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2920-PO

2921-PONeck Circumference (NC) as an Independent Predictor to Metabolic Syndrome (MS)VEER BAHADUR SINGH, PRIYANKA TATER, DEEPAK KUMAR, BABULAL MEENA, SANJAY BENIWAL, SUBASH GAUR, VISHNU KUMAR SAINI, KULVINDER SINGH, SREEHARI DESHMUKH, Bikaner, India

MS is a cluster of metabolic abnormalities including glucolipid metabolism disorders, overweight, insulin resistance and hypertension. Obesity, espe-cially central obesity, is the predominant determinant of MS. Recently NC, as a simple and time-saving anthropometric measurement, was identifi ed as an index of central obesity and a promising potential predictor for MS. The present study aims to investigate whether NC independently contributes to the prediction of the risk of MS. 200 patients with MS were enrolled justify-ing NCEP-ATP-III criteria and relevant exclusion criteria after approval from Institutional Ethics Committee of S.P. MC, Bikaner. Anthropometric indices, biochemical and clinical parameters were measured. NC was measured midway of neck between mid cervical spine and mid anterior neck with

subject standing with head in Frankfort horizontal plane. Receiver operat-ing characteristic, partial correlation and logistic regression analyses were employed for evaluation. In the present study, majority of the subjects were greater than 60 years of age accounting for 44% of the total study popula-tion. Males accounted for 68% (n=136) and females for 34% (n=34). Mean age of the study population was 57.7 ± 13.46 years, NC was positively cor-related TG (r=0.358), TC (r=0.143) and LDL-C (r=0.186), Waist circumference (r=0.552) and negatively correlated with HDL-C (r=-0.301), SBP (r=0.017), DBP (r=-0.106) and fasting blood glucose (FBP r=0.054). In the present study 94% of the patients had abnormal NC. In patients with 3 components of MS (n=47), abnormal NC was found in 42 patients (89.36%). In patients with 4 components of MS (n=87), abnormal NC was found in 81 patients (93.11%). In patients with fi ve components of MS (n=66), all but one had abnormal NC. NC of ≥ 37 cm in males and ≥ 34 cm in females were best correlated with risk of MS. NC can be a useful tool as an anthropometric measurement in patients of MS though larger studies are required to validate its clinical implications.

2922-POVisceral to Subcutaneous Fat Ratio Is a Determinant of Beta-Cell Dysfunction along the Span of Glucose Metabolism ImpairmentFRANCESCA SANTILLI, MARIA TERESA GUAGNANO, CRISTINA SBORGIA, ER-MANNO ANGELUCCI, MARICA MACCARONE, PAOLA G. SIMEONE, VIRGINIA FEDERICO, GIULIANA LARONGA, MARIKA LEO, DEBORA ZONA, VALERIANA FAL-ZANO, CARMEN PULLARA, MARIA GOLATO, LAMBERTO MANZOLI, GIOVANNI DAVI, ARMANDO TARTARO, AGOSTINO CONSOLI, Chieti, Italy

Adipose tissue infl ammation represents a key link between obesity and both insulin resistance and type 2 DM (T2DM), and is likely to infl uence beta cell function through adipokine release.

We examined the distribution of liver fat content, visceral and subcutane-ous adiposity (VAT and SAT) in 53 obese subjects with different degrees of glucose tolerance: prediabetes [impaired fasting glucose (n=13), impaired glucose (n=8) or both (n=12)] and newly diagnosed T2DM (n=20)], in order to assess between-group differences and whether the VAT/SAT distribution and degree of non-alcoholic fatty liver disease (NAFLD) may be associated with insulin sensitivity and beta-cell function.

Insulin sensitivity, as assessed with the Matsuda Index, and beta-cell function, as assessed by Insulin Secretion-Sensitivity Index-2 (ISSI-2) were determined from frequently sampled oral glucose tolerance test, and VAT, SAT and Intrahepatocellular lipid content (IHCL) by magnetic resonance im-aging.

Patients with prediabetes were comparable with diabetic individuals for the majority of clinical characteristics, including body mass index (BMI), waist circumference (WC), insulin resistance, SAT and NAFLD, whereas T2DM was associated with higher VAT (p=0.004), HbA1c (p<0.0001) and decreased beta cell function (p=0.001). On multiple regression analysis, homeostasis model assessment and VAT/SAT ratio were the only signifi cant predictors of beta cell function (ISSI-2), along the span of glucose metabolism impairment (beta=-0.347, p=0.013 and Beta=-0.307, p=0.028), independently of age, gender, BMI, WC, NAFLD, C-reactive protein, Matsuda index, lipid profi le.

We conclude, with the limits of the cross-sectional nature of this study, that the prevalent distribution of fat in the visceral compartment discrimi-nates, among obese subjects with comparable BMI and insulin resistance, those with more rapid decline in beta cell performance, at risk of overt T2DM.

ISLET BIOLOGY—APOPTOSIS

2923-POStriving to Continually Enhance Distribution of Human Islets through Integrated Islet Distribution Program (IIDP)JOYCE C. NILAND, JAMES CRAVENS, JANICE SOWINSKI, BARBARA OLACK, ALI NAJI, CHENGYANG LIU, JOHN KADDIS, Duarte, CA, Philadelphia, PA

The Integrated Islet Distribution Program (IIDP), provides high quality hu-man islets for diabetes research. With increasing demand for human islets and a desire to improve investigator satisfaction, the IIDP has undertaken recent program enhancements. Based on results of an IIDP initiated 2014 shipping study, a media specifi cally designed for post isolation islet recovery and a new 4°C shipping method to reduce cell degradation during transport are being used. IIDP distribution centers now upload images of isolated is-lets during the broadcast to allow potential investigators to view the size, purity and morphology of the human islets awaiting distribution. Pancreatic tissue is now being collected for histological processing and staining (he-

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ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND POSTNATAL GROWTH

motoxylin/eosin and anti-insulin/glucagon/amyloid). Representative images of the stained sections and accompanying morphological evaluation will be uploaded on the IIDP website. Investigators may request histology sections for their research. Further, IIDP requires center reporting of Islet Index (re-fl ects islet size distribution), purity (dithizone staining), viability (inclusion/exclusion dyes), sterility (aerobic, anaerobic, fungal and sentinel tests) and potency (Glucose Stimulated Insulin Secretion (GSIS) assay). Since January 2010, 112.3 million human islets have been distributed. The mean properties of pure and impure broadcasts respectively are as follows: Islet Index 1.2, 1.1; % Purity 85.3, 53.3; % Viability, 92.0, 89.5; Potency-Stimulation Index, 3.0, 2.7; Sterility, 99.1% of broadcasts were sterile. To further enhance the program, the IIDP is currently evaluating the isolation of islets from type 1 diabetic (T1D) donors. Offers for pancreata from T1D donors are rare and retrievable Islet Equivalents (IEQ), can be limited. However, the IIDP recently succeeded in distributing 60,000 IEQs from a T1D child isolated at University of Pennsylvania to 10 investigators for critical T1 research.

Supported By: National Institutes of Health

2924-POGlutathione’s Reduced Form Protects Beta Cells from Destruction Caused by Diabetogenic LigandsGABIT G. MEYRAMOV, KLAUS-DIETER -. KOHNERT, AISULU A. KIKIMBAEVA, OL-IVIA-NEZRYN J. DUPONT, AIDAR M. AITKULOV, ZAURE T. KYSTAUBAEVA, GUL-MIRA M. TYKEZHANOVA, LUDMILA G. TURGUNOVA, ELENA M. LARYUSHINA, AIZHAN G. ABDRAIMOVA-MEYRAMOVA, GULMIRA O. ZHUZBAEVA, ALTYNAI S. SHAYBEK, KARLIGASH A. ZHUMASHEVA, SAYAGUL S. TYRSHANOVA, Kara-ganda, Kazakhstan, Karlsburg, Germany, Astana, Kazakhstan

Glutathione Reduced form contain SH-group (GSH) possess ability to bind- ing with ligands.

Aim of work: To study effect of GSH on action of diabetoge-nic zincbinding ligands Diphenylthiocarbazone (DZ) and 8-para (toluenesul-phonylamino) quino-line (8PTSQ). Groups of rabbits: 1) injection of DZ 49,3-50,8 mg/kg 2) GSH 970-1020 mg/kg +DZ 48,9-51,6 mg/kg 10 min later; 3) GSH 985-1010 mg/kg+8PTSQ 47, 5-49, 7 mg/kg; 4) Glutathione Oxidized form (GOx) 980-1015 mg/kg+DZ 50, 9-51, 6 mg/kg. Blood Glucose level (BG)-3 times per 6 days. Other animals killed 10 min past 2nd injection.

Histology: aldehydefucshine method; insulin immunohistochemistry (IG); vital by DZ and 8PTSQ staining of Zn+2-ions in B-cells with measuring of absorbance past injection of DZ (DZA) and 8PTSQ (IF).

Results: Group 1: BG: 5.4±0.7 mM before and 16.2±2.5 mM 6 days lat-er; histology: necrosis and destruction of 85-90% B-cells; IG 1.07 ±0.03 (intacts-1.89±0.04); 2 animals early killed: binding of almost all amount of Zn+2-ions in B-cells by DZ: DZA 1.95±0.04 (intacts-1.00±0.02). Group 2: BG: 5.2±0.5 mM before and 6.2±0.6 mM 6 days later; histology; islets without changes; 3 animals early killed: binding of 7-12% of Zn+2-ions in B-cells: DZA 1.09±0.02 (intacts-1.00±0.03). Group 3: BG: 5.5 ±0.7 mM before and 6.8±0.9 mM 6 days later; histology: not marked necrobiosis in 5-10% of B-cells; 3 animals early killed: binding of 5-10% of Zn+2-ions in B-cells by 8PTSQ: IF 1.12±0.03 (intacts-1.97±0.07). Group 4: BG: 4.8±0.5 mM before and 14.8±2.6 mM 6 days later; histology: necrosis and destruction of 80-85% B-cells; IG 1.09±0.02 (intacts-1.87±0.05); 2 animals early killed: binding of almost all amount of Zn+2-ions in B-cells by DZ: DZA 1.92±0.05 (intacts-1.00±0.03).

Conclusions: 1) GSH protect B-cells of binding Zn+2-ions by diabetogenic ligands, of destruction B-cells and of developing diabetes; 2) GOx not con-tain SH-group, not protect Zn+2-ions in B-cells of binding by DZ that result destruction of cells and of developing of diabetes.

Supported By: Family of Professor Gabit G. Meyramov

ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND POSTNATAL GROWTH

2925-POSingle-Cell RNA-Seq Reveals Alpha-Cell Maturation Stages in Pan-creas DevelopmentDIANA E. STANESCU, KYOUNG JAE WON, DORIS A. STOFFERS, Philadelphia, PA

The heterogeneous nature of the developing pancreatic epithelium and the low abundance of endocrine progenitors limits the information derived from traditional expression studies. The advent of single-cell transcriptomics allows the identifi cation of important differences between cells otherwise sorted together using lineage markers. To characterize the transcriptomic landscape of pancreatic endocrine cells we performed single-cell RNA-seq of e13.5 mouse pancreata using Fluidigm C1. 76 individual libraries were gen-erated. The data were analyzed by Monocle to sort cells in an order accord-

ing to their progression through differentiation and to identify differentially expressed genes. Four cell states were identifi ed: two appear to be early and terminal developmental stages, and two may represent intermediate stages. Glucagon (Gcg) was the major differentially expressed gene across the Monocle pseudo-differentiation stages, suggesting that Gcg+ cells at e13.5 are in different stages of maturation. A set of 152 genes had a similar expression profi le to Gcg. Gene ontology (GO) analysis of this set showed enriched terms such as “cytoplasmic membrane-bounded vesicle” and “sig-naling and peptide hormone processing.” Cells with very high Gcg transcript expressed other pancreatic hormones (insulin, ghrelin, peptide YY), albeit at lower levels, indicating that plurihormonal expression is a normal develop-mental stage in the maturation of α cells. Overall, Gcg+ cells expressed a variety of pro-endocrine factors (Sox9, MafB, Ngn3, Nkx2.2, FoxA2, Rfx6), and acinar lineage factors (Nr5a2, Rbpj, Cpa1, Gata4). At least at a transcrip-tional level, mouse pancreatic epithelial cells at e13.5 can concurrently ex-press factors specifi c to different cell fates. Our study brings a new aware-ness of the maturational heterogeneity of mouse pancreatic endocrine cells at e13.5. It establishes the utility of single-cell RNA-Seq as an unbiased tool for analyzing pancreatic endocrine progenitors.

2926-POS 100-Positive Cells in the Human Islets of LangerhansALEXANDRA E. PROSHCHINA, YULIYA S. KRIVOVA, VALERIY P. CHERNIKOV, VAL-ERIY M. BARABANOV, SERGEY V. SAVELIEV, Moscow, Russian Federation

The current studies suggest that in the pancreas the structures of the nervous system are the primary target of the autoimmune attack in diabe-tes type one (DM1). Experiments on NOD (nonobese diabetic) mice have re-vealed an autoimmunization against neuronal and glial cell antigens (S-100, GFAP, GAD) at early stages of DM1. In order to characterize cells containing S 100-like immunoreactivity in the islets of Langerhans, we examined the autopsied pancreatic specimens from human fetuses, infants and adults us-ing immunochemistry and electron microscopy. Two types of S 100-positive cells, which differ in shape and localization, were detected in the human islets of Langerhans.

Small oval, spindle or triangular cells with processes were revealed on the margin of the islets. The glial nature of these cells was confi rmed by electron microscopy. In the fetal and children’s pancreata, glial cells were often seen in the forming pancreatic islets. It has been suggested that these cells are a part of neuro-insular complexes and may be involved in the pancreatic islets morphogenesis.

S 100-positive cells, which were similar in shape and localisation with other endocrine cells, were found within the islets. We propose that these cells may play a role in synchronization of islets calcium-dependent hormone release. These fi ndings suggest novel neural-islet regulatory mechanism and therefore advance basic knowledge of islet neurobiology.

Supported By: Russian Foundation for Basic Research (15-04-03155)

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ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND POSTNATAL GROWTH

2927-POActivation of Pancreatic Progenitors during Isolation of Porcine Neonatal Pancreatic Cell ClustersJYUHN-HUARNG JUANG, CHEN-YI CHEN, WAN-JUNG CHANG, CHANG-YI CHEN, CHIEN-HUNG KUO, WAN-CHUN LI, Taoyuan City, Taiwan, Taipei, Taiwan, Hsinchu, Taiwan

Generation of β cells ex vivo that are suitable for transplantation paves the way to correct beta-cell defi cit in diabetic patients. While multiple step-wise protocols to differentiate human stem cells into functional β cells were proposed and early commitment of pluripotent cells into pancreatic progenitors is fairly effi cient, the β cell maturation triggers are still largely unknown. Previous studies showed that in vitro cultivated porcine neonatal pancreatic cell clusters (NPCCs) exhibited primarily epithelial progenitor-like phenotype in vitro and could successfully ameliorate hyperglycemia in diabetic mice revealing a useful tool to explore islet maturation promoters. Current study sought to identify molecular profi le of NPCCs upon isolation to better defi ne their cell properties. Histological analysis showed 3.4%, 7.6%, 8.6% and 11.4% of NPCCs are insulin+ in 1-, 2-, 3- and 4-day cultures, respectively, whereas semi-quantitative RT-PCR detected increased insulin and decreased exocrine enzyme carboxypeptidase B (CPB) mRNA expression over 4-day cultivation implying gradual enrichment of β cell population in culture. Strikingly, highly proliferating (30.1% Ki67+ cells in fi rst 4-day cul-tures compared to 16.1% Ki67+ cells in 1-day old pig pancreas) pancreatic progenitors stained for Pdx1 (83.7%, 72.7%, 77.3% and 77.1% in 1-, 2-, 3- and 4-day cultures, respectively, compared to 51.9% in 1-day old pig pancreas) and Sox9 (respect 78.0%, 79.7%, 80.0% and 81.3% in 1-, 2-, 3- and 4-day cultures compared to 6.8% in 1-day old pig pancreas) were quickly induced after isolation indicating the pancreatic precursors could be propagated in vitro. In summary, our results showed that islet precursors could be simply activated during NPCC isolation. NPCC-derived progenitors maintain their pluripotency in culture providing a useful platform to examine insulintropic and maturation-promoting effects of candidate molecules to direct human pluripotent cells into functional β cells.

Supported By: NSC102-2314-B-182A-012-MY3

2928-PO

2929-POThe Effects of Betatrophin on Beta-Cell Size and Glucose Homeo-stasis in Mouse Models of DiabetesTIMOTHY H. KING, EMILY PELTO, SICHEN LIU, LINDSEY GLENN, MICHELLE B. TREVINO, SO HYUN PARK, YUI MACHIDA, CIRIACO VILLAFLOR, WOJCIECH GRZESIK, MARGARET A. MORRIS, JERRY L. NADLER, YUMI IMAI, Norfolk, VA

The reduction of islet size and function is a major culprit in the progression of T1D and T2D. Betatrophin, a liver secreted hormone, has been implicated as possibly having a role in increasing islet size and improving glycemic con-trol. We set out to determine the effects of Betatrophin on islet area and glu-cose homeostasis in various mouse diabetic models including C57BL/6J (BL6) treated by low dose Streptozotocin (STZ) and NOD (non-obese diabetic). Our previous studies showed that the activation of 12-lipoxygenase (12-LO) con-tributes to islet loss in diabetes through the production of infl ammatory lipid mediators. To evaluate the effects of Betatrophin and 12-LO suppression, 12-LO defi cient NOD (12-LO KO) mice were also studied. We successfully overexpressed Betatrophin in the liver of BL6 mice (p < 0.05 vs. GFP control) using an adenovirus delivered via tail injections (1.1x109 IFU/mouse). All mice

remained healthy during the 2-week experimental period and the mice over-expressing Betatrophin showed a slight trend of reduced blood glucose (BG) levels during a glucose tolerance test (GTT), but no differences in BG dur-ing an Insulin Tolerance Test (ITT). In the STZ induced mice overexpressing Betatrophin there was a trend of reduced blood glucose during GTT and ITT, and these mice showed signifi cantly reduced BG at harvest time (p < 0.05 vs. GFP). The Betatrophin treated mice also showed signifi cantly increased islet area (p < 0.05 vs. GFP), but circulating insulin levels were unchanged. In young pre-diabetic NOD mice, Betatrophin administration resulted in a trend of increased serum triglycerides and serum insulin, as well as slightly increased Ki67 staining within islets. In older diabetic NOD mice (glucose > 300) Betatrophin did not improve glycemic control. However in 12-LO KO NOD mice, Betatrophin aided in glucose lowering. These results indicate the effi cacy of Betatrophin in improving glucose homeostasis is seen in some but not all diabetic mice, and can be increased with 12-LO inhibition.

Supported By: Eastern Virginia Medical School

2930-PONovel Genes Involved in Islet Adaptation during Pregnancy in MiceJENNY VIKMAN, OLOF ASPLUND, PETTER STORM, PETTER VIKMAN, LEIF GROOP, Malmö, Sweden

Pregnancy as well as obesity is associated with increased insulin resis-tance that is accompanied by an adaptation of β-cells and β-cell mass to com-pensate for the increased demand of insulin. The underlying mechanism(s) that drives this β-cell adaptation during pregnancy is largely unknown.

This study has aimed to closely investigate global changes in gene expres-sion that occurs during pregnancy using RNA sequencing. RNA was isolated every third day from gestational day 3.5 and throughout pregnancy until day 4 post pregnancy in NMRI mice and compared to non pregnant mice.

We found 32 genes (Table 1) that were globally signifi cant with an ad-justed P-value of ≤0.05. We could confi rm that Tryptophan hydroxylase 1 (Thp1) was upregulated (p=0.040) whereas Cystic fi brosis transmembrane conductance regulator (Cftr) and Synaptotagmin 10 (Syt10) are two novel genes identifi ed to be upregulated during pregnancy (p= 0.0047 and p= 0.0065 respectively). Only two of the 32 genes were down regulated. The major changes occurred at day 9.5 and day 18.5 where the highest numbers of signifi cant genes were identifi ed.

This is the fi rst study to use RNA sequencing to identify novel genes that drive the β-cell adaptation during pregnancy. We could identify several genes that were globally signifi cant and follow these changes throughout pregnancy.

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ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETION COUPLING AND METABOLISM

ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETION COUPLING AND

METABOLISM

2931-POScreening by RMCE-based Generation of MIN6 Cell Transfectants Identifi es Novel Genes Important for Glucose-stimulated Insulin SecretionHISAMITSU ISHIHARA, ASAMI FURUKAWA, AYA TANAKA, YUICHIRO OTSUKA, MINAMI KOSUDA, SUGURU YAMAGUCHI, Tokyo, Japan

Despite extensive research efforts, a precise picture of how glucose itself and its metabolites initiate insulin secretion in a concentration-dependent manner has been unveiled. To identify candidate genes important for glu-cose-stimulated insulin secretion, we have conducted screening of differ-entially expressed genes identifi ed by microarray analyses between highly glucose-responsive and less responsive MIN6 subclones. For this purpose, we have introduced MIN6 cells expressing a tetracycline-regulatable artifi -cial transcription factor Tet3G with a platform containing zeocin resistant cDNA fl anked with a wild-type and mutated fl ippase recognition targets (FRTs) under a splice acceptor sequence. The platform has been placed at an unknown site and trapped an unknown transcript, allowing expression of the zeocin gene. These MIN6 cells have been used to generate stable transfectants overexpressing genes of interest by means of the recombi-nase-mediated cassette exchange (RMCE) method. When these cells were transfected with a plasmid expressing fl ippase and a plasmid containing the blasticidin resistant gene and a gene of interest under tetracycline-respon-sive promoter fl anked with FRTs, more than 90% of blasticidin-resistant cells have expressed the gene. Suffi cient numbers of cells expressing the desired gene have been produced within one month after transfection. More than 50 trasfectants harboring differentially expressed genes have been created, and numbers of transfectants are being increased. We have examined ef-fects of doxycycline-induced expression of these genes on insulin secretion, and identifi ed several genes, including Gprc5c and Rab19, which modulated glucose-concentration dependence of insulin secretion. Our strategy is a promising step towards understanding of the molecular mechanism of glu-cose sensing by pancreatic β-cells.

Supported By: Japan Society for the Promotion of Science

2932-POPericentrin Regulates β-Cell Insulin Releases through F-actinYANPING GONG, YUAN ZU, LIJUAN WAN, CHUNLIN LI, Beijing, China

To investigate the effect of pericentrin (PCNT) on insulin release, PCNT expression was studied in IR mice, and MIN6 cells were exposed to glucose stimulation, PCNT siRNA, or ERK inhibitor. The results revealed that PCNT highly expressed in metabolism regulating organs (Fig 1A), and cytoplas-mic expression was only enriched in islet cells. PCNT expression reduced obviously in IR mice β cells (Fig. 1B), glucose-stimulated and PCNT siRNA intervened MIN6 cells (Fig. 1C, 1D), which linearly related with cytoplasmic insulin level. Linear decreases of F-actin and PCNT expressions were fur-ther confi rmed in MIN6 cells intervened by PCNT siRNA at different time points (Fig. 2A) and ERK inhibitor (Fig. 2B). So the abnormal insulin secretion observed both in vivo and in vitro was associated with decreased PCNT ex-pression, and F-actin was the target of PCNT regulation.Figure 1.

Figure 2.

Supported By: National Natural Science Foundation of China (81373458)

2933-PO4-Hour (h) High Glucose-infused Overload Injured Insulin Secretion and Insulin Granules of the Islet β-Cells in MiceDIEN YAN, YINGSHENG ZHOU, XIUYING GAO, YINAN ZHAO, Beijing, China

The aim of our study was to clarify whether short-term, high glucose-infused overload injured insulin secretion and insulin granules of the islet β cells in mice. To achieve it, the right jugular vein was catheterized in 12 weeks male C57BL/6. 25% high glucose solution was intravenously infused by the hyperglycemia clamp. The blood glucose (BG) increased to16.7mmol/L in 15min and sustained in the following 2 or 4 h. Then intraperitoneal in-jection glucose tolerance test (IPGTT) was performed respectively. In 4h high glucose-infused mice(4h-HG), BG levels obviously at 15, 30, 60,120min (P<0.05) were higher than those in 2h high glucose-infused mice(2h-HG), however, the serum insulin was lower at only 15min (P<0.05). Islets were also isolated from mice anesthetized with sodium pentobarbital (80 mg/kg). Static glucose-stimulated insulin secretion (GSIS) study showed, in only 4h-HG mice, the secreted insulin concentration at 2.8 and 16.7mmol/L(mM) stimulus (P<0.05) strikingly reduced, and insulin content of the islets sig-nifi cantly decreased only at 16.7mmol/L (P<0.05). Electron microscopy (EM) found that less insulin granules and swelled mitochondria appeared in 4h-HG mice. In conclusion, 4 h high glucose-infused overload injured insulin secretion and insulin granules of the islet β cells in mice.

Supported By: National Natural Science Foundation of China (81041024)

2934-POThe Activity of Hepatocyte Nuclear Factor-1 Alpha Is Regulated by the E3 SUMO Ligase PIASyINGVILD AUKRUST, ALBA KACI, LISE BJØRKHAUG GUNDERSEN, PÅL R. NJØL-STAD, Bergen, Norway

Hepatocyte nuclear factor-1 alpha (HNF-1A) is a transcription factor regu-lating the expression of liver- and pancreas-specifi c genes. Mutations in HN-F1A encoding HNF-1A is the cause of maturity-onset diabetes of the young (HNF1A-MODY). Post-translational modifi cation of HNF-1A has so far been poorly investigated. Although SUMOylation is a post-translational modifi ca-

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tion involved in many cellular processes, a possible role in the regulation of HNF-1A function is presently unknown. Here, we show that HNF-1A is SUMOylated both in vitro and in cellulo. Moreover, we found that the level of SUMO-1 conjugation of HNF-1A is increased in HeLa cells in the presence of the SUMO E3 ligase; protein inhibitor of activated STAT (PIASy). We further identifi ed, by luciferase reporter assays in Min6 cells, that HNF-1A transac-tivation through binding a rat albumin promoter was reduced when co-trans-fecting with human SUMO-1 and Ubc9 (E2). Transactivation was reduced even further when PIASy was transfected to the Min6 cells. Interestingly, although PIASy increases the expression level of HNF-1A protein in Min6 cells, it seems to act as a transcriptional repressor of HNF-1A, which may be important for the role of HNF-1A in glucose uptake and insulin secretion.

Supported By: University of Bergen; Helse Vest; Kristian Gerhard Jebsen Foun-dation; Research Council of Norway

2935-PO

ISLET BIOLOGY—SIGNAL TRANSDUCTION

2936-PORepression of Insulin Gene Transcription by Estrogen Receptor in Pancreatic Beta CellsTAKASHI SEKIDO, YOHSUKE OHKUBO, KEIKO SEKIDO, SHIN-ICHI NISIO, SATORU SUZUKI, MITSUHISA KOMATSU, Matsumoto, Japan, Fukushima, Japan

Estrogens (E2) have diverse effects. Recently it has reported that E2 re-ceptor α (ERα) activates insulin synthesis with E2 in INS-1, rat insulinoma cells. Herein, we have demonstrated that ERα and E2 represses insulin gene transcription in HIT-T15 hamster insulinoma cells. We show using RT-PCR and western blot analyses that ERα is expressed in insulinoma cell lines as well as rat pancreatic beta cells. Treatment of HIT-T15 insulinoma cells with E2 led to the decreased insulin mRNA levels. Transient transfection assays with HIT-T15 cells revealed that ERα repressed transcriptional activity of rat insulin II promoter both in a ligand-dependent and -independent manner in contrast to the previous conclusion from INS-1 rat insulinoma cells. N-ter-minal A/B domain of the ERα is not necessary for the repression, however, the repression was not observed in the presence of mutant forms of ER lack in DNA-binding domain. Moreover introduction of mutations in the second zinc fi nger of the ER (C227S) abolished the repression. Deletion analyses of the insulin promoter showed that the region of negative estrogen respon-siveness within the rat insulin II promoter was localized at nucleotides from

-238 to -144bp relative to the transcription start site. The repression was not mediated by an ERE-like sequence or AP-1 site in the promoter. Instead, PDX-1and BETA2/E47 binding sites appear to be involved. Expression plasmids for Gal4 DBD fusions of these transcription factors were created and co-trans-fected with ER expression vector and UAS reporter plasmid. ER repressed the activity of BETA2 in a E2-dependent manner in HIT-T15 cells. These re-sults imply that estrogen has a variety of physiological effects in pancreatic beta cells, not only the activation but also the repression of insulin promoter. Thus it is possible to speculate that E2 or other related hormones/chemicals might deteriorate beta cell function under particular situations leading to the development of diabetes mellitus.

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