors. insulin secretion and an increased rate of apoptotic β-cells. Addition of Pharmacological inhibition of IL-12 signaling by the immunomodulator, IL-1Ra prevented the deleterious effects in islets, suggesting that they are lisofylline, protected human islets from dysfunction induced by PICs. partly mediated by Interleukin-1β. AngII signaling acts via NF-κB in islets Collectively, these studies suggest a previously unexplored role for IL-12 and in INS-1E cells, and inhibiting NF-κB activation protects the cells from directly in beta cells. Importantly, the data indicate that modulators of IL-12 detrimental consequences of pro-longed AngII exposure. signaling may have utility in direct β-cell protection in both type 1 and type Taken together, these studies support the hypothesis that the RAS is one 2 diabetes. of the auto-, para- or endocrine systems that infl uence islet function and Supported by: DOD 093521 mass and therefore constitutes a therapeutic target for the treatment of diabetes. 1942-P Estrogen Protects Pancreatic β Cells from Cytokine Mediated Cell 1944-P Death Inhibition of Fatty Acid Translocase Cluster Determinant 36 (FAT/ RITA SHRIDHARANI, SIMON PROSSER, SCOTT PAVLETICH, MARY KALDUNSKI, CD36) Prevents Glucotoxicity and Glucolipotoxicity in INS-1 Cells POLLY HANSEN, JOHN CORBETT, MARTIN HESSNER, SANJAY KANSRA, KYU-CHANG WON, YONG-WOON KIM, YE-JIN SEO, JI-SUNG YOON, SO-YOUNG Milwaukee, WI PARK, JONG-YEON KIM, HYOUNG-WOO LEE, Daegu, Republic of Korea Type 1 Diabetes (T1D) is an autoimmune disease that culminates in the The mechanisms of glucotoxicity and glucolipotoxicity involve several death of insulin secreting pancreatic β-cells, resulting in life-long dependence transcription factors and are, at least in part, mediated by generation of on external insulin. Islet cell invading macrophages and leukocytes release chronic oxidative stress. Several recent studies supported that the CD36 cytokines that orchestrate pancreatic β-cell death. Cell culture models and increases uptake of free fat acid in high glucose state in several cells. islets isolated from rodents and humans demonstrate the pro-apoptotic Yet, little is known about the relation between glucotoxicity and CD36 in effect of interleukin 1β (IL-1β), either by itself or in combination with the islets. The purposes of the present study were to determine whether interferon γ (INFγ) and tumor necrosis factor (TNF). Cytokines activate NFκB prolonged exposure of pancreatic islets to glucotoxic or glucolipotoxic signaling pathway, resulting in the transcriptional induction of inducible condition disrupts CD36 or increases CD36, we also wanted to evaluate a nitric oxide synthase (iNOS). Subsequent generation of nitric oxide (NO) is role of CD36 in pancreatic islets against glucotoxicity and glucolipotoxicity. the effector molecule that induces cell death. We checked intracellular peroxide levels, insulin transcription factors, Estrogen (E2) is known to have an anti-diabetogenic effect. However, insulin mRNA, glucose stimulated insulin secretion (GSIS) and CD36 of the the exact mechanism by which E2 protects pancreatic β-cell death remains pancreatic islets (INS-1 cells, rat islets). INS-1 cells were transfected of CD36 to be determined. Our objective was to determine whether E2 can directly siRNA for inhibition of CD36 in INS-1 cells. The intracellular peroxide levels antagonize cytokine activated NFκB signaling. Using rat insulinoma cell of the pancreatic islets were increased in the high glucose (30 mM glucose in lines, RINm5F and INS832/13 cells, we demonstrate that the ability of IL- INS-1 cell or 50 mM ribose in rat islets) media compared to 5.6 mM glucose 1β to suppress cell viability was signifi cantly reversed by E2. Further, in media. The insulin mRNA levels and GSIS were decreased in INS-1 cells these cells E2 blocked IL-β activated NFκB phosphorylation, and subsequent exposed to high glucose media compared to 5.6 mM glucose media. The induction of iNOS. The ability of E2 to antagonize IL-β activated NFκB was insulin mRNA levels, PDX-1 and GSIS were decreased in INS-1 cells exposed reversed by the global anti-estrogen ICI 182780, demonstrating estrogen to glucolipotoxic condition (30 mM glucose and 0.5 mM palmitate) compared receptor (ER) requirement for the protective effect. Finally, in rat islets, E2 to 5.6 mM glucose media. And, at INS-1 cells exposed to glucotoxic or blocked IL-β induced iNOS. Human T1D is modeled by the spontaneously glucolipotoxic condition (30 mM glucose and 0.5 mM palmitate), CD 36 was diabetic BioBreeding (BB) rat, BB DRlyp/lyp rat. Like human T1D, progression signifi cant increased compared to normal glucose media at 12 hours. After of DRlyp/lyp rats to diabetes is characterized by induction of IL-1 regulated transfection of CD36 siRNA, CD 36 was signifi cantly decreased compared to genes, and that treatment of DRlyp/lyp rats with human recombinant IL-1RA control and insulin mRNA and GSIS were signifi cantly increased compared delays disease onset. Treatment of DRlyp/lyp rats, with physiological levels to without CD36 siRNA in INS-1 cells. These results suggest that the infl ux of E2, delayed disease onset. Thus, we conclude that targeting ER mediated of fatty acid to islet at early high glucose condition can be increased by signaling in pancreatic β-cells is a viable therapeutic approach to preventing increasing of CD36. So, at islets exposed to high glucose and high fatty

POSTERS pancreatic β-cell death. We propose identifi cation and development of acid may be exacerbating beta cell dysfunction via increasing of CD 36 and Islet Biology/

Insulin Secretion lead compounds from a panel of selective ER modulators (SERMs), with the inhibition of CD36 may be prevent glucotoxicity and glucolipotoxicity in INS- objective of preserving pancreatic β-cell mass in vivo. 1 cells.

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A524 ISLET BIOLOGY—APOPTOSISCATEGORY

1945-P 1947-P Investigating Endoplasmic Reticulum Redox Status under Various Oxidative Stress Promotes Islet Amyloid Deposition Conditions That Induce Endoplasmic Reticulum Stress in Live MICHAEL MORCOS, SAKENEH ZRAIKA, STEVEN E. KAHN, Seattle, WA Pancreatic β-Cells Type 2 diabetes and chronic hyperglycemia are associated with oxidative IRMGARD SCHUIKI, ALLEN VOLCHUK, Toronto, ON, Canada stress in islet β cells. Islet amyloid, a pathological feature of type 2 diabetes, Endoplasmic reticulum (ER) stress is ultimately caused by an accumulation contributes to loss of β-cell mass but its relationship to oxidative stress of misfolded, unfolded or aggregated proteins in the ER. Cells respond to is not fully understood. Previously we showed that amyloid deposition ER stress by activating the unfolded protein response. Since accurate increases oxidative stress in islets; however, extended exposure to amyloid- quantifi cation of misfolded or unfolded proteins is not feasible, ER stress induced oxidative stress may also increase amyloid deposition in a positive is detected by measuring either the activation of ER stress sensors or the feedback manner. Thus, oxidative stress may promote amyloid deposition induction of downstream genes. Measuring ER stress this way however, is and loss of β-cell mass. an indirect and operational measure that breaks down when the unfolded To determine whether the induction of oxidative stress promotes islet protein response system is disabled or non-functional. Moreover it is diffi cult amyloid deposition, we used our in vitro model where human islet amyloid to determine if the ER stress response is actually successful in alleviating polypeptide transgenic (T) mouse islets develop amyloid in a glucose- ER stress and re-establishing normal ER homeostasis. Recently, a redox dependent manner and non-transgenic (NT) islets are incapable of developing sensitive GFP (eroGFP) was used in yeast to monitor the oxidation status amyloid. T and NT islets were incubated in 11.1 or 16.7 mM glucose for 7 days of the ER in cells experiencing ER stress. Chemical inducers of ER stress with or without 0.5 mM glyceraldehyde, a known oxidative stressor (n=5 per including dithiothreitol and tunicamycin altered the redox potential in the group). Oxidative stress, amyloid deposits and β-cell area were visualized ER, which is normally oxidizing, to a more reduced state; thus eroGFP is a using nitrotyrosine, thiofl avin S and insulin staining respectively. potential optical “ER stress sensor” in living cells. Here we used an INS-1 Glyceraldehyde treatment increased oxidative stress in both 11.1 and insulinoma cell line stably expressing both the eroGFP (ER stress sensor) 16.7 mM glucose-cultured islets. In 16.7 mM glucose-cultured T islets, and a mCherry protein driven by the GRP78 promoter (unfolded protein treatment with glyceraldehyde did not signifi cantly increase amyloid response sensor). Cells were exposed to a variety of stress conditions and deposition (% amyloid area/islet area: 2.9±0.8% vs. 2.7±0.5%; p=0.6), nor monitored using live cell imaging, fl ow cytometry and biochemical assays did it change β-cell area (% insulin area/islet area: 66.1±2.0% vs. 67.2±2.4%; (XBP1 splicing). As expected, treatment of the cells with the reducing agent p=0.2). These fi ndings suggest that in islets with amyloid-induced oxidative dithiothreitol caused a decrease in the oxidation state of the ER accompanied stress, further induction of oxidative stress with glyceraldehyde does by an increase in XBP1 splicing. Unexpectedly however, other treatments not promote additional amyloid formation or β-cell loss. Conversely, in such as tunicamycin, thapsigargin, elevated free fatty acids or high glucose 11.1 mM glucose-cultured T islets, where amyloid deposition is normally had essentially no infl uence on the ER redox status despite inducing ER minimal, glyceraldehyde treatment increased amyloid deposition (0.3±0.1% stress. Thus, unlike in yeast, ER stress is not associated with signifi cant ER vs. 1.2±0.3%; p=0.02) and reduced β-cell area (71.1±0.8% vs. 68.6±0.7%; redox changes and suggests that the pancreatic β-cell ER redox potential is p=0.01). As expected, NT islets did not form amyloid under all conditions. well maintained even in the presence of ER stress. We conclude that induction of oxidative stress under conditions of minimal islet amyloid deposition promotes amyloid formation and thereby may ultimately contribute to loss of β-cell mass in type 2 diabetes. 1946-P Morphometric Analysis of Pancreatic α-Cells in Japanese Non- Obese Patients with Type 2 Diabetes Mellitus—A Relevance of the Morphologic Changes to Incretin Action 1948-P MASAYUKI MIUCHI, JUN-ICHIRO MIYAGAWA, KOSUKE KONISHI, ETSUKO NAGAI, Pancreases of Carboxyl-Ester Lipase MODY Patients Exhibit Irregu- NORIKO KITAMURA, YUZO YANO, SHUZO SHIOTA, JURI TAKEHARA, HUMIHIRO lar Clusters of Islets and Infl ammatory Fat Infi ltrates OCHI, YOSHIKI KUSUNOKI, MASARU TOKUDA, KAZUKI MURAI, TOMOYUKI HELGE RAEDER, DAG HOEM, JIANG HU, HEIKE IMMERVOLL, PAL R. NJOLSTAD, KATSUNO, TOMOYA HAMAGUCHI, MITSUYOSHI NAMBA, Nishinomiya, Japan ANDERS MOLVEN, ROHIT N. KULKARNI, Boston, MA, Bergen, Norway Glucagon-like peptide-1 (GLP-1) has many physiological functions benefi cial Carboxyl-ester lipase MODY (maturity-onset diabetes of the young of the treatment for type 2 diabetes mellitus (T2DM). Among them, the type 8, MODY8) is a human monogenic disease model of pancreatic beta suppression of glucagon secretion from α-cells is thought to be important in cell failure and exocrine dysfunction characterized by severely defective addition to the incretin effect. However, little is known about the morphologic insulin secretion (Raeder, Nat Gen, 2006 and Raeder, Diabetes, 2007). The and functional changes of α-cells in the pancreas of T2DM. We investigated mechanism(s) for beta cell failure is however unknown. To understand the the morphological changes and the expression of GLP-1 receptor (GLP-1r) in disease mechanism, we used immunohistochemistry to examine pancreatic pancreatic α-cells in Japanese non-diabetic subjects (non-DM) and patients tissue from two MODY8 patients (one autopsied pancreas and one specimen with T2DM. From 32 non-obese subjects (15 non-DM and 17 T2DM), we made from a patient undergoing surgery for pancreatic adenocarcinoma). We formalin-fi xed paraffi n embedded pancreatic tissues from surgical operations observed fat cells and fi brous tissue replacing normal pancreatic acinar or autopsies (head, body and tail portions respectively). Using sections tissue that surrounded scattered irregularly shaped islets (Fig 1a; Patient, immunostained with glucagon and GLP-1r, α-cell size (μm2), α-cell area of hematoxylin staining, Fig1b; Patient, insulin staining). The fat and fi brous the islet (%), the relative α-cell area (%), and relative α-cell number (/cm2) tissue was however infi ltrated with leucocytes (CD45+cells) adjacent to were measured and calculated by morphometric analyses. Pancreatic α-cell islets (Fig1c; Patient, Fig 1d; Control). We observed an average of 3.7±4.1 size was signifi cantly enlarged (p<0.01) in T2DM and this was remarkable CD45+ cells adjacent to islets (adjacent defi ned as <5 nuclear diameters). especially in the pancreas head portion. In T2DM, relative α-cell area of the These fi ndings suggest that active infl ammation is potentially linked to the islet (p<0.001), relative α-cell area of the pancreas (p<0.001) and relative beta cell defi ciency in these patients. Fur thermore, we obser ved the presence α-cell number (p<0.001) were signifi cantly increased. In addition, both relative of insulin+ cells in ducts suggesting potential foci of beta cell regeneration α-cell area of the islet and that of the pancreas were correlated positively with in an attempt to compensate for the insulin defi ciency (Fig1e; Patient, ducts, HbA1c (p<0.001, p<0.05, respectively) and the level of fasting plasma glucose Fig 1f; Patient; 17 (6%) insulin positive cells and 14 (5%) Ki67 positive cells of (FPG) (p<0.001, p<0.01, respectively). In both non-DM and T2DM, we could all 305 duct cells counted in the focal area shown). We did not observe islet observe GLP-1r-positive pancreatic α-cells. These results suggest that the cell replication (but insulin+/Ki67+ cells in the ducts) or an increase in islet increased number of pancreatic α-cells contributes in part the pathogenesis cell apoptosis using TUNEL (not shown). Together these data suggest either of the development of T2DM. In addition to “incretin effect” (augmentation a functional suppression of islet function and/or a reduced beta cell mass of deteriorated insulin secretion from β-cells), the suppression of glucagon secondary to leucocyte infi ltrating activity as the potential underlying cause secretion from α-cells might contribute much more than we expect to the of beta cell defi ciency that requires insulin treatment in the patients. improvement of glycemic control in T2DM. POSTERS Islet Biology/ Insulin Secretion

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A525 ISLET BIOLOGY—APOPTOSISCATEGORY

Using Real Time PCR we have established that the NOX1 gene is expressed in primary islets and beta cells. Further, beta cell dysfunction induced by a pro-infl ammatory cytokine cocktail (TNFα, IL-1β and IFNγ) associates with increased NOX expression. Stimulation of primary mouse islets with pro- infl ammatory cytokines (PICs) induced NOX1 expression by 18.8 ± 5.8 fold at four hours. This pathway was confi rmed in human donor islets where NOX1 expression increased 7.1 ± 3 fold following treatment with PICs. To confi rm this as a beta cell response, homogeneous beta cell lines INS-1 (rat) and βTC-3 (mouse) were studied. Upon PIC stimulation, the expression of NOX1 increased 87±0.6-fold in INS-1 cells and 60±12-fold in βTC3 cells respectively. The lipoxygenase enzyme 12-LO has been reported to be upstream of NOX activation in a platelet oxidation model. The activity of 12-LO (ALOX12) is implicated in mediating beta cell dysfunction driven by PICs. An inhibitor of 12-LO activity, that protected human islets from PIC-induced dysfunction, reduced PIC induced NOX1 activation by 50 ±10 % in primary mouse islets. Data is from replicates greater or equal to three, p<0.05. Generation of ROS by NOX pathways may be a relevant component to induced oxidative damage in beta cells. Selective inhibitors of ROS generation mediated by NOX1 in beta cells would be candidate agents to promote beta cell survival.

1951-P Serum Levels of Pancreatic Stone Protein/reg1A in HNF1A-MODY May Indicate Increased Beta-Cell Destruction during Disease Pro- gression SIOBHAN BACON, PEYH KYITHAR, CAROLINE BONNER, SYED R. RIZVI, KAVITA KAUR PANNU, ROLF GRAF, JOCHEN H.M. PREHN, MARIA M. BYRNE, Dublin, Ireland, Zurich, Switzerland Maturity-onset of diabetes of the young(MODY) is characterized by autosomal-dominant inheritance and an insulin secretory defect.Mutations 1949-P in the transcription factor hepatocyte nuclear factor 1 alpha(HNF1A-MODY) Pioglitazone Induces Islet Adrenomedullin Expression and Attenu- represent the commonest form.HNF1A-MODY carriers have reduced pancreatic ates β-Cell ER Stress volume suggesting a decline in beta-cell mass due to apoptosis.HNF1A-MODY YASUHARU OHTA, MANABU KONDO, MASAYUKI HATANAKA, KATSUYA being monogenic makes it a useful model in studying beta-cell dysfunction. TANABE, MASAYOSHI YAMAMOTO, HIROKO NAKABAYASHI, YUKIO TANIZAWA, Previous studies from our laboratory demonstrated enhanced beta-cell Ube, Japan apoptosis induced by suppression of endogenous HNF1A function resulted in Selective β-cell loss and severe diabetes develop in Wfs1-/- mice with mild transcriptional activation of pancreatic stone protein/regenerating protein(PSP/ obesity (Wfs1-/- Ay/a). Pioglitazone (Pio) reduce diabetes incidence in humans reg) gene in beta-cells. PSP/reg was discovered in an unbiased screening of a and remarkably preserves pancreatic β-cells in Wfs1-/- Ay/a mice. The regenerating-islet-derived cDNA library from 90% depancreatized rats and purpose of the present study is to elucidate molecular mechanisms whereby stimulates beta-cell regeneration. In our previous study, the induction of PSP/ Pio prevents cell death as a direct action on β-cells. Gene expression reg during beta-cell apoptosis required the activation of apoptosis-specifi c profi les of islets were analyzed using microarray in Ay/a mice treated with proteases(capases), suggesting that PSP/reg maybe an indicator of beta- or without Pio for 4 wks. One of the upregulated genes was adrenomedullin cell destruction.Proof of concept work suggested that serum levels of PSP/ (AM). Real-time RT-PCR confi rmed signifi cant upregulation by 1.4-fold. When reg are elevated in HNF1A-MODY subjects. We sought to clinically validate cellular localization of AM was determined in pancreas by fl uorescence this in 31 subjects with HNF1A-MODY.These subjects were compared to immunohistochemistry, moderate level of expression was detected in controls consisting of fi rst-degree relatives from MODY pedigrees and a α-cells at any ages. Contrary to a previous report, expression in PP cells historic value based on 31 non-diabetic subjects.PSP was analyzed using was much less than in α-cells. Low level expression was detected in β-cells ELISA technique.HNF1A-MODY subjects had signifi cantly higher fasting serum at 8 wk of age. Oral administration of Pio for 4 wks induced AM expression PSP/reg1A than controls. We analyzed whether PSP/reg1A in HNF1A-MODY in β-cells. AM expression in islet cell lines was also studied by real-time subjects correlated with clinical phenotype.A positive correlation was noted RT-PCR. AM mRNA expression was 1.7-fold higher in αTC1 cells compared between PSP/reg1A levels, age of the patients(rs=0.38,p=0.04) and duration of to that in MIN6 cells. Forty-eight hrs culture in the presence of 10μmol/l diabetes(rs=0.36,p=0.05).We observed a negative correlation between fasting Pio signifi cantly increased AM mRNA expression in both αTC1 (1.3-fold) and insulin levels and PSP/reg1A(rs=-11,p=0.02) and no correlation with HbA1c.We MIN6 (2.3-fold) cells. AM induction by Pio through the activation of PPARγ demonstrated that PSP/reg1a maybe a clinical indicator of beta-cell apoptosis was confi rmed by ChIP assay. Chromatin materials from MIN6 cells cultured that increases with age and duration of diabetes in HNF1A-MODY.This protein in the presence or absence of Pio were immunoprecipitated, followed by may provide information on disease progression and on the response to anti- PCR using primers encompassing PPAR responsive element like regions. Pio diabetic treatment regimes, particularly those aimed at protecting pancreatic stimulated PPARγ binding to a specifi c region within the AM promoter. Then beta-cells. mechanism whereby AM protect β-cells was investigated. MIN6 cells were cultured in medium containing thapsigargin in the presence or absence of 1952-P 100nmol/l AM peptide for 48 h. AM signifi cantly suppressed Caspase-3 ST18: A Novel Transcription Factor That Regulates Pancreatic beta cleavage and thapsigargin-induced apoptosis. The results suggest that Pio Cells Mass and Function induce AM expression in islets through direct PPARγ activation. In turn, AM CYNDI HENRY, JEAN BUTEAU, Quebec, QC, Canada via autocrine and paracrine mechanism prevents β-cell death by attenuating Introduction: The insulin-secreting pancreatic beta cell plays a central ER stress. role in the maintenance of glucose homeostasis and in the pathogenesis of diabetes. Indeed, both type 1 and type 2 diabetes are characterized by a 1950-P reduction in functional beta cell mass. It is therefore important to identify Pro-Infl ammatory Cytokines Induce the Expression of the NADPH the molecular mechanisms involved in the maintenance of beta cell mass Oxidase, NOX1, in Primary Islets and Beta Cell Lines and function. A genomic study conducted in our laboratory identifi ed ST18 JESSICA WEAVER, DAVID A. TAYLOR-FISHWICK, Norfolk, VA (Suppression of tumorigenicity 18) as a potentially important gene in beta

POSTERS Sensitivity to reactive oxygen species (ROS) is a feature of beta cell cells. The ST18 gene encodes a transcription factor belonging to the zinc fi nger Islet Biology/

Insulin Secretion pathogenesis. Induced mitochondrial dysfunction and ER stress are protein family whose biological function remains poorly characterized. considered key drivers of oxidative stress in beta cells. An alternative Objective: Our project aims to investigate the expression, activity and pathway to generating ROS involves the NADPH Oxidase proteins (NOX). potential role of ST18 in the regulation of beta cell mass and function.

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A526 ISLET BIOLOGY—BETACATEGORY CELL—DEVELOPMENT

Methods: The expression of ST18 was evaluated by quantitative PCR Overexpression of PKCdelta in MIN6 beta cells using an adenoviral based and immunohistochemistry. Its activity was investigated in INS cells by system, resulted in increased expression of Nix mRNA and protein levels. electrophoretic mobility shift assay. ST18 gain- and loss-of-function was PKCdelta overexpression also induced MIN6 cell death as assessed by achieved by transfection with pCMV-ST18 and specifi c siRNAs, respectively. propidium iodide staining. Possible effects on apoptosis and necrosis were Apoptosis was measured by TUNEL. Proliferation was evaluated by BrdU evaluated by inhibitors of these two processes, DEVD and Cyclosporine A incorporation. Finally, the effect of ST18 on insulin secretion was studied (CsA), respectively. DEVD and CsA additively prevented PKCdelta-induced by ELISA. MIN6 cell death. On the other hand, autophagy inhibitor did not prevent Results: Our results indicate that ST18 is broadly expressed in human PKCdelta-induced MIN6 cell death, indicating that PKCdelta causes tissues. However, in the pancreas, ST18 expression is restricted to endocrine apoptotic and necrotic but not autophagic beta cell death. Since PKCdelta cells. Our results also show increased expression and activity of ST18 in beta induces Nix expression and beta cell death, Nix expression was reduced cells after exposure to cytotoxic concentrations of fatty acids (lipotoxicity) or using a lentivirally introduced shRNA in the MIN6 cells that overexpress cytokines. ST18 overexpression induced beta cell apoptosis and decreased PKCdelta. Preventing the increase in Nix inhibited PKCdelta-induced MIN6 proliferation. Conversely, knockdown of ST18 prevented the deleterious cell death, suggesting that the effects of PKCdelta in causing beta cell effects of palmitate and cytokines on beta cell proliferation and survival. death are mediated by an increase in Nix expression. Finally, PKCdelta was Finally, ST18 inhibited insulin secretion. overexpressed in islets isolated from Nix-/- and control wild type (WT) mice. Conclusion: In this study, we characterized ST18 as a negative regulator of PKCdelta increased cleaved caspase-3 levels by Western blot in WT but not beta cell mass and function. Nix-/- islets providing additional evidence that the beta cell death promoting Supported by: Canadian Institutes of Health Research actions of PKCdelta are mediated by Nix. PKCdelta induces apoptotic and necrotic beta cell death in a Nix- 1953-P dependent manner. These results establish the PKCdelta-Nix pathway as a SUMO1 Protects Against IL-1β-Induced Apoptosis, but Not IL-1β- critical mediator of apoptosis and programmed necrosis in beta cells. Mediated Calcium Dysfunction in β-Cells CATHERINE HAJMRLE, JOCELYN E. MANNING FOX, PATRICK E. MACDONALD, & 1955-P Edmonton, AB, Canada Role of CXCR3 in the beta Cell Proliferation in Type 1 Diabetes The infl ammatory cytokine interleukin-1β (IL-1β) induces detrimental YOSHIFUMI YAMADA, YOSHIAKI OKUBO, AKIRA SHIMADA, YOICHI OIKAWA, effects on pancreatic islets through activation of the nuclear factor κB (NFκB) SATORU YAMADA, SHOSAKU NARUMI, KOUJI MATSUSHIMA, HIROSHI ITOH, pathway. The small-ubiquitin related modifi er 1 (SUMO1) protein is a post- Tokyo, Japan translational modifi er of numerous target proteins (called SUMOylation), and We previously reported that blockade of CXCL10, the serum level of reduces NFκB activation in a variety of cell types. We therefore investigated which was signifi cantly higher in type 1 diabetes patients, by induction the ability of SUMO1 to prevent IL-1β mediated calcium dysfunction and cell of anti-CXCL10 antibodies in non-obese diabetic (NOD) mice, resulted in a death. Treatment of mouse islets, infected with control adenovirus, with IL- delayed diabetes onset due to β cell proliferation. Because both CXCL10 1β (5 ng/ml) impaired glucose-stimulated calcium oscillations by 47%. This and CXCR3 were expressed on β cells, we surmised that antibodies might detrimental effect of IL-1β was not changed upon expression of SUMO1, accelerate the proliferation of pancreatic β cells by blocking of CXCL10- where calcium oscillations were reduced by 58% (P < 0.05). The effects of CXCR3 interaction loop. Recently, however, it was reported that CXCL10 IL-1β on calcium were most pronounced in the presence of metabolic stress was associated with the suppression of β cell proliferation through Toll-like (i.e. at 25 mM glucose). The combination of IL-1β and hyperglycemia further receptor 4 (TLR4) but not CXCR3, so it is a controversial issue which receptor reduced the amplitude of calcium oscillations (by 71%). Again, SUMO1 over- CXCL10 controls β cell proliferation through. In this study, to clarify the role expression was unable to modify the effect of IL-1β+high glucose, where of CXCR3 in β cell proliferation, we generated CXCR3-defi cient NOD-scid a 73% reduction in calcium oscillations was observed. Similar data was mice by crossing NOD-scid and CXCR3-defi cient NOD mice, and injected obtained in human islets, suggesting that SUMO1 is unable to prevent the diabetogenic splenocytes from a diabetic female wild type NOD mouse calcium-signaling dysfunction induced by IL-1β alone or in combination with intraperitoneally into either female CXCR3-defi cient NOD-scid or NOD-scid hyperglycemia. Surprisingly however, expression of SUMO1 in an insulin- recipients, and compared the diabetes incidence and β cell proliferation secreting cell line (INS-1) was able to completely protect these cells from between two recipient groups. Upon the adoptive transfer of diabetogenic IL-1β-induced cell death (P < 0.05), associated with a 43% reduction in spleen cells, CXCR3-defi cient NOD-scid recipients showed lower incidence cleaved-caspase 3. Conversely, over-expression of the deSUMOylating of diabetes than NOD-scid recipients (CXCR3-defi cient NOD-scid 7.7% vs. enzyme SENP1, which cleaves SUMO1 from it’s targets, sensitized cells to IL- NOD-scid 42.1%, p<0.05). CXCR3-defi cient NOD-scid recipients had many 1β-induced cell death (P < 0.05), and increased cleaved-caspase 3 levels by Ki67 positive β cells, resulting in an increase of β cell mass in comparison 220% (P < 0.05). Thus, although SUMO1 is not able to prevent IL-1β-induced with NOD-scid recipients. We did not detect a signifi cant difference of TLR4 dysfunction in pancreatic islets, it prevents IL-1β-induced cell death through expression level or vascularization in the pancreata between two recipient apoptosis. groups. Taken together, the signals of the suppression of pancreatic β cells Supported by: JDRF are mediated through CXCR3 but not TLR4 in our system.

ISLET BIOLOGY—BETA CELL—DEVELOPMENT & 1956-P Glucose Sensing through Glucokinase Regulates Islet Cell Neo- [See also: Presidents Poster 471-PP, page A131.] genesis TAKESHI MIYATSUKA, MICHAEL S. GERMAN, Osaka, Japan, San Francisco, CA The size of the beta-cell population is a critical determinant of diabetes Guided Audio Tour: Beta Cell Development (Posters 1954-P to 1960-P), risk. The number of islet cells generated during fetal development depends see page 11. on the rate at which new islet cells are generated from Neurogenin3 & 1954-P (Ngn3)-expressing progenitor cells and the rates of proliferation of the islet PKCdelta Induces Pancreatic beta Cell Death in a Nix-Dependent progenitors and mature islet cells. We previously reported a mouse model Manner “Ngn3-Timer” that allows the purifi cation and characterization of Ngn3- KEI FUJIMOTO, TAKASHI SASAKI, KAZUNORI UTSUNOMIYA, GERALD W. DORN II, expressing progenitor cells from more differentiated endocrine cells during KENNETH S. POLONSKY, Shinjyuku-ku, Japan, Tokyo, Japan, St. Louis, MO, Chicago, IL embryonic development of the pancreas. In a screen for genes specifi cally Pancreatic beta cell apoptosis and necrosis are implicated in reduced expressed in these progenitors, we found that endocrine progenitor cells beta cell mass in diabetic animal models. Recently, we have reported that expressed glucokinase mRNA at levels comparable to differentiated pro-death BH3-only like protein Nix induces apoptotic and necrotic beta cell endocrine cells, and also expressed other glucose sensing components. death. In Pdx1-haploinsuffi cient diabetic mice, Nix expression was increased Expression of these glucose-sensing genes in endocrine progenitor cells and Nix ablation normalized glucose tolerance, pancreatic islet architecture, was still detected in Ngn3 null embryos. In cultured embryonic pancreatic beta cell mass, and insulin secretion. However, the signal transduction buds, the number of endocrine progenitor cells markedly increased with POSTERS pathways responsible for the increase in Nix and its effects on the beta increasing glucose concentration or with treatment with the glucokinase Islet Biology/ cell are not known. Our preliminary data suggest that PKCdelta induces Nix activator CpdA; and this increase was still seen in buds from Ngn3 null Insulin Secretion expression in mouse insulinoma MIN6 cells. Thus, the aim of this study was embryos, showing that glucose sensing through glucokinase functions to elucidate the molecular link between PKCdelta and Nix in beta cells. independently of Neurog3-mediated endocrine specifi cation. These fi ndings

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A527 ISLET BIOLOGY—BETACATEGORY CELL—DEVELOPMENT

reveal a novel role of glucokinase and glucose sensing in the neogenesis nodes of mice receiving combination therapy of vorinostat and LSF ± MK-626 of Ngn3-expressing endocrine progenitors during pancreas development, versus diabetic controls. We conclude that combination therapies with LSF, suggesting a potential application of glucokinase activation in future cell MK-626, and vorinostat diminish insulitis, possibly by increasing regulatory therapy for diabetes. T cell populations, and enhance β cell mass. Our study also points to the TM was supported by a JDRF Advanced Postdoctoral Fellowship (10- novel effi cacy of histone deacetylase inhibitors in combination with anti- 2010-561). This work is supported by the Larry L. Hillblom Foundation, Nora infl ammatory and growth promoting agents in T1D. Eccles Treadwell Foundation, JDRF and NIH/NIDDK. Supported by: JDRF Advanced Postdoctoral Fellowship (10-2010-561) & 1959-P Pancreatic Islet and Progenitor Cell Surface Markers with Cell & 1957-P Sorting Potential Combination Effects of Sitagliptin and Losartan on the Neogenesis JACOB HALD, THOMAS GALBO, CLAUDE RESCAN, LOUISE RADZIKOWSKI, and Differentiation of Pancreatic Progenitor Cells ANNE E. SPRINKEL, HARRY HEIMBERG, JONAS AHNFELT-RØNNE, JAN JENSEN, JUAN LIANG, PO SING LEUNG, Hong Kong, China RAPHAEL SCHARFMANN, GERARD GRADWOHL, CHRISTIAN STOECKERT, JR., Enhancing the availability of insulin-producing cells from pancreatic JAN N. JENSEN, OLE D. MADSEN, Gentofte, Denmark, Måløv, Denmark, Brussels, progenitor cells (PPCs) is a strategy to curing diabetes. GLP-1 mimetics and Belgium, Cleveland, OH, Paris, France, Illkirch, France, Philadelphia, PA DDP-4 inhibitors stimulate insulin secretion, enhancing β-cell proliferation The aim of the study was to identify surface bio-markers and corresponding and differentiation. Meanwhile, the angiotensin II type 1 receptor (AT1 antibody tools that can be used for the imaging and immuno-isolation of receptor) has a negative role in stem cell development and its blockade may the pancreatic beta-cell and its progenitor stages in mouse and man. This be benefi cial to β-cell differentiation and regeneration. We hypothesized may prove essential to obtain therapeutic grade beta-cells via stem cell that the combination of the AT1 receptor blocker, losartan with the DPP- differentiation. 4 inhibitor, sitagliptin may have synergistic or additive effects on the Using bioinformatics-driven data mining we generated a target gene differentiation and regeneration of PPCs. We tested this hypothesis by (1) in list encoding putative plasma membrane proteins specifi cally expressed vivo insulin defi cient mice induced by STZ for chronic treatment (70 days) and at distinct stages of the developing pancreas and islet beta cells. In-situ acute treatment (within 14 days), and (2) in vitro PPCs derived from human hybridization and immunohistochemistry were used to further prioritize and fetal pancreas. In vivo, after chronic treatment, pancreatic insulin mRNA identify candidates. was increased to 1.2-, 3.0-, 43.0- folds in sitagliptin(S)-, losartan(L)- and During pancreas development Seizure related 6 homolog like 2 (Sez6l2), combo(C)-treated mice, respectively, compared with vehicle(V)-treated. Low density lipoprotein receptor-related protein 11 (Lrp11), Dispatched The β-cell-to-total islet area ratio was reduced from 0.71±0.02 in normal homolog 2 (Disp2), and Solute carrier family 30 (zinc transporter), member 8 mice to 0.07±0.14 in hyperglycemic mice treated with vehicle, while S-, L- (Slc30a8/ZnT-8), are found on the surface of early islet cells and are absent and C-treatment enhanced the ratio to 0.25±0.03, 0.21±0.04, 0.35±0.01, in Neurogenin 3 (Neurog3) mutant mice while Discoidin domain receptor respectively. OGTT showed the glucose area under curve was decreased family, member 1 (Ddr1) and Delta/notch-like EGF-related receptor (Dner) are by 6.8%, 6.2% and 34% in S-, L- and C-treated mice, respectively, compared expressed in early pancreatic progenitors. The expression pattern of Ddr1 with the V-treated. After acute treatment, expression of NGN3 protein was overlaps with the early Pancreatic and duodenal homeobox 1 (Pdx1)/NK6 detected in ductal cells, which was increased in all groups, especially in homeobox 1 (Nkx6.1)-positive multipotent progenitor cells from embryonic combo group. Meanwhile, islet-like cell clusters (ICCs) were detected in day 11 while DNER expression in part overlaps with Neurog3+ cells. In ductal cells, together with a signifi cant rise in the combo group. In vitro, the adult pancreas Sez6l2, Lrp11, Disp2 and Slc30a8/ZnT-8 but also FXYD PPCs were treated with sitagliptin,losartan or the combo during PPCs domain-containing ion transport regulator 2 (Fxyd2), Tetraspanin 7 (Tspan7), differentiation into ICCs: expression of PDX-1 and NKX6.1 were enhanced and Transmembrane protein 27 (Tmem27), retain an islet specifi c expression concomitantly with an increase of PPARγ, a key transcriptional regulator while Ddr1 is undetectable. In contrast Dner is expressed at low levels in of PDX-1, in the combo group. These in vitro and in vivo data indicate that peripheral mouse and human islet cells. Re-expression of Ddr1 and up- combination of sitagliptin and losartan may have an additive effect on the regulation of Dner is observed in duct ligated pancreas. Antibodies to Dner differentiation of PPCs into mature β-cells via mediation, at least in part, of and Disp2 have been successfully used in cell purifi cation application. PDX-1 and PPARγ expression. Extracellular epitopes of Sez6l2, Lrp11, Disp2, Ddr1 and Dner have Supported by: Merck been identifi ed as useful tags by applying specifi c antibodies to visualize pancreatic cell types at specifi c stages of development. Furthermore, & 1958-P antibodies recognizing Disp2 and Dner are suitable for FACS mediated cell Combination Therapy with Anti-Infl ammatory, Growth-Promoting, purifi cation. and Pro-Differentiation Agents Reduces Insulitis and Increases β Cell Mass in Diabetic NOD Mice & 1960-P SUSANNE M. CABRERA, SARAH A. TERSEY, JERRY L. NADLER, RAGHAVENDRA Glucose-Responsiveness of Mature Beta Cells Is Dependent on G. MIRMIRA, Indianapolis, IN, Norfolk, VA MafA Function In type 1 diabetes (T1D), there is insuffi cient islet β cell mass to maintain AGNES JERMENDY, ILHAM EL KHATTABI, AMEDEO VETERE, JENNIFER LOCK, glucose homeostasis, but evidence indicates that β cell regeneration/ YONG MONG BEE, CRISTINA AGUAYO-MAZZUCATO, ARUN SHARMA, SUSAN replication continues to occur despite ongoing autoimmunity. Thus, BONNER-WEIR, Boston, MA approaches that simultaneously mitigate β cell death while enhancing β MafA, a glucose-responsive transcription factor expressed in beta cells has cell differentiation and growth may allow for restoration of functional β cell been implicated in functional beta cell maturation and regulation of genes mass in T1D. In this study, we hypothesized that combinations of an anti- involved in insulin synthesis and secretion. We have previously demonstrated infl ammatory agent (lisofylline (LSF)), a growth-promoting agent (MK-626, that the knock down of MafA function in adult rat islets by adenoviral a DPP-IV inhibitor), and a pro-differentiation agent (vorinostat, a histone expression of dominant-negative MafA (Ad-CMV-DNMafA-IRES-GFP) resulted deacetylase inhibitor) would reduce insulitis and enhance β cell mass in in a dysfunctional beta cell phenotype, with decreased expression of key beta- NOD mice. Diabetic NOD mice were treated for 4 weeks with combinations cell metabolic genes and transcription factors including Pdx1 and NeuroD1, of LSF, MK-626, and vorinostat and compared to untreated controls. After 4 and blunted glucose stimulated insulin secretion (GSIS). weeks, insulin and TGF-β1 levels were determined by ELISA. The pancreata Here we explored the possibility that the signifi cant down-regulation were stained for insulin and BrdU, allowing for determination of β-cell mass, of Pdx1 and NeuroD1 contributed to the loss of GSIS in the dysfunctional insulitis scoring, and β cell proliferation. Regulatory T cell populations in rat islets after Ad-DNMafA infection. Adenovirus-mediated reconstitution the spleen and pancreatic lymph nodes were determined by fl ow cytometry. of Pdx1 and NeuroD1 expression was carried out in freshly isolated, In each treatment group, there were responders and non-responders handpicked adult rat islets that were totally dispersed to optimize infection as refl ected by fi nal glucose and insulin levels. Mice receiving vorinostat and then passively reaggregated. Ad-GFP served as control. With nearly alone, MK-626 alone, and the triple combination of vorinostat, MK-626, and 90% infection at 72 hours, co-infection with AdNeuroD1 or AdPdx1 could LSF trended towards greater β-cell mass than diabetic controls. Insulitis not rescue glucose-stimulated insulin secretion in DNMafA-expressing

POSTERS was quantifi ed by degree of mononuclear cell infi ltration of the islets; islets. Of note, insulin content was similar in untreated and adenovirally- Islet Biology/

Insulin Secretion experimental groups trended towards less pervasive invasion compared infected islets, irrespective of the over-expressed genes. Then, to test the to diabetic controls. Flow cytometry suggested increased populations of role of these transcription factors in dysfunctional beta cells, we studied CD4+CD25+Foxp3+ regulatory T cells in the spleen and pancreatic lymph isolated islets from GK rats with impaired glucose tolerance (blood glucose:

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A528 ISLET BIOLOGY—BETACATEGORY CELL—DEVELOPMENT

GK 136±5 vs Wistar-Kyoto controls 111±3 mg/dl, p=0.003). In GK islets, transcription factors (Pdx-1, Sox9, Ngn3 and Nkx6.1), which are associated insulin and MafA mRNA were decreased (41%, p<0.001 and 57%, p=0.02; with β-cell growth and function, was signifi cantly increased (p<0.05-0.01). respectively) but NeuroD1 and Pdx1 mRNA levels unchanged compared with On a high-fat diet β-cell specifi c c-Kit transgenic mice exhibited signifi cantly control Wistar-Kyoto islets. When MafA over expression was titrated to improved glucose tolerance, insulin secretion and sensitive (p<0.05). Taken obtain near physiological MafA levels in GK islets, their insulin secretion together, these data demonstrate that c-Kit receptor tyrosine kinase is was improved compared to control and Ad-GFP-infected GK islets. Thus, involved in the regulation of glucose metabolism and contributes to the glucose-responsiveness of the mature beta cells is dependent on adequate maintenance of β-cell function. MafA function and not that of Pdx1 or NeuroD1. Supported by: Canadian Institutes of Health Research

1961-P 1963-P β Cell Proliferation and Expansion of β Cell Mass Were Independent Competency of Sox9-Expressing Cells in the Mouse Pancreas of Glucokinase in 60% Partial Pancreatectomized Mice BRUCE A. ADAMS, YASUHIRO KOSAKA, FRANCIS C. LYNN, ZHONGMEI LI, YU TOGASHI, JUN SHIRAKAWA, AKINOBU NAKAMURA, YASUO TERAUCHI, MICHAEL S. GERMAN, San Francisco, CA, Vancouver, BC, Canada Yokohama, Japan The progenitor cells of the developing pancreas express the transcription We previously demonstrated that, in β-cell-specifi c glucokinase haplo- factor Sox9, and it persists in the duct and centroacinar cells of the adult insuffi cient (Gck+/-) diabetic mice, a model of impaired insulin secretion in pancreas. Sox9 comprises part of a network of regulators that maintain response to glucose, IRS-2 in β cells was not up-regulated in response to progenitor gene expression and support differentiation into other mature cell high-fat diet-induced insulin resistance, which resulted in impairment of β cell types. A subset of these Sox9+ progenitor cells activates the Neurogenin3 adaptive proliferation. By contrast, activation of IRS-2/Akt/PDX-1-mediated (Ngn3) gene, which drives their differentiation into endocrine cells. insulin signaling pathways were reportedly implicated in adapted expansion Although it is known that the competence of these Sox9+ progenitor cells to of β cells in partial pancreatectomized mice. In this study, we investigated generate different endocrine cell types changes during fetal development, the role of glucokinase in the increment of β cell mass following partial the mechanisms that control this competence remain uncertain. Here we pancreatectomy by using Gck+/- mice. Eight-week-old male wild-type (WT) set out to characterize the competency windows of Sox9+ cells using two or Gck+/- mice were subjected to 60% partial pancreatectomy (Px) or sham transgenic models: 1) the Sox9-EGFP line that allows medium-length labeling operation (SHAM). We evaluated β cell proliferation at 6 days after surgery of cells based on the relatively long half-life of GFP, and 2) an inducible Sox9 (Day 6), glucose tolerance at Day 14, and β cell mass at Day 18. Casual blood line (Sox9-rtTA:tetO-H2BGFP) that allows doxycycline inducible labeling of glucose levels of Px Gck+/- mice demonstrated overt hyperglycemia compared Sox9-expressing cells. to SHAM Gck+/- mice, whereas both Px and SHAM WT mice maintained In the two reporter lines, we compared the reporter protein expression euglycemia. However, glucose tolerance and serum insulin levels during oral with native Sox9 expression by IHC and mRNA quantifi cation from cells glucose tolerance test showed no signifi cant differences between Px Gck+/- purifi ed by fl uorescent-activated cell sorting from developing and adult mice and SHAM Gck+/- mice at Day 14. Both Px WT mice and Px Gck+/- mice pancreas. During the peak of endocrine cell genesis at embryonic day 15.5, showed signifi cant increases in BrdU incorporation in β cells, compared to fl uorescent cells from the Sox9-EGFP line could be separated into two general SHAM animals at Day 6 (fi gure). Furthermore, Px signifi cantly expanded β cell populations based on their level of GFP expression: cells expressing high mass in both genotypes at Day 18 (fi gure). These results suggested that β levels of GFP and enriched for mRNA encoding endocrine lineage markers; cell adaptation following Px was independent of glucokinase. We have also and cells expressing lower levels of GFP and enriched for exocrine markers. noted that β cell mass tended to increase in IRS-2-defi cient mice receiving With the Sox9-rtTA:tetO-H2BGFP mice we could further separate the Sox9 Px. In conclusion, pancreatectomized mice give rise to the compensatory β expressing cells from differentiating cells that had recently shut off Sox9 cell proliferation mediated by the different mechanisms from that of β cell expression. These models then allow us to compare the gene expression hyperplasia in response to high-fat diet-induced insulin resistance. characteristics and transcriptomes of pancreatic progenitor cells during specifi c competency windows. Supported by: L.L. Hillblom Foundation, Nora Eccles Treadwell Foundation, NIH/ NIDDK, JDRF 3-2005-902 & CDA to B.A.A., JDRF 3-2004-276 & 10-2007-86 to F.C.L. ADA-Funded Research

1964-P IBCAP, Intestine-Specifi c Secretory Protein with Incretin-Like Activity TOMOTAKA YOKOO, HIROAKI SUZUKI, HITOSHI SHIMANO, KAZUHISA WATANA- BE, MASANOBU KAWAKAMI, NOBUHIRO YAMADA, YASUSHI OKAZAKI, HIDEO TOYOSHIMA, Hidaka, Japan, Tsukuba, Japan, Saitama, Japan Nowadays, therapeutic strategies for diabetic patients are facing big changes due to the success of incretin related drugs. Obviously, discovery 1962-P of additional factors bearing fundamental roles and/or effects in the β-Cell Specifi c C-Kit Receptor Over-Expression Transgenic Mice controls of energy metabolisms may uncover another stage. As one of these Show Improved β-Cell Function and Resistance to High-Fat Diet- candidates, we have been working with IBCAP (intestine derived beta-cell Induced Diabetes augmenting promoter), a novel intestine-specifi c secretory protein identifi ed JINMING LI, ZHI CHAO FENG, BRYEN A. TURCO, SUI-POK YEE, RENNIAN WANG, by ourselves using the Oligo-cap Signal Sequence Trap (Oligo-cap SST) London, ON, Canada, Farmington, CT strategy developed in our laboratory. We demonstrated that IBCAP have It has been shown that c-Kit, a receptor tyrosine kinase, and its ligand, promotive effect on insulin secretion, and adenovirus-mediated expression stem cell factor (SCF), are important in a number of cell types to control of IBCAP in STZ treated type 1 diabetes model mice decreased the blood a variety of cellular processes including haematopoiesis, gametogenesis glucose concentration and increased the area of pancreatic β-cells. Also, and pancreatic β-cell survival and maturation. We previously reported that IBCAP transgenic (Tg) mice was shown to have dramatically increased c-KitWv/+ male mice display early onset of diabetes; however, the underlying pancreatic islets, while blood glucose concentration and OGTT test were mechanism by which c-Kit regulates β-cell function remains unknown. To normal compared with littermate mice. These results demonstrated further delineate this mechanism, we have generated RIP-c-Kit(h) transgenic that IBCAP is a novel intestine-specifi c secretory protein with incretin- mice in C57BL/6J background to specifi cally target c-kit overexpression in like activity, which can activate glucose dependent insulin secretion and β-cell and characterize glucose metabolism and β-cell function. The aim of also can increase pancreatic β-cells. Therefore, IBCAP should be another the present study is to examine whether a β-cell specifi c c-Kit overexpression potential therapeutic target for diabetes and pancreas β-cell regeneration. would have physiological and functional implications in β-cell function in Now, we have generated IBCAP-KO mice and will be presenting the results both normal and high-fat diet-induced diabetic status. On a normal diet β-cell at this meeting. POSTERS

specifi c c-Kit transgenic mice display mild improvement of glucose tolerance Supported by: Ministry of Education, Culture, Sports, Science and Technology Islet Biology/ and glucose-stimulated insulin secretion at 8 weeks of age as compared to of Japan Insulin Secretion wildtype littermates (p<0.05). They also display an increase of β-cell mass along with an increase of β-cell proliferation (p<0.05). The expression of

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A529 ISLET BIOLOGY—BETACATEGORY CELL—POSTNATAL GROWTH

1965-P The microarray analysis and qRT-PCR revealed that normal hepatic markers INGAP Induces Duct Cell Proliferation In Vitro and beta Cell Forma- (C/EBP-β, G6PC, AAT and GLUI) were downregulated from week 1 and the tion in Normal Non Diabetic Mice immature hepatic marker, AFP, was upregulated, suggesting a reversion to an RAHUL KAPUR, SIF G. RØNN, JOHN P. MOGENSEN, ALLAN E. KARLSEN, R. SCOTT immature hepatic phenotype. The gene profi ling also revealed upregulation HELLER, Gentofte, Denmark, Måløv, Denmark of other major pancreatic transcription factors, including Foxa2, Lmx1.1, HNF1 The Reg3 family of peptides (INGAP and HIP) has been hypothesized to homeobox B and Pbxip1, as well as synaptophysin, chromogranin A and stimulate beta cell neogenesis in the pancreas. These peptides have been secretogranin II. The data suggests that the switching off of liver markers shown to cause an increase in beta cell mass in multiple animal models, causes a shift towards an immature hepatic phenotype allowing for partial reverse STZ induced diabetes in mice, reduce HbA1c levels and improve transdifferentiation of liver tissue and the reversal of T1D. arginine stimulated insulin secretion in human clinical studies. In this study, 1967-P we have tested various peptides of the Reg3 family. In vitro we examined cell Synergizing Genomic Analysis with Biological Knowledge To proliferation in the HPDE cell line (pancreatic ductal cell line) by measuring Identify Novel Genes in Pancreatic Development Edu incorporation and changes in cell impedance using the Xcelligence SUPARNA A. SARKAR, CATHERINE E. LEE, HANNAH TIPNEY, ANIS KARIMPOUR- technology. At 72 hrs post treatment, INGAP increased proliferation by 24%, FARD, JASON D. DINELLA, KIRSTINE JUHL, LAWRENCE HUNTER, JAN KRAUS, Tyr-INGAP by 27%, HIP by 27% (n=6) as compared to a non peptide control JOHN C. HUTTON, Aurora, CO, Whitehouse Station, NJ (all p values < 0.05) as measured by Edu incorporation. The Xcelligence The mechanism of differentiation of pancreatic beta cells is a fundamental platform supported the conclusions of the Edu data. In vivo, we assessed enigma during development. An expression dataset for mouse pancreatic their ability to induce neogenesis and increases in beta cell numbers in non development was created on Affymetrix MOE 430_2 chips and analyzed diabetic, female 9 week old NMRI mice after injection of the peptide for 5 from E12.5-E18.5. A novel computational approach for analyzing genome- days. We see a greater than 2 fold increase in small insulin positive cluster scale data in the light of existing knowledge, built on three broad classes volume (clusters of 4 or less insulin positive cells in extra-islet tissue) post of algorithms: reading, reasoning and reporting was undertaken. Integrated 5 days of treatment in INGAP treated mice as compared to our saline and information about genes, gene products and their interactions from dozens scrambled INGAP peptide treated control animals (p<0.05), however we do of databases and via text mining from the literature was combined with not see this in HIP treated mice (n=7 for each condition). the analysis of complex,high-throughput microarray data. We identifi ed a sub network of 33 genes associated with regulation of transcription during pancreatic development. We validated the expression of Cystathionine Beta Synthase (Cbs), a key enzyme in the trans-sulfuration pathway that catalyzes the irreversible, serine dependant conversion of homocysteine to cystathionine. In the general population, an elevated level of homocysteine is recognized as an independent risk factor for developing cardiovascular diseases and metabolic syndrome. Cbs expression was documented by Q-RTPCR in mouse embryos (E12.5-E17.5) and adult CD1 islets. Cbs expression was decreased in ngn3 KO mice as compared to WT at E12.5 and Q-RTPCR performed on cDNA extracted from cell lines showed that Cbs was minimally expressed or excluded from alpha TC and ductal cell lines. In situ hybridization demonstrated that Cbs was expressed in the early pancreatic epithelium at E12.5. Immunohistochemical localization of Cbs was also noted in the epithelial duct like progenitor cells (E12.5). The cells staining with higher intensity were localized to the tips of the budding/branching epithelium and were later restricted to the developing acinar cells. CBS was also cloned from human pancreas and 4 isoforms were documented. To our Real time Q-PCR on whole pancreatic samples from mice (n=5 for each knowledge the expression of Cbs has not been previously documented in condition) post 5 days of treatment, demonstrated a tendency towards developing mouse pancreas. We conclude that the analysis of microarray increased levels of beta cell markers such as Ngn3, Nkx6.1, Nkx2.2 and data in combination with background knowledge can expedite the discovery Insulin in INGAP treated animals. Taken together these results indicate of novel genes that regulate early pancreatic differentiation. that the INGAP pentadecapeptide causes formation of small beta cell Supported by: NIH K01DK080193, JDRF 1-2008-1021 clusters in extra-islet pancreatic tissue via upregulation of factors which are responsible for beta cell development in the embryo. ISLET BIOLOGY—BETA CELL—POSTNATAL GROWTH

1966-P [See also: Presidents Poster 472-PP, page A131.] Kinetics of Pancreatic Transdifferentiation in the Livers of Non Obese Diabetic (NOD) Mice Following Lentiviral Delivery of Furin- Cleavable Insulin Guided Audio Tour: Postnatal Beta Cell Growth (Posters 1968-P to 1974-P), ANN M. SIMPSON, MICHELLE R. BYRNE, BINHAI REN, BRONWYN A. O’BRIEN, see page 11. Sydney, Australia Type I diabetes (TID) results from the autoimmune destruction of the pancreatic beta (β) cells. Insulin therapy cannot achieve precise physiologic & 1968-P control of blood glucose concentrations and chronic complications develop. Low Levels of NO Mediate Growth and Proliferation of Human Islets: Gene therapy is one strategy being examined to cure TID. Using a novel Involvement of PKA/CREB, GSK-3/β-Catenin and mTOR Signaling surgical technique that isolates the liver from the circulation we introduced Pathways furin-cleavable human insulin (INS-FUR) to the livers of diabetic NOD mice. We HAYTHAM ALY, NIDHI ROHATGI, CONNIE A. MARSHALL, MICHAEL L. MCDANIEL, normalized blood glucose for 150 days (experimental endpoint), and noted some St. Louis, MO endocrine transdifferentiation of the liver, with storage of insulin in granules, Our previous studies demonstrated that regulation of the Wnt/GSK-3/β- and expression of some β-cell transcription factors (Pdx1, Neurod1, Neurog3, catenin and mTOR signaling pathways are necessary for increasing DNA Nkx2-2, Pax4). The aim of the current study was to investigate the kinetics of synthesis, cell cycle progression and proliferation of adult human β-cells, pancreatic transdifferentiation in diabetic NOD mice after the delivery of INS- whereas in rodent β-cells, nutrient activation of mTOR was suffi cient. FUR. Mice were sacrifi ced 1 and 2 weeks and 5 months post-transduction. We demonstrated that treatment of human islets with LiCl engaged Wnt The livers were excised and RNA extracted for (RT-PCR, quantitative RT-PCR signaling by inhibiting GSK-3 and increasing β-catenin nuclear translocation. [qRT-PCR]) and microarray gene expression studies. Initially, the expression of Recently, we discovered that the free radical nitric oxide (NO) and PKA the β-cell transcription factors, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4, Nkx6.1, signaling were also involved in addition to Wnt/GSK-3/β-catenin and mTOR Pax6, and mouse insulin 1 in the 1 and 2 week and 5 month samples were in these regenerative processes. G

POSTERS compared to expression levels in untreated pancreas and untreated liver. Treatment of human and rodent islets with aminoguanidine (AG) or L-N - Islet Biology/ monomethyl arginine (L-NMMA), inhibitors of nitric oxide synthases (NOS),

Insulin Secretion The results showed that all of the transcription factors were upregulated as compared to untreated liver. Interestingly, mouse insulin 1 was found to be signifi cantly blocked the positive effects of both LiCl and glucose on DNA upregulated in the 2 week and 5 months samples only. synthesis and reduced glucose-induced S phase accumulation. Treatment

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A530 ISLET BIOLOGY—BETACATEGORY CELL—POSTNATAL GROWTH of human and rodent islets with LiCl, dose-dependently increased gene mice. The mRNA for the following cyclins and E2F were also increased in expression of iNOS and nNOS. Furthermore, measurements of nitrite islets from insulin-resistant animals compared to controls: Cyclin B1 (fold formation and iNOS and nNOS protein levels in islets indicated that LiCl increase, ob/ob: 2.67+0.25, p<0.001; HFD: 5.06+1.57, p<0.05), Cyclin D1 (ob/ induced low levels of NO relative to cytokine-induced levels. ob: 2.01+0.26, p<0.05; HFD: 2.36+0.24, p<0.01), Cyclin E1 (ob/ob: 3.15+0.22, In addition, treatment of human and rodent islets with H89, a specifi c p<0.001; HFD: 1.89+0.36, P<0.05) and E2F1 (ob/ob: 2.60+0.26, p<0.01; HFD: inhibitor of cAMP-dependent protein kinase (PKA), signifi cantly inhibited 3.13+0.69, P<0.05). The expression of the CDK inhibitors, p19, p27, and p57, LiCl-induced DNA synthesis to basal levels, and treatment of human and the rate of apoptosis, as assessed by TUNEL assay and apoptosis-related islets with Exenatide moderately increased human islet DNA synthesis. In gene expression (Bad, Bid, Bcl2, and Bclx), were similar in insulin-resistant agreement with these data, forskolin increased the phosphorylation of GSK- and control mice. These results suggest that cell cycle regulators play a role 3 and CREB and led to an increase in mRNA expression of the CREB target in the islet response to insulin resistance and that the CDK1/cyclins complex genes, NR4A2 and IRS2. Moreover, forskolin increased iNOS and nNOS gene is a key signaling pathway in beta cell growth in insulin-resistant mice. and protein expression. These results suggested that PKA engages Wnt signaling by phosphorylation of GSK-3 and activates insulin/mTOR signaling & 1971-P through CREB and increased transcription of IRS2. The Role of Focal Adhesion Kinase (FAK) in Pancreatic β-Cells Overall, low physiological levels of NO and crosstalk between the PKA/ ERICA P. CAI, STEPHANIE A. SCHROER, MARINA CASIMIR, XIAO QING DAI, DIANA CREB, GSK-3/β-catenin and mTOR signaling pathways are required for CHOI, SHUNYAN LU, CATHERINE HAJMRLE, ALIYA F. SPIGELMAN, PATRICK E. human islet regenerative processes. MACDONALD, MINNA WOO, Toronto, ON, Canada, Edmonton, AB, Canada Supported by: NIH ADA-Funded Research Focal contacts serve as a junction for interactions between the extracellular matrix (ECM), integrins and the cell cytoskeleton. Focal adhesion kinase & 1969-P (FAK) acts as adaptor at this junction to coordinate cell motility and survival. Increased beta-Cell Replication and Islet Neogenesis in Subjects Previous studies have shown an important role of FAK in facilitating glucose with Insulinoma uptake and in enhancing insulin signalling in metabolic tissuessuch as the ROBERT A. RITZEL, JOHANNES SCHOTT, FRANK BERGMANN, PETER P. NAW- liver and muscle. Furthermore, ECM signalling has been shown to improve ROTH, Munich, Germany, Heidelberg, Germany survival of cultured β-cells through enhancing FAK activity. However, the Type 2 diabetes is characterized by insulin defi ciency, reduced beta-cell physiological role of FAK in pancreatic β-cells is unclear. To defi ne the in vivo mass due to apoptosis and insuffi cient beta-cell regeneration. It is largely role of FAK in β-cells, we took a genetic approach using rat insulin promoter unknown how insulin affects beta-cell turnover in vivo. Subjects with (RIP)-driven Cre-loxP recombination system to generate β-cell specifi c FAK inadequately elevated insulin secretion from endogenous sources (e.g. knockout mice (FAK-KO). These mice had normal body weight, but increased insulinoma) are a good model to address this question. Therefore we analyzed blood glucose levels and impaired glucose tolerance without changes in healthy pancreatic tissue either directly adjacent to (close) or 3-4 cm away insulin sensitivity compared to wildtype littermates. They also showed (distant) from a histologically and clinically confi rmed benign insulinoma (n= reduced β-cell mass that was associated with decreased proliferation and 10 cases) and compared it to pancreatic tissue from organ donors (control increased apoptosis, as assessed by Ki67 and cleaved caspase 3 expression, group, n=20 cases). Using morphometric analysis we determined beta-cell respectively. Consistent with this, basal Ca2+ levels were somewhat replication (Ki-67 staining), islet neogenesis (pancreatic duct-cells positive increased in isolated FAK-KO islets, although glucose stimulated Ca2+ for insulin), beta-cell apoptosis (TUNEL staining) and fractional pancreatic responses and β-cell Ca2+ channel activity was normal. β-cells from FAK- beta-cell content (immunohistochemistry for insulin). Beta-cell replication KO mice did however show impaired exocytotic responses at the single-cell in insulinoma cases is about 3-fold increased in comparison to control level, and this was consistent with a defect in glucose-stimulated insulin (p<0.01), particularly in tissue directly adjacent to an insulinoma (p<0.05 secretion as assessed by perfusion studies. Together, these data show that for close versus distant). There also is a 2-fold increase in the percentage FAK is an essential regulator of pancreatic β-cell viability, proliferation and of exocrine ducts with insulin positive cells (10.9±2.3 / 8.7±2.9 % versus function, likely through controlling cytoskeletal reorganization and vesicle 4.6±0.5 %; p<0.01 / p<0.05 for close / distant versus control), while there traffi cking/exocytosis in addition to insulin signal transduction pathways is no difference between close and distant locations (p=n.s.). Beta-cell within β-cells. apoptosis is unchanged in insulinoma cases compared to control (p=n.s.), Supported by: Canadian Institutes of Health Research while fractional beta-cell content is reduced (1.0±0.2 / 0.9±0.1 % versus 2.3±0.2 %; p<0.001 for close / distant versus control, respectively). Taken & 1972-P together there is evidence to suggest that secretory products of insulinoma Trefoil Factor 2 (TFF2) Induces Proliferation of Pancreatic β Cells tissue stimulate beta-cell replication and differentiation of progenitor KAZUKI ORIME, Yokohama, Japan cells into beta-cells. However, there apparently is an adaptive reduction Trefoil factor 2 (TFF2) is reportedly expressed in gastrointestinal mucous of fractional beta-cell content in the pancreas of insulinoma cases. Based neck cells and implicated in anti-apoptosis, migration, and the gastric on the present data further studies are justifi ed to test whether exogenous protection. TFF2 is also expressed in pancreatic β cells, however the role insulin therapy induces beta-cell regeneration in type 2 diabetes. of TFF2 in pancreatic β cells has not been elucidated. In this study, we Supported by: Deutsche Forschungsgemeinschaft (Ri 1055/3-1) investigated whether overexpression of TFF2 affected insulin secretion or proliferation in murine islets and INS1 (832/13) β cells. The endogenous & 1970-P TFF2 expression in islets was confi rmed with quantitative real-time PCR Increased Expression of Cell Cycle Genes in Islets from Insulin- and immunohistochemistry in mice. To investigate the effects of TFF2 in β Resistant Mice cell, we constructed recombinant adenovirus vectors containing either TFF2 CHUNHUA DAI, COURTNEY THOMPSON, ALVIN C. POWERS, Nashville, TN cDNA (AdTFF2) or LacZ cDNA (AdLacz). AdTFF2 increased expression levels Pancreatic islets adapt to insulin resistance by increasing insulin of TFF2 mRNA and proteins in a dose-dependent manner, peaking at 48 production and beta cell mass. Since beta cell mass is controlled by a hrs after infection in INS1 cells. Recombinant TFF2 proteins were localized balance of proliferation and apoptosis, we examined beta cell proliferation in cytoplasmic granular compartment in INS1 cells. Glucose stimulated and cell cycle gene expression in two insulin-resistant models – mice with insulin secretion (GSIS) showed no signifi cant differences between AdTFF2- leptin defi ciency (ob/ob) and mice on a high fat diet (HFD). Compared to and AdLacZ-transfected cells in both INS1 cells and islets. By contrast, control mice, both types of mice with insulin resistance had an increase overexpression of TFF2 in INS1 cells led to signifi cant increase in BrdU in average islet area [ob/ob vs wt/wt: 4wks, 25,872+2,074 vs 14,752+696; incorporation in a virus dose-dependent manner. Islets transfected with 8wks, 43,404+2,514 vs 20,311+1,108; 16wks, 58,236+3131 vs 22,015+1493 AdTFF2 also exhibited a 2.3 fold increase in BrdU incorporation, compared μm2, n=130 islets/genotype, p<0.001; HFD vs chow diet: 19 wks old (16wks to islets transfected with AdLacZ. TFF2 signifi cantly increased the mRNA on HFD), 24,943+1,946 vs 14,740+959 μm2; n=120 islets/genotype, p<0.001]. expression of cyclin A2, D1, D2, D3, E1, and PDX-1 in isolated islets, compared The ob/ob mice had increased beta cell replication rate as refl ected by to LacZ or non-transfected control. Furthermore, the phosphorylation of Akt Ki67 staining (ob/ob vs wt/wt: 1wk, 21.9+1.5 vs 15.2+1.6; 4wks, 12.2+0.6 vs and ERK1/2 was up-regulated in MIN6 cells stimulated with TFF2 peptide 5.5+0.3; 8wks, 4.6+0.7 vs 2.4+0.4; 16wks, 2.7+0.1 vs 1.8+0.5% Ki67+ beta (500 nM). In conclusion, we identifi ed an involvement of TFF2 in β cell POSTERS

cells, n>3000, p<0.05). By quantitative RT-PCR for cell cycle genes, the proliferation. TFF2 may activate Akt or MAPK signaling pathway, raising the Islet Biology/ mRNA level of CDK1 was 2.5- in ob/ob (4wks, n=5 mice) and 3.4-fold higher possibility for TFF2 as a novel target for β cell proliferation. Insulin Secretion in HFD islets (19 wks, n=3 mice) compared to controls (p<0.01) but the mRNA for CDK2, 4, and 6 were similar in ob/ob mice, mice on HFD, and control

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A531 ISLET BIOLOGY—BETACATEGORY CELL—POSTNATAL GROWTH

1975-P & 1973-P Beta Cell Function and Proliferation in Response to Small Molecule Activating AMPK and Downregulating mTOR with a PPAR-γ Sparing Glucokinase Activator under Various Conditions TZD Decreases Insulin Resistance & Mediates Human β-Cell Rest & AKINOBU NAKAMURA, YU TOGASHI, KAZUKI ORIME, KOICHIRO SATO, JUN Restoration of Insulin Content SHIRA KAWA, YASUO TERAUCHI, Yokohama, Japan NIDHI ROHATGI, HAYTHAM ALY, CONNIE A. MARSHALL, ROLF KLETZIEN, JERRY We previously demonstrated that small molecule glucokinase (GK) R. COLCA, MICHAEL L. MCDANIEL, St. Louis, MO, Kalamazoo, MI activator (GKA) was able to improve glucose metabolism and had an effect The objective of this study is to develop treatment strategies that on beta cell proliferation as well as beta cell function in mice. However, in increase adult β-cell proliferation in vitro, while maintaining insulin content human studies, one of the GKA compounds was reported to be the lack of and function. We have shown previously that activation of the Wnt/GSK- durability although good glucose lowering effi cacy was observed. A better 3/β-catenin pathway by pharmacologic inhibition of GSK-3 with nutrient understanding of the impact of GKA on metabolic pathways is necessary activation of mTOR was required to increase human β-cell proliferation to determine whether GK activation is a viable treatment target in human. in vitro. Importantly, human islets also displayed insulin resistance, due We therefore investigated the changes in GKA-stimulated gene expression to chronic activation of mTOR/S6K1 signaling that resulted in a negative regarding beta cell function, proliferation and an involvement of oxidative feedback of the insulin-signaling pathway and a loss of insulin content. In stress in mouse islets with various genetic backgrounds. GKA, but not this study, we show that a novel PPAR-γ sparing thiazolidinedione, MSDC- gliclazide, was able to up-regulate insulin receptor substrate-2 (Irs2) mRNA 0160, improves insulin sensitivity by reducing the negative feedback and expression in isolated islets of wild-type mice. GKA signifi cantly increased restores insulin content in human islets in vitro. Cyclin D2, Pdx1, Glut2, GK, insulin-1 and insulin-2 mRNA expressions. Thus, treatment of human islets with MSDC-0160 activated AMPK and Meanwhile, oxidative stress signifi cantly inhibited up-regulation of Irs2 resulted in a partial reduction in S6 phosphorylation, a downstream target and Pdx1 induced by GKA, although these expressions were restored by of mTOR/S6K1. This reduction in S6K1 decreased insulin resistance and pre-administration of α-tocopherol plus ascorbate. Expressions of these allowed IGF-1 to phosphorylate Akt and GSK-3. MSDC-0160 treatment also genes were not increased by GKA in isolated islets of db/db mice. In Irs2- signifi cantly increased expression of the insulin and the anti-apoptotic bcl-2 knockout (Irs2-/-) mice, glucose tolerance was improved shortly after the GKA genes, and a reduction of the pro-apoptotic bax gene. In addition, MSDC- treatment. GKA increased glucose-stimulated insulin secretion from islets, 0160 signifi cantly decreased the levels of cleaved caspase-3 due to exposure but failed to increase beta cell mass in Irs2-/- mice in vivo. Although 3 days’ to elevated glucose. administration of GKA increased the BrdU incorporation ratio in certain wild- Next, we investigated the effect of MSDC-0160 on maintaining insulin type mice, there was no such increase in the value in Irs2-/- mice treated with content and secretion. Human islets treated for 4 days under proliferative GKA. Pdx1 expression was signifi cantly increased by GKA in isolated islets conditions (GSK-3 inhibitor- LiCl, IGF-1 and 8 mmol/l glucose) displayed a of Irs2-/- mice. These results suggest that GK activation by GKA increases signifi cant reduction of insulin content. Treatment with MSDC-0160 alone Irs2 expression, thereby leading to beta cell proliferation via cell cycle dose-dependently prevented the loss of insulin content. However, co- signaling, and that GKA can stimulate beta cell function independently of incubation of LiCl and MSDC-0160 did not increase DNA synthesis and Irs2. Oxidative stress prohibited the effects of GKA on the changes in gene had only modest effects on restoring insulin content. Due to the inability expression regarding beta cell function and proliferation. of human islets to simultaneously promote proliferative signaling and maintain insulin stores, we developed a strategy of alternating a pro-growth stimulatory phase with a resting phase. This strategy induces human β-cell 1976-P Deciphering Functional Maturation Codes for Pancreatic Islet Cells proliferation followed by treatment with MSDC-0160 to allow restoration WITHDRAWN of insulin content. CHONG WEE LIEW, JOSHUA W.K. HO, JOEL CHICK, JENNIFER HOLLISTER- Supported by: NIH ADA-Funded Research LOCK, JOAN QC XI, PETER J. PARK, STEVEN GYGI, RICHARD MAAS, ROHIT N. KULKARNI, Boston, MA A major challenge for the successful treatment of both type 1 and type & 1974-P 2 diabetes is to generate a renewable source of insulin that resembles the Maternal High Fat Diet during Gestation and Lactation Affects mature pancreatic islet in maintaining tight glycemic control. Recent reports Glucose Metabolism and Pancreatic β Cell Function in Mature indicate that by use of protocols that mimic normal pancreatic development Offspring in Mice or which employ small molecules, insulin-producing cells can be derived via HISASHI YOKOMIZO, TOYOSHI INOGUCHI, NORIYUKI SONODA, MASAKAZU the directed differentiation of human embryonic stem (ES) cells. However, FUJII, YASUTAKA MAEDA, EIICHI HIRATA, TOMOAKI INOUE, KUNIHISA KOBA- these approaches can be relatively ineffi cient, and in vitro differentiated YASHI, RYOICHI TAKAYANAGI, Fukuoka, Japan insulin-producing cells are not optimally glucose-responsive - a major It is well known that intrauterine environment has a signifi cant infl uence limitation for cell-replacement therapy. Neonatal mouse islets are known on the health of postnatal offspring. In fact, undernutrition in dams during to respond poorly to glucose until after a defi ned maturation process, which pregnancy produces mature offspring who are at a high risk of developing generally occurs a few weeks postpartum. To gain further insight into this obesity and diabetes epidemiologically. Here we examined whether maternal maturation process, mouse islets were isolated from pups at postpartum high fat diet may have an effect on glucose metabolism and islet function (P) day 2, P4, P7, P14, P21, P28 or P35 and compared to adult mature islets in mature offspring. Female and male C57/BL6J mice were mating and fed from 8 to 12-week old mice. For comparison, in vitro differentiated pancreatic either a control diet (CD) or high-fat diet (HFD) during gestation and lactation. homeodomain protein 1 (Pdx1) positive and insulin-positive cells obtained After weaning, all offspring were fed CD from 3 to 6 weeks age, and then from the directed differentiation of mouse ES cells were isolated using were fed CD or HFD from 6 to 20 weeks age, resulting in four groups: C-C, FACS. RNAs extracted from all samples were hybridized to Illumina Mouse C-H, H-C, H-H combination with maternal and offspring diet. Body weight BeadChips-WG6. Samples at each time point in the in vivo study were also of male and female offspring exposed to a maternal HFD was greater from subjected to proteomic analyses using label-free quantitative tandem mass 4 to 6 weeks than in offspring exposed to a maternal CD, and from 6 to spectrometry. Integrative bioinformatic analyses against revealed global 20 weeks there was a signifi cant difference only between H-H and C-H in changes in gene and protein expression within the fi rst week postpartum, female. Intraperitoneal glucose tolerance test at 14 and 20 weeks showed followed temporally by more subtle but consistent changes. We identifi ed that the blood glucose (BG) was higher in H-C and H-H than in C-C and C-H novel expression dynamics for various transcription factors and signaling respectively in male, and BG was higher in H-H than in C-H in female. Insulin pathways that are linked to functional maturation in postpartum islets. secretion was higher in H-C and H-H than in C-C and C-H at 14 weeks in both Consistent with in vitro data, cell cycle proteins were highly expressed at P7, male and female. At 20 weeks, insulin secretion was higher in H-C and H-H in while genes coding for proteins in the endoplasmic reticulum, Golgi apparatus, female, while it was lower in H-C and H-H in male. HOMA-R was increased mitochondria, or involved in secretory granule storage and transport were in H-C and H-H than in C-C and C-H in both male and female. At 20 weeks, differentially up-regulated from P2 to P28. Our fi ndings should enable further insulin content and mRNA levels of PDX-1 in isolated islets were increased rational design of an in vitro differentiation and maturation protocol for the in H-C and H-H in female, while they were decreased in H-C and H-H in male. effi cient generation of functional pancreatic islet cells. To investigate the mechanism underlying a gender difference in pancreatic β cell function, oxidative stress in islets was evaluated by immunostaining POSTERS Islet Biology/ of 8-hydroxy-deoxyguanosine (8-OHdG). Staining intensities of 8-OHdG was Insulin Secretion increased in H-C and H-H only in male. In conclusion, maternal HFD induced glucose intolerance in mature offspring, via induction of insulin resistance and deterioration of β cell function with a gender difference.

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A532 ISLET BIOLOGY—BETACATEGORY CELL—POSTNATAL GROWTH

1977-P 1979-P Identifi cation of Regulatory Elements and Chromatin Domains in the Transcriptional Regulation of Mig-6 by PPARγ in Beta Cells MIN6 Insulinoma Cell Line YI-CHUN CHEN, DHANANJAY GUPTA, E SCOTT COLVIN, PATRICK T. FUEGER, MICHAEL L. STITZEL, PRAVEEN SETHUPATHY, PETER S. CHINES, DANIEL S. Indianapolis, IN PEARSON, YOICHIRO SHIBATA, NISC COMPARATIVE SEQUENCING PROGRAM, Epidermal growth factor receptor (EGFR) signaling plays crucial roles GREGORY E. CRAWFORD, FRANCIS S. COLLINS, Bethesda, MD, Durham, NC in pancreatic beta cell transdifferentiation and proliferation. Mitogen- Characterizing the epigenome of the beta cell is an important goal for inducible gene-6 (Mig-6; also known as Gene 33, Errfi 1, and RALT), a negative understanding gene regulation in insulin-producing cells. We have recently feedback inhibitor of EGFR, has been shown to regulate EGFR-mediated cell completed genome-wide epigenomic analysis of human pancreatic islets proliferation in human bronchiolar epithelial cells and mouse liver progenitor and are now extending this work to other cell types. Here, we report the cells. Likewise, our previous work suggests Mig-6 overexpression inhibits generation of genome-wide chromatin state maps in the commonly utilized proliferation of primary rat islets. Peroxisome proliferator-activated receptor mouse insulinoma cell line MIN6 to identify transcriptional regulatory gamma (PPARγ) is an important regulator in diverse biological processes elements and regulatory chromatin domains. We have conducted DNase- including beta cell differentiation, lipid metabolism, glucose homeostasis, seq and ChIP-seq analysis of histone H3 modifi cations in these cells at low and, like Mig-6, inhibits beta cell proliferation. The transcriptional regulation passage (passage 20) under standard culture conditions (25mM glucose, 100 of Mig-6 is likely orchestrated by multiple transcription factors. The Mig- uM 2-mercaptoethanol). Using this approach, we identifi ed 115,701 putative 6 promoter contains a potential PPARγ response element (PPRE). Thus, we regulatory and 19,612 putative promoter elements. Approximately 10% of hypothesized that PPARγ could directly regulate Mig-6 gene expression at promoters were enriched for both histone H3K4me3 and H3K27me3 and the transcriptional level. We fi rst observed that Mig-6 mRNA level was therefore appear to be “poised” for active transcription. Combined analysis upregulated by the PPARγ agonist troglitazone (Trog) in 832/13 cells, a rat of active histone modifi cations, CTCF binding, and repressive histone insulinoma-derived beta cell line. Moreover, the proliferation of 832/13 modifi cations indicates that several hundred CTCF sites function as barrier cells was decreased by 40% after 72-h Trog treatment. To study the direct elements in MIN6. transcriptional regulation of Mig-6 by PPARγ, we performed electrophoretic Comparing these chromatin maps with those we have previously generated mobility shift assays. Nuclear extracts from 832/13 cells were incubated with in human islets identifi es important similarities and differences. Both MIN6 a fl uorescently-labeled, double-stranded PPRE probe, and a specifi c band was and human islets utilize the same β cell specifi c promoter at multiple observed in a non-denatured SDS-PAGE gel. Importantly, whereas unlabeled genes including Gck and Hnf4a. In contrast, several actively transcribed probe was able to compete against the labeled probe in a concentration- euchromatic regions in human islets reside in repressive chromatin regions dependent manner, a mutated, unlabeled probe failed to do so. We further in MIN6, including the type 2 diabetes-associated loci Tcf7l2, Hnf1b, and confi rmed PPARγ-specifi c binding by forming a supershifted complex upon Camk1d/Cdc123 loci. adding a PPARγ-specifi c antibody. In summary, Mig-6 is a negative regulator The extensive chromatin profi ling of early passage MIN6 under basal of beta cell proliferation. PPARγ regulates Mig-6 transcription by directly culture conditions completed in this study should serve as an important binding to the Mig-6 promoter thus raising the possibility that PPARγ- resource to investigate the epigenetic effects of physiologic and pathologic mediated impairments in beta cell proliferation are mediated by Mig-6. In stimuli on β cells. It has also identifi ed key differences in chromatin structure conclusion, this work potentially describes a novel aspect of PPARγ signaling in diabetes-associated regions between human islets and MIN6 cells, which in pancreatic beta cells. may have important consequences for their functional analysis. Supported by: NIH grant DK078732

1978-P 1980-P Rapid α- and δ-Cell Regeneration Following a Single Diabetic Dose Trefoil Factor 3 Is Not Required To Establish Functional Beta Cell of Streptozotocin Injection in Mice Mass In Vivo YANQING ZHANG, YUAN ZHANG, ROBERT N. BONE, WANXING CUI, JI-BIN ANGELINA M. HERNANDEZ, KRISTYN JEFFRIES, E. SCOTT COLVIN, PATRICK T. PENG, GENE P. SIEGAL, HONGJU WU, Birmingham, AL FUEGER, Indianapolis, IN Streptozotocin selectively destroys insulin-producing β-cells, and thus has Trefoil factor 3 (TFF3), a growth factor secreted from epithelial cells of been widely used to generate Type 1 Diabetes animal models. The islet of various tissues, is known to be important for regeneration and proliferation Langerhans within the pancreas plays a pivotal role in maintaining glucose of cells, including pancreatic beta cells. Indeed, adenovirally-mediated homeostasis. Among the pancreatic endocrine cells, β-cell destruction is overexpression of TFF3 in primary rat islets increases proliferation of beta clearly documented following STZ administration. The non-β cell population cells by 5-fold in vitro, and thus delivery of TFF3 may be one way to combat in the islets following STZ treatment, however, has received little attention. the decline in functional beta cell mass that occurs in both major forms of The objective of this study was to examine this non-β cell population in diabetes. The role of TFF3 in establishing functional beta cell mass, however, islets following a single diabetes-inducing dose of STZ in mice. In this study, has not been determined. We hypothesized that an absence of TFF3 would adult C57BL/6 mice were injected with a single diabetes-inducing dose result in decreased beta cell mass and, therefore, impaired glucose of STZ, monitored for hyperglycemia development, and their pancreases tolerance. To this end, a TFF3 knockout (KO) animal mouse model was assessed for select non-β cell population by immunohistochemistry assays constructed to determine whether the loss of TFF3 resulted in a pancreatic at multiple specifi c days post-injection. The distribution of the major phenotype. Intraperitoneal glucose tolerance tests (1.5 g glucose/kg body pancreatic endocrine cells was analyzed. Since rapid expansion of glucagon- weight) were performed on wild-type (WT) and TFF3 KO C57Bl/6J mice producing α-cells and somatostatin-producing δ-cells was observed, in vivo after a 5-h fast (n = 5-6/genotype). Mice were sacrifi ced the following day BrdU incorporation was employed to assess cell proliferation. Ki67 staining and pancreata were harvested. Immunofl uorescent staining for insulin and was also used to identify the proliferating cells. We found the number of glucagon revealed no overt alterations in islet architecture. Beta cell cross- α- and δ-cells rapidly increased in the islets, which was fi rst detected at sectional area was determined by immunohistochemistry using an insulin Day 2 post-STZ and peaked at Day 6. In addition, the normal core-to-mantle antibody on 3 sections per animal. Unexpectedly, TFF3 KO mice displayed zone organization of β- and non-β cells changed into a more intermingling comparable beta-cell area (0.63 ± 0.08 vs. 0.63 ± 0.07% islet cross-sectional distribution within the islets. In vivo BrdU incorporation assays suggested cell area for WT and KO, respectively), yet had signifi cantly improved glucose proliferation was involved in the regeneration of α- and δ-cells. Furthermore, tolerance (area-under-the-curve: 35596.25 ± 2022.3 vs. 29203.5 ± 833.2 mg/ Ki67 showed both α- and δ-cells had the capacity of self duplication. Taken dL x 120 min for WT and KO, respectively) compared to WT mice. In summary, together, our study provided evidence that pancreatic α- and δ-cells in adult lack of TFF3 does not produce any morphological changes in beta cell area mice can rapidly regenerate, and both self-duplication and regeneration of pancreata from mice fed a standard chow diet; however, its absence from progenitor cells play a role in α- and δ-cells regeneration. may stimulate a compensatory mechanism of improved islet function, Supported by: JDRF 27-2009-378; NIH R01DK081463 thereby enhancing glucose tolerance. Thus, whereas TFF3 is not required for establishing functional beta cell mass in vivo, it remains to be determined whether compensatory beta cell mass expansion requires TFF3. Supported by: NIH grant DK078732. POSTERS Islet Biology/ Insulin Secretion

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A533 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

ISLET BIOLOGY—BETA CELL—STIMULUS- SECRETION COUPLING AND METABOLISM & 1983-P Voltage-Gated Potassium Channels in Pancreatic β-Cells Are [See also: Presidents Posters 473-PP to 475-PP, page A131.] Regulated Via Phosphorylation/Dephosphorylation and Divalent Cations MASASHI YOSHIDA, SHIHO YAMATO, MASANORI NAKATA, SAN-E ISHIKAWA, Guided Audio Tour: Stimulus-Secretion Coupling I (Posters 1981-P to TOSHIHIKO YADA, MASANOBU KAWAKAMI, MASAFUMI KAKEI, Saitama, Japan, 1987-P), see page 13. Shimotsuke, Japan ATP-sensitive potassium channel (KATP channel) is a determinant of & 1981-P resting membrane potential in pancreatic β-cells at lower glucose levels. Stimulus-Induced S-Nitrosylation of Syntaxin 4 Impacts Insulin Closure of the channel is a consequence of elevation of ATP/ADP ratio upon Granule Exocytosis glucose stimulation. In normal individual, however, plasma glucose level DEAN A. WISEMAN, MICHAEL A. KALWAT, EUNJIN OH, DEBBIE C. THURMOND, at fasting state is stabilized between 5 and 6 mM. At this situation it is Indianapolis, IN unlikely that alteration of activity of KATP channels contributes to regulation Glucose-stimulated insulin release from a pancreatic islet beta (β) cell of insulin secretion to keep plasma glucose level constant because ATP/ADP involves increased levels of reactive oxygen and nitrogen species. While ratio is unchangeable at such a glucose concentration. Thus, a fi ne-tuning of this is a normal occurrence, under pathophysiological conditions such as insulin secretion to maintain the plasma glucose level in normal state should chronic hyperglycemia and infl ammation, insulin exocytosis fails and the be explained with a factor other than KATP channels. We therefore examined mechanistic reason for failure is unknown. Given their dysfunction under whether the voltage-gated potassium channel (Kv channel) is regulated by conditions of nitrosative stress serving as a mechanistic basis for insulin metabolism in rat pancreatic β-cells or Kv2.1-expressed HEK293 cells by secretory dysfunction, we hypothesized that exocytotic proteins might be using whole-cell patch clamp technique. Under the following conditions at the targets of S-nitrosylation. Using a biotin-switch technique for isolating external and internal sides, respectively, in the presence of 2.8 mM glucose S-nitrosylated proteins we identifi ed the plasma membrane t-SNARE protein 2+ 2+ 2+ and 5 mM MgATP (intracellular Ca concentration, [Ca ]i, is 16 nM with Ca - Syntaxin 4 as one specifi c target of this modifi cation. The cellular content EGTA buffer), in the absence of glucose and MgATP, and in the presence of of S-nitrosylated Syntaxin 4 increased acutely within 5 min of glucose- 11.2 mM glucose and 5 mM AMPPNP or 1 μM FCCP; Kv-channel currents were stimulation, corresponding to the time at which Syntaxin 4 activation was increased at the membrane potentials between -40 mV and -10 mM. Kv2.1- detectable. S-nitrosylation was mapped to Syntaxin 4 residue Cys141, channel current expressed in HEK293 cells did not change in the presence located within the Hc domain predicted to increase accessibility for 2+ 2+ of 10 mM total ATP and 5 mM total Mg at the cytosolic side ([Ca ]i 16 nM, v-SNARE interaction. A C141S-Syntaxin 4 mutant resisted S-nitrosylation 2+ [Mg ]i 0.1 mM), whereas the currents increased at the above potential range induced in vitro by the nitric oxide donor compound GSNO, failed to exhibit upon the continuous dialysis with 0 mM ATP at cytoplasm ([Ca2+]i, 16 nM glucose-induced activation and VAMP2 binding, and failed to potentiate 2+ and [Mg ]i, 3.2 mM). Exposure of 10 U/ml alkaline phosphatase or 1.6 μM insulin release akin to that of wild-type Syntaxin 4. Strikingly, S-nitrosylation Ca2+ into cytoplasm through the patch pipette produced similar Kv-channel of Syntaxin 4 could be induced by acute treatment with infl ammatory current increases. The resting membrane potentials were hyperpolarized cytokines (TNFα, IL-1β, and IFNγ), coordinate with inappropriate Syntaxin from -20.2 ± 1.4 mV at 16 nM Ca2+ (n=8) to -33.2 ± 1.8 mV at 1.6 μM (n=5, 4 activation and insulin release in the absence of the glucose stimulus, P<0.0001) that were measured in the presence of 100 μM tolbutamide consistent with nitrosative stress and dysfunctional exocytosis, preceding without Ca2+ at external side in β-cells. We conclude that Kv2.1 channel is the cell dysfunction and death associated with more chronic stimulation mediated by glucose metabolism, phosphorylation/dephosphorylation and (24 hr). Taken together, these data indicate a signifi cant role for reactive internal divalent cations. nitrogen species in the insulin exocytosis mechanism in β-cells and expose a potential pathophysiological exploitation of this mechanism to cause & dysfunctional exocytosis. 1984-P Pancreatic Islets Produce and Release Active Glucagon-Like Supported by: NIH: DK087811, HL090198 (DAW); DK067912 (DCT); AHA: Peptide 1 (GLP-1) 10PRE3040010 (MAK) XIAOYAN (NINA) LI, YAN ZOU, VIJAY B.G. REDDY, LAN GE, JEAN-LUC V. TRAN, LIYANG WANG, PAUL A. FISCHER, BEI B. ZHANG, ANDREW D. HOWARD, YUN- & 1982-P PING ZHOU, Rahway, NJ, Shanghai, China Amplifi cation of Glucose Stimulated Insulin Secretion by Activating Glucagon-like peptide 1 (GLP-1), an incretin hormone essential for glucose the Free Fatty Acid Receptor GPR40 in beta-Cells and Isolated Pan- homeostasis, is believed to be produced mainly by the enteroendocrine L-cells creas in the gut. However, the glucagon receptor knockout (Gcgr-/-) mice have over GUIDO HASCHKE, VIKTORIA DIETRICH, ELISABETH DEFOSSA, SIEGFRIED ten-fold increased circulating GLP-1 level, and we recently observed signifi cant STENGELIN, ANGELA DUDDA, ANDREAS W. HERLING, Frankfurt, Germany production and release of active GLP-1 in pancreatic islets of Gcgr-/- mice Glucose homeostasis depends critically on insulin release from endocrine using immunocytochemistry staining and ELISA assays specifi c for active GLP- pancreatic beta-cells in healthy subjects. Multi-factorial pathways modify 1. Here, we further reported that removal of 50% pancreas in Gcgr-/- mice the pattern of insulin secretion, e.g. long-chain free fatty acids (FFA). The reduced plasma levels of total and active GLP-1 and glucagon by half relative pleiotropic effects of FFA can act on different pathways in the beta-cell and to sham-operated mice, whereas the same procedure only reduced plasma are important for energy supply and survival. However, in type 2 diabetic glucagon, but not GLP-1 levels in WT mice. To study the chemical identity of patients, the plasma levels of FFA and glucose are elevated and result in an the GLP-1 molecule produced in pancreatic islet, we profi led the presence of impaired insulin secretion. various proteolytic products of proglucagon in cell lysates by LC-MS analysis. One physiological role of FFA is the binding to a specifi c receptor on the The acid ethanol extract of the Gcgr-/- islets contained much greater content beta-cell, the G-Protein-Receptor 40 (GPR40). GPR40 mediates a glucose- of glucagon relative to WT islet lysate, consistent with the α-cell mass dependent increase in insulin secretion by elevating the intracellular Ca2+ expansion in Gcgr-/- mice. Interestingly, both active forms of GLP-1, GLP-1 (7- concentration. SAR1 has been identifi ed as a potent and selective GPR40 37) and GLP-1 (7-36)-NH2 were readily detected in the Gcgr-/- islet lysate, but 2+ agonist (EC50<0.1µM). In vitro Ca -Imaging studies in islet cells and isolated not the WT islets. Finally, we confi rmed the production and release of active perfused pancreas in rats were performed to demonstrate the amplifi cation GLP-1 from Gcgr-/- islets with ELISA assay. LC-MS analysis of islet lysates of the glucose dependent insulin secretion in these models. SAR1 clearly reveals active form GLP-1 (7-36)-NH2 existed in both type 2 diabetic and demonstrated a glucose dependent mechanism in increasing the intracellular non-diabetic subjects. Together, these data provided further evidence for the Ca2+-signal and a resulting increase in insulin secretion under elevated production of GLP-1 in pancreatic islets under the conditions of Gcgr ablation glucose concentrations. The observed GPR40 mediated increase in insulin or rapid islet expansion. secretion by SAR1 was superior to a parallel stimulation with the saturated long-chain FFA palmitate in the isolated perfused pancreas. After repeated stimulation of GPR40 with SAR1 under high glucose levels the insulin response was stable inducible and no desensitization of the POSTERS

Islet Biology/ receptor could be observed. These results indicate a positive and benefi cial Insulin Secretion effect of the selective GPR40 agonist SAR1 to modulate the glucose dependent insulin response in type 2 diabetic patients with disturbed FFA and glucose metabolism.

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A534 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

& 1985-P & 1987-P Gelsolin Functions in Glucose-Stimulated Insulin Secretion through Glucotoxicity to Pancreatic Islets with Different Excitability In Association with Syntaxin 4 Vitro MICHAEL A. KALWAT, WEI LUO, DEAN A. WISEMAN, DEBBIE C. THURMOND, ZHIYU WANG, MARIA S. REMEDI, COLIN G. NICHOLS, St. Louis, MO Indianapolis, IN, Piscataway, NJ Pancreatic beta cell dysfunction plays a crucial role in type 2 diabetes SNARE proteins mediate vesicle exocytosis and have an important role in pathophysiology. It manifests as reduced beta cell mass and insulin biphasic insulin secretion. The plasma membrane SNARE protein Syntaxin 4 defi ciency. The effect of high blood glucose on beta-cells (glucotoxicity) has (Syn4) is required for both phases of insulin secretion and has been shown long been proposed as one of the causes, however, the underlying mechanism to interact with F-actin in a glucose-dependent fashion. Here we report for beta cell dysfunction remains unclear. Elevated glucose inevitably leads the existence of a novel type of Syn4 binding partner, Gelsolin, a calcium- to enhanced electrical activity and elevated [Cai] in WT islets, but this link dependent F-actin severing and capping protein, identifi ed in a directed is removed in KATP-channel gain-of-function (KATP-GOF) and KATP-channel yeast two-hybrid screen for Syn4 interacting proteins. Although Gelsolin knockout (KATP-KO) mice, which exhibit permanently basal and permanently has already been implicated as a positive effector of glucose-stimulated elevated levels of [Cai], respectively. We compared the responses of isolated in insulin secretion, its linkage to the exocytosis machinery has remained islets from WT, KATP-GOF and KATP-KO mice to incubation in high or low incomplete. Toward this goal, we validated that the interaction between glucose, to assess the role of beta cell excitability in the islet response to Gelsolin and Syn4 was direct in vitro, and went on to map the Gelsolin hyperglycemia, without the interference of systemic changes associated binding site within Syn4 to residues 39-70; this Syn4 peptide was suffi cient with hyperglycemia in vivo. Isolated islets were cultured in low (5 mM) to confer Gelsolin binding. Syn4-Gelsolin complexes were observed in co- vs high (30 mM) glucose CMRL complete media. Insulin content and islet immunoprecipitation assays from clonal MIN6 beta cell lysates, dissociating survival were followed. WT islets lost insulin content signifi cantly in high upon acute glucose-stimulation, suggesting the Syn4-Gelsolin complex to be glucose as compared to low glucose condition. In high glucose, islets lost glucose-regulated. To test the importance of this complex, the 39-70 residue insulin in an early rapid phase (day 1-3), followed by a slow phase (day 4-9). In Syn4 peptide was fused to GFP (GFP-39-70) and introduced into MIN6 cells by low glucose, insulin content decreased only very slowly. Only a similar slow both transient transfection and adenoviral transduction, with both methods decrease was observed in KATP-GOF islets in high or low glucose conditions, yielding evidence of competitive inhibition and/or disruption of endogenous but in KATP-KO islets, insulin content decreased rapidly and signifi cantly even Syn4-Gelsolin binding. Disruption of endogenous Syn4-Gelsolin complexes in low glucose; high glucose decreased insulin content further in KATP- KO corresponded to decreased glucose-stimulated insulin secretion, although islets. Notably, the decrease in insulin content observed in WT islets was was without effect upon KCl-induced depolarization. Notably, both isolated recoverable when high glucose was replaced with low glucose, over 3 islets and MIN6 beta cells expressing GFP-39-70 showed elevated basal days. No difference in islet survival was observed between low and high insulin secretion, and pilot data show the inappropriate and de-regulated glucose conditions in any genotype islets over 10 days. These data lead us to activation of Syn4 under unstimulated conditions in cells expressing the conclude that: in vitro, 1) hyperglycemia decreases islet insulin content, but competitive GFP-39-70 peptide. Taken together, these results are suggestive does not change islet survival over 10 days; 2) insulin content response to of a regulatory barrier role for the Syn4-Gelsolin complex in controlling insulin hyperglycemia correlates with beta cell excitability, suggesting that 3) it is a granule fusion and inappropriate insulin release in the absence of stimuli. direct, reversible, result of hyperstimulation and hypersecretion. Supported by: AHA 10PRE3040010 to MAK and R01 DK-076614 to DCT Supported by: NIH ADA-Funded Research & 1986-P II Impaired Glucose and GLP-1-Stimulated Insulin Secretion in Pancreas- Guided Audio Tour: Stimulus-Secretion Coupling (Posters 1988-P to Specifi c Tcf7l2 null Mice 1995-P), see page 13. GABRIELA DA SILVA XAVIER, GUY A. RUTTER, London, United Kingdom Polymorphisms in the TCF7L2 gene are associated with an increased & 1988-P risk of type 2 diabetes and decreased pancreatic beta-cell function. We Enhanced Insulin Sensitivity Underlies Long-Term Remission in have previously shown that silencing of Tcf7l2 gene expression in clonal Response to Early Sulfonylurea Therapy in a Mouse Model of KATP- mouse beta cells and mouse pancreatic islets leads to dysregulated insulin Induced Neonatal Diabetes Mellitus secretion and the altered expression of genes involved in insulin release. MARIA S. REMEDI, ARPITA K. VYAS, PAUL W. HRUZ, COLIN G. NICHOLS, St. Louis, Here we have generated and studied mice deleted for Tcf7l2 throughout the MO pancreas (pTcf7l2 KO). Gain-of-function KATP channel mutations cause Neonatal Diabetes Mellitus Mice bearing conditional Tcf7l2 allelles (Genoway), where exon 1 is (NDM). In an experimental mouse model (xNDM), hyperglycemia progressively fl anked by loxP sites, were back-crossed onto a C57BL/6 background. worsens over time, accompanied by secondary loss of insulin content (Remedi Selective deletion of Tcf7l2 in the pancreas was achieved by breeding with et al., 2009; Cell Metab. 9, 140-51). Chronic sulfonylurea treatment avoids PDX1-Cre deleter mice (Prof. D. Melton). Real-time quantitative PCR (qPCR) diabetes and maintains insulin content if initiated prior to disease onset, but revealed Tcf7l2 transcript levels below the level of detection in pTcf7l2 KO is ineffective after diabetes has developed. In human NDM, sulfonylureas can islets. Insulin 2 (Ins2) and GLP-1 receptor (Glp1r) gene expression were also replace insulin therapy, but dosing and effi cacy are highly variable. To gain decreased by 15.4±4.8% and 42±0.08% (n=3 islet preparations), respectively, insight into drug dosing and effi cacy in NDM, we treated xNDM mice with in islets from pTcf7l2 KO vs wild-type mice. high-dose of the sulfonylurea glibenclamide for 6 days only at the beginning Intraperitoneal glucose tolerance tests (1g/kg glucose) were conducted of disease induction. As we previously reported (Remedi and Nichols, 2010; on 8-week old mice starved for 16 h. Peak blood glucose at 15 min was not Diabetes 59, Suppl. 1, A454), ∼70% of mice develop severe diabetes after different between pTcf7l2 KO mice (14.4±0.14 mmol/l, n=6) and wild-type treatment cessation, but surprisingly, ∼30% maintain near-normal blood glucose littermate controls (13.5±1.06 mmol/l, n=3). Similar data were obtained in indefi nitely. We initiated a second group of animals on high-dose sulfonylureas, mice at 12 and 16 weeks of age. and then titrated the dose daily to maintain blood glucose <200mg/dl. Again, By contrast, insulin secretion from pTcf7l2 KO mouse islets was impaired;17 ∼30% of the animals weaned themselves off the drug, maintaining insulin vs 3 mM glucose stimulated insulin secretion by 2.12±0.1 (p≤0.05) and content and normal blood glucose indefi nitely. In response to bolus injection 3.9±0.1-fold (p≤0.05) in pTcf7l2 KO and wild-type islets, respectively (n=3 of insulin, these xNDM responder animals exhibited a prolonged period of mice per genotype). The stimulation of insulin secretion in response to 100 glucose lowering relative to the control animals, suggesting enhanced insulin nM GLP-1 at 17 mM glucose was also impaired in pTcf7l2 KO islets (1.04±0.01 sensitivity, a compensatory mechanism that could account for maintained blood fold) compared with wild-type islets (1.28±0.02 fold, p≤0.05). glucose in the face of only basal secretion. To more directly examine peripheral We conclude that TCF7L2 is necessary for the normal regulation of insulin insulin sensitivity, hyperinsulinemic-euglycemic clamps were performed in secretion. pTCF7L2 KO mice should provide a useful model for studies of the control and xNDM responder mice. Under steady-state conditions, peripheral role of this factor in the regulation of islet hormone secretion in vivo. glucose disposal was markedly increased (by ∼30%) in xNDM responder mice Supported by: Wellcome Trust, MRC and EU (FP7, IMIDIA) compared to control littermates. The results imply that there is a critical window early in disease onset, during which compensatory mechanisms can POSTERS develop, switching the disease from permanent to an essentially transient Islet Biology/ form. We propose that such a mechanism underlies the fact that the same Insulin Secretion mutation can cause either transient or permanent NDM in humans. Supported by: NIH ADA-Funded Research

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A535 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

& 1989-P & 1991-P Impaired Glucose Tolerance, Insulin Sensitivity and Biphasic Transgenic Mice with beta Cell-Specifi c p8 Overexpression Display Insulin Release in Doc2b Knockout Mice Enhanced Insulin Secretory Function upon High Fat Diet-Induced LATHA RAMALINGAM, EUNJIN OH, ALEXANDER J. GROFFEN, DEBBIE C. Obesity THURMOND, Indianapolis, IN, Amsterdam, The Netherlands INGO H. PILZ, KATHARINA LAUBNER, GÜNTER PÄTH, BIANCA DUFNER, NATALIE Exocytosis of intracellular vesicles, such as insulin granules, is carried RIMMER, JOCHEN SEUFERT, Freiburg, Germany out by SNARE and SM (Sec1/Munc18) proteins. An additional regulatory OBJECTIVE: p8 a putative architectural transcription factor is acutely protein, Doc2b (Double C2 domain), has recently been implicated in induced by pancreatitis and protects acinar tissue from infl ammator y damage. exocytosis events crucial to glucose homeostasis, namely insulin secretion Within the endocrine pancreas, own previous work has demonstrated in vitro from MIN6 β-cells and GLUT4 vesicle exocytosis in 3T3-L1 adipocytes. that p8 is a glucose-induced mediator of beta cell proliferation and reduces However, if Doc2b is required for these processes in primary tissues or STZ-induced beta cell apoptosis in an acute and transient manner. Here we for maintenance of glucose homeostasis in vivo remains unexplored. To investigated (I) STZ-induced endogenous p8 gene expresion in beta cells in address this, a Doc2b knockout (KO) mouse model was used. First assessed vivo and (II) generated and characterized transgenic mice with pancreatic by intraperitoneal glucose tolerance test, male Doc2b (-/+) and (-/-) KO mice beta cell-specifi c p8 overexpression. exhibited signifi cant impairments when compared to wild-type littermate Methods: (I) p8 gene expression was characterised in C57Bl/6 wild type mice. Doc2b (-/+) and (-/-) KO mice also showed defi ciencies in serum insulin (wt) mice after a single injection of STZ (250 µg/g body weight) by qPCR. levels following an acute stimulation with glucose, suggesting defects in (II) C57Bl/6 transgenic (tg) mice with beta cell-specifi c p8 overexpression insulin secretion. Furthermore, insulin tolerance testing revealed both Doc2b were generated by pro-nucleus injection of a rat insulin promoter I driven p8 (-/+) and (-/-) KO mice to exhibit profound peripheral insulin resistance. To expression cassette. Tg mice and wt littermates were fed with normal and link these whole-body impairments to mechanistic defects at the tissue high fat diet (HFD) chow for 10 weeks and then tested by intra-peritoneal level, exocytosis events were examined in primary cells from these mice. glucose tolerance test (ipGTT) and insulin tolerance test (ipITT). Weight and In perifusion analyses, islets isolated from both Doc2b (-/+) and (-/-) KO blood glucoses were measured weekly. mice showed attenuated insulin release during both fi rst & second phases. Results: (I) Injection of STZ in wt mice resulted in a strong induction of These data support the concept of glucose intolerance resulting from insulin endogenous p8 mRNA expression in beta cells at 6 h followed by a decline insuffi ciency. To explain the insulin resistant phenotype, we are currently to baseline levels after 24 h. (II) As compared to wt littermates, tg mice analyzing GLUT4 translocation in the skeletal muscle of these mice. Given gained signifi cantly more body weight under HFD, suggesting a p8 mediated that no signifi cant differences in body or tissue weights were noted, the improved beta cell function in tg mice. At 10 weeks of HFD, tg mice displayed data suggest that the paucity of Doc2b exerted specifi c effects upon the a signifi cantly improved glucose tolerance upon ipGTT acompanied by an SNARE/SM protein machineries in these exocytotic processes. Detailed improved insulin secretory response. Insulin sensitivity was not signifi cantly analyses of the SNARE-SM-Doc2b interactions within islet β-cells, skeletal affected in tg animals upon ipITT. muscle and adipocytes isolated from these mice are underway to determine CONCLUSION: Endogenous p8 is a beta cell proliferative and protective if the requirement for Doc2b in these diverse cell types is of a conserved protein that is acutely upregulated upon beta cell injury in vivo. Transgenic or distinct nature. Regardless, our data indicate that diminished Doc2b beta cell specifi c overexpression of p8 improves insulin secretory function abundance could be an important risk factor for increased susceptibility for in response to HFD. p8 may represent an attractive target to improve beta development of type 2 diabetes and β-cell failure. cell function in diabetes mellitus. Improved by ectopic p8. Investigations are Supported by: 2R01DK067912 being carried out to determine effects of ectopic p8 on beta cell mass under normal vs. HFD and in response to STZ. & 1990-P Supported by: DFG (PA 1663/2-1) Replenishment of TCA Intermediates in Pancreatic β-Cells Defi cient in ARNT/HIF1β Expression Rescues the Defective Insulin Secretion RENJITHA PILLAI, JAMIE W. JOSEPH, Kitchener, ON, Canada & 1992-P Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible Overexpression of ERO-1β in Pancreatic β Cells Causes ER Stress factor (HIF)-1β is a transcription factor that was shown to be dramatically and Impaired Glucose Tolerance in Mice reduced in human type 2 diabetic islets. We have shown that knockdown MOTOHARU AWAZAWA, KOHJIRO UEKI, KAZUMA KANEKO, MITSURU OHSUGI, of ARNT/HIF1β impairs biphasic insulin secretion and glucose utilization, MASATO KOIKE, YASUO UCHIYAMA, TAKASHI KADOWAKI, Tokyo, Japan without affecting glucose oxidation or ATP/ADP ratio in rat pancreatic ER oxidoreductin (ERO) 1-like β (ERO1lβ) is a pancreas-specifi c disulfi de beta-cells. We have also reported that ARNT/HIF1β knockdown leads oxidase, which is upregulated in response to ER stress and promotes protein to a signifi cant reduction in the glucose-stimulated increase in pyruvate folding in pancreatic β cells. It has recently been demonstrated that ERO1lβ and TCA intermediates indicating that anaplerosis is an important target promotes insulin biogenesis and thus contributes to physiological glucose of this transcription factor. The goal of the present study was to further homeostasis, while it is unknown how ERO1lβ is involved in the pathogenesis characterize the secretory defects of beta-cells defi cient in ARNT/HIF1β of type 2 diabetes. Here we show that ERO1lβ is upregulated in pancreatic β using previously defi ned nutrient and non-nutrient insulin secretagogues. cells of db/db mice in early ages. β cell-specifi c ERO1lβ transgenic (βERO1lβ- We show that although siRNA mediated knockdown of ARNT/HIF1β Tg) mice showed upregulation of unfolded protein response genes including inhibits glucose-stimulated insulin secretion (GSIS) in beta-cells, the BIP, CHOP and TRB3. Electron microscopy showed markedly enlarged ER in secretory response to low glucose and depolarizing concentrations of KCl the β cells of βERO1lβ-Tg mice, indicating that the β cells were subjected and diazoxide was unaffected, suggesting that Ca2+-associated events in to severe ER stress. Interestingly, despite these distinct changes, βERO1lβ- stimulus-secretion coupling were unaffected. However, in the presence Tg mice showed mild glucose intolerance with slightly impaired insulin of high glucose and KCl and diazoxide or high glucose and glibenclamide, response. Microscopy showed that the islet mass was maintained with no the secretory response of ARNT/HIF1β knockdown cells to glucose was signs of apoptosis in β cells, suggesting that these cells were resistant to signifi cantly reduced suggesting that the defects mediated by ARNT/HIF1β ER stress-induced apoptosis. Mature insulin content was decreased in the involve the KATP channel independent pathway of insulin release. We also β cells overexpressing ERO1lβ, which accounted for the impaired insulin assessed the insulin response of ARNT/HIF1β knockdown cells to amino response. ER associated degradation remained unchanged, as evidenced by acids and found that the secretory response to leucine and glutamine was pulse chase analysis in MIN6 β cells. These data collectively suggest that intact in these cells. Finally, we show that treatment of beta-cells defi cient ERO1lβ overexpression caused ER stress with impaired insulin maturation. in ARNT/HIF1β with membrane permeable TCA intermediates dimethyl- These data indicate previously unknown roles of ERO1lβ in pancreatic β alpha-ketoglutarate and dimethyl malate restores GSIS nearly completely, cells in the pathogenesis of type 2 diabetes. indicating that the reduction in insulin secretion may be attributed to lack of suffi cient substrate fl ow through the TCA cycle. Our data strongly suggests the importance of ARNT/HIF1β in maintaining the ability of beta-cells to increase anaplerosis in response to glucose and this plays a key role in the POSTERS Islet Biology/ KATP-channel independent pathway of insulin secretion. Insulin Secretion Supported by: CIHR, CFI

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A536 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

& 1994-P A Possible Role for Neurexin in the Docking of Insulin Granules at the β-Cell Membrane MERRIE MOSEDALE, SONYA EGODAGE, REI C. CALMA, STEVEN D. CHESSLER, La Jolla, CA β cells express many protein constituents of the neurotransmitter exocytotic machinery including a family of transmembrane, synaptic adhesion molecules called neurexins (NRXNs). In the brain, α-NRXNs help organize the neuronal exocytotic machinery via intracellular interactions with exocytotic proteins. We hypothesized that α-NRXNs play a comparable role in β cells, participating in insulin granule docking and exocytosis. Plasma membrane purifi cation, western blot analysis and immunostaining were used to determine that NRXN protein is expressed on the β-cell surface and is not present in other islet cell types. We used absolute RT- qPCR to demonstrate that NRXN1α and 2β are the most abundant NRXN isoforms in the β cell, with transcript levels comparable to those in brain. Using immunoprecipitation, we determined that NRXN1 interacts with CASK, Syntaxin1, Munc18 and Granuphilin in the INS-1E β-cell line. To test whether NRXNs are involved in insulin secretion, we used siRNA to knockdown the expression of NRXN1 in INS-1E β cells and observed a 54% increase (p<0.05) in insulin secretion at high glucose. We confi rmed this effect was restricted to the regulated secretory pathway by observing no change in the constitutively-secreted protein alkaline phosphatase (SEAP) at high glucose after cotransfection of SEAP with NRXN1 siRNA. Using western blot analysis, we observed a 33% and 40% decrease in NRXN1 protein levels (p<0.05) after 1h of glucose stimulation in non-transfected and NRXN1-overexpressing INS-1E β cells respectively. This effect could be prevented by treatment with lysosomal inhibitors. We conclude that NRXN1 is a component of the β-cell secretory machinery. NRXNs are involved in the exocytosis of insulin granules from β cells, possibly by organizing the exocytotic machinery and/or by facilitating the docking of granules at the β-cell surface. Increased insulin secretion after NRXN1 gene silencing and the decrease in NRXN1 levels observed during glucose stimulation suggest that NRXN1 contributes to the negative regulation of insulin release. Future work will determine the exact role of NRXNs in the docking, priming and fusion steps of insulin granule exocytosis.

& 1995-P Inducible Expression of Monocarboxylate Transporter 1 (Mct1/ Slc16a1) in the Beta Cell of Transgenic Mice: A Model of Exercise- & 1993-P Induced HyperInsulinism Glucagon-Like Peptide-1 (GLP-1) Regulates miR-132 and miR-212 TIMOTHY J. PULLEN, GUY A. RUTTER, London, United Kingdom Expression in Pancreatic β-Cells in a PKA-Dependent Manner The plasma membrane monocarboxylate transporter MCT1 is expressed JIN SHANG, LIYANG WANG, YUE FENG, XIAOLAN SHEN, CHRISTOPHER V. at vanishingly low levels in beta cells but at high levels in all other tissues. JOHNSON, MARY RABAGLIA, MUFADDAL SONI, ALAN D. ATTIE, MARK KELLER, Activating mutations in the MCT1 promoter are linked to Exercise-Induced HANS E. HOHMEIER, CHRISTOPHER B. NEWGARD, YUN-PING ZHOU, ANDREW HyperInsulinism (EIHI), where circulating pyruvate produced during exercise D. HOWARD, Rahway, NJ, Madison, WI, Durham, NC inappropriately stimulates insulin secretion, causing hypoglycaemia. While Glucagon-like peptide-1 (GLP-1) enhances pancreatic β-cell function and EIHI patients exhibit increased MCT1 expression in fi broblasts, there is no survival, but the underlying mechanism remains incompletely understood. direct evidence that MCT1 is overexpressed in patient beta cells and that We found that two closely related microRNA species, miR-132 and miR-212, this is directly responsible for unmasking an effect of pyruvate on insulin were robustly up-regulated by GLP-1 in the rat insulinoma INS-1-derived secretion. Here, we have generated a transgenic mouse model to examine cell line 832/3, as well as in isolated mouse or rat islets. Furthermore, the impact of beta cell-restricted, inducible overexpression of MCT1 on overexpression of miR-132 or miR-212 signifi cantly enhanced glucose- pyruvate or glucose tolerance. stimulated insulin secretion (GSIS) in 832/3 cells. To determine if these Double transgenic (DT) mice were generated expressing MCT1 cDNA observations occur in vivo, C57Bl/6N lean mice were infused with Exendin-4, under a tetracycline-regulated promoter, and the reverse tetracycline a GLP-1 analog, for 48 hours by osmotic mini-pumps that were implanted transactivator under rat insulin promoter control. Before doxycycline subcutaneously. Pancreatic islets were isolated and miRNA expression treatment, intraperitoneal injection of pyruvate (0.5 g/kg) led to identical levels measured. Exendin-4 signifi cantly reduced glucose excursion and excursions in blood glucose in DT and wild-type (WT) mice. Thus, blood increased insulin secretion during an oral glucose challenge, demonstrating glucose (± SEM) 30 min after pyruvate challenge was: DT = 10.37 ± 0.45 the physiological effect of the infused Exendin-4. Similar to what was mmol/l, n=13; WT = 9.90 ± 0.58, n=5; p=0.573. However, following doxycycline observed ex vivo, Exendin-4 signifi cantly and dose-dependently induced treatment, DT mice displayed signifi cantly lower blood glucose at this time the expression levels of miR-132 and miR-212, but not of miR-16. GLP-1 point: DT = 7.18 ± 0.40 mmol/l, n=13; WT = 8.78 ± 0.51 mmol/l, n=5; p=0.039. and increases in intracellular cAMP levels have been reported to enhance Correspondingly, doxycline-treated DT mouse islets responded to 10 mmol/l GSIS in β cells via an interplay of different signaling pathways including pyruvate with a 2.8-fold stimulation of insulin secretion, whereas untreated PKA and Epac2. Epac2 (exchange protein 2 directly activated by cAMP) is a islets were unresponsive, indicating that overexpression of MCT1 in beta novel cAMP sensor recently shown to play a PKA-independent role in insulin cells is suffi cient to allow exogenous pyruvate to elicit a large stimulation of secretion. We asked whether the GLP-1 mediated induction of miR-132 and insulin secretion. By contrast, transgene induction had no effect on insulin miR-212 involves PKA or Epac2 signaling pathways. The effect of GLP-1 on secretion stimulated by 17 mmol/l glucose: +Dox = 0.42% ± 0.034 total islet miR-132/212 expression in 832/3 cells was blocked by PKA inhibitor H-89 insulin released; -Dox = 0.43% ± .072; n=3. In conclusion, expression of MCT1 in beta cells replicates the key feature (10 μM), whereas the Epac specifi c activator, 8-pCPT-2’-O-Me-cAMP-AM (10 POSTERS of EIHI, and highlights the importance of this transporter’s absence from Islet Biology/

μM) did not alter miR-132/212 expression either in the presence or absence Insulin Secretion of GLP-1. In summary, our fi ndings support a model linking GLP-1 to improved these cells for the normal control of insulin secretion. β-cell function: GLP1R→cAMP→PKA→miR-132/212→insulin secretion. Supported by: Wellcome Trust

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A537 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

1996-P measured the secreted insulin in the medium by ELISA and found that the Acute and Chronic Free Fatty Acid Treatment Differentially Impact IH treatment attenuated GIS. Real-time RT-PCR revealed that the levels of β-Cell Function in Mouse Islets Lacking Leptin Receptors insulin mRNAs in HIT-T15 cells and islets were unchanged by IH, suggesting TOMOAKI MORIOKA, JOHN F. DISHINGER, KENDRA R. REID, ROBERT T. that IH causes dysfunction of GIS system. To see what is wrong in the system, KENNEDY, MASAAKI INABA, ROHIT N. KULKARNI, Osaka, Japan, Ann Arbor, MI, we measured the mRNA levels of Glut2, GK, SUR1, and Cav1.2 in IH-treated Boston, MA islets and found they were similar to those in normoxia-treated islets. In We have previously reported that the pancreas-specifi c leptin receptor contrast, the mRNA level of CD38 in IH-treated islets was signifi cantly lower (ObR) knockout (KO) mouse model exhibits enhanced glucose-induced insulin than that in normoxia-treated islets (39% of the controls). To investigate secretion (GSIS) in vivo on regular diet and impaired GSIS following high-fat whether IH inhibits CD38 transcription, HIT-T15 cells were transiently diet feeding. We have also reported that islets isolated from the KO mice transfected with reporter plasmid containing human CD38 promoter. We exhibit greater insulin secretion induced by glucose or GLP-1. To investigate measured luciferase activities and found that the transcriptional activity of a potential association between leptin signaling and free fatty acid (FFA) CD38 was attenuated by IH. As CD38 was reported to play an essential role effects in β-cell, we treated single, size-matched islets isolated from control in GIS, it is quite possible that the IH-induced GIS attenuation is mediated or pancreas-ObR KO mice with acute or chronic palmitate (0.5 mM) and by the down-regulation of CD38. To verify this possibility, we prepared CD38 2+ 2+ expression vector and transfected it into HIT-T15 cells. The transfection of measured intracellular Ca concentration ([Ca ]i) by ratiometric fl uorescence and insulin release by serial immunoassay on a micro-fl uidic chip. Islets from CD38 expression vector recovered the IH-induced attenuation of GIS. These 2+ results indicate that IH stress attenuates GIS through the suppression of both genotypes exhibited ∼3-fold greater [Ca ]i and a signifi cant insulin secretion in response to glucose (3 to 8 mM). The simultaneous presence of CD38 transcription. It is quite possible that the cyclic change of hypoxia- palmitate in the incubation media for 10 min (acute treatment) resulted in a reoxygenation, which occurs in SAS patients, induces beta cell dysfunction 2+ via CD38 down-regulation, resulting in glucose intolerance and type2 greater increase in [Ca ]i in the KO islets (control; 5.4, KO; 73.4 % increase; n=5, p<0.01) and insulin release (control; 7.5, KO; 70.6 % increase; n=5; diabetes. p<0.05) compared to controls. On the contrary, while chronic treatment with palmitate (48 hours) prior to glucose treatment impaired GSIS by 21.0% in 1999-P control islets, indicating lipotoxic effects of fatty acid, the KO islets showed Beta-Cell Function Indexes from the Oral Glucose Tolerance Test Are an even greater impairment in GSIS (47.9% decrease). However, chronic Not Determined Entirely by Pancreatic Beta-Cell Area in Korean 2+ palmitate treatment decreased glucose-induced [Ca ]i to the same extent in SUNGHWAN SUH, MI YEON KIM, SANG-MAN JIN, HYE WON JANG, KYU YEON islets from both genotypes (control; 38.5, KO; 35.9 % decrease; n=5; p=NS). HUR, JAE HYEON KIM, JAE HOON CHUNG, YONG-KI MIN, MYUNG-SHIK LEE, In summary, an acute increase in FFAs acts as a nutrient signal to enhance MOON KYU LEE, KWANG-WON KIM, Seoul, Republic of Korea β-cell function; however, chronic exposure of FFAs generates a lipotoxic Simplifi ed indexes of pancreatic β-cell function using the oral glucose signal that is amplifi ed in islets lacking ObR. tolerance test (OGTT) have been suggested. Several studies have reported the accuracy of metabolic tests as a measure of pancreatic β-cell mass. 1997-P Here, we investigated how well the OGTT β-cell function index refl ects An Open Innovation Phenotypic Drug Discovery Platform To Identify pancreatic β-cell area in Korean patients. The study consisted of 45 patients Glucose Dependent Insulin Secretagogues who underwent pancreatectomies. Before operation, a standard 75-g OGTT KAREN L. COX, CHRISTOPHER MOXHAM, FRANCIS WILLARD, SABA HUSAIN, was performed. Of the 45 patients, 22 had diabetes, 12 had impaired glucose Indianapolis, IN tolerance (IGT), 1 had impaired fasting glucose (IFG), 4 had combined IGT and The Lilly Phenotypic Drug Discovery (PD2) Initiative is a novel open IFG, and 6 had normal glucose tolerance. Immunohistochemical staining was innovation program that seeks to engage researchers worldwide in the performed, and β-cell function indexes were calculated with the OGTT data identifi cation of novel compounds that have potential to be developed into and compared with pancreatic β-cell area. drug candidates for areas of unmet medical need like Type 2 Diabetes. To this end, we have established a PD2 assay module aimed at discovering novel glucose-dependent insulin secretagogues. Utilizing the INS-1E/G insulinoma cell line, we have validated a 384-well insulin secretion assay (Z’>0.5) to identify glucose-dependent insulin secretagogues. Screening results for the fi rst 4691 compounds submitted to PD2 have yielded hit rates of 2% with a median potency of 4 uM. The overwhelming majority (92%) of active compounds exhibit glucose-dependent activity. Using an assay to monitor K+-channel activity, we demonstrate that the active compounds do not behave as K+-channel modulators, thus differentiating them from current standard of care agents such as the sulfonylureas and glinides. Moreover the compounds do not appear to be structurally similar to glucokinase activators or imidazolines, nor do they appear to be acting via biogenic amine receptors known to promote insulin secretion. The compounds are not promiscuous secretagogues in that they do not promote growth hormone or ApoE secretion. Lastly, several compounds elicit glucose-dependent insulin secretion in isolated rat islets. In summary, PD2 has enabled the identifi cation of novel glucose-dependent insulin secretagogues that elicit a biological profi le that is distinct from current agents.

1998-P Attenuation of Glucose-Induced Insulin Secretion by Intermittent Hypoxia Via Down-Regulation of CD38 HIROYO OTA, SHINJI TAMAKI, ASAKO ITAYA-HIRONAKA, AKIYO YAMAUCHI, SUMIYO SAKURAMOTO-TSUCHIDA, SHIN TAKASAWA, HIROSHI KIMURA, Kashihara, Japan Sleep apnea syndrome (SAS) is a highly prevalent sleep disorder characterized by cyclic intermittent hypoxia (IH). Although accumulating evidence suggests the association of IH and type2 diabetes independently on body mass index and waist circumference, the direct effects of IH on glucose–induced insulin secretion (GIS) are elusive. Hamster insulinoma POSTERS

Islet Biology/ HIT-T15 cells and isolated rat islets were exposed either to 64 cycles/24

Insulin Secretion h of IH (5 min hypoxia/10 min normoxia), or normoxia for 24 h. After the treatment, HIT-T15 cells and islets were incubated in RPMI1640 medium containing 5.5 mM glucose, or 22 mM glucose for 1 h in normoxia. We

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A538 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

The pancreatic β-cell area showed a relationship with a homeostasis 2002-P model assessment β-cell function (r = 0.358, P < 0.05), disposition index (r = FoxO1 Contributes to Beta-Cell Compensation 0.336, P < 0.05), fasting glucose (r = -0.359, P < 0.05), and the C-peptide/ DAE HYUN KIM, SNADRA SLUSHER, TING ZHANG, SUZANNE BERTERA, HENRY glucose30 min ratio (r = 0.319, P < 0.05). However, there was no single best DONG, Pittsburgh, PA parameter among several OGTT β-cell function indexes for refl ecting Beta-cell compensation is critical for overcoming insulin resistance and pancreatic β-cell area in Korean. Some OGTT β-cell function parameters maintaining normoglycemia in subjects with metabolic syndrome. Failure showed a signifi cant relationship with pancreatic β-cell area, but there was in beta-cell compensation contributes to insulin insuffi ciency and overt no single best parameter for refl ecting the pancreatic β-cell area in Korean. diabetes. To understand the mechanism of beta-cell compensation, we This study implies that the regulation of blood glucose level requires both an characterized the role of the forkhead transcription factor FoxO1 in beta-cell adequate number and proper function of β-cells. function in transgenic mice, in which FoxO1 is selectively expressed from the rat insulin promoter (RIP). RIP-directed FoxO1 transgenic expression resulted 2000-P in islet hypertrophy, accounting for augmented glucose-stimulated insulin Chronic Free Fatty Acids but Not Glucose Decrease Matrix Metallo- release. This effect contributed to improved glycemic control, rendering RIP- proteinase-9 in Islets FoxO1 transgenic mice refractory to high fat-induced impairment in glucose KATHRYN ASTON-MOURNEY, SAKENEH ZRAIKA, DANIKA SVY, STEVEN E. tolerance. When challenged with multiple low doses of streptozotocin (50 mg/ KAHN, REBECCA L. HULL, Seattle, WA kg per day for 5 consecutive days), RIP-FoxO1 transgenic mice, as opposed to Matrix metalloproteinase-9 (MMP-9) is an extracellular enzyme that is control littermates, were protected from developing streptozotocin-induced upregulated in several tissues in type 2 diabetes and has been implicated diabetes. This effect is attributable to FoxO1-mediated induction in anti- in the development of diabetic complications. However, in the islet MMP-9 oxidative function in islets. is thought to have benefi cial effects, including increasing vascularization Our data demonstrate that beta cell-specifi c FoxO1 production enhanced following islet transplantation and reducing amyloid deposition, thus a the ability of islets to compensate for insulin resistance in response to high decrease may be detrimental. In fact, microarray data show that in islets from fat feeding as well as for streptozotocin-elicited oxidative stress. subjects with type 2 diabetes, MMP-9 mRNA levels are decreased by 34% (0.66 ± 0.13 type 2 diabetes vs. 1.00 ± 0.04 control, p<0.05, n=4). The aim of 2003-P this study was to determine whether the decrease in islet MMP-9 that occurs GLP-1 Rescues Insulin Secretion from Priming Protein Munc13-1 under diabetic conditions is due to chronic high glucose or free fatty acids. We Defi cient Mouse Islet β-Cells Primarily by Recruitment of Newcomer fi rst sought to confi rm, by real-time PCR, whether MMP-9 is decreased in islets Granules in Both First and Second Phase Secretion in diabetes. We used an animal model of type 2 diabetes, the db/db mouse, LI XIE, DAN ZHU, HERBERT Y. GAISANO, Toronto, ON, Canada which is characterized by obesity, hyperglycemia and hyperlipidemia. Islets Glucose-stimulated insulin secretion from pancreatic islets follows a were isolated from db/db and control db/+ mice at 16 weeks of age and MMP-9 biphasic pattern consisting of a robust fi rst-phase (∼6 min) and a longer mRNA measured. MMP-9 mRNA was decreased 36% in db/db islets compared lasting low-level second-phase insulin secretion. This biphasic insulin to control db/+ islets (0.64 ± 0.07 db/db vs. 1.0 ± 0.14 db/+, p<0.05, n=8). Next release pattern is perturbed in patients with type2 diabetes, resulting in we determined whether MMP-9 was decreased in vitro by high glucose or high abolished fi rst-phase and blunted second-phase insulin secretion; and we fatty acids. Mouse islets susceptible to glucose-induced beta-cell dysfunction reported this to be correlated to reduced islet levels of priming (Munc13- (DBA/2) were cultured for 48 hrs in 11.1, 22.2 or 33.3 mM glucose. High glucose 1, Munc18a) and SNARE proteins. Glucagon-like peptide-1 (GLP-1) bypasses did not decrease MMP-9 mRNA (1.0 11.1 mM vs. 1.38 ± 0.65 22.2 mM vs. 1.32 these insulin secretory defects of type2 diabetes through cAMP signaling ± 0.43 33.3 mM, p=0.49, n=3). Mouse islets susceptible to free fatty acid- pathways. We had reported that haplodefi cient Munc13-1+/- mouse exhibited induced beta-cell dysfunction (C57BL/6) were cultured for 48 hrs with and impaired biphasic insulin secretion, causing glucose intolerance mimicking without 1 mM palmitate. Palmitate decreased MMP-9 mRNA by 67% (0.33 ± type2 diabetes. Interaction between Munc13-1 and PKA substrate RIM is 0.11 palmitate vs. 1.0 control, p<0.05, n=3). critical for GLP-mediated rescue of exocytotic defects in Munc13-1-defi cient In conclusion, MMP-9 mRNA is decreased in islets from db/db diabetic islet β-cells. To further determine the precise exocytotic events mediated mice and by chronic palmitate but not high glucose. Thus, the hyperlipidemic by GLP-1 in rescuing insulin secretion, we employed total internal refl ection milieu of type 2 diabetes may contribute to decreased MMP-9 in the islet. fl uorescence (TIRF) microscopy technique to examine Munc13-1 defi cient This may in turn contribute to the islet lesion in diabetes including impaired mouse b-cells. First, we show that reduced fi rst phase secretion caused by vascularity and amyloid deposition. Munc13-1 defi ciency is attributed to reduced fusion of pre-docked granules and also newcomer granules. Reduced second phase secretion caused 2001-P by Munc13-1 defi ciency is attributed entirely to a reduction in newcomer Combination of an SGLT2 Inhibitor (BI 38335) with Linagliptin: Bene- granules. This means that Munc13-1 is a priming protein for both docked fi cial Effects on Islet Function and Insulin Sensitivity in db/db Mice granules and newcomer granules. Second, we found no difference in density LIHUA CHEN, THOMAS KLEIN, PO SING LEUNG, Hong Kong, China, Biberach, of docked granules at resting state between Munc13-1+/+ and Munc13-1+/- Germany β-cells confi rming that the defect caused by Munc13-1 defi ciency is not the Dipeptidyl peptidase (DPP)-4 inhibitors enhance incretin action and inability for granule to dock but rather its fusion competence after docking pancreatic beta-cell function. Sodium-glucose co-transporter (SGLT2) on the membrane. Third, GLP-1 rescue of biphasic insulin secretion caused inhibitors are promising therapies for type 2 diabetes due to their potential by Munc13-1 defi ciency is attributed mainly to increased recruitment of for glycemic control, independently of insulin levels. This study evaluated the newcomer granules in both fi rst and second phase secretion. effects of linagliptin (DPP-4 inhibitor) and BI 38335 (SGLT2 inhibitor), alone or in Supported by: Canadian Diabetes Association Postdoctoral Fellowship Award combination, on glucose metabolism, islet cell function, and insulin sensitivity and CIHR grant in mice. Diabetic C57BL/KsJ db/db and non-diabetic m+/db mice received 2004-P linagliptin 3 mg/kg; and/or BI 38335 1 mg/kg, or vehicle, once daily by gavage, Histamine Receptor H3 Regulates the Functions of Pancreatic β-Cells for 8 weeks. Glucose homeostasis and insulin sensitivity were assessed, in TADAHO NAKAMURA, TAKEO YOSHIKAWA, NAOYA NOGUCHI, HIROSHI addition to islet function, β/α cell ratios, and expression of protein or mRNA OKAMOTO, AKIRA SUGAWARA, KAZUHIKO YANAI, Sendai, Japan markers related to infl ammation and islet immune cells. Serum triglycerides Histamine has been implicated in a variety of biological processes such (TG) were also measured. After 8 weeks of combination therapy, signifi cant as smooth muscle contraction, gastric acid secretion and sleep-waking improvements were observed in glucose metabolism vs monotherapy: blood cycle. Histamine exerts its diverse effects through the interactions with four glucose levels fell by 46%; islet glucose-stimulated insulin secretion increased different subtypes (H1- H4) belonging to the superfamily of G-protein coupled by 4-fold; glucose tolerance improved by 55%; and insulin sensitivity increased receptors (GPCRs). Recent studies have revealed that this biogenic amine by 46%. In addition, serum TG were reduced by 62%, with combination therapy was also involved in appetite and satiety by regulating the hypothalamic vs monotherapy. Combination therapy was also associated with normalization neuronal activities, indicating that histamine played an important role of islet β/α cell ratios towards non-diabetic levels; decreases in islet immune in energy homeostasis. However, the effects of histamine on peripheral cell markers (eg CD53, CD68); and suppression of infl ammation (eg, IL-6, TNFα, metabolic tissues including pancreatic β-cells remained to be elucidated. In POSTERS MCP-1). The fi ndings of this study show that combination therapy with BI the present study, we aimed to reveal the functions of histamine receptors Islet Biology/ 38335 and linagliptin may have at least additive benefi ts on islet cell function in insulin secretion and β-cell proliferation. Insulin Secretion and insulin resistance, leading to improved glycemic control in db/db mice. First, we found that histamine receptor H3 (H3R) gene, encoding GPCR Supported by: Boehringer Ingelheim Pharmaceuticals, Inc. coupled to inhibition of adenylate cyclase via Gi/o proteins, was predominantly

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A539 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

expressed among histamine receptors in mouse pancreatic islets and MIN6 of mammals, and SMP30/GNL knockout (KO) mice display symptoms of cells using RT-PCR analysis. The H3R protein expression in the β-cells was scurvy when fed a vitamin C (VC) defi cient diet. The aim of this study was to confi rmed by western blotting and immunohistochemical analysis. Next, assess the role of VC on insulin secretion using SMP30/GNL KO mice as a we examined the effect of H3R in insulin secretion from MIN6 cells using model of VC defi ciency. Male SMP30/GNL KO mice and wild-type mice (WT) imetit, a selective H3R agonist. Imetit remarkably inhibited glucose-induced were divided into two groups: VC suffi cient and VC insuffi cient. Starting at insulin secretion in a dose-dependent manner, while basal secretion with 8 weeks of age, all groups had free access to water containing 1.5 or 0.0375 imetit was preserved. Imetit also decreased insulin secretion in response g/L of VC for 8 weeks. VC contents in the liver and serum of VC insuffi cient to KCl, which elicited Ca2+ infl ux and induced insulin secretion. However, SMP30/GNL KO mice (KO-) were signifi cantly lower than VC suffi cient imetit did not affect the increase in intracellular Ca2+ concentration induced SMP30/GNL KO mice (KO+). Supplementation of VC did not affect glucose by KCl, suggesting that imetit was involved in insulin secretion at the level tolerance in WT. In intraperitoneal glucose tolerance test, the blood glucose downstream of Ca2+ infl ux. In addition, we investigated the effect of imetit levels at 30 min were signifi cantly higher in KO+ than in WT. On the other on the proliferation of MIN6 cells and found that imetit attenuated BrdU hand, in KO-, signifi cantly high blood glucose levels were persisted until incorporation in MIN6 cells after insulin stimulation. 120 mins after glucose load (120 mins; KO- 241.1 ± 42.2 vs WT 131.3 ± 18.4 In this study, we have demonstrated for the fi rst time that H3R activation mg/dl, P < 0.01). Serum insulin levels in KO- were signifi cantly decreased at with imetit, an H3R agonist, inhibited insulin secretion. Imetit also inhibited 30, 60, 120 mins. The insulin tolerance test revealed high peripheral insulin increase in BrdU incorporation, suggesting H3R has an inhibitory effect on sensitivity in KO+. In the static incubation study of islets, insulin secretion β-cell proliferation. These lines of evidence might indicate that H3R plays an in response to 20 mM glucose for 60 mins was signifi cantly decreased only essential role in β-cell functions. in KO-. Furthermore, islet ATP contents at 20 mM glucose were signifi cantly decreased only in KO-. These data indicate that the site of VC action lies 2005-P between the metabolism of glucose and ATP production by mitochondrial Impaired Glucose Stimulated Insulin Secretion (GSIS) Is Mediated oxidative phosphorylation. A defi ciency of SMP30 itself impairs the distal by Interleukin-1β (IL-1β) Induced Inhibition of Mitochondrial Cyto- portion of the insulin secretion pathway in VC suffi cient condition. chrome C Oxidase GENYA AHARON-HANANEL, ANN SAADA, CARMIT MANTZUR, FIONA VERNEA, 2007-P ITAMAR RAZ, SARAH WEKSLER-ZANGEN, Jerusalem, Israel Metabolic Deceleration of the Pancreatic β-Cell: A Common Mode Cytokines were suggested to impair β-cell function via the production of of Action for Insulin-Sensitizing Agents? nitric oxide (NO), a potential inhibitor of cytochrome C oxidase (COX). Cohen JULIEN LAMONTAGNE, MARIE-LINE PEYOT, ÉRIK JOLY, S.R. MURTHY MADIRAJU, diabetes-sensitive rats (CDs) maintain normal GSIS on regular diet (RD). MARC PRENTKI, Montreal, QC, Canada When fed high sucrose low copper (Cu) diet (HSD), they develop blunted Thiazolidinediones (TZD) and metformin (Metf) are insulin-sensitizing GSIS associated with peri-islet infi ltration of macrophages secreting IL1-β. agents which have a protective effect on pancreatic β-cells. The latter may be The resistant (CDr) rats maintain normal GSIS on either diet. Recently we a consequence of improved insulin sensitivity but may as well stem from non- observed that the inhibitory effect of IL1-β on the GSIS of CDrRD and CDsRD genomic actions on β-cells. For example, both compounds acutely activate islets may be prevented by the iNOS inhibitor, L-NOARG. Our study aimed AMP-kinase in various cell types including β-cells. We recently presented to examine the interaction between impaired COX activity, defective GSIS evidences that the TZD pioglitazone (Pio) acutely inhibits glucose-induced and whether Cu, a co-factor for COX, could prevent the inhibitory effect insulin secretion (GIIS) in rat islets and INS 832/13 cells, and causes “metabolic of IL1-β. Freshly isolated CDsRD islets stimulated by 16.7 mmol/l glucose deceleration” of β-cells: it reduces GIIS and key metabolic pathways at exhibited a 2 fold lower GSIS capacity and reduced COX activity (black vs. intermediate, but not basal or elevated glucose. Looking for a possible stripe columns in Fig 1A & B) compared to CDrRD. 24h pre-incubation with general mode of action for insulin-sensitizing agents on β-cells, we expanded IL1-β (10U/ml) induced 49% reduction of the GSIS (Fig 1A) and 12% decrease our studies to Metf and investigated the effect of Pio on in vivo glucose in COX activity (Fig 1B) in CDrRD islets. In CDsRD islets, IL1-β induced a homeostasis. We treated acutely INS 832/13 cells with Metf (5mM, ≤210 complete inhibition of GSIS (Fig 1A) and a marked reduction in COX activity min) and measured GIIS and parameters of energy metabolism (glucose and (Fig 1B). Pre-incubation with the combination of IL1-β and Cu (0.4umol/l- free fatty acid (FFA) oxidation and mitochondrial membrane potential (ΔΨm)). 0.7umol/l) prevented the reduction in GSIS (Fig 1A) and restored COX activity We also monitored the effect of an oral administration of a single-dose of Pio (Fig 1B) in CDrRD and CDsRD islets. The genetically determined low GSIS (30mg/kg) on glucose homeostasis in Wistar rats through hyperglycemic (HGC) capacity and reduced COX activity of CDsRD islets underlies the sensitivity and hyperinsulinemic-euglycemic clamps (HIEC). In INS 832/13 cells, Metf, alike to the deleterious effect of IL1-β. Cu may exerts its protective effect by Pio, caused a right shift in glucose sensitivity for GIIS, reduced both glucose acting directly as a necessary COX co-factor or indirectly by preventing the and FFA oxidation, and partially dissipated ΔΨm. In vivo, Pio reduced insulinemia IL1-β mediated NO inhibition. Our study implies a direct link between low Cu during HGC with no change in insulin clearance or insulin sensitivity (according concentrations, reduced COX activity and diminished GSIS. to HGC and HIEC), indicating an inhibitory effect on GIIS. Thus, Metf and Pio have acute and direct effects on the β-cell, causing its metabolic deceleration and an inhibition of GIIS at submaximal glucose concentrations. In addition we show in vivo that Pio acutely inhibits GIIS independently of changes in insulin sensitivity and clearance. The new data support the concept of “metabolic deceleration” which may be, along with direct reduction of GIIS, involved in the mechanisms whereby widely used oral antidiabetic agents reduce insulin resistance and hyperinsulinemia. Mild metabolic deceleration may represent a new avenue to treat type 2 diabetes. Supported by: CIHR, CDA and FRSQ

2008-P Metabolism of Amylin and Insulin in Insulinoma Cells with Reduced Insulin-Degrading Enzyme JANET FAWCETT, JENNIFER L. LEVY, WILLIAM C. DUCKWORTH, Phoenix, AZ 2006-P Many patients with Type 2 diabetes (T2DM) have islet amyloid deposits Insulin Release from Pancreatic Islet in Vitamin C-Defi cient Sene- consisting of amylin. These deposits may contribute to β-cell dysfunction. scence Marker Protein-30/Gluconolactonase Knockout Mice Amylin is produced in non-diabetics but amyloid does not form so additional TAKAFUMI SENMARU, GOJI HASEGAWA, AKIHITO ISHIGAMI, YOSHITAKA factors seem to be required. Inhibition of insulin-degrading enzyme (IDE) KONDO, HIROSHI OKADA, MAI ASANO, MASAHIRO YAMAZAKI, MICHIAKI increases formation of amyloid deposits. The effect of this on insulin release FUKUI, NAOTO NAKAMURA, Kyoto, Japan, Chiba, Japan, Tokyo, Japan is unknown. In addition to insulin IDE degrades amylin and changes in this Senescence marker protein-30 (SMP30), which functions enzymatically as enzyme may cause accumulation of amyloid deposits. Genetic studies have

POSTERS gluconolactonase (GNL), is an androgen-independent factor that decreases suggested that IDE is a T2DM susceptibility gene. One way that reduced Islet Biology/

Insulin Secretion with aging. Recent studies have revealed that a reduction of SMP30 IDE activity may lead to or exacerbate T2DM is to alter islet metabolism. expression could account for the age-associated deterioration of cellular IDE was reduced by siRNA in cultured RIN-m5F and βTC-6 cells. Insulin and functions. On the other hand, GNL is involved in L-ascorbic acid biosynthesis amylin degradation, insulin secretion and formation of amyloid deposits were

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A540 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM measured. In RIN cells IDE mRNA levels were reduced ∼ 80%. Protein levels stimulation of ATP and calcium infl ux compared to no palmitate (control; were reduced 65%. Control cells degraded 21±6 % of added insulin; this was table). Similarly, in C57BL/6.NEP-/- islets exposed to palmitate, basal insulin reduced to 9±4% in cells with reduced IDE. Similar results were seen with secretion was increased and glucose-stimulated ATP levels reduced. In degradation of amylin. Incubation with amylin increased amyloid deposits. contrast however, in C57BL/6.NEP-/- islets, glucose-stimulated insulin Reducing IDE enhanced this effect signifi cantly. RIN cells (control vs IDE siRNA) secretion and calcium infl ux remained unchanged in the presence or absence released 2.2±0.7 vs 4.3±0.9ng/ml (P<0.01) insulin in the absence of glucose. of palmitate. Glucose (4mM) increased insulin release in the control and reduced IDE cells (2.9±0.4 vs 5.6±0.9ng/ml). As the release of insulin from RIN cells is minimally responsive to glucose we also analyzed the effect of reducing IDE on the metabolism of βTC-6 cells. An IDE knockdown of 85±2% of control was achieved. Insulin degradation was reduced 85±6%. βTC-6 cells are more responsive to glucose for insulin secretion. Glucose (2mM) increases insulin secretion from 107±17 to 156±25ng/ml in control cells. This stimulation was diminished by reducing IDE (130±21 vs. 147±29ng/ml). These results further suggest that IDE is involved in the removal of amylin by β-cells and that alterations seen in IDE activity inhibit the glucose stimulated insulin release. This would lead to insulin In summary, the absence of neprilysin selectively protects against resistance and as IDE has been suggested to be a susceptibility gene for T2DM palmitate-induced reductions in glucose-stimulated insulin secretion and this could be a mechanism by which this could occur. calcium infl ux, but not palmitate-induced reductions in glucose-stimulated ATP levels. Thus, the mechanism for protection conferred by neprilysin 2009-P deletion involves a step(s) in the insulin secretory pathway distal to ATP Microarray Analysis of Isolated Human Islet (HI) Transcriptome production but at or proximal to calcium infl ux. Consequently, in states and the Role of the Ubiquitin-Proteasome System (UPS) in Type 2 of elevated FFAs, interventions to reduce islet neprilysin levels may be Diabetes (T2D) Beta Cell Dysfunction benefi cial for β-cell function. MARCO BUGLIANI, ROBIN LIECHTI, HWANJU CHEON, LORELLA MARSELLI, MARA SULEIMAN, IOANNIS XENARIOS, MYUNG-SHIK LEE, PIERO MARCHETTI, 2011-P Pisa, Italy, Lausanne, Switzerland, Seoul, Republic of Korea Potential Role of Direct K-ATP Channel Opening in the Anti-Diabetic Beta cell failure is crucial to the onset and progression of T2D. Limited Actions of Rimonabant and Ibipinabant information is however available on the molecular phenotype of islet cells in CHRISTOPHER J. LYNCH, DAVID J. HEAL, SHARON C. CHEETHAM, KEITH DICKIN- human T2D. We performed microarray analysis of HI prepared from T2D and SON, PETER-COLIN GREGORY, MICHAEL FIRNGES, STEPHANIE GOSHORN, control (Ctrl) individuals and then focused on the UPS, the major intracellular DANIA REICHE, LECHOSLAV TURSKI, JOCHEN ANTEL, Hershey, PA, Nottingham, pathway for protein degradation. United Kingdom, Hannover, Germany Overall, pancreata/islet preparations from 15 T2D and 20 Ctrl individuals We sought to understand the mechanism underlying the weight loss with similar clinical characteristics were studied. Gene expression was independent effects of two cannabinoid 1 receptor (CB1) inverse-agonists evaluated by microarray analysis (Affymetrix HG U133A) and quantitative which improve insulinemia following chronic treatment. Pursuing an open RT-PCR of selected genes. Ubiquitinated proteins were assessed by approach we performed glucose tolerance tests (GTT) and measured plasma immunohistochemistry and proteasome activity (trypsin-like) was measured insulin concentrations not only after chronic, but also after acute treatments by a luminescent assay. The effects of a proteasome inhibitor (Z-Leu-Leu- in either Zucker rats, other rodent models of obesity, CB1 or SUR1 KO mice. By Leu-Al) were also assessed. surprise we observed in acute studies a CB1-independent, SUR1 dependent Microarray analysis showed 1,345 probe sets differently expressed in diazoxide/NN414 (K-channel openers, KCOs)-like effect of rimonabant and T2D islets (59 up- and 1,286 downregulated). They identifi ed 1,230 genes, ibipinabant on glucose tolerance, with decreasing peak plasma insulin involved in many HI structures and functions, including the UPS. Gene during a GTT. These compounds dose-dependently inhibited GSIS with a ∼5- ontology and KEGG analyses identifi ed 22 biological processes and 15 fold better potency than diazoxide and equal or similar potency to NN414. pathways, respectively, related to genes downregulated in T2D. Gene set For further substantiation of the initial hypothesis we performed glucose enrichment analysis showed that 195 and 42 gene sets were positively and stimulated insulin secretion (GSIS) in perifused rat islets and MgATP- negatively enriched in T2D. Again, these evaluations pinpointed consistent dependent allosteric reglation of 3H-glibenclamide binding in membranes alterations of the UPS in T2D HI. We therefore performed quantitative RT- from HEK293 cells stably over-expressing human Kir6.2/SUR1. In addition PCR experiments, that demonstrated signifi cant downregulation of UBE2K, we studied distomers and eutomers of Ibibinabant (inverse agonist) as well PSMB7 and UCHL1 (genes of the UPS) in T2D HI. Immunohistochemistry as agonists (WIN55212, CP99540) in those assays. We conclude that the revealed greater ubiquitin accumulation and reduced proteasome activity investigated CB1 inverse agonists directly bind to and allosterically regulate (4364±1975 vs 8748±4238 RLU/ng DNA, p<0.02) in T2D HI. Z-Leu-Leu-Leu- Kir6.2/SUR1 K-ATP channels at the KCO site like known KCOs. This could Al suppressed proteasome activity of Ctrl HI by 80-90% (p<0.01), which account for their acute effects on GSIS and insulinemia and supports the was associated with lower glucose-stimulated insulin secretion (45±7% of idea that CB1 blockers chronically improve insulinemia in obese animals, untreated islets, p<0.01). at least partially, through a CB1-independent, SUR1-dependent mechanism These results show the presence of many changes in HI transcriptome shared with KCOs. in T2D and underline the importance of UPS alterations as a mechanism Supported by: Grants from Solvay Pharmaceuticals and NIH DK084428, associated to beta cell dysfunction in human T2D. DK062880 ADA-Funded Research Supported by: European Community, IMIDIA project 2012-P 2010-P PSP/Reg and hsCRP Serum Levels May Discriminate HNF1A from Neprilysin Defi ciency Protects Against Palmitate-Induced Impair- HNF4A-MODY ments in Insulin Secretion and Calcium Infl ux Independent of ATP MA P. KYITHAR, SIOBHAN BACON, CAROLINE BONNER, JASMIN SCHMID, ROLF Levels GRAF, JOCHEN H.M. PREHN, MARIA M. BYRNE, Dublin, Ireland, Zurich, Switzerland SAKENEH ZRAIKA, Seattle, WA PSP/reg (Pancreatic stone protein/regenerating protein) is a protein of Chronically elevated free fatty acids (FFAs) impair β-cell function. We the Reg family that is produced in β-cells during injury and regeneration. have shown that the protein neprilysin is synthesized in islets, its activity We have shown that the induction of PSP/reg occurs during the process of increases with FFA exposure, and its deletion protects against FFA-induced β-cell apoptosis, and that elevated serum PSP/reg levels can be detected reductions in glucose-stimulated insulin secretion. Thus, we now sought to in subjects with Hepatocyte Nuclear Factor (HNF) 1A-maturity onset determine the mechanism for protection conferred by neprilysin deletion. diabetes of the young (MODY) and type 1 diabetes (Bonner et al., Diabetes, C57BL/6 and C57BL/6.NEP-/- (neprilysin defi cient) mouse islets (n=5 per 59(11):2799-808). As genetic testing is expensive it is important to identify group) were cultured for 48 hours in the absence or presence of 1 mM biomarkers that may discriminate between different subtypes of MODY. POSTERS

palmitate after which basal and glucose-stimulated insulin secretion, ATP Previous studies have indicated that serum levels of highly-sensitive Islet Biology/ levels and calcium infl ux were measured. C-reactive protein (hsCRP), a known HNF1A target gene, are signifi cantly Insulin Secretion In C57BL/6 islets, palmitate exposure increased basal and decreased reduced in HNF1A-MODY subjects. We here demonstrate that PSP/reg stimulated insulin secretion, and this was associated with reduced glucose levels may be able to discriminate HNF4A-MODY from HNF1A-MODY. Serum

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A541 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM

PSP/reg levels were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY In insulin-secreting MIN6 cells, Rho GTPase Cdc42 was activated by glucose subjects. The two groups were matched in age, BMI, diabetes duration, stimulation in a concentration-dependent manner, while it was not activated blood pressure, lipid profi le and aspirin and statin use. PSP/reg levels were by potassium stimulation. GIIS in Cdc42-knocked down pancreatic islets was signifi cantly lower in HNF4A subjects when compared to HNF1A subjects reduced signifi cantly compared with that in control islets. Immunoprecipitation (10.82±1.45 vs. 14.86±1.24 ng/ml, P=0.024). Experimental studies in INS-1 with anti-Cdc42 antibody revealed that Cdc42 activated by glucose stimulation cells inducibly expressing a dominant-negative (DN)-HNF4A confi rmed interacts with N-WASP (Neural Wiskott Aldrich Syndrome Protein) in MIN6 that PSP/reg induction required a functional HNF4A. Serum hsCRP levels cells. N-WASP is known to promote actin polymerization through activation determined by immunonephelometry were signifi cantly higher in HNF4A- of actin nucleation factor Arp2/3 complex. To clarify the role of N-WASP- MODY than in HNF1A-MODY subjects (1.74±0.94 vs. 0.46±0.12 mg/L, mediated actin dynamics in insulin secretion, we constructed N-WASP mutant P=0.002). Again, expression of the CRP gene was signifi cantly reduced in (N-WASPΔWA), which lacks the actin-binding region WA, and introduced the INS-1 cells expressing a DN-HNF1A, but was normal in cells expressing a mutant together with AcGFP1-labeled actin into isolated pancreatic β-cells by DN-HNF4A. Parallel measurements of serum PSP/reg and hsCRP levels were adenovirus-based gene transfer. When visualized by total internal refl ection able to discriminate HNF1A- and HNF4A-MODY subjects. fl uorescence microscopy (TIRFM), the fl uorescence intensity of AcGFP1-actin at plasma membrane was enhanced in wild-type N-WASP-introduced β-cells in the presence of high concentrations of glucose, while it was not enhanced in N-WASPΔWA-introduced β-cells. αIn addition, in pancreatic islets infected with adenovirus carrying N-WASPΔWA, GIIS was reduced signifi cantly compared with that in wild-type N-WASP-introduced islets. Furthermore, knockdown of p34-Arc, a component of Arp2/3 complex, in pancreatic islets caused a signifi cant decrease in GIIS. We then performed perifusion experiment using MIN6 cells infected with adenovirus carrying N-WASPΔWA. Interestingly, the second phase of GIIS was specifi cally impaired in these cells. Taken together, the results indicate that Cdc42/N-WASP signaling is activated by glucose stimulation and that it is involved in the second phase of GIIS In conclusion, our study suggests that serum PSP/reg & hsCRP levels may through regulation of actin dynamics. be of clinical use in distinguishing HNF1A from HNF4A MODY subjects. 2015-P Role of Derlin-1 in Processing and Expression of β-Cell KATP Channels 2013-P FANG WANG, FEI-FEI YAN, SHOW-LING SHYNG, Portland, OR, Beijing, China Regulation of Mitochondrial ATP Synthesis and Export by Phosphate The β-cell ATP-sensitive potassium (KATP) channel regulates insulin secretion in Permeabilized Insulin-Secreting Cells by linking glucose metabolism with membrane potential and consists of four KYU-SANG PARK, XIANGLAN QUAN, RANJAN DAS, SHANHUA XU, ANDREAS sulfonylurea receptor 1 (SUR1) subunits and four pore-forming Kir6.2 subunits. WIEDER KEHR, CLAES B. WOLLHEIM, Wonju, Republic of Korea, Geneva, Switzerland The number of KATP channels in the plasma membrane determines the sensitivity Mitochondria generate ATP and metabolites essential for β-cell of β-cells to glucose, yet the molecular mechanisms that control surface metabolism-secretion coupling. Their function in turn is regulated by ionic expression of KATP channels are poorly understood. Previously we showed that changes in the cytosol. Here, we have investigated the effects of cytosolic in the rat β-cell line INS-1, KATP channel proteins undergo endoplasmic reticulum inorganic phosphate (Pi) on the mitochondrial electrochemical gradient (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway and and the synthesis of ATP in α-toxin permeabilized rat insulinoma INS- that inhibition of proteasome function leads to an increase in channel surface 1E cells. Mitochondrial matrix pH was monitored using the pH sensitive expression. Derlin-1 is a protein involved in retro-translocation of misfolded or probe mitoAlpHi. The mitochondrial membrane potential was recorded unassembled substrate proteins destined for polyubiquitination and degradation ratiometrically using the lipophilic dye JC-1. To assess adenine nucleotide by cytosolic proteasomes. In the present study, we investigated whether translocase (ANT) activity, ATP levels were measured both in the supernatant Derlin-1 plays a role in ERAD, and in turn, biogenesis effi ciency of KATP channels. (released ATP) and after lysis of the permeabilized cells (stored ATP) using We show, by mass spectrometry, that Derlin-1 and an associated protein p97 a luciferase assay. Both stored and released ATP were substantially are present in the KATP channel interactome affi nity purifi ed from INS-1 cells increased by succinate, glycerol-phosphate or TMPD/ascorbate, substrates expressing exogenous Kir6.2 and FLAG-tagged SUR1. Interactions of both SUR1 respectively for the TCA cycle plus complex II, complex II only and and Kir6.2 with Derlin-1 and p97 in INS-1 cells were further confi rmed by co- cytochrome c. Substrate-induced increases in released but not in stored immunoprecipitation. In metabolic pulse-chase and western blot experiments, ATP were abolished by removal of extramitochondrial ADP or by inhibition of overexpression of Derlin-1 led to a decrease in the signal of the complex- ANT with atractyloside. ATP release thus strictly depends on ANT activity. glycosylated SUR1, the abundance of which refl ects the number of assembled Remarkably, removal of extramitochondrial Pi blocked ATP release. Under channels that have exited the ER. Correspondingly, there was a decrease these conditions, substrates still increased stored ATP, indicating suffi cient in surface expression of KATP channels. In contrast, knockdown of Derlin-1 by intramitochondrial Pi available for ATP synthesis. Raising extramitochondrial shRNA resulted in increased processing of SUR1 from the core-glycosylated Pi from the usual 1 to 10 mM only had small effects on stored while greatly to the complex-glycosylated form and a corresponding increase in surface enhancing released ATP. The driving force for ANT is the electrical potential expression of KATP channels. Our results show that Derlin-1, by being involved across the inner mitochondrial membrane, which was markedly increased in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis effi ciency by P , on top of substrate-induced hyperpolarization. Through its proton i and surface expression of KATP channels. We propose that physiological or dependent uptake, Pi also caused a moderate reduction of the pH gradient pathological changes in Derlin-1 expression level may affect glucose-stimulated across the inner mitochondrial membrane. Inorganic arsenate (As ), mimicked i insulin secretion by altering surface expression of KATP channels. the actions of Pi on the electrical potential and matrix pH. As Asi is not substrate for ATP synthesis, stored ATP was not enhanced while nucleotide 2016-P release was stimulated. These results demonstrate a novel role for cytosolic Small Molecules Targeting CAMP Generation Restores β-Cell P in metabolism-secretion coupling by reinforcing mitochondrial ATP export i Function in Islets from Type 2 Diabetic Patients through an action on the mitochondrial membrane potential. ALEXANDER BALHUIZEN, SARHEED MUHAMMED, RAJESH KUMAR, ALBERT SALEHI, Malmö, Sweden Accumulating data suggest that nitric oxid (NO) produced by inducible 2014-P NOS (iNOS) play an essential role in the β-cell dysfunction and apoptosis. Role of Cdc42/N-WASP Signaling-Regulated Actin Dynamics in The main purpose of this study was to investigate whether the expression Insulin Secretion of iNOS in the islets of diabetic subjects could be modulated by small TADAO SHIBASAKI, EITAI UENISHI, HARUMI TAKAHASHI, HITOMI HAMAGUCHI, molecules which increase cAMP generation by targeting adenylate cyclase MASAO TATEBE, YUTAKA OISO, SUSUMU SEINO, Kobe, Japan, Nagoya, Japan and phosphodiesterase. Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, iNOS expression was analyzed by confocal microscopy, Western blot and

POSTERS how actin dynamics is regulated by intracellular signaling is not known. Here Islet Biology/ qPCR in islets from both human type 2 diabetic and non-diabetic donors. Hormone

Insulin Secretion we demonstrate that actin dynamics is regulated by Cdc42/N-WASP signaling, secretion was determined with RIA. Cell viability was measured with ELISA. which is activated by glucose stimulation, and that the dynamics participate in Confocal microscopy revealed that iNOS is expressed in pancreatic islet temporal regulation of glucose-induced insulin secretion (GIIS). cells (insulin, glucagon and somatostatin) of type 2 diabetic subjects and that

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A542 ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETIONCATEGORY COUPLING AND METABOLISM no iNOS was detected in islets from normal human donors. iNOS mRNA and upregulated enzymes, by RNA interference reduced GIIS to about 75% of control protein expression was markedly higher in type 2 diabetic islets vs normal treatment. Insulin secretion induced by KCl and cell proliferation were, however, human islets. The hormone secretory pattern of type 2 diabetic pancreatic unaffected in MIN6 cells with RNA interference. These data indicate that sulfate islets incubated at different glucose concentrations showed a reduced insulin groups of HS (possibly 3-O-sulfate groups modifi ed by 3-O-sulfotransferase) play secretion response to glucose. Cell viability assay showed that islet cells of important role(s) in the insulin secretory pathway, selectively through upstream type 2 diabetic subjects are more sensitive to long-term culture in glucotoxic of membrane depolarization in β-cells. environment. Islets incubated with PDE inhibitors IBMX or Rolipram showed Supported by: KAKENHI (20590312), Japan an increased insulin secretion while islets incubated with Zaprinast displayed a less increase then islets incubated with Rolipram or IBMX. 2019-P Our data shows that long term hyperglycaemia seen in human type 2 SUMO Modulation of Incretin Responsiveness in Hyperglycemia diabetic patients, based on their HbA1c, might result in the expression of SINDHU RAJAN, MICHAEL S. THOMPSON, LOUIS H. PHILIPSON, Chicago, IL iNOS and thus infl uencing b-cell viability in the pancreatic islets consequently Incretin-based therapy is becoming central to the treatment of Type-2 diabetes. could disrupt the normal b-cell function. Our data also shows that small The gut incretin hormones, GLP-1 and GIP (Glucose-dependent insulinotropic molecules targeting adenylate cyclase and phosphodiesterase could be polypeptide), contribute to post-prandial insulin secretion. However, patients important players to restore β-cell function in type 2 diabetic patients. with Type-2 diabetes can exhibit a substantially impaired response to GLP-1 and lack of response to GIP treatment, compared to non-diabetic controls. Here, 2017-P we show that the expression of astress responsive protein, SUMO-1 (Small Stimulation-Secretion Coupling in Human Primary Islet Beta Cells Ubiquitin- related modifi er) isup-regulated 3-6 fold in mouse islets exposed DALIANG LI, SHIUHWEI CHEN, CALLAN KAUT, WEN-HONG LI, Dallas, TX to high glucose and in human islets from Type-2 diabetic patients. Activation Human islet beta cells show important differences from rodent beta of the incretin signaling pathway stimulates adenylate cyclase, resulting in cells in terms of cytoarchitecture, electrical activity, cellular signaling an increase in cytosolic cAMP. Here, we show that SUMO-1 down-regulates and metabolism. Studies on stimulation-secretion coupling in primary incretin receptor signaling. Spatial and temporal changes in cAMP signaling human beta cells have been hampered by two major obstacles: (1) Limited following agonist stimulation of the incretin receptors were studied using a FRET availability of human tissues; and (2) Lack of a sensitive, non-invasive based biosensor where binding of cAMP induces a conformational change in imaging assay for monitoring the release of native insulin granules with high the protein that results in progressive loss of FRET. Over-expression of SUMO- spatial and temporal resolution. Towards fi nding a solution to overcome the 1 caused 5 and 3.7 fold reduction in FRET ratio when treated with a GLP-1 second problem, we recently have developed a fl uorescence imaging assay receptor agonist in mouse insulinoma (MIN6) cells and mouse primary beta cells for tracking insulin secretion in fully intact beta cells. The assay is based on respectively. Similarly, stimulation of the GIP receptor in MIN6 cells expressing a new fl uorescent Zn2+ sensor, ZIMIR (Zinc Indicator for Monitoring Insulin SUMO-1 resulted in 3 fold reduced FRET ratio. Covalent and non-covalent Release), which displays more than 70-fold fl uorescence enhancement modifi cation by SUMO resulted in impaired forward traffi cking of the GLP-1 when it binds Zn2+, a divalent cation that is co-released with insulin during receptor to the cell membrane with accumulation of the receptors in the ER. exocytosis. When added to cells at low micromolar concentration, ZIMIR We conclude that hyperglycemia-induced over-expression of SUMO-1, results was taken up by cells rapidly both in culture and throughout dissected in intracellular retention of the incretin receptors and in this way contributes islets. Upon cellular uptake, ZIMIR selectively detects local Zn2+ elevation to the down-regulation of receptor signaling. Understanding the mechanisms immediately above plasma membrane with very high sensitivity. The probe of incretin receptor down-regulation is likely to result in improved therapeutic is applicable to both wide fi eld and laser scanning fl uorescence microscopy. strategies for treating diabetes. As an initial effort to systematically study insulin release in primary human Supported by: NIDDK, Merck IISP beta cells, we dissociated human islets and cultured the resulting primary cells (or cell clusters) for ∼ 1 day before imaging, attempting to characterize 2020-P their insulin release dynamics in several aspects: (1) The spatiotemporal The Effects of SK-0403, a Highly Selective DPP-4 Inhibitor, on the profi le of insulin release at subcellular resolution after stimulating cells Amelioration of Glucose Intolerance in Haploinsuffi ciency of Gluco- with secretagogues such as KCl, carbachol, and glucose; and examining kinase Mice on a High-Fat Diet insulin release sites with respect to cell morphology and cell-cell contact; (2) KEIZO NAKAYA, NAOTO KUBOTA, ISEKI TAKAMOTO, TETSUYA KUBOTA, HISAYUKI 2+ Moment-to-moment correlation of cellular signaling events (Ca transition for KATSUYAMA, HIROYUKI SATO, SHINJI HASHIMOTO, MORITAKA GOTO, TAKAHITO example) with insulin secretory activity by multi-color imaging; (3) Dissection JOMORI, KOHJIRO UEKI, TAKASHI KADOWAKI, Tokyo, Japan, Mie, Japan 2+ 2+ of the relative contribution of Ca infl ux and intracellular Ca release to the Haploinsuffi ciency of glucokinase (GckKO) mice with impaired insulin 2+ overall Ca activity and insulin release in primary human beta cells. secretion on a high-fat (HF) diet have been reported to develop diabetes Supported by: JDRF, NIH and Welch Foundation because of a lack of β cell hyperplasia in response to insulin resistance via impaired up-regulation of IRS-2. In this study, we investigated the long-term 2018-P effects of SK-0403 on these mice as an animal model of type 2 diabetes Sulfation Fine Structure of Heparan Sulfate Is Implicated in the linked to obesity. HF diet-fed wild-type (HF-Wild) mice were used as a Regulation of Insulin Secretion control. HF-Wild and HF-GckKO mice were treated with two doses of SK- IWAO TAKAHASHI, KAZUAKI OHASHI, NAUSHEEN J. SHERVANI, KOJI NATA, 0403 as dietary admixture (0.05% and 0.3%) for 10 weeks. 0.3% SK-0403 Yahaba, Japan signifi cantly decreased the body weight gain with suppression of food intake Heparan sulfate (HS) is linear polysaccharides consisting of repeating in both genotypes. 0.3% SK-0403 also ameliorated insulin resistance and disaccharide unit backbone onto which is superimposed a complex pattern of glucose intolerance in HF-GckKO mice, being similar to those observed in modifi cations, most notably addition of sulfate groups. The produced polymorphic HF-Wild mice, indicating that the effects of SK-0403 on the suppression of sulfated sequence motifs are responsible for binding to a variety of signaling body weight gain and amelioration of insulin sensitivity were independent molecules and modulating their biological functions. Recently, we found HS of glucokinase. On the other hand, the body weight and food intake after is localized exclusively around β-cells in islets of adult mice and required for 0.05% SK-0403 treatment were unchanged in both genotypes. Insulin islet morphogenesis, β-cell proliferation and insulin secretion. Depletion of HS sensitivity was also unchanged by 0.05% SK-0403 treatment, suggesting in β-cells using the technique of knockout mouse or enzymatic removal of HS that the amelioration of insulin resistance by 0.3% SK-0403 treatment might decreased glucose-induced insulin secretion (GIIS). However the contribution be largely dependent on suppression of body weight gain. 0.05% SK-0403 of sulfate fi ne structure in HS to β-cell function is still unclear. In the present improved glucose tolerance with increment of insulin secretion in both study, we fi rst examined GIIS after removal of sulfate groups by sodium chlorate genotypes. Moreover, 0.05% SK-0403 signifi cantly increased β-cell mass (a competitive inhibitor of glycosaminoglycan sulfation). We next examined the via up-regulation of IRS-2 protein levels and phosphorylation of CREB in the ef fect of sulfate groups to GIIS by silencing expression of sulfotransferases. GIIS in islets of both genotypes. However, the effects of SK-0403 on the increment the sodium chlorate-treated mouse pancreatic islets and MIN6, insulin-secreting of insulin secretion and up-regulation of IRS-2 and P-CREB of islets in HF- mouse insulinoma cell line, showed about 60% and 30% decrease compared GckKO mice were impaired as compared with in HF-Wild mice, suggesting to the controls. The sodium chlorate-treated MIN6 cells showed a decrease in that the effects of SK-0403 were, at least in part, dependent on glucose POSTERS cell proliferation compared to the controls. Importantly, addition of exogenous metabolism via glucokinase in the β-cells. In conclusion, high dose of SK- Islet Biology/ soluble HS in culture recovered GIIS of the MIN6 cell treated with sodium 0403 improved glucose tolerance with suppression of body weight gain and Insulin Secretion chlorate. Sodium chlorate-treatment upregulated some of sulfotransferase amelioration of insulin resistance, whereas low dose of SK-0403 improved mRNAs detected in MIN6 cells. Silencing of a 3-O-sulfotransferase, one of these glucose tolerance with increased insulin secretion.

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A543 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

2021-P Vitamin D Modulation of Pancreatic Islet Renin-Angiotensin System & 2023-P Function and Glucose Homeostasis in Vitamin D Defi cient Mice Effects of Small FFA1 Receptor Agonists in Insulin Secreting Cells QIANNI CHENG, BARBARA J. BOUCHER, PO SING LEUNG, Hong Kong, China, SUSANNE ULLRICH, MICHAEL PFLEIDERER, KATHRIN LIEBSCHER, FELICIA London, United Kingdom RANTA, KATJA NOACK, GISELA DREWS, ELISABETH CHRISTIANSEN, HANS- Serum 25(OH)-vitamin D levels relate inversely to blood glucose concen- ULRICH HÄRING, TROND ULVEN, Tuebingen, Germany, Odense, Denmark tration and vitamin D insuffi ciency is associated with human hyper tension High concentrations of free fatty acids (FFAs) are known to enhance glucose- and diabetes. The islet renin-angiotensin system (RAS) modulates glucose induced insulin secretion, whereas chronic exposure of insulin secreting cells homeostasis, inappropriate activity of the RAS reducing islet function. to FFAs induces apoptotic cell death (lipotoxicity). The insulinotropic effect Meanwhile, renal renin production increases in kidneys lacking functional of FFA depends on the activation of free fatty acid receptor 1 (FFA1/GPR40) vitamin D receptor pathways. We, therefore, hypothesize that vitamin which couple to phospholipase C. However, lipotoxicity is rather due to a D insuffi ciency in mice causes alterations in islet renin and RAS activity, cellular accumulation of FFA metabolites. The aim of the present study was contributing to islet dysfunction. In this study, the relationships between to investigate the effects of FFA1 activation by small receptor agonists on vitamin D status, local islet RAS activity and islet beta-cell function were glucose-dependent insulin secretion and apoptotic cell death and to analyze examined by assessing islet RAS component expression, islet architecture, the underlying molecular mechanism. and glucose homeostasis in diet-induced vitamin D defi cient mice compared The synthetic small agonist (TUG-469, 10 µM) which contains a para- to normal chow diet-treated controls. We found that vitamin D defi cient diet- substituted dihydrocinnamic acid moiety and binds specifi cally to FFA1 treated mice exhibited normal growth, with low circulating 25(OH)D3 and enhanced insulin secretion in INS-1E cell at stimulatory (12 mM) but not 1,25(OH)2D3 concentrations but increased glycemia and impaired glucose at basal (2.8 mM) glucose. When INS-1E cells became glucose-insensitive tolerance while major islet RAS components exhibited over-activation at during aging, TUG-469 still signifi cantly increased insulin release at high but 2+ both mRNA and protein levels, especially of renin, a rate-limiting enzyme of not at low glucose. In parallel, TUG-469 increased [Ca ]i only at 12 mM but the RAS. Furthermore, the increased islet size and decreased transcription not at 2.8 mM glucose. Surprisingly, stimulation of secretion by TUG-469 of insulin receptor and its substrates observed in vitamin D defi cient islets was not inhibited by ryanodine (1 and 100 µM) which inhibits mobilisation 2+ indicated diminished local islet insulin signaling and repletion with vitamin D of Ca from ryanodine-sensitive stores nor by xestospongin (1 and 10 µM) 2+ 2+ improved glycemic control in vitamin D defi cient mice. Although relationships which inhibits Ca release from IP3-sensitive intracellular Ca stores. between islet RAS and vitamin D metabolism are not yet fully understood, When INS-1E cells were cultured for prolonged time with TUG-469 (10 µM these data provide support for the postulate that modulation of islet function for 2 d) no increase in apoptotic cell death was measured by TUNEL or cleaved by vitamin D acts, at least in part, through physiological regulation of the local caspase-3 staining. In contrast, palmitate signifi cantly augmented apoptotic islet RAS system, most markedly through suppression of renin secretion. cell death of INS-1E cells. Moreover, islet cells from FFA1 KO mice revealed to Supported by: Research Grants Council of Hong Kong be more sensitive to palmitate than islet cells from wild type mice suggesting that FFA1 activation does not augment but even protects from apoptosis. In isolated human islets which express FFA1 TUG-469 (10 µM) stimulated ISLET BIOLOGY—SIGNAL TRANSDUCTION insulin secretion by 60 % and was, thus, as potent as palmitate. These results reveal that selective activation of FFA1 is a suitable therapeutic target to [See also: Presidents Posters 476-PP to 477-PP, page A132.] increase insulin secretion without adverse effects on β-cell survival. Supported by: BMBF and DFG (UL140/7-2;GRK1302/1); Danish Council (09- 070364) Guided Audio Tour: Signal Transduction (Posters 2022-P to 2029-P), & 2024-P see page 13. The Nutrient Sensor, MTORC1, Mediates ADRP Expression and & 2022-P Formation of β-Cell Specifi c Lipid Droplets O-GlcNAcylation of Carbohydrate Responsive Element Binding ANGELA CHIU, JOSEPH SCHOBER, THERESA WEBER, PHUONG NGUYEN, MARK Protein (ChREBP) and Thioredoxin Interacting Protein (TxNIP) Contri- LUER, CONNIE MARSHALL, MICHAEL L. MCDANIEL, ANDREW GREENBERG, butes to Glucotoxicity Mechanisms in Pancreatic β Cells GUIM KWON, Edwardsville, IL, St. Louis, MO, Boston, MA GAËLLE FILHOULAUD, JEAN GIRARD, CATHERINE POSTIC, Paris, France Lipid droplet formation and its role in β-cell function and growth in In pancreatic β-cells, chronic hyperglycemia enhances oxidative stress response to nutrient overload were studied in rat islets. and lipogenesis leading to increased apoptosis and an alteration of insulin Treatment of rat islet cells with elevated glucose and FFAs resulted secretion. ChREBP is a key transcription factor that mediates the effect of in increases in the size and number of lipid droplets in β-cells in a time- glucose on glycolytic and lipogenic gene including L-pyruvate kinase (LPK) and concentration-dependent manner. The role of the nutrient sensor, and fatty acid synthase (FAS). Interestingly, ChREBP also mediates the mammalian target of rapamycin complex 1 (mTORC1), on the process and transcription of TxNIP, a molecular mediator of glucose toxic effects in effects of lipid droplet formation was assessed. A combination of elevated 3 pancreatic β cells by increasing oxidative stress and apoptosis. When β glucose and FFAs markedly increased mTORC1 activation, [ H] thymidine cells are chronically exposed to elevated glucose concentrations, several incorporation, and islet size compared to elevated glucose or FFAs alone. metabolic pathways are turned on, including O-GlcNAcylation (O-GlcNAc), Importantly, lipid droplets accumulated only in β-cells but not α-cells, in an a post-translational modifi cation of proteins, which contributes to the mTORC1-dependent manner. Based on these fi ndings, we postulated that deterioration of β cell mass or function. Indeed, sustained O-GlcNAcylation translation of adipocyte differentiation related protein (ADRP), known to levels have been implicated as a pathogenic contributor to glucose toxicity stabilize lipid droplets, was regulated by mTORC1. Western blot analysis and insulin resistance, hallmarks of type 2 diabetes. O-GlcNAc is a dynamic indicated a correlation between mTORC1 activity and ADRP expression. modifi cation regulated by a balance between the O-GlcNAc Tranferase Under conditions of nutrient overload, a signifi cant loss of insulin secretion (OGT), the unique enzyme allowing the fi xation of the sugar on proteins and content was observed. mTORC1-mediated lipid droplet formation and the O-GlcNAcase (OGA) responsible for the de-O-GlcNAc process. The preserved partially insulin secretion in response to an intermediate level of aim of our study was to determine whether a modulation of the O-GlcNAc nutrient overload, but not at a higher level. These fi ndings indicate that lipid pathway affects β cell function through a modifi cation of ChREBP activity. droplets exert a protective role in β-cells under physiological or intermediate O-GlcNAc levels were decreased in a pancreatic β-cell line (INS1) via levels of nutrients, whereas chronic nutrient overload reduces the capacity adenoviral approaches, by either overexpressing OGA or inhibiting OGT of lipid droplets to regulate intracellular FFAs. In conclusion, mTORC1 using shRNA. OGA overexpression or OGT inhibition, effi ciently decreased through nutrient regulation exerts a pivotal role in mediating β-cell specifi c global levels of O-GlcNAcylated proteins as well as the one of ChREBP, formation of lipid droplets through the expression of ADRP. resulting in a 50% decrease in LPK, FAS, ACC and TxNIP RNA and protein content. Interestingly, we also report, for the fi rst time, that TxNIP itself is & 2025-P O-GlcNAcylated under 20mM glucose and directly interacts with OGT. In Differential Roles of Insulin Receptor Substrates in the Regulation addition, incubation of primary islets with a pharmacologic inhibitor of OGA, of Pancreatic β-Cell ER Stress

POSTERS results in 1,6 fold increase in TxNIP expression. Taken together, our results Islet Biology/ CHONG WEE LIEW, JYOTSNA SHANKAR, SINDHU RAJAN, LOUIS PHILIPSON,

Insulin Secretion reveal that O-GlcNAc represents an important contributor of glucose toxicity ROHIT N. KULKARNI, Boston, MA, Chicago, IL in β cells by regulating ChREBP and TxNIP activities. Insulin/IGF1 receptor signaling pathways play diverse roles in β-cell biology. Supported by: ANR DIABO-GLYC Although insulin receptor substrate (IRS) proteins are highly homologous

For author disclosure information, see page 785. & Guided Audio Tour poster ADA-Funded Research

A544 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION and signal via many similar tyrosine-phosphorylation motifs, the decrease different mouse models of insulin resistance to show that expression of FFAR2 in β-cell mass in IRS2 knockout (KO) mice in contrast to hyperplastic islets and FFAR3 protein in mouse islets is altered during the development of insulin in IRS1KOs suggest complementary, rather than redundant, roles for the resistance. During and after pregnancy, peak expression of FFAR2 occurred on substrates in regulating β-cell mass. To investigate the signaling role of the day 13.5 of gestation. In contrast, peak expression of FFAR3 was on day 16 of substrates in secretory β-cells, in the context of endoplasmic reticulum (ER) gestation through post-partum day 1. For mice on a high fat diet (started at 5 homeostasis, we examined ER stress markers in IRS1KO or IRS2KO β-cells weeks), the expression of FFAR2 was not altered when islets were isolated at under basal or stressed (induced by expression of the C96Y Akita mutant weeks 8, 12, 16, and 20; whereas the expression of FFAR3 was upregulated proinsulin that is known to induce ER stress due to a failure to fold. IRE1a, at week 8 and returned to control levels at weeks 12, 16 and 20. In db/db a central regulator of ER stress, was signifi cantly up-regulated in IRS1KOs and ob/ob mice, islets were isolated at weeks 8 and 20. Expression of both (∼6 fold, n=3) in contrast to an attenuated effect in IRS2KOs (∼3 fold) under FFAR2 and FFAR3 protein in islets was increased at week 20 but minimal basal growth conditions. Interestingly, expressing C96Y further increased changes in expression occurred at week 8. To defi ne the role of the ligands for IRE1a expression only in IRS1KO (∼9 fold). Spliced XBP1 was signifi cantly up- these receptors, short chain fatty acids (SCFAs), in glucose stimulated insulin regulated in both KO cells (∼1.7 fold) in the basal state. However, expression secretion (GSIS) in mouse and human islets, islets were treated with 1 mM of C96Y proinsulin increased spliced XBP1 only in IRS1KOs but not IRS2KOs. acetate for 30 min, which inhibited (P < 0.05) GSIS from mouse islets by ∼65%. Unlike the IRE1a sensor, the protein synthesis regulator during ER stress Studies with human islets showed a similar pattern with SCFAs inhibiting (PERK), and its downstream target, ATF4, were signifi cantly upregulated only GSIS. In summary, we have observed that expression of FFAR2 and FFAR3 in in IRS1KO cells basally but unaffected by expression of C96Y. Surprisingly, islets is altered in different insulin resistant states, suggesting a possible role in the basal state, expression of CHOP, a target of ATF4, was up-regulated in metabolic changes associated with insulin resistance. over 12-fold in IRS2KO cells compared to a smaller increase in IRS1KOs (∼8 Supported by: Endocrine Fellows Foundation fold) suggesting an ATF4-independent regulation of CHOP in IRS2KOs. The molecular chaperone, BiP was signifi cantly up-regulated in both KO cells in & 2028-P the basal state; however, unlike other ER stress markers, its expression was Human delta Cells Are Regulated by Paracrine Signals from beta up-regulated by over-expression of either WT or C96Y in IRS2KO cells (∼3.8 and alpha Cells fold) while BiP expression was selectively increased further (∼6-fold) upon ALBERTO FACHADO, JUDITH MOLINA, OVER CABRERA, RAYNER RODRIGUEZ- expressing C96Y only in IRS1KO cells. Our data indicate differential roles for DIAZ, MIDHAT H. ABDULREDA, CAMILLO RICORDI, ALEJANDRO CAICEDO, PER- IRS1 and IRS2 in the maintenance of β-cell ER homeostasis. OLOF BERGGREN, Miami, FL, Stockholm, Sweden The delta cell of the islet of Langerhans secretes somatostatin to inhibit & 2026-P the secretion of the islet hormones insulin and glucagon and thus indirectly Regulation of Deoxyhypusine Synthase by p38 MAPK Links Cytokine impacts glucose metabolism. Despite its infl uence on islet hormone output, Signaling to the Translation of iNOS in Pancreatic Islets the delta cell has been barely investigated, in particular in the human islet. YURIKA NISHIKI, BERNHARD MAIER, RAGHAVENDRA G. MIRMIRA, Indianapolis, IN Little is known about the nutrient, paracrine, or nervous signals that regulate The pathogenesis of islet beta cell dysfunction in type 1 and type 2 the activity of the delta cell. We fi rst investigated how human delta cells diabetes includes infl ammatory signaling induced by cytokines, leading respond to changes in glucose concentration by measuring somatostatin acutely to production of inducible nitric oxide synthase (iNOS) and nitric secretion with perifusion assays. Somatostatin secretion increased oxide. In prior studies, we showed that the translational factor eIF5A concentration dependently with a threshold between 1 and 3 mM glucose was necessary for the binding, nucleocytoplasmic shuttling, and eventual concentration. The concentration dependency was similar to that of insulin translation of the mRNA encoding inducible nitric oxide synthase (iNOS) in responses but the dynamics were different, that is, somatostatin responses islets, and that knockdown of eIF5A blocked cytokine-mediated translation were slower. Interestingly, somatostatin secretion also increased when the of iNOS mRNA. This function of eIF5A activity depends exclusively on a glucose concentration was decreased. This “off” response paralleled that posttranslational modifi cation known as hypusine, which is catalyzed by the of glucagon secretion. Increases in somatostatin secretion could further be enzyme deoxyhypusine synthase (DHS). In response to cytokines, we show elicited by activating neurotransmitter receptors for glutamate, acetylcholine, that DHS translocates from the cytoplasm to the nucleus in INS-1 beta cells, GABA or serotonin, but not for adrenaline. Modulating GABA and glutamate presumably causing hypusination of a nuclear pool of eIF5A. Using pulse receptors respectively with the allosteric modulators allopregnolone labeling of cytokine-treated INS-1 cells and rat islets with 3H-spermidine (a and cyclothiazide increased somatostatin secretion, indicating that substrate of DHS), we studied the regulation of hypusination by the mitogen- endogenously released GABA and glutamate affected delta cell function. activated protein kinases (MAPKs). We identifi ed that hypusination activity Because in the human islet GABA is most likely released from beta cells and in INS-1 cells and primary islets was induced ∼2-fold by cytokine exposure, glutamate from alpha cells, these neurotransmitters may act as paracrine suggesting that hypusination is upregulated during acute stress. Whereas signals that stimulate delta cell secretion. By using immunohistochemistry cytokines cause a 103-104 -fold induction of iNOS mRNA in INS-1 cells on human pancreatic sections we found that most delta cells were indeed and islets that is unaffected by inhibition of any of the MAPKs (p38, JNK, contacted by beta and alpha cells and are thus ideally located for paracrine ERK via MEK), the induction of iNOS protein was inhibited 60% by the p38 interactions. Based on our results, we propose that human delta cells are MAPK inhibitor PD169316. This apparent inhibition of iNOS translation was activated to secrete somatostatin when beta or alpha cells are stimulated. accompanied by a block in the cytokine-induced hypusination activity, raising This arrangement provides negative feedback on insulin and glucagon the possibility that p38 MAPK activates DHS. Accordingly, treatment with secretion and likely avoids exacerbated islet hormone secretory responses PD169316 or mutation of the putative MAPK site within DHS led to a defect to plasma fl uctuations in glucose concentration. in the cytoplasmic to nuclear translocation of the enzyme, thereby precluding Supported by: NIH Grant R56DK084321 its hypusination of nuclear eIF5A. In conclusion, our results suggest a novel pathway linking infl ammation-induced production of iNOS protein through & 2029-P p38 MAPK phosphorylation of the hypusination enzyme DHS. PBK Is Required for Glucose Responsiveness of Pancreatic β-Cells Supported by: NIH R01 DK60581 through Activation of RhoGTPases JIA NIE, CHAO SUN, GUANGMING YE, OMAR FARUQUE, CHUNMING CHENG, & 2027-P ZHIJIE CHANG, WANNIAN YANG, XIAO HAN, YUGUANG SHI, Hershey, PA, The Expression of Two Novel GPCRs, FFAR2 and FFAR3, in Mouse Beijing, China, Danville, PA, Nanjing, China Islets Is Altered in Insulin Resistant States Loss of glucose responsiveness of pancreatic β-cells is a major defect BRIAN T. LAYDEN, VIVEK DURAI, SONIA SHAH, MARSHA NEWMAN, WILLIAM in type 2 diabetes. This is underscored by the high remission rate of type L. LOWE, Chicago, IL 2 diabetes after bariatric surgery which restores glucose sensitivity of Type 2 diabetes results when insulin resistance occurs in the setting of β-cells by stimulating the secretion of GLP1 and other incretins. However, pancreatic beta cell dysfunction. Recently, we observed that two novel G the molecular mechanisms underlying glucose responsiveness of islet protein coupled receptors, free fatty acid receptor 2 (FFAR2) and -3 (FFAR3), β-cells are largely unknown. Here we show that PBK, a pancreas and brain are expressed in mouse islets. Specifi cally, we observed expression of these specifi c kinase, is required for glucose responsiveness of islet β-cells. POSTERS

receptors using messenger RNA, Western blot, and immunohistochemical PBK greatly enhances glucose-stimulated insulin secretion (GSIS) and the Islet Biology/ analyses in pancreatic beta cells in mouse and human islets. Because of their effect of incretins on GSIS. PBK kinase activity is signifi cantly stimulated Insulin Secretion unclear role in islets, we set to test the hypothesis that insulin resistance in response to GSIS and treatment with exanatide, a GLP1 analogue. alters the expression of these receptors in islets. In this study, we used Glucose and exanatide also stimulates dephosphorylation of Thr443, a

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A545 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

newly identifi ed auto-inhibitory phosphorylation site of the PBK kinase. We [Ca2+] fl uctuations of greater amplitude. Thus, these results suggest also identifi ed PAK1 and GDIα as novel substrates and mediators of PBK’s important roles for K2P channels in regulating the β-cell membrane potential, effect on GSIS. PAK1 and GDIα required for GSIS by regulating cytoskeletal which may underlie anesthetic affects on blood sugar and modulate glucose remodeling as the effector and inhibitor of RhoGTPases, respectively. PBK induced insulin secretion. directly binds to and phosphorylates PAK1 and GDIα at key regulatory sites required for GSIS, leading to the activation of RhoGTPases, including Cdc42 2032-P and Rac1. Consequently, ablation of PBK in mice leads to hypoinsulinemia, Endothelial Activation Is an Early Feature of the Diabetic Islet in defective GSIS, defi ciency of insulin secretory granules, and abnormal islet C57BL/6.db/db Mice morphogenesis. These fi ndings identifi ed PBK as a key regulator of glucose ADAM C. OTTLEY, REBECCA L. HULL, Seattle, WA responsiveness and insulin biogenesis of islet β-cells. Endothelial dysfunction, characterized by defi ciency in the vasodilator Supported by: NIH (DK076685, Y.S) nitric oxide (NO), increased vasoconstriction and endothelial cell activation, underlies the development of diabetic microvascular complications in 2030-P several tissues. We have shown that key enzymes involved in endothelial Activation of iPLA2β—Results in Phosphorylation of p38 MAP NO production are dysregulated in the islet in diabetes, and that this occurs Kinase in β-Cells early in the pathogenesis of the disease. The aim of this study was to HAOWEI SONG, MARY WOHLTMANN, MIN TAN, JACK H. LADENSON, JOHN determine whether markers of endothelial cell vasoconstriction or activation TURK, St. Louis, MO also occur in islets from a mouse model of type 2 diabetes. Studies with insulinoma cells and mice in which iPLA2β expression level Islets were isolated from C57BL/6.db/db mice and db/+ littermate controls is genetically manipulated indicate that iPLA2β and its hydrolysis product at 8 weeks of age (n=6) and FACS-purifi ed islet endothelial cells and beta arachidonic acid (AA) participate in regulating activity of β-cell Kv2.1 cells were also obtained from 8 week old db/+ control mice (n=3). mRNA delayed rectifi er channels. Kv2.1 activity has been reported to be attenuated levels were determined for the vasoconstrictor endothelin-1 (ET-1), the cell by phosphorylation, and three Kv2.1 p38 MAP Kinase (MAPK) consensus adhesion molecule e-Selectin, and the proinfl ammatory cytokine interleukin-6 sites have been demonstrated by MS to undergo regulated phosphorylation. (IL-6). mRNA levels were determined by real time PCR, using 18S rRNA as the AA stimulates p38 MAPK activity in several cell lines, and we examined endogenous control and expressed relative to db/+ controls. whether p38 activation mediates downstream events in iPLA2β signaling As expected, db/db mice were obese (56.5±0.83 vs. 32.4±0.83 g, p<0.001) in β-cells. and hyperglycemic compared to db/+ controls (414±54 vs. 191±13 mg/dl, With an antibody that recognizes a doubly phosphorylated peptide p<0.001). In samples from db/+ mice ET-1, e-Selectin and IL-6 were expressed (p-Thr180/p-Tyr182) in activated p38, we demonstrated that AA activates INS- in islet endothelial cells but were at or below the limit of detection in beta 1 cell p38 and that this form of p38 correlates with iPLA2β expression level in cells. When comparing db/db and db/+ islets ET-1 levels did not differ genetically modifi ed INS-1 cells and mouse islets, although p38 protein level (1.0±0.2 vs. 1.0±0.2). In contrast, both e-Selectin and IL-6 mRNA levels were is unaffected. Incubation of INS-1 cells with D-glucose at concentrations signifi cantly increased in db/db islets (5.3±0.6 vs. 1.0±0.1 and 1.8±0.1 vs. that stimulate insulin secretion or with thapsigargin under conditions that 1.0±0.2, p<0.005 for both). induce ER stress results in increased p38 phosphorylation in a manner that In summary, islets from 8-week old db/db diabetic mice do not exhibit correlates with iPLA2β expression and is blocked by the iPLA2β inhibitor increased levels of the vasoconstrictor ET-1, but show endothelial-specifi c BEL. Either BEL or the p38 inhibitor PD16916 inhibits glucose- and forskolin- increases in e-Selectin and IL-6. Taken together with our fi ndings regarding induced insulin secretion by INS-1 cells or islets, and either compound also the NO synthetic pathway, our data show that changes in vasoactive suppresses thapsigargin-induced ceramide accumulation in β-cells, which properties and endothelial activation are features of the islet in the db/db is known to be dependent on iPLA2β action. These observations suggest mouse model of type 2 diabetes. that p38 MAPK phosphorylation occurs downstream of iPLA2β activation in β-cells and may play a role in iPLA2β-dependent signaling. 2033-P Supported by: R37-DK34388, P41-RR00954, P60-DK20579, and P30-DK56341 Enhancement of GLP-1-Facilitated Glucose-Stimulated Insulin Secretion in Non-Human Primate Islets by Alogliptin 2031-P JULIE M. CARROLL, CHARLES T. ROBERTS, Beaverton, OR Anesthetic Modulation of Pancreatic B-Cell Two-Pore Domain Modulation of incretin action is an important approach in the treatment of Potassium Channels Alters Islet Electrical Activity and Calcium type-2 diabetes. Current therapies include the use of GLP-1 analogs resistant Infl ux to proteolysis by the dipeptidyl peptidase DPP-4 and small-molecule PRASANNA K. DADI, LOUIS H. PHILIPSON, DAVID A. JACOBSON, Nashville, TN, inhibitors of DPP-4, which increase levels of endogenous GLP-1 and GIP. Chicago, IL DPP-4 is expressed in many tissues, including porcine pancreatic alpha cells. The anesthetic compounds lidocaine and halothane have been shown to The presence of DPP-4 in islets suggests that DPP-4 inhibitors could exert induce perturbations in human islet hormone secretion resulting in aberrant islet-autonomous effects on beta-cell function in addition to their systemic glucose homeostasis. However, the mechanism(s) responsible for anasthetic effects on circulating incretin levels. While direct effects of DPP-4 inhibitors modulation of islet hormone secretion remain undefi ned. Two-pore-domain on islet function have been inferred from analysis of serum parameters, no potassium (K2P) channels are regulated by anesthetic compounds and published reports have investigated potential DPP-4 effects on isolated play a role in setting the membrane potential from where action potential islets. In this study, we analyzed the expression of DPP-4 protein and the (AP) fi ring occurs in many neurons. Therefore, we tested if lidocaine and effect of the novel DPP-4 inhibitor alogliptin on glucose-stimulated insulin halothane regulate islet K2P channels and modulate β-cell membrane secretion (GSIS) in isolated islets from male and female juvenile and adult potential and Ca2+ entry. β-cell voltage clamp recordings detected K2P-like rhesus macaques, whose islet architecture closely approximates that of currents, which closely resembled cloned K2P channels TASK-1 and TASK-3. human islets in comparison to rodents, and which, unlike human islets, can The β-cell K2P-like currents are inhibited by external acidifi cation, activated be isolated immediately following euthanasia. DPP-4 protein was present in by external alkalinization, activated by halothane, and inhibited by lidocaine. NHP islets as evidenced by Western immunoblotting with a human-specifi c Furthermore the K2P currents regulate islet β-cell membrane potential antibody. In 3/6 males and 1/3 females whose islets exhibited robust GSIS causing hyperpolarization when activated by halothane and depolarization in the absence of GLP-1 or alogliptin, no signifi cant additional effect of GLP- when inhibited by lidocaine (6.2 +/- 1.5 mV). The resulting anesthetic induced 1 or alogliptin was evident. In one juvenile male with modest GSIS, GLP-1 changes in β-cell membrane potential induce signifi cant alterations in Ca2+ treatment resulted in a signifi cant enhancement of GSIS, with no additional infl ux; increasing peak Ca2+ infl ux with lidocaine induced depolarization effect of alogliptin. In 2/6 adult males and 2/3 adult females with no GSIS (10.1 +/- 2.5%), which would be predicted to increase insulin secretion, per se, GLP-1 alone increased GSIS, and this effect was increased by addition and decreasing islet Ca2+ infl ux with halothane induced hyperpolarization, of alogliptin. All animals had normal glucose levels at the time of necropsy, which would be predicted to decrease insulin secretion. To further assess but varied in islet glucose sensitivity ex vivo. These data demonstrate that the role of K2P channels in regulating islet electrical activity we utilized alogliptin exerts an islet-autonomous effect through enhancement of GLP-1 a pharmacological inhibitor of the K2P channels TASK-1 and TASK-3, action, and that this effect is most pronounced in islets that exhibit intrinsic

POSTERS isobutylalkylamide (IBA). Islets treated with 10 µM IBA show signifi cantly glucose insensitivity. Islet Biology/

Insulin Secretion altered glucose induced AP fi ring with increased AP amplitude (9.4 +/- 1.7 Supported by: Investigator Initiated research grant from Takeda Pharmaceuticals mV). Additionally 10 µM IBA modulates glucose induced islet Ca2+ infl ux similar to the changes observed with lidocaine treatment causing increased

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A546 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

2034-P 2036-P Exendin-4 Upregulates the Experession of Wnt4, a Novel Regulator Involvement of p66Shc in FFA-Mediated Beta-Cell Death of Pancreatic beta-Cell Proliferation ANNALISA NATALICCHIO, FEDERICA TORTOSA, ROSSELLA LABARBUTA, MAURA CHARLOTTE HELLER, WERNER A. SCHERBAUM, SVEN SCHINNER, Düsseldorf, ROBERTA ORLANDO, ANNA LEONARDINI, ANGELO CIGNARELLI, ROMINA Germany FICARELLA, PIERO MARCHETTI, SEBASTIO PERRINI, LUIGI LAVIOLA, FRANCESCO The Wnt-signalling pathway regulates beta-cell functions. It is not known GIORGINO, Bari, Italy, Pisa, Italy how the expression of endogenous Wnt-signalling molecules in beta-cells The adaptor protein p66Shc mediates apoptosis and oxidative stress in is regulated. Therefore, we investigated the effect of antidiabetic drugs multiple cell types. Chronically increased free fatty acid (FFA) levels induce and glucose on the expression of Wnt-signalling molecules in beta-cells. beta-cell dysfunction and death, thus contributing to the pathogenesis Furthermore, we examined effects of Wnt4 on beta-cells. of type 2 diabetes. The aim of this study was to investigate the potential Using Ins-1 cells as well as primary murine beta-cells we found exendin-4 regulatory effects of FFA on the p66Shc protein in rat insulinoma cells INS-1. (10nM) to signifi cantly increase the expression of Wnt4 in beta-cells on Exposure of INS-1 cells to 0.5 mM palmitate induced a selective 2- to-3-fold the mRNA level (2.5fold) and on the protein level(threefold; p<0.001). The increase in both mRNA and protein levels of p66Shc, evaluated by qRT-PCR effect of exendin-4 on the expression of Wnt4 was signifi cantly inhibited and immunoblotting, respectively (p<0.05), without affecting the other Shc by exendin-(9-39) (a competitive GLP-1 receptor antagonist). The effect protein isoforms; furthermore, palmitate enhanced p66Shc phosphorylation of exendin-4 on the Wnt4 mRNA expression was dose-dependent with on Ser36 (p<0.05). The effects on p66Shc were associated with a 2.5-fold the strongest stimulation at a concentration of 10 nM. In time-response increase in cell apoptosis, evaluated by measuring cytosolic oligosomes experiments we found a signifi cant effect of exendin-4 on the expression (p<0.05). When INS-1 cells were pretreated with pifi thrin-a, an inhibitor of of Wnt4 after incubation for four hours and this effect was maintained even the tumor suppressor protein p53, the palmitate-induced increase in p66Shc after long term stimulation over four weeks. expression was completely abrogated (p<0.05), suggesting the involvement Treatment with insulin (10µM), tolbutamide (100µM), rosiglitazone of p53 in the FFA-p66Shc pathway. INS-1 cells with adenovirus-mediated (100nM), metformin (500µM) and glucose (5.5mM, 11mM, 16.7mM) for 24 overexpression of p66Shc (Ad/p66Shc) were next studied. Ad/p66Shc INS- h had no effect on the Wnt4 expression. The expression of Frizzled-4, LRP5, 1 cells showed increased rates of basal and palmitate-induced apoptosis, Wnt10b and TCF7L2 was not regulated by any of the stimuli. In transient as compared with wild-type or mock-transduced cells (p<0.05). p66Shc transfection assays using a Wnt-responsive reporter gene Wnt4 antagonised overexpression was also associated with increased phosphorylation of the activation of canonical Wnt-signalling in beta-cells. Stimulation with p66Shc on Ser36, both under basal conditions and following exposure to exogenous Wnt4 had no effect on the glucose-stimulated insulin gene palmitate (p<0.05). By contrast, overexpression of a phosphorylation- expression or on insulin secretion in beta-cells. However, knocking down defective p66Shc protein, in which Ser36 had been mutated to Ala, did not (siRNA) Wnt4 decreased beta-cell proliferation markedly to 34.7% of affect basal and reduced palmitate-induced apoptosis (p<0.05). In conclusion, controls. in INS-1 cells, the FFA palmitate triggers the p53-mediated upregulation of These data demonstrate that stimulation with the GLP-1 receptor agonist the p66Shc protein. This is associated with increased cell apoptosis, which exendin-4 increases the expression of Wnt4 in beta-cells. Wnt4 modulates requires p66Shc phosphorylation on Ser36. Thus, p66Shc acts as a novel canonical Wnt-signaling and acts as regulator of beta-cell proliferation. This signaling intermediate in the FFA-mediated beta-cell damage, and may suggests a novel mechanism through which GLP-1 can regulate beta-cell represent a potential therapeutic target to prevent the deleterious effects proliferation. of lipotoxicity on pancreatic beta-cells.

2035-P 2037-P Identifi cation of Related Proteins to PDE3B in Pancreatic β-Cells Modulation of the Pancreatic Islet Transcriptome by Glucose and EMILIA KAROLINA HEIMANN, VINCENT C. MANGANIELLO, LENA STENSON, Intralipid Infusions in 6-Month Old Rats EVA DEGERMAN, Lund, Sweden, Bethesda, MD GHISLAINE FONTÉS, BADER ZARROUKI, ONDREJ SEDA, GHISLAIN ROCHELEAU, Cyclic nucleotide phosphodiesterases (PDEs) are enzymes with the VINCENT POITOUT, Montreal, QC, Canada function to hydrolyze cyclic AMP (cAMP) and cyclic GMP (cGMP) to their We have previously shown that simultaneous infusion of glucose inactive 5´derivatives. Earlier studies in our group have shown that (GLU) and Intralipid (IL) in 6-month old rats leads to insulin resistance and PDE3B, a member of the PDE3 family, has an important role in pancreatic reduced insulin secretion in vivo despite an increase in beta cell mass and β-cells with regard to the regulation of insulin secretion and whole body proliferation. The aim of the study was to determine the effects of GLU energy homeostasis. Here we aim to further study the role of PDE3B and and IL infusions on the global pattern of gene expression in islets. A 72-h identify related proteins within the same signaling network in β-cells. The intravenous infusion of GLU, IL, GLU+IL, or saline control (SAL) was performed pancreatic gene expression profi le for the transgenic RIP-PDE3B mouse in 6-month old Wistar rats (n=5 in each group). Islets were isolated and RNA model, exhibiting either a 2-fold (RIP- PDE3B/2) or a 7-fold (RIP- PDE3B/7) extracted for microarray analysis using Affymetrix rat exon 1 probe sets. β-cell specifi c overexpression of PDE3B, has been compared to C57Bl/6 Selected genes were confi rmed by RT-PCR. Out of the 27,342 probe sets, mice. We show that Osteopontin (OPN) differs in a gene-dose dependent 791 genes in the GLU+IL group (434 genes were up- and 357 were down- manner between RIP-PDE3B/2 and RIP-PDE3B/7 islets compared to C57Bl/6 regulated), and 700 in the GLU group (404 up- and 297 down-regulated) were islets. OPN is differentially expressed by several tissues and is involved signifi cantly modulated compared to SAL. Amongst these, 95 genes were in diabetes and infl ammatory diseases. PDE3B as well as OPN-protein modulated only by GLU+IL and 4 genes only by GLU. IL infusions had modest interaction network were derived from STRING database and integrated by effects on gene expression (20 genes modulated), but when co-infused Cytoscape to fi nd a relationship between these genes. We found that PDE3B with GLU, IL strongly potentiated the GLU effects on gene expression. IL and OPN are connected through protein-protein interaction. To investigate if amplifi cation of GLU effects was confi rmed by RT-PCR for key genes: Cyclin OPN expression is related to cAMP-signaling pathways in pancreatic β-cells, D1 (fold from SAL): GLU: 1.28 ± 0.04; GLU+IL: 1.44 ± 0.05; PreINS2: GLU: 0.75 we stimulated INS-1 (832/13) cells with agents relevant to β-cell function. ± 0.05; GLU+IL: 0.47 ± 0.03; PDX-1: GLU: 0.79 ± 0.06; GLU+IL: 0.61 ± 0.03. The results show that OPN expression is induced by insulin and the cAMP- Gene set enrichment analysis using Ingenuity Pathways software indicated elevating agent forskolin. We conclude that OPN and PDE3B are connected that the canonical pathways most highly regulated by GLU and GLU+IL were through protein-protein interaction and that OPN expression is induced related to proliferation (e.g. Mitotic Roles of Polo-like Kinase, Cell Cycle:G2/M in response to stimuli relevant to β-cell function. Studies are ongoing to DNA Damage), N-Glycan Biosynthesis, and signalling pathways, whereas few elucidate if PDE3B and OPN have related interaction partners and if this genes involved in apoptosis, endoplasmic reticulum stress, or infl ammation interaction is essential for β-cell signaling. This is important for exploring were responsive to the infusion. In conclusion, this global transcriptomics the underlying regulatory mechanisms in islets as well as for identifying analysis in an in vivo model of nutrient excess confi rm that excessive fatty- potential drug targets in type 2 diabetes. acid levels have little effect on beta-cell function under normoglycemia, but are particularly deleterious under hyperglycemia. Further, our results indicate that the most regulated pathways in this model are related to cell proliferation and function rather than cellular stress.

Supported by: NIH CDA POSTERS Islet Biology/ Insulin Secretion

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A547 ISLET BIOLOGY—SIGNALCATEGORY TRANSDUCTION

2038-P 2040-P Potential Role of Cytosolic Phospholipase A1 in the Regulation of Role of Forkhead Box O1 (FOXO1) in Glucose-Induced Beta Cell Insulin Secretion from β-Cells Failure SHIYING SHAO, BING XIE, GUODONG LI, Singapore, Singapore YUZHEN LIANG, JINGSI LUO, HE HUANG, MENGXIA ZHANG, DAN LIU, LEPING Lysophospholipids constitute a group of important lipid mediators, among FENG, NING XIA, Nanning, China, Guilin, China which lysophosphatidic acid (LPA) exhibits multiple biological functions Forkhead box O (FoxO) plays an important role in glucose metabolism, e.g. platelet aggregation, smooth muscle contraction and cell proliferation. tumor suppression, and anti-oxidant defense. Among FoxO isoforms, FoxO1 LPA may evoke its effects through G-protein-coupled receptors followed links the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt cascade to the by activation of various signaling pathways such as phospholipases C expression of genes that regulate cell growth, survival, and metabolism. and D, and mitogen-activated protein kinase. LPA can be generated via Since glucotoxicity leads to beta-cell dysfunction and apoptosis, we studied hydrolysis of phosphatidic acid (PA) by phospholipase A1 (PLA1) and A2. the mechanism by which hyperglycemia induces apoptosis and proliferation This study investigated the possible role of a cytosolic PLA1 with a strong of beta-cells, especially related to the role of the FoxO1 activation. Pancreatic preference for PA (PA-PLA1) in insulin-secreting cells. The activity of this β-cells INT1 were cultured with various concentrations of glucose in DMEM PA-PLA1 was originally found in the supernatant fraction of bovine testis (5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mM, respectively). After culture for and brain. We observed high expression of PA-PLA1 in isolated rat islets and 120 h, insulin secretion was evaluated by radioimmunoassay, apoptosis also in insulin-secreting INS-1 β-cells as determined by real-time RT-PCR by Hoechst33342 assay, and cells proliferation by MTT. In addition, the and immuno-blotting. PA-PLA1 expression in isolated pancreatic acinar cells FoxO1 expression was examined by semi-quantitative RT-PCR and the was undetectable. The function of PA-PLA1 in β-cells was assessed after phosphorylation of FoxO1 was analyzed by Western blot. Our results showed its knockdown by small RNA interference. Transfection of INS-1 cells with that increasing glucose concentrations (5.6-11.1mmol/l) in cell culture led a mixed pool of 3 siRNA oligos targeting PA-PLA1 mRNA for 3 days reduced to early increase of phospho-FoxO1 expression that preceded insulin its expression by 80%. PA-PLA1 knockdown caused a decrease of LPA secretion, cell proliferation, and reduction of cell apoptosis. However, when levels in INS-1 cells as determined by thin-layer chromatography, but had no glucose concentrations were over 11.1mmol/l, the opposite results were effect on the cell proliferation as assessed by fl ow cytometry. Importantly, observed. The decrease of Phospho-FoxO1 expression was associated with high glucose (11.1-22.2 mM)-stimulated insulin secretion was signifi cantly the decreased cell proliferation, impaired insulin secretion, and increased reduced by >75%, with a severer inhibition of the late phase secretion. apoptotic rate of pancreatic β-cells. Increasing glucose concentrations Moreover, insulin secretion induced by the sulfonylurea glibenclamide from 5.6-27.6 mM resulted in the increase in FoxO1 expression in beta- and by membrane potential depolarization with high K+ was markedly cells. Taken together, our results suggest that FoxO1 activation plays an diminished. However, PA-PLA1 knockdown did not alter glucose-stimulated important role in glucose-induced apoptosis and the phosphorylation of- insulin release in the presence of forskolin and GLP-1 which can increase FoxO1 reduced apoptosis, promoted beta-cell survival, and increased insulin cAMP levels. Glucose metabolism was lowered after PA-PLA1 knockdown. secretion, which suggest that Fox01 could be a potential therapeutic target PA-PLA1 knockdown inhibited glucose-induced increase of cAMP levels for preserving beta-cells in type 2 diabetes. but an addition of forskolin or GLP-1 could rescue such inhibition. Our data Supported by: The National Natural Science Fund No.30860116 suggest that PA-PLA1 might play an important role in the regulation of insulin secretion in β-cells via a signaling pathway implicated with cAMP. 2041-P Supported by: NMRC of Singapore Under Oxidative Stress P38MAPK Regulates MafA Protein Stability through Threonine 134 and Not Threonine 57 2039-P ILHAM EL KHATTABI, ARUN SHARMA, Boston, MA Regulation of the Inducible Nitric Oxide Synthase Gene (iNOS) Transcription factor MafA regulates function and maturation of pancreatic by Cytokines Requires Phosphorylation of the p65 Subunit of NF- β-cells. We previously showed that p38MAPK regulates MafA stability ΚB at Ser276 and Ser536 and Signal Transducer and Activator of under both normal and oxidative stress conditions, while GSK3 regulates Transcription 1 (STAT1) at Tyr701 MafA stability only under normal culture conditions. Simultaneous mutation SUSAN J. BURKE, DANHONG LU, THOMAS C. BECKER, RACHEL M. BELLICH, of Threonines(T) 57 and 134 of MafA to Alanines(A) was required to prevent JAMISON NORWOOD, MICHAEL D. KARLSTAD, PATRICIA L. BROWN, STEVEN C. p38MAPK-mediated degradation under normal conditions. Here we examined MINKIN, JR., JOHN BIGGERSTAFF, JASON COLLIER, Knoxville, TN, Durham, NC whether these p38MAPK sites also regulate degradation of MafA under The pro-infl ammatory cytokines IL-1β and γ-IFN decrease functional islet oxidative stress. Additionally, since T57 is recognized by GSK3, we examined β-cell mass by upregulating specifi c genes, such as iNOS, that impair function whether p38MAPK phosphorylates this site in β-cells. Mutation of the and diminish viability. However, the underlying mechanisms controlling priming kinase site in MafA (Serine(S) 65 to S65A) prevents phosphorylation expression of the iNOS gene in response to cytokines are not completely by GSK3, at T57 and other sites. We observed that T57 in S65A mutant was understood. Therefore, we tested the hypothesis that cytokine-mediated still responsive to p38MAPK-mediated degradation in MIN6 cells. Next, control of the iNOS gene requires signaling through p65 and STAT1. In 832/13 MIN6 cells were cotransfeced with MafA and Ubiquitin (Ub) expression rat insulinoma cells, 6h exposure to 1ng/mL IL-1β increased iNOS mRNA levels plasmids to determine whether p38MAPK degrades MafA under normal approximately 100,000-fold, an effect that is potentiated 5-fold by 100U/mL culture condition via proteasomal pathway. Immunoprecipitation of MafA γ-IFN. siRNA-mediated suppression of p65 (75%) or STAT1 (91%) blocked the pulled-down Ub, and the presence of p38MAPK inhibitor or MafA with cytokine-mediated induction of iNOS mRNA levels by 71% and eliminated the mutations in p38 MAPK sites (T57T134AA) reduced the amount of pulled- potentiation by γ-IFN, respectively. A promoter luciferase construct containing down Ub. To examine the role of these sites in oxidative stress mediated 1kb of the proximal iNOS gene promoter was induced approximately 100- degradation of MafA, MIN6 cells transfected with Wild type (WT) and fold by 6hr exposure to IL-1β; this induction is also potentiated 2-3 fold by different MafA mutants were cultured in the presence of an oxidant, tert- γ-IFN and was blunted 92% and 66% by siRNA duplexes targeting p65 and Butyl hydroperoxide. Unlike normal culture condition where p38MAPK STAT1, respectively. Individual mutations of the proximal and distal NF-ΚB mediated degradation was prevented only when both T57 and T134 were sites present within the 1kb region impaired the IL-1β induction by 63% and mutated, degradation of MafA under oxidative stress could be prevented by 93%, respectively, while mutation of the STAT1-binding site within the same mutation of only T134. These results suggest the presence of two distinct promoter region only reduced the potentiation by γ-IFN. MafA degradation pathways, T57 phosphorylated by either p38MAPK IL-1β induced rapid phosphorylation (within 15min) of p65 at Ser276 or GSK3 is degraded via the same downstream processes, while MafA and Ser536, which correlated with p65 nuclear translocation (measured phosphorylated at T134 by p38MAPK is degraded by another pathway. via immunofl uoresence), while γ-IFN induced phosphorylation of STAT1 Under oxidative stress, the pathway involved in the degradation of T57 is at Tyr701. Overexpression of S276A or S536A p65 mutant constructs, inhibited, while that for phosphorylated T134 degradation is maintained which retain nuclear localization capability, impaired the ability of p65 to or enhanced. We suggest that preventing p38MAPK degradation of drive expression of either a generic NF-ΚB responsive promoter or iNOS MafA through T134 would improve β-cell dysfunction caused by diabetic gene promoter in both the basal state and in response to IL-1β. Moreover, milieu. ADA-Funded Research overexpression of Y701F STAT1 eliminates the γ-IFN-mediated potentiation

POSTERS of the iNOS gene. Islet Biology/

Insulin Secretion Taken together, we conclude that specifi c phosphoacceptor sites within p65 and STAT1 are required for maximal induction of the iNOS gene in response to cytokines.

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