ISLET BIOLOGY—APOPTOSIS 2161-P - Diabetes...A556 For author disclosure information, see page 829....

A556

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

Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS

ISLET BIOLOGY—APOPTOSIS


2161-PCirculating Demethylated Insulin DNA as a Potential Early Indicator of β-Cell Loss in a Non-Human Primate Model of Pre-DiabetesEITAN M. AKIRAV, LAUREN A. KENNA, PAUL KIEVIT, DAVID M. HARLAN, KEVIN GROVE, CHARLES T. ROBERTS, Mineola, NY, Beaverton, OR, Boston, MA

Both type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are caused by islet dysfunction and eventual β-cell failure. In T2DM pathogenesis, β-cell dysfunction takes years to progress, providing an opportunity for intervention prior to the development of frank hyperglycemia. However, current biomarkers of diabetes, such as C-peptide and HbA1c, only provide a measure of β-cell function late in the disease process. New biomarkers that are evident earlier in the disease process are needed to facilitate preemptive preventative strategies. Our previous studies have shown that the β-cell failure underlying T1DM can be monitored by measuring demethylated β-cell insulin (deMeth-β) DNA that is released into the circulation; e.g., we have reported serum deMeth-β DNA as a biomarker of β-cell loss in murine T1D models and in T1D patients. In this study, we adapted our assay for the rhesus macaque. We utilized our well-characterized non-human primate (NHP) model of diet-induced obesity (DIO) to determine deMeth-β DNA’s utility as an early T2DM biomarker. Three adult male NHPs (10-13 yrs old) were fed a high-fat, high-fructose diet (HFFD; 35% of calories from fat) and demonstrated signifi cant weight gain and the gradual onset of insulin resistance. Pre-diabetes was determined by an increase in insulin secretion during a glucose tolerance test (GTT), indicative of early β-cell dysfunction. Serum analysis from these NHPs found that, compared to levels prior to the start of the HFFD, deMeth-β DNA levels more than doubled with DIO. This increase in deMeth-β DNA coincided with increased insulin secretion during the GTT. These data suggest that an increase in circulating deMeth-β DNA levels may serve as a biomarker of early β-cell dysfunction in pre-T2DM.

Supported by: NIH (R24DK093437), (P510D011092)

2162-PBlocking IL-1 Receptor Reduces Amyloid Formation and Enhances Beta-Cell Survival and Function in Cultured Human IsletsYOO JIN PARK, KATHRIN MAEDLER, ZILIANG AO, NOOSHIN SAFIKHAN, GARTH L. WARNOCK, LUCY MARZBAN, Vancouver, BC, Canada, Bremen, Germany

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to progressive beta-cell dysfunction and death in type 2 diabetes, as well as in cultured and transplanted human islets. We recently showed that impaired prohIAPP processing due to beta-cell dysfunction potentiates hIAPP aggregation and that endogenously formed hIAPP aggregates induce upregulation of the Fas cell death receptor in beta cells. In this study, we examined the effects of blocking IL-1 receptor (IL-1R) on islet amyloid formation and amyloid-induced Fas upregulation in beta cells. Isolated human islets were cultured with or without IL-1R antagonist anakinra (Kineret) for up to 7 days. IL-1 beta was released from human islets during culture in a time-dependent manner associated with progressive beta-cell dysfunction and death. There was no signifi cant change in islet IL-1 alpha release during 7 days culture. hIAPP aggregates were present in cultured (but not freshly isolated) human islets as assessed by immunolabelling for insulin and hIAPP oligomers (or thiofl avin s). Increase in islet IL-1 beta release preceded amyloid formation. Anakinra-treated islets had lower beta-cell apoptosis, greater insulin response to elevated glucose and insulin content than non-treated cultured islets, which was associated with lower amyloid formation and beta-cell Fas expression. Adenoviral siRNA mediated suppression of amyloid formation in human islets during culture did not have any marked effect on islet IL-1 beta release but reduced beta-cell IL-1 beta and Fas immunoreactivity. Anakinra did not have any signifi cant effect on amyloid formation in transduced islets. In summary, these data suggest that: 1. IL-1 beta release from human islets during culture is associated with progressive amyloid formation and Fas upregulation; 2. IL-1R antagonists such as anakinra reduce amyloid formation and amyloid-induced Fas upregulation in beta cells during islet culture.

Supported by: CIHR

2163-PEvaluation of 18F-TTCO-Cys40-Exendin-4 for In Vivo Imaging Pancre-atic IsletsZHANHONG WU, INDU NAIR, IVAN TODOROV, JOSEPH M. FOX, ZIBO LI, FOUAD KANDEEL, Duarte, CA, Newark, DE, Los Angeles, CA

Currently, clinical verifi cation of beta-cell abnormalities can only be obtained indirectly via metabolic studies, which typically is not possible

until a signifi cant deterioration in islet function has already occurred. The development of non-invasive imaging methods to assess pancreatic beta-cells could therefore offer great potential for early detection of beta cell dysfunction prior to the clinical onset of type 1 diabetes and type 2 diabetes. Recently, 18F-TTCO-Cys40-Exendin-4 has been developed as a promising PET agent for transplanted islet imaging. In this study, we evaluated this agent for PET imaging of native pancreas islets in rat model.

SD rats were treated with streptozotocin (STZ) to induce beta cell damage. The pancreata of normal and STZ-treated rats were collected at 1 hr post injection of 18F-TTCO-Cys40-Exendin-4. Beta-cell mass within pancreas was determined by both ex vivo PET scans and morphometry using insulin-immunostained pancreas sections.

The pancreas uptake of normal rats was signifi cantly higher than that of the STZ-treated group, which was especially clear in ex vivo PET image (Fig 1, n=3, p<0.01). The pancreas uptake of 18F-TTCO-Cys40-Exendin-4 was 0.171 ± 0.007 and 0.052 ± 0.006 %ID/g for normal and STZ treated rats, respectively. Apparently, the reduced islet mass caused by STZ destruction was successfully refl ected on microPET images. In conclusion, 18F-TTCO-Cys40-Exendin-4 demonstrated the potential for imaging islets in situ.

Supported by: JDRF

2164-PMicroRNA Expression Profi ling Reveals Disease-Associated Sig-natures in Type 1 Diabetes Cellular ModelsAMMIRA S. AL-SHABEEB AKIL, ALLIE FINLAY, ANAND W. HARDIKAR, CHI NAM IGNATIUS PANG, MARC R. WILKINS, WILLIAM D. RAWLINSON, MARIA E. CRAIG, Randwick, Australia, Camperdown, Australia

Human enterovirus (HEV) infections, specifi cally Coxsackievirus B (CVB), demonstrate β-cell tropism and are associated with type 1 diabetes (T1D). microRNAs (miRNAs) function as translational repressors and important regulators of key biological processes. We hypothesise that CVB infection of β-cells and human islet derived precursor cells (HIPCs) alters miRNAs abundance, thereby regulating gene expression, inducing pro-infl ammatory cytokines and β-cell death. Human islets and HIPCs were infected with CVB 3, 4 and 5. Human miRNAs (n=756) were quantifi ed using the TaqMan Array. miRBase and miRWalk algorithms were used to predict miRNA gene targets. Spearman’s correlation coeffi cient was used to calculate the pairwise correlation in miRNA levels between each pair of miRNAs. Hierarchical clustering was used to determine groups of miRNAs with similar expression patterns following EV infection. R software was used for analyses and for creating heat maps. We identifi ed 21 microRNAs associated with T1D candidate genes that were increased > 10 fold (relative to uninfected controls, p<0.05) following CVB infection of human islets. In the HIPCs, 23 miRNAs were differentially-expressed after CVB5 infection. Many of the miRNAs target genes that control cytokine production and signalling (eg IL-2, IL-2RA, IL-10, PTPN22), T cell receptor signalling (PRKCQ, RASGRP1), immune response to viral infection (TNFAIP3) and apoptosis (TYK2). Heat maps demonstrated two clusters: 8 miRNAs increased by CVB3, 4, 5) and 13 miRNAs (CVB3 only). Analysis of interactions between miRNAs with >10 fold higher expression post CBV infection and a human T1D protein network showed miRNAs mainly target positive regulatory motifs in highly connected scaffolds. In conclusion, CBV infection of human islets and HIPCs leads to dysregulation of multiple microRNAs. This appears to disrupt the protection of cellular integrity, with alterations in the immune response, ultimately leading to β-cell death.

2165-PFeed-Forward Regulation of NADPH Oxidase-1 in Beta-CellsJESSICA R. WEAVER, DAVID A. TAYLOR-FISHWICK, Norfolk, VA

Beta cells are highly susceptible to reactive oxygen species (ROS) which are elevated by pro-infl ammatory cytokines (PICs) along with other diabetogenic stimuli. NADPH oxidase-1 (NOX1) is an enzyme that generates ROS. NOX1 is expressed in beta cells and is upregulated following exposure to PICs. We have also shown that NOX1 is upregulated by 12-lipoxygenase (12-LO) activity

A557

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

Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


in beta cells. 12-HETE (a product of 12-LO activity) upregulates NOX1 13±3-fold (p<0.01) and a selective inhibitor of 12-LO blocks PIC-stimulated NOX1 80±5% (p<0.01) in human islets and INS1 beta cells. Inhibitors of NOX activity reduce cellular ROS and the expression of NOX1. This observation suggests a feed-forward regulation of NOX1 in beta cells involving ROS elevation. Anti-oxidants (GEE, BHA) inhibited PIC-induced NOX1 26±11%, 29±15% (p<0.05) and pro-oxidants (H202, pyocynin) directly elevated NOX1 expression 9.3±2.1, 2.8±0.3 fold (p<0.05) in beta cells. The redox-responsive kinase, Src-kinase, regulated NOX1. An inhibitor of Src-kinase, PP2 blocked NOX1 expression induced by PICs or H202 in beta cells (52±8.5% and 48±5.4% resp, p<0.001). This was not seen with a structurally related but inactive compound PP3. Functionally, inhibition of NOX1 with a selective pyrazolopyridine dione, or inhibition of Src-kinase with PP2, preserved function and survival in beta cells exposed to PICs. Induction of apoptosis by PICs, measured by caspase-3 activation and Yo-Pro-1 fl uorescence microscopy was blocked by PP2, 95±2.5% (p<0.01). Uncoupling of glucose stimulated insulin secretion by PICs was preserved by PP2 and NOX1 inhibition. Collectively, these data indicate NOX1 upregulation is an important component in PIC-induced beta cell dysfunction. A feed-forward regulation of NOX1 and ROS production could rapidly result in beta cell pathology. Targeted inhibition of NOX1 feedback could open a new strategy to preserve and protect beta cell mass in the presence of elevated PICs.

Supported by: CDMRP (PR093521)

2166-PPax4 Expression Maintains the β-Cell Mass in a Mouse Model of Experimental Autoimmune DiabetesANABEL I. ALVAREZ-MERCADO, JOSÉ M. MELLADO-GIL, CARMEN M. JIMENEZ-MORENO, NADIA COBO-VUILLEUMIER, ANDREAS SPYRANTIS, JORGE VALLE-JO-ORTEGA, PETRA I. LORENZO, PAOLO MEDA, BERNHARD BOEHM, BENOIT R. GAUTHIER, Sevilla, Spain, Ulm, Germany, Geneva, Switzerland

We previously demonstrated that Pax4 overexpression in adult β-cells blunts the development of hyperglycemia in mice exposed to repeated streptozotocin challenges. The aim of the current study was to evaluate the protective role of Pax4 in a model of experimental autoimmune diabetes, and to monitor changes in β-cells mass using non-invasive in vivo imaging technology. To this end, we generated BPTL mice which bear the following transgenes: 1) RIP-B7.1, that facilitates the development of an autoimmune attack after vaccination with preproinsulin cDNA, 2) TRE/CMV Pax4, a strong, tetracycline inducible promoter, 3) RIP-rtTA that allows the selective expression of Pax4 in β-cells exposed to doxycycline and 4) MIP-LUC, that permits monitoring the β-cell mass through non-invasive in vivo bioluminescence imaging (BLI). In the absence of Pax4 induction, preproinsulin-vaccinated BPTL mice revealed a rapid decrease in BLI signal, reaching undetectable levels by day 28, which coincided with the development of severe hyperglycemia. At this time, immunohistochemistry (IHC) confi rmed the complete loss of β-cells with insulitis greater than 55% of islet volume. In contrast, after Pax4 induction, the BLI signal in vaccinated BPTL mice increased transiently, reaching 200% of the initial signal by day 21, and decreasing thereafter to 50% by day 28 onwards. Consistent with BLI signal, IHC performed at day 63 revealed the preservation of 60% of islet β-cell mass with a concomitant reduction in insulitis. Intriguingly, these animals exhibited fl uctuations in blood glucose levels, which ultimately resulted in persistent hyperglycemia by day 63. The results suggest that although Pax4 can adapt the β-cell mass in response to autoimmunity, it does not prevent the impairment of β-cell function, explaining the hyperglycemia. Thus, future diabetes therapies should focus on β-cell regeneration and increased survival, as well as on inhibiting the insulitis, which may be central to β-cell dysfunction.

Supported by: Junta de Andalucia (PI-0727-2010); ISCIII/FEDER (PI10/00871)

2167-PMatrix Metalloproteinase-9 (MMP-9) Cleaves Islet Amyloid Poly-peptide (IAPP) into Non-Toxic Fragments, Thereby Reducing Amy-loid Toxicity in INS-1 CellsDANIEL T. MEIER, LING-HSIEN TU, TANYA SAMARASEKERA, PETER J. MAREK, DANIEL P. RALEIGH, STEVEN E. KAHN, Seattle, WA, New York, NY

Human IAPP (hIAPP) is a 37 amino acid peptide containing fi ve amino acids between residues 24 and 28 that render it prone to aggregate as amyloid fi brils. hIAPP fi bril formation in turn contributes to increased β-cell apoptosis and reduced β-cell mass in type 2 diabetes. We have shown that MMP-9 is a metalloproteinase expressed in islets which cleaves synthetic hIAPP between residues 15/16 and 25/26, potentially leading to the generation of non-amyloidogenic fragments. We therefore investigated the amyloidogenic

and cytotoxic potential of the hIAPP fragments (1-15, 1-25, 26-37 and 16-37) resulting from MMP-9 cleavage. Aggregation kinetics revealed that fragments 1-15, 1-25 and 26-37 do not form amyloid fi brils. In contrast, fragment 16-37, which still contains the amyloidogenic sequence, rapidly formed fi brils without an initial lag phase. However, further cleavage of hIAPP fragment 16-37 by MMP-9 yielded two additional fragments (16-25, 26-27), both of which were non-amyloidogenic. To test whether the aggregation properties of these hIAPP fragments resulted in altered β-cell toxicity, all fragments were incubated with INS-1 cells at a concentration of 60 µM for 24 h. Cell viability following treatment with non-amyloidogenic rodent IAPP or hIAPP fragments 1-15, 1-25 and 26-37 did not differ from vehicle-treated cells (p>0.80 for all vs vehicle, n=4). In contrast, full length hIAPP and fragment 16-37 were toxic reducing cell viability by 90±0.005% and 80±0.043%, respectively (p<0.05 for both vs vehicle, n=4). In conclusion, MMP-9 cleavage of hIAPP yields three non-toxic fragments and one that is still amyloidogenic and cytotoxic. However, the latter can be further degraded by MMP-9 into non-amyloidogenic fragments, thus limiting β-cell toxicity. Activation or up regulation of MMP-9 in islets might be a strategy to prevent amyloid formation and β-cell loss in type 2 diabetes.

2168-P

2169-PBeta-Cell Compensation: Form Follows FunctionJOON HA, LESLIE SATIN, RICHARD BERTRAM, ARTHUR SHERMAN, Bethesda, MD, Ann Arbor, MI, Tallahassee, FL

It is generally accepted that type 2 diabetes results when insulin secretion fails to compensate for the increased demand imposed by insulin resistance. However, the mechanism of compensation has been mysterious. Thus, it is not clear whether compensation consists of increased beta-cell function (secretion per cell),increased beta cell mass or both. In addition, overt hyperglycemia, the most obvious putative trigger for compensation, is not typically observed. Here we extend an existing model for the regulation of beta-cell mass (Topp et al, J Theor Biol. 206:605, 2000), incorporating the workload hypothesis of Dor and colleagues (Porat et al, Cell Metab. 13:44,0 2011), to propose there is a hierarchy of responses in which enhanced beta-cell function precedes and in turn drives increased beta-cell mass. Acutely, beta cells dose dependently increase their insulin release according to the prevailing glucose concentration. However, persistent post-prandial hyperglycemia, which constitutes a small increase in average daily glucose but a large increase in average daily workload, shifts the beta-cell dose response curve to the left. If high workload persists for an extended period, beta-cell mass in diabetes-resistant individuals slowly expands, allowing workload and post-prandial glucose to return to normal. If insulin resistance is extreme, or accompanied by an impaired ability to increase mass, chronic hyperglycemia results. We show that a mathematical model based on these core principles can account for a variety of observed scenarios for the progression from euglycemia to diabetes or its reversal in patients.

2170-PThe Cytoprotective Effect of Nrf2 Pathway Activation on Human Pancreatic β-CellsYUICHI MASUDA, SHIRI LI, CLARENCE E. FOSTER, NOSRATOLA D. VAZIRI, HIRO-HITO ICHII, Orange, CA, Irine, CA

Nrf2 signaling pathway plays a central role in cell defense against oxidative stress. Islets are known to contain low level of antioxidants. It is known islets are exposed to signifi cant oxidative stress during isolation and transplant. Activation of this pathway induces up-regulation of numerous genes encoding antioxidant and phase II detoxifying enzymes. However, little is known regarding a role of this pathway in human islet. In this study, we investigate the effect of Nrf2 pathway activation on human islets those were obtained from cadaveric donors. After treatment with Nrf2 activator (NRA: a bardoxolone methyl analog), Nrf2 translocation, mRNA expression, and protein of anti-oxidants were evaluated. Cyto-protective

WITHDRAWN

A558


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


effect of NRA on human β cells against oxidative stress was evaluated by FACS. Infl ammatory cytokines/chemokines from islets were measured in supernatant. The translocation of Nrf2 from cytoplasm to nucleus was observed within 2 hours after NRA treatment. The mRNA expression of anti-oxidants, including GCLC (1, 1.7, 3.0 times), HO-1 (1, 3.5, 10.3), and NQO1 (1, 2.6, 5.0), was signifi cantly increased in dose-dependent manner (0, 250, and 1000nM of NRA). Hydrogen peroxide was used to induce oxidative stress. After dissociation into single cells, cells were stained with 7-AAD, TMRE, and Newport Green (NG) and analyzed by FACS. The percentage of viable β cells (7AAD-, NG+, TMRE+) was compared to evaluate the cyto-protective effect of NRA. The proportion of β cells and their viability in NRA-treated was higher than in vehicle-treated cells. NRA decreased production of cytokines/chemokines including IL-1β, IL-4, IL-6, and MCP-1. In conclusion, in vitro treatment of human islets with NRA signifi cantly increased expression of anti-oxidants and conferred protection against oxidative stress in β cells. These results indicate fortifi cation of antioxidant defense system by Nrf2 activation in human islets may be a promising strategy to improve the outcome in clinical islet transplantation.

Supported by: JDRF

2171-PThree-Dimensional Collagen Matrix Restores Reduced Levels of PKB Phosphorylation Associated With Amyloid Formation in Cul-tured IsletsYUN ZHANG, GARTH L. WARNOCK, ZILIANG AO, NOOSHIN SAFIKHAN, AZIZ GHAHARY, LUCY MARZBAN, Vancouver, BC, Canada

Human islet transplantation provides a feasible approach for treatment of type 1 diabetes (T1D) but is currently limited by insuffi cient donors and loss of beta cells during pre-transplant culture and in islet grafts. Aggregation of human islet amyloid polypeptide (hIAPP), a hallmark of pancreas in T2D, also occurs during islet culture and post-transplantation. hIAPP aggregates contribute to beta-cell death in all three conditions. Protein kinase B (PKB) signaling pathway plays a key role in the regulation of beta-cell survival, function and proliferation. We used transformed beta cells, human and transgenic mouse islets to: 1. examine the effects of exogenous and endogenous hIAPP aggregates on PKB phosphorylation; 2. test if embedding islets in collagen matrix can restore reduced PKB phosphorylation associated with hIAPP aggregation and thereby enhance islet survival/function during pre-transplant culture. Phospho-PKB levels, assessed by immunolabelling and Western blot, were markedly lower in hIAPP-treated INS-1 beta cells compared to those treated with non-fi brillogenic rat IAPP and non-treated cells. This was associated with reduced proliferation and increased apoptosis. hIAPP-expressing mouse islets formed hIAPP aggregates during culture and had markedly lower beta cell phospho-PKB than wild-type islets, both of which were prevented by the amyloid binding dye Congo red. Human and hIAPP-expressing mouse islets embedded in collagen matrix had signifi cantly lower hIAPP aggregation and beta-cell death (assessed by quantitative immunolabelling), higher phospho-PKB, insulin response to glucose, and islet insulin content than free-fl oating cultured islets. In summary, these data suggest that: 1) exogenously applied and endogenously formed hIAPP aggregates reduce PKB phosphorylation in islet beta cells; 2) collagen matrix restores reduced phospho-PKB associated with hIAPP aggregation in cultured islets.

2172-PNegative Regulation of the Beta-Cell Mass via Sympathetic Signal-ingHECTOR MACIAS, MICHAEL GERMAN, San Francisco, CA

We have investigated the role of sympathetic signaling in the control of the β-cell mass. Sympathetic signaling was found to potentiate β-cell apoptosis via the inhibition of MAPK signaling. We began by treating the β-cell line, MIN6, with Norepinephrine and Galanin, products deposited by sympathetic neurons, and fi nd that they induce apoptosis. Further, Norepinephrine initiates apoptosis via ADRA2A, and not ADRA2B. This was evident as Guanfacine, an ADRA2A specifi c agonist, and not 3F8, an ADRA2B specifi c agonist, initiated apoptosis. Pretreatment with the ADRA2A specifi c antagonist, Rauwolscine, abrogated Guanfacine induced apoptosis. ADRA2A and GALR1 are Gi/o-coupled GPCRs. Pretreatment with the Gi/o inhibitor, Pertussis Toxin, abrogated Guanfacine and Galanin induced apoptosis. β-cells under duress elevate MAPK signaling to alleviate and prevent the apoptosis program. We fi nd MAPK family members, Raf, MEK and ERK1/2 are dephosphorylated upon Guanfacine and Galanin treatment. Members of other signaling modules, such as β-Catenin, NF-κB, and AKT are left unperturbed. Pretreatment with Pertussis Toxin

prevented the inhibition of MAPK signaling upon sympathetic stimulation. Next, we found that Guanfacine and Galanin inhibit cultured mouse and human islet MAPK signaling. Given the protective nature of MAPK signaling, we sought to determine whether the abrogation of MAPK signaling upon sympathetic stimulation could potentiate β-cell apoptosis. We subjected cultured mouse islets to both sympathetic signaling and ER stress and fi nd that the level of apoptosis is dramatically higher upon treatment with both the ER stress inducing compound, Thapsigargin, and Guanfacine, than with either compound alone. Further, this synergism in apoptosis induction was observed upon the use of Thapsigargin, Brefeldin A, or Tunicamycin in MIN6. These fi ndings reveal a mechanism that might act to prune and restrict endocrine growth during embryogenesis, as well as enable Type 1 and Type 2 diabetes disease progression.

2173-PFormation and Detection of Peroxynitrite in β-CellKATARZYNA A. BRONIOWSKA, JOHN A. CORBETT, Milwaukee, WI

Infl ammatory cytokines impair pancreatic β-cell function by stimulating the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Nitric oxide acts as a signaling molecule at low levels, but in large quantities, it causes cell death. Nitric oxide rapidly reacts with superoxide to generate the highly reactive oxidant, peroxynitrite. Pancreatic β-cells have the capacity to generate superoxide, produced by NADPH oxidase or as a byproduct of mitochondrial respiration, but direct evidence of peroxynitrite formation has not been demonstrated. While the induction of iNOS is closely associated with β-cell death under infl ammatory conditions, it is not known whether the damaging species is nitric oxide or peroxynitrite.

Here, we examined the generation of peroxynitrite in β-cells (INS 832/13) and in isolated islets using coumarin-7-boronate, selective indicator of peroxynitrite. Treatment of INS 832/13 cells with IL-1β stimulates iNOS induction, nitric oxide production and decreases viability. When cytokine-treated INS 832/13 cells and isolated islets are exposed to phorbol ester, an NADPH oxidase activator, peroxynitrite is not detected. In contrast, addition of phorbol ester to LPS-activated macrophages results in peroxynitrite generation as monitored in real time with boronate probe. Nitric oxide donor causes concentration-dependent decrease in viability, inhibition of mitochondrial aconitase and depletion of ATP in INS 832/13 cells. Interestingly, when nitric oxide-treated β-cells are exposed to menadione, a compound that generates superoxide, we observe a decrease in nitric oxide-induced toxicity and preservation of ATP levels and aconitase activity. The effects of menadione and nitric oxide on glucose-stimulated insulin secretion are currently being evaluated. Taken together, our data indicate that nitric oxide mediates the toxic effects of cytokines and that terminally differentiated quiescent β-cells do not produce peroxynitrite in contrast to cells of innate immune system.

Supported by: R01-DK52194, R01-AI44458

2174-PActivation of PPARβ/δ Protects β-Cells from Lipotoxic Apoptosis by Upregulating the Expression of GLP-1RYAN YANG, XIUJUN LI, NANWEI TONG, Chengdu, China

We previously showed that activated peroxisome proliferator-activated receptor (PPAR)β/δ can protect pancreatic β cells against lipotoxic apoptosis. However, the underlying molecular mechanism remained unclear. Glucagon-like peptide-1 receptor(GLP-1R) has been reported to exhibit a protective effect against lipotoxic apoptosis in β cells. In the present study, we proposed that PPARβ/δ activation suppressed apoptosis and improved β cells function impaired by fatty acids via upregulating the expression of GLP-1R. Isolated rat islets and the rat insulin-secreting INS-1 cells were treated with and without a PPARβ/δ agonist GW501516 (GW) in the presence or absence of palmitate (PA). Apoptosis was assessed by DNA fragmentation and fl ow cytometry. The expression of GLP-1R in INS-1 cells was assessed and further confi rmed in islets. Akt,Bcl-2,Bcl-xl,caspase-3 signal molecules were assayed by western and quantifi cation of PCR. Our results showed: First, PA induced β-cells apoptosis in a dose-dependent manner and impaired glucose-stimulated insulin secretion (GSIS). However, GW protected β-cells from apoptosis and repaired GSIS in the same lipotoxic environment. Second, activation of PPARβ/δ enhanced the expression of GLP-1R under lipotoxic conditions, whereas GLP-1R activation had no effect on PPARβ/δ and further demonstrated that GLP-1R was the target facor of PPARβ/δ. Third, silencing in GLP-1R expression attenuated the protective action of GW against lipotoxic apoptosis and augmenting GSIS impaired by PA. Finally, GW exerted its anti-apoptotic effect partly by interfering with GLP-1R-mediated Akt/Bcl-2,Bcl-xl/Caspase3 signaling pathways. Therefore, this study confi rmed

A559


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


that a potential mechanism for the anti-apoptotic action of PPARβ/δ was provided by revealing its transcriptional regulation of GLP-1R, implicating a new therapeutic target for protecting pancreatic β-cells from lipotoxicity.

2175-PSet 7/9 Regulates Cytokine-Induced Expression of iNOS in Beta-Cells through Histone Modifi cationKYOKO KUDO-FUJIMAKI, TAKESHI OGIHARA, YOSHIO FUJITANI, HIROTAKA WATADA, Tokyo, Japan

Set7/9 is an enzyme to induce methylation of lysine 4 in histone 3 (H3K4), and accelerates the formation of euchromatin. Set7/9 is enriched in islets, and contributes upregulation of beta cell specifi c genes, including ins1 and slc2a. On the other hand, set7/9 reportedly binds to transcription factor, nuclear factor κ-B (NF-κB) to modify its transcriptional activity in response to proinfl ammatory cytokines, thus controls infl ammatory genes in non-beta cells. Given that infl ammation is involved in the pathophysiological changes of islet not only in type 1 but also in type 2 diabetes, the aim of this study is to elucidate the role of set7/9 on the response to proinfl ammatory cytokines in islets. The treatment with cytokine cocktail (IL-1beta, TNF-alpha and INF-gamma) induced the expression of inducible nitric oxide synthase (iNOS) which is attenuated by the treatment of siRNA for set7/9 both in mouse beta cell lines, betaTC3 cells and MIN6 cells. In betaTC3 cells, cytokine-induced nuclear translocation of NF-κB was not altered by the treatment of siRNA for set7/9, however, set7/9 translocated from cytosol into nucleus by the treatment with cytokine cocktail. Consistent with previous reports, set7/9 was co-precipitated with p65, a subunit of NF-κB in betaTC3 cells. In addition, ChIP assay demonstrated that the treatment with cytokine cocktail augmented the methylation of H3K4 in nos2 (iNOS) gene promoter region, which was attenuated by set7/9 knockdown. Our results indicate that set7/9 at least in part regulates nos2 expression through histone modifi cation. While set7/9 play essential role on the expression of the beta cell specifi c genes, it may also mediate delirious effect on glucose metabolism to promote cytokine-induced iNOS expression in beta cells.

2176-PATF6β Depletion Increases Susceptibility of Pancreatic β-Cells to Endoplasmic Reticulum Stress-Induced ApoptosisTANYA ODISHO, LILING ZHANG, ALLEN VOLCHUK, Toronto, ON, Canada

Endoplasmic reticulum (ER) stress has been implicated as a causative factor in the development of pancreatic β-cell dysfunction and death resulting in type 2 diabetes. Activating transcription factor 6 (ATF6) is a critical component of the Unfolded Protein Response in cells undergoing ER stress. As unfolded and misfolded proteins accumulate in the ER, ATF6 is activated and contributes to the induction of various ER chaperones and components of the ER-associated degradation pathway, which assist in maintaining ER homeostasis in the cell. We have previously demonstrated that the ATF6α isoform is required for pancreatic β-cell survival under basal and ER stress conditions. However, no studies to date have investigated the role of the ATF6β isoform in pancreatic β-cells. Using an ATF6β-specifi c antibody we detected expression of full-length ATF6β in various insulinoma cell lines and rodent islets and the induction of the active form (ATF6βp60) under ER stress conditions. However, knockdown of ATF6β using siRNA in INS1 832/13 insulinoma cells did not affect mRNA or protein induction of known ER stress response genes (Grp78, Grp94, Herp, Hrd1) in response to tunicamcin-induced ER stress. Thus, ATF6β does not appear to be required for the induction of genes known to be induced by ATF6α, although insulinoma cells defi cient in ATF6β had a compensatory increase in ATF6α levels. Interestingly, knockdown of ATF6β increased the susceptibility of β-cells to ER stress-induced apoptosis, as monitored by the levels of pro-apoptotic markers (cleaved caspase 3, PARP, and CHOP). Together, our results suggest that ATF6β is not required for induction of major ER stress response genes, but is required to maintain cell survival in β-cells undergoing ER stress. Thus, ATF6β has a pro-survival role as part of the ER stress response, although the exact complement of genes regulated by this ER stress response sensor is unknown and the subject of current investigation.

2177-PDifferential Expression of GLUT2 in Pancreatic Islets Affects Strep-tozotocin Sensitivity in NOD and NOR MiceSEVIM KAHRAMAN, CIGDEM AYDIN, GULSUM OZLEM ELPEK, ERCUMENT DI-RICE, AHTER DILSAD SANLIOGLU, Antalya, Turkey, Boston, MA

Type 1 Diabetes (T1D) is an autoimmune disease characterized by select-ive destruction of pancreatic β cells. Non-obese diabetic (NOD) mice are most preferred animal models in T1D research, and non-obese diabetes

resistant (NOR) mice are considered as proper controls. Previously, we showed that streptozotocin (STZ), a diabetic agent, caused a more severe diabetic phenotype in 10 wk old female NOR mice than age-matched NOD mice. While NOR mice were hyperglycemic two days after a single dose of STZ (150 mg/kg) injection with a high level of weight loss at day 4 and poor survival, NOD mice displayed prominent hyperglycemia only at day 7 and had high survival rates. Aiming to investigate the underlying mechanism, we hypothesized that the difference in STZ sensitivities could be due to differential GLUT2 receptor expression levels in β cells, through which STZ is taken inside to cause DNA damage. First we assessed the β cell content of STZ-given animals by insulin-glucagon double staining and found that NOR mice had nearly half the β cell content compared to NOD mice at day 2 (NOR 66%; NOD 35%; n=3, p<0.05). Pancreatic sections were then double stained with anti-GLUT2 and anti-insulin for GLUT2 expression levels, following with measurement of mean fl uorescence intensities (MFI), calculated from anti-GLUT2 relative to anti-insulin stained areas. Although the MFIs were similar in NOD and NOR islets before STZ injection, NOR islets displayed increased MFIs shortly after STZ injection. Fluorescence intensities were 2-fold higher in NOR compared to NOD islets at day 4 (NOR 162.5±31.9; NOD 81.1±9.2 MFI). We also checked GLUT2 expression levels by DAB staining. Intensity scoring revealed weak staining in NOD and NOR islets at day 0, and strong staining only in NOR islets at day 4 (NOR 3.0±0.0; NOD 0.75±0.25; n=3, p<0.05). These results suggest that NOR control strain is highly sensitive to STZ toxicity as a result of increased STZ transport due to elevated expression levels of GLUT2 in pancreatic β cells.

2178-PERp57 Protects Autophagy Defi cient Beta-Cells from Cellular Dam-ageERIKO YAMAMOTO, TOYOYOSHI UCHIDA, HIROKO ABE, KOJI KOMIYA, AKEMI HARA, TAKESHI OGIHARA, SATORU TAKEDA, YOSHIO FUJITANI, HIROTAKA WATADA, Tokyo, Japan

Beta-cell specifi c autophagy defi cient mice exhibited hypoinsulinemia and hyperglycemia associated with mitochondrial dysfunction. The dysregulated autophagy may possibly be involved in diverse aspects of the pathogenesis of type2 diabetes mellitus. To comprehensively fi nd molecular targets of autophagy in β cell, we compared protein expression profi le in the islets of beta-cell specifi c autophagy defi cient mice to that of control (Atg7f/f) islets by 1- dimensional electrophoresis. We identifi ed ERp57 (PDIA3) signifi cantly increased in the islets of beta-cell specifi c autophagy defi cient mice, which is the protein disulfi de isomerase localized predominantly in endoplasmic reticulum (ER) to maintain ER quality control. Immunohistochemistry showed that ERp57 was expressed more abundantly in β cell of beta-cell specifi c autophagy defi cient mice than in that of Atg7f/f. To investigate the role of ERp57 in autophagy defi cient β cells, we generated tetracycline-inducible Atg7 knock-down (Atg7KD) rat insulinoma INS-1 cells and ERp57 was signifi cantly increased in Atg7KD than in Atg7 wild type (WT) INS-1 cells. Then, we additively reduced ERp57 using siRNA in both INS-1 cells to investigate cell viabilities by sub-G1 analysis. The suppression of ERp57 caused to increase sub-G1 population in Atg7KD INS-1 cells, but did not affect on sub-G1 population in Atg7WT INS-1 cells. These data suggested that ERp57 might be induced by autophagy defi ciency in β cells and ERp57 had protective effects on autophagy defi cient β cells against apoptotic cell death.

2179-PMolecular Mechanism of β-Cell Dysfunction in Obese Diabetic b/db Mice: Implication of the Islet Infl ammation due to Residual Lep-tin SignalMASASHI SHIMODA, YUKIKO KIMURA, KAZUHITO TAWARAMOTO, MITSURU HASHIRAMOTO, KOHEI KAKU, Okayama, Japan

The db gene homozygous (db/db), but not the ob gene homozygous (ob/ob), mice develop a marked hyperglycemia with severe pancreatic β-cell damage. To clarify the molecular mechanism underlying db gene-associated β-cell damage, we compared Islet morphology, β-cell function, and gene expression profi les specifi c for pancreatic islet cells between 2 strains of mice at early stage of life, demonstrating the similar mild metabolic disorder. Glucose tolerance measured by ipGTT, insulin sensitivity by ipITT, plasma triglyceride level, and glucose-stimulated insulin secretion from islets were not different each other at 6 weeks of age. The β-cell mass and function were also not different. The cDNA microarray assay and real time RT-PCR of islet cells demonstrated a signifi cant upregulation of genes associated with infl ammatory cytokines (il1β, tgfβ1) and profi brosis (col1a1, Fn1) in db/db mice. In contrast, the mRNA levels of cell differentiation-related Mafa

A560


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


and Pou3f4, cell proliferation-related Ccnd1, and cell apoptosis-related Cad were not different between 2 mouse strains. F4/80-positive ratio in the islet was signifi cantly higher in db/db than in ob/ob mice. Both CD4 and CD8a cell positivity in the islet was comparable in 2 strains of mice and very few. The residual leptin signal was confi rmed by the positive gene expression of functional short-form leptin receptor (Ob-Rs) in the islet core area of db/db, but not ob/ob mice. Recombinant mouse leptin increased the islet il1β, il6 and tgfβ1 gene expressions dose-dependently in both db/db and ob/ob mice. The double-staining of the islets by anti-insulin/anti-phospho-p38MAPK revealed the accelerated p38MAPK pathway in β-cells of db/db compared with ob/ob mice. The present results strongly suggest that the precedent infl ammation and fi brosis of the islets caused by leptin signal through the residual Ob-Rs deteriorates pancreatic islet structure and β-cell function in db/db mice.

2180-PMulti-Parameter High-Content Screening Identifi es Families of Se-creted Factors that Prevent Beta-Cell DeathYU HSUAN CAROL YANG, JAMES D. JOHNSON, Vancouver, BC, Canada

Adult pancreatic islet cells secrete and respond to factors beyond the well characterized hormones. Our systematic analysis of these factors began with a compiled list of secretagogues and receptors that are expressed in adult mouse or human islets, which led to our discovery of novel paracrine signaling loops within the islets. We have previously focused on the pro-survival role of axon guidance factors during neuronal development, including the Netrin and SLIT family of ligands and their respective receptors, Neogenin/UNC5A-D and ROBO1-4. To rank the pro-survival affects of individual factors in our list along with those found in the literature, we have generated a library of 206 recombinant proteins/peptides and conducted a parallel comparison of these factors for their ability to prevent cell death under different diabetes-related stress conditions (cytokine exposure, ER stress and serum withdrawal). Following treatments, dispersed mouse islet cells were assessed using high-content kinetic imaging for nuclear morphology and cell number with Hoechst 33342 DNA dye and different stages of cell death with propidium iodide and AlexaFluor647-conjugated AnnexinV. Fluorescently labelled mouse beta-cells were used for hit confi rmation. We were able to identify factors that prevented cell death only at early time points and those with pro-survival effects persisting throughout the entire time course. Amongst the top ranked factors that prevented cell death were members of the Bone Morphogenetic Protein and Wingless families. Generation of self-organizing maps allowed for the selection of factors that displayed pro-survival roles under all of the stress conditions tested. Since diabetes results from a defi ciency in functional beta-cell mass, our efforts of identifying factors that prevent beta-cell death will assist in novel therapeutic developments.

Supported by: JDRF; Johnson & Johnson; SCN; CIHR; NSERC

2181-PBid in Human Islet Cell DeathMUGDHA JOGLEKAR, PRERAK TRIVEDI, THOMAS W. KAY, ALICIA J. JENKINS, ANANDWARDHAN A. HARDIKAR, HELEN E. THOMAS, Sydney, Australia, Fitzroy, Australia, Oklahoma City, OK

Type 1 diabetes results from autoimmune destruction of pancreatic beta cells by apoptotic mechanisms. Bid is a BH3-only protein of the Bcl-2 family that is activated in both extrinsic (death receptor-induced) and intrinsic (Bcl-2-regulated or mitochondrial) cell death pathways in mouse beta cells. We have previously demonstrated that Bid-defi cient mouse islets are protected from Fas ligand-mediated as well as granzyme B-mediated apoptosis in vitro. Silencing Bid therefore has therapeutic potential to inhibit both cytotoxic T cell-activated pathways of cell death in beta cells. However, it is not yet known if Bid plays similar central role in human beta cell death. We therefore aimed to test the role of Bid in human islet cell apoptosis using siRNA knockdown.

Freshly isolated human islets were dispersed into single cells, cultured overnight and then transfected with bid siRNA. We also forced the expression of siRNA for FADD (FAS-associated via death domain), a molecule in the extrinsic cell death pathway upstream of Bid. Using FITC-labeled non-specifi c siRNA, we found that the transfection effi ciency varies from 40-60%. We observed a 60-80% reduction in bid transcript as compared to untransfected and scramble siRNA transfected controls. Exposure of bid siRNA transfected cells to apoptotic stimuli (cytokines + FasL) in vitro did not result in signifi cant protection from cell death as determined by DNA fragmentation analysis using fl ow cytometry, whereas FADD knockdown was protective. Our results suggest that Bid independent pathways are

responsible for human islet cell death. Current work is focused on confi rming whether the amount of Bid knockdown is suffi cient to prevent granzyme-B-dependent cytotoxic T cell-mediated death of human islet cells.

If successful these studies would have signifi cant impact on present understanding of beta cell death in progression and treatment of diabetes.

Supported by: JDRF

2182-PThe Protective Effect of APPL1 on β-CellsXIAOWEN LI, SHIHONG WANG, LING LI, KAIDA MU, CHEN WANG, WEIPING JIA, Shanghai, China

APPL1 is an adaptor protein playing a critical role in regulating adiponectin signaling and insulin signaling. We previously reported that APPL1 knockdown in insulin-secreting INS-1 cells inhibited glucose-stimulated insulin secretion (GSIS), indicating a role of APPL1 in β-cell function regulation. Addition of adiponectin has been reported to lower free fat acid-induced β-cell death. However, whether APPL1 is involved in anti-apoptotic effects in β-cells is not clear. The aim of the study was to determine if APPL1 has any protection on pancreatic β-cells. To this end, INS-1 cells were used and APPL1 was overexpressed with adenovirus encoding APPL1. We found that incubation of INS-1 cell to IL-1β (30U/ml) in combination with TNF-α (20 ng/ml) for 48h induced cell apoptosis [18.78±1.12% (IL-1β+TNF-α) vs. 1.65 ± 0.60% (Control), P<0.01, n=4], impaired 2h GSIS [18.35±3.50 (IL-1β+TNF-α) vs. 61.08±4.20 (Control) µg/mg protein·2h, P<0.01, n = 5] compared with control cells. Moreover, APPL1 protein expression decreased in cytokines treated cells [relative expression: 1.31±0.16 (IL-1β+TNF-α) vs.2.08±0.23 (Control), P<0.05, n = 4]. To determine the potential roles of APPL1 in β-cell apoptosis, we treated INS-1 with adenovirus encoding APPL1 and found that cytokines-induced cell death was decreased in INS-1 cells upon overexpressing of APPL1 [10.75±0.67% (APPL1) vs. 18.78±1.12% (GFP), P <0.01, n = 4]. Furthermore, GSIS in INS1 expressing APPL1 was recovered compared with GFP groups after treatment of IL-1β and TNF-α [32.03±6.73 (APPL1) vs. 23.02±5.95 (GFP) µg/mg protein·2h, P < 0.05, n = 5]. In conclusion, APPL1 in INS1 cells has a protective effect on cytokines induced β-cell apoptosis.

2183-PIncreases in Glucose Concentrations Promote Beta-Cell Autophagy via mTOR-Independent MechanismROBERT N. BONE, SASANKA RAMANADHAM, Birmingham, AL

Type 2 diabetes mellitus (T2D) is a metabolic disorder characterized by hyperglycemia and associated with insulin resistance and beta-cell death. Apoptosis is a well-studied feature of beta-cell death, but a less well-studied beta-cell process is autophagy. Autophagy is an endogenous process of cellular component degradation in response to intra- and extracellular signals; including ER stress, oxidative stress, lipids, and starvation. Autophagy is thought to be a benefi cial process; however, dysregulation of autophagy could be detrimental to the beta-cell in times of stress. Macroautophagy (MA) is distinguished by the formation of the autophagosome, which surrounds components to be degraded and shuttles them to the lysosome for degradation. Microtubule-associated protein light chain 3 (LC3) is known to be present on the autophagosomes and serves as a marker of the autophagosome. We believe dysregulation of autophagy due to hyperglycemia and hyperlipidemia negatively impact autophagy. Our studies in the INS-1 beta-cell line suggest that autophagy is dysregulated by features of T2D. Cells challenged with high glucose have shown a reduction in initiation of MA, as evidenced by decreased LC3-II. Addition of the fatty acid palmitate to culture medium has also shown that the initiation of MA is reduced. Here, investigation into the possible mechanism of reduced MA centered on mTOR and protein kinase A (PKA), known inhibitors of MA. We found that phosphorylation of the mTOR downstream element p70-S6kinase was unchanged after increasing glucose, suggesting an mTOR independent pathway. PKA downstream element CREB phosphorylation was reduced in 22mM glucose, but at 33mM glucose was similar to 11mM glucose control. Interestingly, addition of Exendin-4, a drug used for treatment of T2D and known inducer of the PKA pathway, further reduced MA initiation. Our results suggest that MA is negatively dysregulated during hyperglycemia and is potentially regulated in an mTOR-independent manner.

Supported by: NIH (R01-DK69455)

A561


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2184-PA Novel Long-Acting GLP-1R Agonist, SKL-18287, Protects Against Pancreatic β-Cell DeteriorationKATSURA TSUKAMOTO, HIROSHI KINOSHITA, RYUJI OKAMOTO, SANWA KA-GAKU KENKYUSHO, Inabe, Japan

SKL-18287 is a novel long-acting GLP-1R agonist designed to exhibit DPP-IV resistance and multimer formation for improving stability with several natural amino acid modifi cations of native GLP-1 that is currently being developed as QD and QW GLP-1 mimetic product. We investigated the effects of SKL-18287 on pancreatic β cells using in vitro and in vivo models.

HIT-T15 cells and freshly prepared dispersed C57BL/6J mice islet cells were used in vitro for the measurement of cell survival and growth under various culture conditions including FFA or cytokine mixture induced apoptotic conditions. In HIT-T15 cells, SKL-18287 (10 nmol/L) signifi cantly reduced the cell death induced by 24-hour exposure to FFA or a cytokine mixture. Moreover, SKL-18287 (0.1-10 nmol/L) signifi cantly increased the BrdU incorporation after a 24-hour culture. In dispersed islet cells, SKL-18287 (0.1-10 nmol/L) improved the cell survival (1.36-1.53 fold, p<0.05) compared with non-treated cells during 48-hour primary culture. On the other hand, alloxan-induced diabetic C57BL/6J mice were used to investigate the effects of SKL-18287 in vivo. SKL-18287 (48 nmol/kg/day) was administered for 4 weeks using the osmotic pump implanted under the dorsal skin of diabetic mice. The treatment with SKL-18287 signifi cantly reduced the random-fed blood glucose and glycated hemoglobin levels in diabetic mice compared with the control. Consequently, the pancreatic insulin content measured by ELISA and β cell area measured by immunohistochemistry of the SKL-18287-treated group were signifi cantly improved and at least twice as much as those of the control.

SKL-18287 exhibited a direct protective and growth effect on rodent pancreatic β cells deterioration in vitro under stress conditions. In addition to the direct effect on pancreatic β cells, the strong and sustained blood glucose lowering effect of SKL-18287 may contribute to the maintenance of the pancreatic β cells in alloxan-induced diabetic mice.

2185-PCo-Culturing of Primary Human Islets Together With Islet Endothe-lial Cells Maintains Islet Structure and Extracellular Matrix Integ-rity Ex VivoEITAN M. AKIRAV, Mineola, NY

Islet endothelial cells (iECs) are an integral part of the islets of Langerhans and play a pivotal role in maintaining normal β-cell function and islet integrity. They serve as a source of extracellular matrix (ECM) proteins, such as collagen IV (col-IV) and laminin. Following islet isolation, both iECs and the ECM are largely lost, resulting in islet disintegration and a loss of β-cell function. We previously developed a method using iECs for inducing free-fl oating pseudoislets while improving β-cell function and survival. Here we examined if transformed and primary iECs can maintain islet structure and insulin expression in primary human islets (hIslets). hIslets were cultured with or without the murine iEC line, MS-1. Co-culturing of hIslets and MS-1 cells preserved hIslet integrity and structure. In contrast, hIslets cultured alone formed cell aggregates with increased cell death over time. Confocal imaging of hIslets cultured together with MS-1 cells revealed round islet structures that were positive for col-IV and laminin in and around the islet. In contrast, hIslets cultured alone showed reduced ECM staining. Histomorphic analysis of insulin staining showed higher protein expression in hIslets cultured together with iECs. Next we examined the ability of primary human iECs to preserve hIslet structure and ECM integrity. iECs from hIslets were expanded and enriched using magnetic separation. Co-culturing of islets with iECs preserved islet structure for up to 18 d in culture. In contrast, hIslets cultured alone showed reduce islet integrity. Col-IV and laminin were detected in hIslets cultured with primary iECs. In this report we describe a novel method for culturing free-fl oating hIslets in the presence iECs. This method can preserve islet structure and ECM ex vivo, thus providing a new tool for the in vitro study of human islets over time.

2186-PProtective Role of GLP-1 Analogue on Cholesterol-Induced Apopto-sis via Activation of mTOR in βTC-6 CellsJIAQIANG ZHOU, JIAHUA WU, FENPING ZHENG, HONG LI, Hangzhou, China

The potential role of cholesterol in lipotoxicity and its relationship with apoptosis and dysfunction in pancreatic β-cells has been received attention recently. Despite an increasing number of evidence showed that cholesterol had direct effect on pancreatic β-cells, no studies have ever investigated how to protect β-cells from the potential toxicity of cholesterol. It was reported

that glucagon-like peptide-1(GLP-1 )analogue could protect β-cells from glucolipotoxicity via activation of protein kinase B. The present study was undertaken to investigate whether GLP-1 analogue had a protective role on the toxicity of cholesterol on pancreatic β-cells and its possible mechanism. βTC-6 cells were treated with 5 mmol/l cholesterol for various time, in the presence or absence of 10 nmol/l GLP-1 analogue liraglutide. Compared with control group, cholesterol signifi cantly decreased glucose-stimulated insulin secretion (GSIS), increased the level of cleaved caspase 3 and proportion of apoptotic nuclei. Pretreatment with liraglutide reversed the reduction of GSIS induced by cholesterol, and protected cells from apoptosis. To further investigate the possible mechanism of anti-apoptotic effect of liraglutide, cells were pre-incubated with 10 nmol/l liraglutide, alone or with 1 µmol/l Rapamycin, an inhibitor of mTOR, followed by incubation with 5 mmol/l cholesterol for 6h. The anti-apoptotic effect of liraglutide was absolutely abrogated by pre-incubation with Rapamycin. Furthermore, pre-incubation with 1µmol/l Rapamycin signifi cantly increased cholesterol-induced β-cell apoptosis. These data demonstrated mTOR signaling acting as an essential mediator in protective role of GLP-1 analogue on cholesterol-induced apoptosis in βTC-6 cells.

Supported by: NSFC

2187-PHumoral Factors by Bone Marrow-Derived MSC Enhance Survival of Injured β-Cells by Preserving Akt SignallingCHUNE LIU, LEONIE LANG, CHRISTINA JÄGER, MARLENE SCHMID, JOHANNES BAUMANN, AMIR E. MEHANA, MARCUS ALT, SANDRA SOJKA, NIKOLAOS PERAKAKIS, KATHARINA LAUBNER, GÜNTER PÄTH, JOCHEN SEUFERT, Freiburg, Germany

Our previous work has verifi ed in vitro that human bone marrow-derived MSC with telomerase overexpression (hMSC-TERT) migrate towards injured rat INS-1E β-cells and increase their viability. Both cell lines were considered a valid model since they closely resemble primary rat (plastic-adherent) MSC and islets. Here we investigated whether humoral factors by cocultured hMSC-TERT reduce apoptosis and induce proliferation in INS-1E. Since AKT signalling plays a very central role for survival and proliferation in β-cells, we further analysed whether humoral hMSC-TERT-derived factors rescue p-AKT levels during β-cell injury.

hMSC-TERT were cocultured in inserts with 0.4 µm pores that allow only soluble factors but not cells to pass the membrane. Apoptosis was measured by Annexin-V FACS and caspases 3/7 activity assay. Viability was measured by MTS assay. Active (phosphorylated) p-AKT levels were detected by Western blot. Proliferation was analysed by counting cell numbers and Ki-67+ nuclei.

Cocultured hMSC-TERT signifi cantly reduced ALX and STZ-induced apoptosis in INS-1E. The degree of this reduction was similar to enhancement of viability. LD50 dosages of ALX (6.6 mM) and STZ (0.66 mM) completely deplete p-AKT levels within 2 and 8 h, respectively. This loss of p-AKT was substantially prevented by cocultured hMSC-TERT that have been preactivated over night by injured INS-1E while hMSC-TERT without preactivation have only small effects. Furthermore, chronic coculture of hMSC-TERT enhanced proliferation of INS-1E to signifi cant levels after 5 days. HEK293 human kidney cells and MRC5 human fi broblasts were used as controls to verify specifi city of MSC-mediated effects.

MSC-derived factors promote survival of injured β-cells by reduction of apoptosis via protection of p-AKT levels. Modulation of AKT signalling may further be responsible for increased viability and enhanced proliferation in untreated β-cells.

Supported by: Bundesministeriums für Bildung und Forschung

2188-PExendin-4 Promotes Survival of Mouse Pancreatic β-Cell Line in Li-potoxic Conditions, through the Phosphoinositide 3-Kinase (PI3K)/ PKB Signaling PathwayJIN ZHANG, QIAN WEI, Shenyang, China

Type 2 diabetes is a heterogeneous disorder that develops as a result of relatively inappropriate insulin secretion and insulin resistance. Increased levels of free fatty acids (FFAs) is one of the important factors for the pathogenesis of type 2 diabetes, and contributes to defective β-cell proliferation and increased β-cell apoptosis. Recently, glucagon-like peptide (GLP-1) receptor agonists have been shown to possess an antiapoptotic effect, increasing β-cell mass, and improving β-cell function. However, their effects on β-cells in vitro against lipotoxicity have not been elucidated completely. In this study, we investigated whether the GLP-1 receptor agonist exendin-4 promoted cell survival and attenuated palmitate-induced

A562


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS

ISLET BIOLOGY—BETA CELL—DEVELOPMENT AND POSTNATAL GROWTH

apoptosis in murine pancreatic β-cells (MIN6) exposed to chronic elevated FFAs. Cell survival was assessed by MTT. The percentage of apoptotic cells was determined by fl uorescence microscopy analysis after Hoechst/PI staining and fl ow cytometric assay after Annexin V-FITC/PI staining. Expression of PKB, p-PKB, BAX, BCL-2 Caspase-3 was detected by western blotting. Results showed that exendin-4 promoted cell survival and inhibited apoptosis induced by palmitate (0.4 mM) in MIN6 cells. After 24 h of incubation, exendin-4 caused rapid activation of protein kinase B (PKB) under lipotoxic conditions. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of exendin-4 on MIN6 cells. Exendin-4 also inhibited the mitochondrial pathway of apoptosis and down-regulated Bax in MIN6 cells. Our fi ndings suggest that exendin-4 may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB and inhibition of the mitochondrial pathway.

2189-PEstrogen Protects MIN6 β-Cell from Hypoxic Cell Death via PI3K/Akt PathwayZHIPENG LI, SHUYU YANG, SUHUAN LIU, Xiamen, China

Pancreatic islet transplantation (PIT) offers a physiological treatment for type 1 diabetes (T1D), but the rapid loss of functional islet mass after PIT hinders its application. Hypoxia is a major deteriorate factor and the main cause of islet loss after PIT, and islet β−cells are particularly vulnerable to hypoxia. We have shown that the female hormone 17β-estradiol (E2) favors islet survival and attenuates engraft hypoxia after PIT, but the mechanism remains unclear. Using mouse MIN6 cells, we investigated the effect of E2 on β-cell survival under hypoxic conditions. Apoptosis was measured by caspase 3/7 activity assay, and cell viability was evaluated by CCK-8 cell viability assay. Western blot was used to visualize protein phosphorylation. We observed that hypoxia induced a remarkable increase of β-cell apoptosis and decrease of cell viability, which was attenuated by E2 treatment in a dose dependent manner. E2 protection of β-cell survival from hypoxia was blocked when LY294002, a PI3K/Akt inhibitor, was administrated, suggesting an involvement of PI3K/Akt pathway in E2 protection of β-cell apoptosis from hypoxia. PI3K/Akt plays predominant roles in β-cell function and survival, and in deed, we found here that E2 did acutely activated Akt phosphorylation within minutes and reach the peak at 1h in cultured MIN6 cells. Taken together, we concluded that E2 protects β-cell from hypoxia-induced apoptosis, which involves PI3K/Akt pathway. In addition to the well-known E2 protection of pancreatic β-cell from oxidative stress, infl ammation, and lipotoxicity, we showed here that E2 can also protect β-cell survival from hypoxic injury.

Supported by: NSFC


Guided Audio Tour: Development and Postnatal Growth (Posters: 2190-P to 2197-P), see page 17.

& 2190-PFewer Beta Cells and Insulin Granules in Autopsy Histology of a Pa-tient With Sulfonylurea-Unresponsive KCNJ11 Neonatal DiabetesSIRI ATMA W. GREELEY, MARK C. ZIELINSKI, JAMIE R. WOOD, DONALD F. STEINER, GRAEME I. BELL, LOUIS H. PHILIPSON, MANAMI HARA, Chicago, IL

Here we report the fi rst autopsy of a patient with KATP channel-related neonatal diabetes, in this case due to the G334D mutation in KCNJ11 with associated severe neurodevelopmental disability. Glyburide started at 2 mo (months old) had no effect on exogenous insulin requirements. A meal test at 8 mo confi rmed barely detectable C-peptide despite glyburide of 1.8 mg/kg/day. She developed central hypoventilation and died in her sleep at 24 mo. Immunohistochemical analysis of the whole pancreas (sampling from head to tail) revealed signifi cantly reduced but relatively preserved beta-cell mass (>70% loss compared to 13 age-matched controls; see fi g), but not other cell types. Electron micrographic analysis confi rmed the presence of beta cells with insulin granules; however, beta (more so than alpha) cell destruction was evident with swollen mitochondria and fewer granules with possible crinophagic vacuoles. Mouse models of KCNJ11 diabetes exhibit a similar loss of beta cell mass over time, although our patient’s relatively preserved mass at 24 mo suggests a slow decline. Indeed, those with less severe mutations are capable of sulfonylurea-stimulated insulin secretion much later in life but it remains unclear what factors infl uence preservation of

beta cells. In summary, this rare case with mutated KATP channels suggests that beta cells may be less able to survive when they are capable only of insulin production but not insulin secretion.

Supported by: UC DRTC (P60 DK020595); JDRF (9-2008-177); NIH (UL1RR024999

& 2191-PMechanisms Underlying Compensatory Islet Response to Insulin ResistanceABDELFATTAH EL OUAAMARI, JIAN-YING ZHOU, ERCUMENT DIRICE, CHONG WEE LIEW, JONG-SEO KIM, RICHARD SMITH, WEI-JUN QIAN, ROHIT N. KULKARNI, Boston, MA, Richland, WA

Compensatory islet hyperplasia is a distinct feature of the pre-diabetic insulin resistant state in rodents and humans. We undertook studies aimed at identifying alterations in islet proteins that were relevant for secretory function, apoptosis and proliferation that characterize the adaptive response.

Islets from six-month-old male control, high-fat diet fed (HFD) and obese ob/ob (OB) mice (n=5) were analyzed by liquid chromatography coupled with mass spectrometry. Global label-free quantifi cation using the accurate mass and time tag approach and targeted quantifi cation using selected reaction monitoring were applied to identify ~1,700 islet proteins and quantifi ed by at least two unique peptides with a false discovery rate <1%. Statistically signifi cant alterations in protein abundances were observed for ~450 proteins between groups. The islets exhibited alterations in energy metabolism (e.g. ALDOA, PFKL), oxidative phosphorylation (e.g. ATP5I, COX5A, COX6A), hormone processing (e.g. PC1, PC2) and secretory pathways (e.g. RAB7A, UCN3). An increased expression of molecules involved in protein synthesis (e.g. EIF3 family members) and folding (e.g. ERP29, ERP44) suggests endoplasmic reticulum stress response in insulin resistant islets. Compensatory cell survival and proliferation appeared to be the major mechanisms by which islet-cells responded to increased metabolic needs. Finally, proteome comparison between islets from HFD and OB mice revealed a subset of proteins that were differentially regulated by leptin signaling.

This study represents the fi rst quantitative study of the islet proteome that is focused on the compensatory islet-cell growth in response to insulin-resistance without overt diabetes. The extensive dataset provides a unique resource of candidate proteins that are linked to cell regeneration for undertaking studies aimed at enhancing the functional β-cell mass in diabetic patients.

Supported by: R011DK067536 (to R.N.K.); SFD (to A.E.O.)

A563


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


& 2192-PHistological Analysis of the Whole Human Pancreas in Health and DiseaseMARK C. ZIELINSKI, XIAOJUN WANG, RYOSUKE MISAWA, LING-JIA WANG, PIOTR WITKOWSKI, MANAMI HARA, Chicago, IL

Total beta-cell mass changes during development and aging, as well as under disease conditions. Unlike laboratory animals, a long life span and considerable diversity in humans confound the precise analysis of human pancreas. We examined whole pancreata from head to tail regions in each donor: non-diabetic (n=15; Age: 2 mo-51 yr; BMI: 14-36), patients with T2D (n=8; Age: 26-66 yr; BMI: 21-50; Duration: 3 mo-15 yr) and patients with T1D (n=2; Age: 7 and 43 yr; BMI: 18 and 27; Duration: 5.5 and 15 yr, respectively). Each pancreas was divided into consecutive tissue blocks with preparation alternating between fresh-frozen and paraffi n-embedding. We have applied a large-scale computer assisted image analysis that we developed, which provides representative data in the entire section. Intra-pancreatic analysis in each individual shows a similar pattern of changes in total endocrine cell mass with a gradual decrease from the head to the body region and a marked increase toward the end of the tail region ~2-fold. However, in adults over 20 yr of age (except patients with T1D), inter-subject analysis has revealed signifi cant individual variability in total endocrine cell mass that shows no direct correlation with age, BMI or diabetic conditions including the duration of diabetes. Three cases are shown in the Figure. Further electron microscopic analysis was confi rmatory that the individual variability exceeded phenotypic changes such as cellular fat deposit and vacuole formation.

& 2193-POverexpression of E2F3 Promotes Proliferation of Functional Hu-man Beta-Cells Without Induction of ApoptosisBRIAN RADY, PILAR VACA, YONG WANG, JOSE OBERHOLZER, Chicago, IL

Reversal of diabetes through islet cell transplant is proving safe and effective. However, cadaver organs, the current source of islets for transplant, are too scarce to transplant all those affected by this disease. Mitotic division of beta-cells provides a route to expand transplantable islets. However, adult human beta-cells are particularly resistant to common

proliferative targets and often dedifferentiate during proliferation. Here we show that expression of the transcription factor E2F3 has a role in regulating beta-cell quiescence and proliferation. We found human islets have low levels of pro-proliferative E2Fs (E2F1-3), but an abundance of inhibitory E2Fs (E2F4-6). In proliferative human insulinomas inhibitory E2Fs are absent and E2F3 is highly overexpressed. Using this pattern as a ‘roadmap’ for proliferation, we demonstrate that ectopic expression of E2F3 induced signifi cant expansion of insulin positive cells in both rat and human islets. These cells retained their beta-cell phenotype and did not undergo apoptosis. Our results suggest that E2F4-6 may help maintain quiescence in human beta-cells and identify E2F3 as a novel target to induce proliferation of functional beta-cells. Refi nement of this approach may increase the islets available for cell-based therapies and research and one day could provide in vivo regeneration for individuals developing diabetes.

& 2194-PRole of Adipose Triglyceride Lipase and Lipolysis in the Regulation of Insulin Secretion: Study in β-Cell Specifi c ATGL Defi cient MiceCAMILLE ATTANÉ, MARIE-LINE PEYOT, SHU PEI WANG, GRANT A. MITCHELL, ROXANE LUSSIER, MARCO PINEDA, SR., MURTHY MADIRAJU, ERIK JOLY, MARC PRENTKI, Montreal, QC, Canada

Previous studies have suggested that lipolysis-derived lipid signaling molecules play a role in the regulation of glucose-stimulated insulin secretion. To directly assess the role of lipolysis of β-cell endogenous lipid stores in glucose signaling for insulin secretion, beta-cell specifi c adipose triglyceride lipase (ATGL) defi cient mice were generated (B-ATGL).

Beta-cell specifi c tamoxifen-inducible ATGL deletion in MIP-Cre-ER/ATGL-LoxP mice (B-ATGL KO) was compared to tamoxifen-injected-MIP-Cre-ER and -ATGL-LoxP mice (controls).

Two weeks after tamoxifen treatment in 8 wk-old mice, ATGL protein level was dramatically reduced in B-ATGL KO islets from both sexes. KO male mice showed a decrease in body weight gain compared to the controls (body weight gain of 3.8 ± 0.2g vs 6.2 ± 0.5g at 6 weeks post tamoxifen), which became signifi cant after the 6th week of tamoxifen-injection. Fasting plasma levels of FFA and triglycerides were unchanged in KO mice of both sexes. Male KO mice showed a 60% decrease in insulinemia without altered glycemia in fed and fasted states. They also showed a 70% reduction in plasma insulin levels in response to a glucose challenge and a trend towards glucose intolerance. ATGL deletion led to reduced glucose- and KCl-induced insulin secretion in isolated islets from male mice. However, despite similar ATGL deletion in female KO mice, none of these changes in glucose homeostasis and insulin secretory response were observed in females, suggesting compensatory mechanisms by other lipases.

These results demonstrate that lipolysis via ATGL is involved in fuel- and non-fuel-stimulated insulin secretion, at least in male mice. Female mice are often protected from diabetes in various animal models due to estrogens and a number of compensatory processes. We are currently studying the compensatory mechanisms that occur in B-ATGL KO female mice as they may reveal signaling and compensatory pathways involved in insulin secretion.

Supported by: CDA

& 2195-PEffects of MSDC-0602K and GLP1 on Pancreatic Islets in ZDSD/Pco Rats Challenged With a High Fat DietJERRY R. COLCA, HEITOR F. FERRARI, CHARLES D. MACKENZIE, ROB R. EVER-SOLE, TROY GOBBETT, RICHARD PETERSON, Kalamazoo, MI, East Lansing, MI, Indianapolis, IN

MSDC-0602 is a new insulin sensitizing agent designed to act on the mitochondrial target, mTOT. We have previously shown MSDC-0602 lowered circulating glucose and insulin in a phase 2 clinical trial. Here we asked whether MSDC-0602 would have benefi cial effects on the pancreatic islets in the ZDSD/Pco rat, a model selected for beta cell dysfunction in response to a high fat diet. Since this model to responds to GLP1, we also evaluated the combination of GLP1 treatment with this mTOT modulator. Five matched groups of ZDSD/Pco rats were selected at 14 weeks of age and then treated for 6 weeks. Group 1 was maintained on the standard chow diet while the other 6 groups were switched to the 5SCA (Purina) high fat diet and then given either oral vehicle and saline injection (group 2), oral MSDC-0602 (30 mg/kg) and saline injection (group 3), oral vehicle and exenatide (s.q. 1 µg) (group 4), or oral MSDC-0602 (30 mg/kg) and exenatide (s.q. 1 µg) (group 5). The change in diet caused glucose levels to progressively rise to over 500 mg/dl in the control treated rats ( group 2). Either treatment alone signifi cantly reduced glucose levels over 4 week treatment as compared

A564


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


to the group 2 (control treatment on the high fat diet), but the combination of MSDC-0602 and exenatide prevented any rise in glucose. At sacrifi ce, the pancreas was removed, fi xed, and stained for insulin, PDX1, and Ki67, as an index of proliferation. Both treatments increased the number of islet cells and insulin staining, but the largest increase was group 5 where both treatments were combined. Exenatide increased the number of Ki67 positive cells and the combination with MSDC-0602 increased this further. Interestingly, however, individual cells were either positive for Ki67 or insulin and/or PDX1 but not both, indicating that the individual islet cells were at different stages of differentiation.. These data suggest that the combination of an mTOT modulator with GLP1 therapy may be useful for the restoration of pancreatic islets in diabetes.

& 2196-PHuman Induced Pluripotent Stem Cell-Derived Insulin Producing Cells Correct Hyperglycemia in Diabetic MiceSUDHANSHU P. RAIKWAR, EUNMI KIM, MAN CHUN JOHN MA, DANIEL THEDENS, CHANTAL ALLAMARGOT, WEILIANG XIE, WILLIAM I. SIVITZ, JOHN ENGELHARDT, ANNE KWITEK, NICHOLAS ZAVAZAVA, Iowa City, IA

Human induced pluripotent stem (hiPS) cells recently generated in our laboratory potentially present a novel source of cells for the generation of glucose responsive insulin producing cells (IPCs) that could be used to cure type 1 diabetes. Here, we tested the hypothesis that pancreatic endocrine lineage commitment of hiPS cells leads to the generation of glucose responsive IPCs. The hiPS cells were subjected to a multi-step in vitro differentiation protocol mimicking in vivo pancreatic development to generate IPCs. We reasoned that during human fetal development the pancreas is derived from defi nitive endoderm (DE). Treatment of hiPS cells with Activin A led to the generation of the DE cells. The DE cells were further differentiated and expanded into Pdx1- expressing pancreatic endodermal (PE) cells. Differentiation of the PE cells led to the generation of endocrine progenitors which spontaneously developed into three dimensional pancreatic islet-like clusters. The hiPS cell-derived IPCs secreted higher levels of insulin and were glucose responsive. The therapeutic effi cacy of hiPS cell-derived IPCs was tested in streptozotocin (STZ) induced diabetic Rag2-/-γc-/- mice by transplanting the IPCs under the kidney capsule. The pre-transplant fasting blood glucose levels in the STZ induced-diabetic Rag2-/-γc-/- mice were >400 mg/dl. The fasting blood glucose levels declined to <200 mg/dl 100 days post-transplantation in 60% of the mice. The in vivo fate of the transplanted IPCs was monitored by magnetic resonance imaging (MRI) on day 150 post transplantation. The transplanted IPCs had formed a neo-pancreas-like organ which was clearly visible by MRI. On explantation of the kidneys, IPCs were detectable by immunofl uorescence as evidence that the transplanted cells survived long-term and continued to secrete insulin in vivo. These studies provide a new rationale to advance stem cell research and establish a novel source of IPCs that could be used to treat type 1 diabetes.

Supported by: NIH/NHLBI (R01HLO73015); U.S. Dept. of Veterans Affairs (1I01BX001125-01A1)

& 2197-PExtra-Islet Insulin+Glut2- Progenitor Cells Contribute to β-Cell Plas-ticity in Neonatal Mouse PancreasCHRISTINE A. BEAMISH, BRENDA J. STRUTT, EDITH J. ARANY, DAVID J. HILL, London, ON, Canada

Regeneration of pancreatic β-cells for the reversal of diabetes is dependent on an understanding of their plasticity. Developmentally, islets arise from cells within the ductal epithelium. Dedifferentiation of β-cells towards a proliferative ductal epithelial phenotype and subsequent redifferentiation could be exploited to provide an expanded cell pool for β-cell transplantation.

We hypothesized that β-cells from young (P7) mice would exhibit such plasticity. Culture of neonatal mouse islets on collagen in the presence of EGF and cholera toxin resulted in their reversion to a ductal epithelial phenotype expressing cytokeratin 19 (CK19). RIPCre;Z/alkaline phosphatase (AP)+ mice were used to lineage track β-cell fate during culture. Islets lost markers in vitro including insulin and Pdx1, whilst gaining duct markers. The proportion of AP+ β-cells from islets decreased signifi cantly in culture to 1/500 cells, but the remaining AP+ cells gained expression of CK19. Flow cytometry and recovery of insulin+ cells showed that they had derived from an insulin+Glut2- subpopulation, the majority of which were found in extra-islet aggregates of 1-5 β-cells representing 4% of neonatal pancreas cells. These insulin+Glut2- cells were proliferative in vivo and in vitro by EdU incorporation, and could be differentiated into CK19+ duct cells or neural-lineage cells, including neurons, astrocytes, and oligodendrocytes.

These cells could be subsequently redifferentiated back into insulin+ cells, illustrating lineage plasticity. Insulin+Glut2- cells may represent a multipotent, resident progenitor population which could be targeted for cellular therapies for diabetes.

2198-PHuman Gallbladder Epithelial Cells for Treatment of DiabetesSUBHSHRI SAHU, MUGDHA V. JOGLEKAR, SARANG N. SATOOR, MICHAEL WILLIAMS, NICOLA HETHERINGTON, SUNDY N. YANG, THOMAS LOUDOVA-RIS, GAURANG JHALA, DAVID MARTIN, DHAN THIRUCHELVAM, D.N. REDDY, G.V. RAO, SASIKALA MITNALA, ED STANLEY, ANDREW ELEFANTY, HELEN E. THOMAS, THOMAS W. KAY, DAVID TOSH, ANANDWARDHAN A. HARDIKAR, Pune, India, Camperdown, Australia, Melbourne, Australia, Fitzroy, Australia, Sydney, Australia, Hyderabad, India, Bath, United Kingdom

There has been considerable effort towards identifying newer sources of insulin-producing cells for treatment of diabetes. Gallbladder is a next door neighbor of pancreas and shares several transcription factors with liver and pancreas during embryonic development. We reported for the fi rst time that human gallbladder cells naturally express pancreatic hormones, including insulin. We now present comparative transcriptome and epigenome profi les of human gallbladder and pancreas (n>=20). We also provide evidence suggesting that insulin-producing cells in adult human gallbladders are immature beta-like cells.

TaqMan-based low density arrays were carried out to compare expression profi les of human gallbladder and pancreas isolated from 6 human fetal samples. Although human gallbladder samples express pancreatic gene transcripts at similar or lower levels than human islets, Hes1, is detected at very high levels in gallbladder cells as well as their progeny. Human gallbladder cells proliferate in vitro and lineage tracing studies confi rm that insulin-producing cells can proliferate under such conditions. Targeting Hes1 using dominant negative strategies as well as forced expression of pre-microRNAs targeting Hes1, allow for effi cient differentiation of gallbladder-derived progenitors to pro-endocrine cells in vitro. Gallbladder epithelial cells are capable of secreting insulin in response to glucose and arginine stimulation. Current studies are focused on understanding their role in diabetes by transplanting these gallbladder epithelial cells in experimental diabetic animals.

Our studies demonstrate that human gallbladder epithelial cells produce and secrete insulin at a level that is higher than most differentiated stem / precursor cells. We therefore believe that human gallbladder-derived cells have tremendous potential for cell replacement therapy in diabetes.

Supported by: British Council UK; NHMRC (Australia)

2199-PPartial Pancreatectomy Facilitated β-Cell Proliferation in Glucoki-nase Haploinsuffi cient Mice and IRS-2 Defi cient MiceYU TOGASHI, JUN SHIRAKAWA, KAZUKI ORIME, HIDEAKI INOUE, AKINOBU NA-KAMURA, YASUO TERAUCHI, Yokohama, Japan

The precise mechanisms underlying the β cells proliferation following a pancreatectomy is still obscure. We fi rst investigated the proliferative response of β cells following a 60% partial pancreatectomy (Px) in mice. After a Px, β cell mass of remnant pancreas was 46 ± 8 % relative to that after a sham operation (Sham) on postoperative day (Day) 0. However, β cell mass apparently increased to 56 ± 7 % on Day 6 and reached signifi cant increase up to 62 ± 7 % on Day 18. Furthermore, enhanced gene expression of FoxM1 and cyclin B1, and unchanged expression of IRS-2, cyclin D2, and PDX1 were observed in Px islets on Day 3 and Day 6. Thus, Px drove β cell proliferation within a few days, in contrast to gradual increase of β cell mass in high-fat diet-fed mice. We have previously reported that both β-cell-specifi c glucokinase haploinsuffi cient (Gck+/-) mice and IRS-2 defi cient (IRS-2-/-) mice developed abnormality in compensatory expansion of β cell mass in response to high-fat diet-induced insulin resistance. Hence, Gck+/- mice, IRS-2-/- mice, or wild-type (WT) mice were subjected to a Px or a Sham to assess the role of glucokinase and IRS-2 in β cell proliferation after pancreatectomy. In both Gck+/- mice and IRS-2-/- mice, Px caused β cell expansion and signifi cant increase in β cell proliferation equally to in WT mice. The profi le of gene expression in Gck+/- islets after a Px were similar to that in WT islets, while such alteration was not observed in IRS-2-/- islets. These data suggested that β cell proliferation after a pancreatectomy is independent of glucokinase or IRS-2 and also raised the possibility that β cell proliferation in IRS-2-/- mice is mediated by different modes from those in WT or Gck+/- mice. In this way, a model of partial pancreatectomy in mice can guide further investigation of β cell proliferation via previously unknown mechanisms.

A565


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2200-PGlucose Homeostasis Is Regulated by PERK eIF2alpha Kinase through its Control of Beta Cell Proliferation and Insulin Synthesis and StorageRONG WANG, ELYSE E. MUNOZ, BARBARA MCGRATH, DOUGLAS R. CAVENER, University Park, PA

Insulin synthesis and cell proliferation are under tight regulation in pancreatic beta cells to maintain glucose homeostasis. Dysfunction in either aspect can lead to diabetes. Perk (EIF2AK3) loss of function mutations in humans and mice result in permanent neonatal diabetes that is characterized by insuffi cient beta cell mass and reduced proinsulin traffi cking and insulin secretion. Investigating the molecular basis of these defects in beta cells is hampered by the multiplicity of metabolic and growth defects in individuals bearing loss of function mutations in the Perk gene. To avoid the confounding problems associated with the complete absence of PERK, we have investigated a Perk gene dosage series in mice equivalent 1, 2, and 3 copies of the Perk gene. We discovered that blood glucose levels are modulated as a simple linear relationship to Perk gene dosage. To examine the underlying cellular mechanisms that modulate glucose homeostasis as a function of Perk gene dosage, we examined two parameters: beta cell mass and insulin content per beta cell. We discovered that beta cell mass and insulin content per beta cell are both inversely correlated with Perk gene dosage. Further exploration of the cause of differences in beta cell mass revealed a difference in beta cell proliferation without any apparent change in beta cell death. In addition, we used short-term ablation of PERK to determine the immediate impact of the loss of PERK function by using a highly specifi c PERK inhibitor GSK2606414, IC50 0.4nM1 developed by GlaxoSmithKline. Acute inhibition of PERK resulted in reduced insulin content and reduced insulin secretion in cultured beta cells and mice. In conclusion PERK plays multiple roles in regulating beta cell growth and function that are critically important for the maintenance of glucose homeostasis.

Supported by: NIH (R01DK088140)

2201-PProfi ling Individual Pancreatic Islet Cell Subsets in Health and Dis-easeDAVID M. BLODGETT, JULIE M. CARROLL, SHAKED AFIK, SUSANNE PECHHOLD, MANUEL GARBER, ALVIN C. POWERS, CHARLES T. ROBERTS, DAVID M. HAR-LAN, Worcester, MA, Beaverton, OR, Nashville, TN

The pancreatic Islets of Langerhans, a heterogeneous arrangement of alpha, beta, delta, epsilon, and PP cells, are embedded within pancreatic acinar tissue, which has hampered the determination of cell type-specifi c gene-expression profi les. Since cell surface reagents to sort islet cells into pure populations are not currently available, we developed techniques to separate cellular subsets based upon the hormones they produce. We fi xed dissociated islet cell preparations isolated from human and nonhuman (NHP) pancreata and then stained for intracellular hormone content. Using fl uorescence-activated cell sorting, we isolated >97% pure populations of glucagon-producing alpha cells and insulin-producing beta cells. Fixative-induced crosslinks were removed with protease, which permitted the extraction of total RNA. We then submitted the RNA to library construction, duplex-specifi c nuclease treatment to reduce ribosomal RNA contamination, and paired-end, 91-base pair, next-generation sequencing. We analyzed RNASeq results from distinct alpha cell (2 human, 1 NHP donors), beta cell (3 human, 2 NHP donors), and whole-islet (3 human, 1 NHP donors) populations using the GENE-E matrix visualization and analysis platform. Clustering results showed that the human and NHP alpha, beta, and whole-islet cell populations were grouped together across species, based upon comparative gene-expression patterns of >200 differentially expressed genes. As expected, hormone-specifi c expression can be visualized in each cluster: i.e., alpha cells expressed the most glucagon, beta cells the most insulin, and whole islets the most somatostatin. These results show that RNA can be extracted from purifi ed alpha and beta cells, sequenced, and used to measure differential gene expression patterns among cell types and species. The genetic concordance in gene-expression patterns supports the use of NHP islets as a model system for studying beta cell proliferation and dysfunction in health, development, and disease.

Supported by: NIH/NIDDK (U01DK089572-02), (R24DK093437)

2202-PInfl ammation Accompanies the Deleterious effects of Hyperglyce-mia on Exocrine Reprogramming to Beta-Like CellsCLAUDIA R. CAVELTI-WEDER, WEIDA LI, MARIANNE STEMANN-ANDERSEN, AMY ZHANG, TAKATSUGU YAMADA, SUSAN BONNER-WEIR, GORDON WEIR, QIAO ZHOU, Boston, MA, Cambridge, MA

We have previously shown that injection of a polycistronic adenovirus construct containing MafA/Pdx/Ngn3/Cherry (M3Cherry) into mouse pancreas results in reprogramming of exocrine cells to insulin-producing cells in suffi cient numbers to reverse diabetes. Understanding mechanisms infl uencing this process is important. As hyperglycemia is an important driver of beta-cell replication, but also exerts toxic effects, we assessed effects of glycemia on this in vivo reprogramming.

Rag-/- mice at 6 wks were made diabetic with 180 mg/kg STZ and transplanted with 500 islets from SD-rats, C57-mice, or treated with insulin pellets to create distinct glycemic groups. The M3Cherry construct or a Cherry control were injected into the pancreas 2 wks later. After 10 or 25 days, islet graft removal by nephrectomy (Nx) was performed or animals sacrifi ced. Some mice were followed up to 3 mos after virus injection.

There was an inverse relationship between glycemia and number of reprogrammed cells (r=-0.75, p=0.003). A minimal time window of 25 days of normoglycemia was required for adequate glycemic control after Nx. Besides decreased numbers of reprogrammed cells with hyperglycemia, we observed abundant tubular complex formation and macrophage accumulation, suggestive for increased infl ammation. Compared to normoglycemia, Cherry+ cells were less likely to express insulin with hyperglycemia (% insulin+/Cherry+ cells day 10: hyperglycemia 37.2±4.9%, normoglycemia 57.3±5.8%); these not fully reprogrammed cells were perhaps removed by macrophages. Under optimal reprogramming conditions, however, a subgroup of animals achieved long-term phenotype and function of reprogrammed cells up to 3 mos after virus injection.

In summary, inhibition by hyperglycemia of the induction of insulin+ cells from exocrine cells is associated with infl ammation. Improved understanding of factors infl uencing reprogramming like glycemia might be exploited for translational application.

Supported by: JDRF; NIH; SNF

2203-PAge and Gender Effects on Human Islet Cell CompositionDAVID M. BLODGETT, JULIE M. CARROLL, SUSANNE PECHHOLD, MARCELA BRISSOVA, ALVIN C. POWERS, CHARLES T. ROBERTS, DAVID M. HARLAN, Worcester, MA, Beaverton, OR, Nashville, TN

Based on histological analyses, several groups have shown that the proportions of the unique endocrine cell subsets (alpha, beta, delta) found in human and nonhuman primate (NHP) Islets of Langerhans differ from the proportions in rodents. To date, however, techniques have not existed to effi ciently purify and quantify cellular subsets to allow molecular analyses and correlations with phenotype. We have developed fl ow-cytometry methods to isolate alpha, beta, and delta cells based on dissociation into single-cell suspensions, fi xation and permeabilization, and staining for intracellular hormone content. We analyzed 22 human islet preparations provided by the Integrated Islet Distribution Program. Average age was 37.2 (SD = 16; range = 16-66) years, average BMI was 29.6 kg/m2 (SD = 7.1; range = 19-50), and 41% were female. The average composition of the human islets was 39.9% alpha cells, 54.7% beta cells, and 5.4% delta cells, for a beta:alpha cell proportionality of 1.4 (i.e., 1.4 beta cells for every islet alpha cell). We stratifi ed the beta:alpha cell proportions by gender (females, 1.7; males, 1.4), age (<40, 1.3; >40, 1.8), and BMI (< 25, 1.49; >25, 1.57), suggesting that, when compared to male or younger donors, female and older donors’ pancreatic islets have more beta cells relative to alpha cells. We also found that, compared to rodent islets, human islets display greater donor-to-donor variability and have fewer beta cells relative to alpha cells (1.4 versus 4.0). Similar analysis of NHP islets revealed beta:alpha cell proportionality similar to human (1.8). We conclude that age and gender affect human islet composition. Further, compared to rodent islets, the similarities in human and NHP islet morphology, structure, and composition, support the NHP as a model for studies that are impossible to perform in humans.

Supported by: NIH/NIDDK (U01DK089572-02), (R24DK093437)

A566


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2204-PImpaired Iron Metabolism in Monogenic Ncb5or DiabetesHAO ZHU, WENFANG WANG, HAIPING WANG, Kansas City, KS

The risk of diabetes is increased in conditions of impaired iron metabolism, such as hereditary hemochromatosis and Friedreich Ataxia. This study focuses on the role of iron metabolism in beta-cells by utilizing a monogenic diabetes mouse model with targeted ablation of Ncb5or (NADH cytochrome b5 oxidoreductase).

Ncb5or is a redox enzyme widely expressed in animal cells and capable of reducing heme and non-heme ferric iron in vitro. Our previous studies show that Ncb5or defi ciency increases production of reactive oxygen species, mitochondrial proliferation, and endoplasmic reticulum (ER) stress in beta-cells. The underlying mechanism, however, is unclear.

Our recent data suggest that the onset of diabetes in Ncb5or-null (KO) mice is modulated by dietary iron status, i.e., accelerated under an iron-defi cient diet (<6 ppm) and delayed with an iron-suffi cient control diet (300 ppm). Islets of chow-fed prediabetic KO mice exhibit decreased iron content by ICP-MS measurements. Iron defi ciency in KO beta-cells correlates to their higher mRNA levels of genes involved in iron mobilization and regulation, such as Tfr1, Steap2, Dmt1, Zip14, Fpn1, Irp1 and Irp2, as determined by quantitative RT−PCR. Iron defi ciency in prediabetic KO mice is consistent with their decreased transferrin saturation in serum and anemic phenotypes, which are worsened or attenuated by the iron defi cient or suffi cient diet, respectively. We have recently generated pancreas-specifi c Ncb5or-null (PKO) mice by using the loxP-cre technology. The onset of diabetes in PKO mice starts as early as age 4 weeks, and prediabetic mice exhibit decreased mitochondrial complex I activity and altered mitochondrial morphology in islet beta-cells.

Our fi ndings suggest that Ncb5or maintains iron homeostasis in beta-cells and systematically. Studies are underway to test the hypothesis that Ncb5or functions to mobilize intracellular iron for beta-cell function.

2205-PProlactin as a Therapeutic Adjunct in Reversing Hyperglycemia in NOD MiceCOLIN HYSLOP, SUE TSAI, PERE SANTAMARIA, CAROL HUANG, Calgary, AB, Canada

Type 1 diabetes (T1DM) is caused by autoimmune destruction of β-cells. Immune therapy can blunt the autoimmune attack on β-cells, but the residual β-cell mass at the time diagnosis is often insuffi cient for restoration of normal blood glucose despite cessation of immune destruction, which likely hampers the ability of immune agents to cure T1DM in humans. We hypothesize that using a growth factor to increase β-cell mass while applying an immune modulator to attenuate autoimmune attacks on β-cells will be more effi cacious in restoring β-cell mass and reversing hyperglycemia than an immune agent alone. We chose prolactin because it is a physiologic growth factor that enhances β-cell mass by increasing β-cell proliferation and it increases insulin synthesis in vivo. We will treat diabetic NOD mice with a 5-day course of anti-CD3 ± a 21-day course of prolactin (Prl) and determine the rate of long term diabetes cure. We have treated 18 diabetic mice in the CD3 group and 19 mice in the CD3+Prl group and followed them for 40 weeks after treatment. We found that a higher proportion of mice in the CD3+Prl group had sustained normal blood glucose in comparison to the CD3 group (Kaplan-Meier diabetes free survival, p=0.035). Furthermore, the CD3+Prl group had lower fasting blood glucose and a trend for higher fasting insulin level, although the two groups had similar insulin and glucose tolerance.

Interestingly, of the entire treatment group, 6/18 mice in the CD3 group never experienced diabetes reversal while all 19 mice in the CD3+Prl group experienced a period of normoglycemia. In terms of the immune system, we did not detect a difference in the number of CD4+ or CD8+ T cells, the number of Foxp3+/CD4+ regulatory T cells, or proliferation of CD4+ or CD8+ T cells between the two treatment groups. In conclusion, prolactin is an effective therapeutic adjunct in improving anti-CD3-mediated diabetes cure in NOD mice, and this positive effect is associated with an increase in insulin levels in the prolactin-treated mice.

Supported by: CDA

2206-PLaminin Expression Is Decreased in Islets from db/db Diabetic MiceAMY W. LIU, MICHAEL J. PETERS, JOSHUA R. WILLARD, LEN TRAN, REBECCA L. HULL, Seattle, WA

Islet endothelial cells support normal β-cell function in part through production of laminin, found in extracellular matrix. Laminin subunits α4 or α5 combine with β1 and γ1 to form heterotrimers (denoted 411 and 511 respectively) that interact with integrin receptor isoforms α3β1 or α6β1 on β cells to enhance insulin release. We previously showed that islet endothelial dysfunction in diabetes may contribute to decreased insulin secretion and thus hypothesize that laminin and/or integrin expression is decreased in diabetic islets, a mechanism by which endothelial dysfunction may contribute to impaired insulin secretion. To test this hypothesis, islets were isolated from 8-week-old C57BL/6.db/db and db/+ littermate control mice (Alt) (n=3 per group). mRNA levels of laminin subunits α4, α5, β1, and γ1, which are produced exclusively by islet endothelial cells, were assessed in db/db and Alt islets. Since integrins are made by both β and non-β cells, islet cells were sorted by fl uorescence-activated cell sorting, and expression of integrin subunits α3, α6, and β1 was determined specifi cally in β cells. Body weight and plasma glucose were elevated in db/db compared to Alt mice (p<0.001). db/db islets had decreased laminin α4, β1, and γ1 mRNA levels vs Alt (α4: 0.56±0.12 vs 1.00±0.09; β1: 0.64±0.03 vs 1.00±0.08; γ1: 0.72±0.03 vs 0.99±0.04; all p<0.05); however, laminin α5 was increased (1.95±0.07 vs 1.00±0.02, p<0.05). Integrin α3, α6, and β1 expression was not changed in db/db β-cells (α3: 0.99±0.43 vs 1.00±0.10, p=0.98; α6: 0.67±0.14 vs 1.00±0.11, p=0.44; β1: 1.27±0.25 vs 1.00±0.01, p=0.25).

Our data show decreased expression of laminin α4, β1, and γ1, but not α5. Thus, production of laminin-411 may be decreased in diabetic islets; however, the effect of increased α5 expression on laminin-511 levels is unclear. These data suggest that laminin-integrin signaling between islet endothelial cells and β cells, normally supportive of β-cell insulin secretory function, may be disrupted in diabetes.

Supported by: NIH (DK088082)

2207-PConstruction of Adenovirus Carrying Pax4 and its Regulation to the Differentiation of α-CellsYANQING ZHANG, ROBERT N. BONE, VIVIAN FONSECA, HONGJU WU, New Or-leans, LA, Birmingham, AL

Type 1 diabetes is caused by a cell-mediated autoimmune loss of insulin-producing β-cells in the pancreas. Recent evidence of an inherent plasticity of mature pancreatic endocrine cells has fueled interest into regeneration of β-cells. During normal β-cell development, the cooperation of several transcription factors successively specifi es progenitor cells towards development into β-cells. Of these transcription factors, expression of the paired box 4 (Pax4) gene is mandatory for the development and maturation of β-cells. This study was designed to explore the contribution of Pax4 in α-to-β-cell reprogramming. Successful reprogramming of α- to β-cells via expression of this gene could provide a new avenue to β-cell replacement therapies. In the present study, an adenoviral vector, Ad5-CMV-PAX4, was constructed and its infl uence on αTC1.9 cells was detected in vitro. Following Ad5-CMV-PAX4 infection, the PAX4 transgene expressed effi ciently in αTC1.9 cells. After 7 days postinfection, some PAX4- modifi ed αTC1.9 cells were shown to be insulin positive, while several PAX4 modifi ed αTC1.9 cells were Pdx1 positive by immunofl uorescence staining. We measured insulin, glucagon and Arx expression on 2 days, 4 days, 7 days,10 days post-infection by Western blot or ELISA, we determined that glucagon and Arx expression in Ad5-CMV-PAX4 transfected αTC1.9 cells decreased continually, however, we could not detect insulin expression. Real-time Quantitative PCR results showed the transcription factors Pax4, Nkx6.1, and MafA increased, while the expression of Arx decreased. Taken together, our study provided evidence that Ad5-CMV-PAX4 promote αTC1.9 cells conversion into β-like cells in vitro, indicating that a switch of α- to β-cell fate is possible. However, the β-like cells do not appear to be fully functional.

Supported by: JDRF (27-2009-378); NIDDK (R01DK081463 to H.W.)

A567


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS

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

2208-PHuman Induced Pluripotent Stem Cells from Type 2 Diabetes Pa-tients for In Vitro Drug ScreeningADRIAN K.K. TEO, REBECCA WINDMUELLER, IVAN VALDEZ, ALLISON B. GOLD-FINE, DAVE M. SMITH, ROHIT N. KULKARNI, Boston, MA, Mölndal, Sweden

Human induced pluripotent stem cells (hiPSCs) are generated by the intro-duction of nuclear reprogramming factors OCT4, SOX2, KLF4 and CMYC into somatic cells to induce cell fate reversion. These stem cells are pluripotent and have the capacity to differentiate into virtually all cell types of the body. In the context of diabetes, hiPSCs derived from patients with type 2 diabetes (T2D) and their directed differentiation towards the pancreatic lineage would provide an inexhaustible and unique source of material that retains the genetic make-up of the individual for studying diabetes disease mechanisms.

Here, we report the derivation of hiPSCs from T2D patients and their age- and gender-matched controls using a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the 4 reprogramming factors. These T2D-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers, are able to give rise to derivatives of the 3 germ layers in a teratoma assay, and are karyotypically normal. Both BMP and Wnt signaling are found to synergistically act with Activin/Nodal signaling to promote CXCR4+SOX17+FOXA2+ defi nitive endo-derm (DE) formation from control hiPSCs. Current efforts are still aimed at optimizing directed differentiation protocols to obtain effi cient numbers of DE and PDX1+ pancreatic progenitors. The availability of T2D-hiPSCs-derived DE, pancreatic progenitors, and ultimately glucose-responsive insulin-secreting pancreatic beta-like cells will provide new insights into defects in the endocrine pancreas of T2D patients. The patient-specifi c pancreatic cells will be screened with chemical compound libraries to determine their ability to mature in vitro.

Overall, T2D-hiPSCs will serve as an invaluable resource for studying mechanisms (such as insulin resistance) underlying T2D in relevant cell types and open up new opportunities to derive functional mature beta-like cells.

Supported by: AstraZeneca

2209-PHigh Serum Levels of Adiponectin Ameliorate the Gradual Pancre-atic Islet Regression With Age in Klotho MiceSHUICHI OTABE, XIAOHONG YUAN, TOMOKA FUKUTANI, KAYO TANAKA, KENTO HARA, TSUYOSHI OHKI, TOSHIHIKO HASHINAGA, SATOMI KAKINO, YAYOI KU-RITA, NOBUHIKO WADA, ERI SOEJIMA, HITOMI NAKAYAMA, JUNKO KAMEO, SATOMI SHIGEMOTO, MIKA MIGITA, HONAMI NAKAO, YUJI TAJIRI, KENTARO YAMADA, Kurume, Japan

We previously reported that adiponectin had a life-extending effect in mammals. Meanwhile, it is well known that a presenile mouse strain using insertional mutagenesis of the Klotho gene was developed; mice which are homozygous for the Klotho mutant (Kl/Kl mice) have exhibited rather short life span and several other signs of aging. Hence, to investigate whether hyperadiponectinemia can restore the phenotypes of Kl/Kl mice, we overexpressed human adiponectin gene in Kl/Kl mice. Then, we observed the phenotypes of Kl/Kl mice with hyperadiponectinemia. As Kl/Kl mice are infertile, we crossed fertile Klotho mutant mice (Kl/+ mice) with previously reported human adiponectin transgenic mice with B6 background over 20 generations and made the Kl/+ mice of hyperadiponectinemia with the same genetic background as Klotho mice. The levels of serum adiponectin and the expressions of Klotho protein in several organs in Kl/Kl mice overexpressing adiponectin gene were compared with those of control Kl/Kl mice at the age of 7 weeks. The serumlevels of external human adiponectin were extremely high (95.4±58.1µg/ml). The serum levels of internal mouse adiponectin in Kl/Kl mice with hyperadiponectinemia were signifi cantly higher than those of control Kl/Kl mice (65.7±8.9 vs. 34.6±3.9µg/ml, p<0.001). Concerning lifespan, Kl/Kl mice with hyperadiponectinemia did not signifi cantly prolong their average life span compared with control Kl/Kl mice. In the pathological investigation of mice at the age of 9 weeks, pancreas of control Kl/Kl mice showed that the number and volume of pancreatic islets reduced at H&E staining and that the number of insulin positive cells decreased signifi cantly at immunostaining compared with Kl/Kl mice with hyperadiponectinemia. Adiponectin might restore the insulin secretion in Kl/Kl mice. Type2 diabetes associated with aging might be prevented by hyperadiponectinemia.

ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETION COUPLING AND

METABOLISM

Guided Audio Tour: Stimulus-Secretion Coupling and Metabolism (Posters: 2210-P to 2217-P), see page 15.

& 2210-PProperties of Beta-Cells in Human Type 2 Diabetes (T2D)LORELLA MARSELLI, MARA SULEIMAN, MARCO BUGLIANI, FAROOQ SYED, FRANCESCO OLIMPICO, FABRIZIO SCATENA, DANIELE FOCOSI, FRANCO FILIP-PONI, UGO BOGGI, PIERO MARCHETTI, Pisa, Italy

T2D is due to reduced beta cell mass and function. Little information is available on factors affecting beta cell properties in human T2D and the relationships with disease progression and treatment.

In this study, pancreases were obtained from 58 T2D donors (age: 70±8 yrs, M/F: 33/25, BMI: 27.3±4.0 Kg/m2, diabetes duration - DD: 9±7 yrs); islet (HI) insulin area (IA) was assessed after immunocytochemistry; HI were isolated and ex-vivo insulin release (IR) was evaluated in response to 3.3 mM glucose (G), 16.7 mM G, 100 µM glyburide (gly), and 20 mM arginine (arg).

IA was 56±18%. IR (µU/islet/min) was 0.032±0.011 at 3.3 mM G, 0.053±0.032 at 16.7 mM G, 0.061±0.036 with gly and 0.060±0.033 with arg (all p<0.01 vs 3.3 G); the respective stimulation index (SI) values were 1.6±0.7, 1.9±1.0 (p=0.03 vs G) and 1.9±1.0 (p=0.09 vs G). Age, BMI and DD did not affect IA nor IR. There was a positive correlation between IA and arg induced IR (p<0.05). As for anti-diabetic therapy, 15 subjects (29%) were treated with diet (D) or D plus metformin (D+M), 24 (47%) with sulfonylurea (S) alone or in combination with M (S+M), and 12 (24%) with insulin either alone or associated with oral agents (Ins). The three groups (D+M, S+M and Ins) had similar age and BMI; however, DD was longer (p<0.05) in the Ins group. IA was not signifi cantly different between D+M (48±20%), S+M (64±15%) and Ins (52 ± 17%). Furthermore, IR in response to 16.7 G was higher (p<0.05) in D+M (SI: 2.1±0.7) than S+M (1.5±0.7) and Ins (1.2±0.3). In the D+M and Ins groups, gly and arg elicited signifi cantly higher IR than G stimulation.

In this T2D cohort, age, BMI and DD did not affect IA and beta cell function; the correlation observed between IA and arg-stimulated IR suggests that this latter could be a surrogate of beta cell mass; T2D islets showed better gly- and arg- than G-stimulated IR, in particular with advanced disease; of interest, insulin treatment was associated with beta cell functional impairment rather than with loss of beta cells. If so, T2D therapies should primarily target beta cell functional defects.

Supported by: PRIN (2010-2011)

& 2211-PDirect Measurements of Oscillatory Glycolysis in Pancreatic Islet Beta Cells using a Novel FRET Biosensor for Pyruvate Kinase M2 ActivityMATTHEW J. MERRINS, RICHARD BERTRAM, ARTHUR SHERMAN, MARK A. RIZZO, LESLIE SATIN, Ann Arbor, MI, Tallahassee, FL, Bethesda, MD, Baltimore, MD

Pulses of insulin from pancreatic beta cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) endows beta cells with the ability to generate metabolic oscillations via autocatalytic activation by its product fructose-1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has not previously been made. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; Pyruvate Kinase Activity Reporter), which multimerizes upon binding FBP. When introduced into beta cells of intact islets, PKAR FRET effi ciency increased dose-dependently in response to glucose. This change was rapid (within seconds) and reversible. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, suggesting the sensor responds to FBP activation; the dose-dependence of PKAR on FBP was further confi rmed using purifi ed sensor in vitro. When beta cells were stimulated with 11 mM glucose, PKAR oscillations were out of phase with mitochondrial fl avin autofl uorescence oscillations measured from neighboring cells lacking PKAR, and shifted by one-quarter cycle with respect to NAD(P)H oscillations, which were measured simultaneously. These results indicate that glycolysis in beta cells is oscillatory and that PFK1 is the likely generator of slow islet oscillations. More broadly, the family of PKARs could be useful for separating glycolytic fl ux from other metabolic signaling in islet beta cells, as well as other types of cells.

Supported by: F32DK085960 (to M.J.M.); R01DK46409 (to L.S.)

A568


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

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

& 2212-PMetabolomics of Fatty Acid Potentiation of Glucose Stimulated In-sulin Secretion (GSIS)MAHMOUD ELAZZOUNY, ROBERT T. KENNEDY, CHARLES F. BURANT, Ann Arbor, MI

Acute fatty acid exposure potentiates GSIS in β-cells though the signaling pathways that participate in the potentiation remain unclear. We tested the hypothesis that fatty acids may cause changes in glucose metabolism that lead to augmentation of insulin secretion. We assessed the alteration in the metabolome of INS-1 (832/3) cell following preincubation with palmitic acid (PA, 500 µM)or BSA followed by stimulation with 16 mM unlabeled or U-[13C]glucose for 5-60 minutes. Fatty acid exposure resulted in a 2-fold potentiation of GSIS at 60 minutes and a signifi cant increase in plamitoyl-CoA, which fell by 50% within 5 minutes after addition of glucose. A signifi cant rise in glycerol-3-phosphate (Go3P) found after glucose addition was attenuated by palmitic acid addition. 13C-glucose addition showed the rapid appearance of M+3 isotopomers of glycerolipids, indicating increased fl ux of glucose into the glycerolipids pathway via Go3P. Untargeted metabolomic profi ling also detected the increases in potential signaling molecules including Palmitoyl taurine and palmitoyl glycine. The rapid esterifi cation of lipids was accompanied by a decrease in static levels of glycolytic and pentose phosphate pathway intermediates metabolites and a 2-folds decrease in NADH/NAD+ ratio, presumably due to the consumption of NADH in the conversion of the glycolytic intermediate DHAP to Go3P. The change in redox potential was accompanied by an increase in 13C-isotopomer levels in TCA cycle intermediates and increased oxygen consumption, indicating an increased fl ux of glucose through the TCA cycle. The increase in glucose utilization by palmitic acid was accompanied by decreases in malonyl-CoA levels and AMP-Kinase activation. These studies suggest that acute exposure to fatty acids may potentiate insulin secretion by augmenting the formation of signaling lipids as well as by increasing glycolytic fl ux and increasing glucose utilization in the TCA cycle where additional signaling molecules may be formed.

Supported by: NIH (DK046960 to R.T.K), (DK079084 to C.F.B), (P30DK089530), (P60DK20572), (U24DK097153)

& 2213-PEffect of Vildagliptin on Postprandial Proinsulin Processing in the Beta-Cell of Patients With Type 2 Diabetes MellitusTHOMAS FORST, MARKUS DWORAK, CHRISTINE BERNDT-ZIPFEL, ANDREA LÖF-FLER, INGA KLAMP, MICHAEL MITRY, ANDREAS PFÜTZNER, Mainz, Germany, Nuremberg, Germany

In type 2 diabetes mellitus (T2DM) increasing intact proinsulin levels serve as a marker of declining beta cell function and were found to be associated with an increase in cardiovascular risk. The aim of this study was to compare the effect of Glimepiride versus Vildagliptin on metabolic control and postprandial (pp) proinsulin processing in patients with T2DM.

Forty-four patients (age: 59±8 years; HbA1c: 7.3±0.7 %) were randomized to treatment with Vildagliptin or Glimepiride, added to ongoing Metformin. A standardized test meal was given at baseline and after 12 and 24 weeks of treatment. Insulin, intact proinsulin, and blood glucose values were measured in the fasting state and pp for 300 minutes.

HbA1c and fasting glucose levels improved in both groups without a signifi cant difference between both groups. The area under the curve (AUC) for the pp glucose excursion was lower with Glimepiride 2181±395 compared to Vildagliptin 2451±437 mmol/L*min (p<0.05). Fasting intact proinsulin levels decreased signifi cantly with Vildagliptin from 13.0±9.1 to 8.3±4.7 pmol/L (p<0.05), and remained unchanged with Glimepiride. The AUC for pp intact proinsulin secretion decreased from 7559±4417 to 5494±2987 pmol/L*min with Vildagliptin (p<0.05), while it increased from 7871±3358 to 9277±4402 pmol/L*min with Glimepiride treatment (p<0.05). The AUC for the proinsulin to insulin ratio declined from 361±169 to 279±129 with Vildagliptin (p<0.05), while no change was observed with Glimepiride. Treatment with Vildagliptin decreased body weight by 1.74±3.24 kg (mean±SD) and increased adiponectin levels by 0.73±1.08 ng/mL (p<0.05), while Glimepiride increased body weight by 0.64±1.89 kg without changes in adiponectin.

In summary, compared to Glimepiride, Vildagliptin decreased fasting and pp intact proinsulin and improves the proinsulin to insulin ratio, thereby indicating its potential role in beta cell preservation.

Supported by: Novartis Pharmaceuticals Corporation

& 2214-PModulating Islet Gap Junction Coupling Enhances Insulin Release and Normalizes Glycemia in a Mouse Model of Neonatal Diabetes MellitusLINDA M. NGUYEN, MARINA POZZOLI, RICHARD K.P. BENNINGER, Aurora, CO

Mutations to the KATP channel which reduce the sensitivity of ATP-inhibition can cause neonatal diabetes mellitus (NDM). In mice, expression of ATP-insensitive KATP channels in the islet suppresses glucose-stimulated intracellular free-calcium activity ([Ca2+]i); thereby reducing insulin secretion. Connexin-36 (Cx36) gap junctions are important for regulating islet electrical activity: in the absence of Cx36, β-cells show spontaneous elevations in [Ca2+]i at basal glucose. We hypothesized that gap junctions exacerbate the suppression of [Ca2+]i by ATP-insensitive KATP channels, and a reduction in Cx36 may allow spontaneous elevations in [Ca2+]i to trigger insulin secretion. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels.

In the normal presence of Cx36 following expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500mg/dl and plasma insulin decreased ~5-fold. Islets from these mice showed no glucose-stimulated insulin secretion. A loss of insulin content and mitochondrial dysfunction (reduced NADH response) was also observed; likely due to the prolonged hyperglycemia. In mice lacking Cx36 following expression of ATP-insensitive KATP channels, only a minor (~10mg/dl) elevation in blood glucose was measured and plasma insulin levels were unchanged. Islets from these mice had near-normal glucose-stimulated insulin secretion, and normal mitochondrial function and preserved insulin content compared to control animals.

We therefore demonstrate a novel mechanism by which islet function can be recovered in a model of diabetes. A reduction of islet gap junction coupling allows suffi cient spontaneous [Ca2+]i elevation and insulin secretion which prevents the emergence of diabetes. Such an approach may be used to treat sulfonylurea-insensitive NDM or other monogenic forms of diabetes caused by a defect in the regulation of insulin secretion.


& 2215-PNephrin and Neph1 Directly Affect the Metabolic Function of Insulin Expressing CellsRODRIGO VILLARREAL, DONY MAIGUEL, JONGMIN JEON, FLORIAN GRAHA-MMER, INGO LEIBIGER, JOHANNA GUZMAN, ALBERTO FACHADO, TAE YOO, SANDRA MERSCHER-GOMEZ, GEORGE BURKE, PER-OLOF BERGGREN, TOBIAS HUBER, ALESSIA FORNONI, Miami, FL, Stockholm, Sweden, Freiburg, Germany

Diabetes and prediabetes are major risk factors for cardiovascular morbidity and mortality and are the primary cause of end stage renal disease. Nephrin (NP) and Neph1 (N1) proteins are key components of the glomerular fi ltration barrier and are heavily modulated in diabetes. We previously reported an important role of NP in glucose stimulated insulin release in insulinoma cells and human pancreatic islets in vitro. We now hypothesize that NP and N1 directly affect the metabolic function of pancreatic beta cells.

We studied the metabolic phenotype of mice with a conditional deletion of NP or N1 in pancreatic beta cells. Mice with a conditional deletion of NP (NP KO) but not N1 (N1 KO) demonstrated worse glucose tolerance at baseline when compared to controls RIP-Cre+ littermates (Ctrl). Isolated islets from both N1 KO and NP KO mice showed decreased release of insulin in response to glucose when compared to Ctrl, suggesting an intrinsic metabolic function of Nephrin and Neph1. Immunoprecipitation experiments showed NP and N1 interaction with Insulin Receptor (IR). In order to study if NP and N1 affect IR dependent signaling, we studied Akt and p70S6K phosphorylation in HEK cells transfected with NP/N1. Overexpression of both NP and N1 resulted in an increase of p70S6K and Akt (pT308) phosphorylation that was independent of insulin and of glucose. On the contrary, Akt (pS473) phosphorylation was not affected. Overexpression of both NP and N1 in MIN6 cells also resulted in increased insulin and glucokinase mRNA expression. The effect of both NP and N1 on p70S6K and Akt phosphorylation requires PI3K and mTORC1.

In summary, both NP and N1 affects pancreatic beta cell function in vivo and demonstrated a metabolic function in vitro. NP and N1 may represent a new therapeutic target for treatment of diabetes.

Supported by: AHA

A569


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


& 2216-PmiRNA30a-5p-Mediated Silencing of BETA2/NeuroD Gene Expres-sion Is an Important Initial Event of Glucotoxicity-Induced Beta-Cell DysfunctionJI-WON KIM, SEUNG-HYUN KO, YOUNG-HYE YOU, YU-BAE AHN, KUN-HO YOON, Seoul, Republic of Korea

Aims/hypothesis: The loss of beta-cell function is a critical factor for the development of type 2 diabetes. Glucotoxicity plays a major role in the progressive deterioration of beta-cell function and development of type 2 diabetes mellitus. Here we demonstrate that miR-30a-5p is a key player in early-stage glucotoxicity-induced beta-cell dysfunction.

Methods: The studies that we performed were Northern blots, RT-PCR, and western blots in glucotoxicity-induced primary rat islets and INS-1 cells. we also measured glucose-stimulated insulin secretion and insulin contents. In vivo approaches were used to evaluate the effect of miR-30a-5p in beta-cell dysfunction.

Results: miR-30a-5p expression was increased in beta-cells after exposure to glucotoxic conditions, and exogenous miR-30a-5p overexpression also induces beta-cell dysfunction in vitro. miR-30a-5p directly suppressed the expression of BETA2/NeuroD by binding to a specifi c binding site in its 3’-untranslated region. After BETA2/NeuroD expression was restored by knockdown miR-30a-5p or transfection of the BETA2/NeuroD gene, beta-cell dysfunctions, including decreased insulin content, gene expression and glucose-stimulated insulin secretion, recovered. Glucose tolerance and beta-cell dysfunction were improved upon direct injection of Ad-si30a-5p into the pancreas of diabetic mice.

Conclusions/interpretation: Our data demonstrate that miR-30a-5p-mediated direct suppression of BETA2/NeuroD gene expression is an important initiation step of glucotoxicity-induced beta-cell dysfunction.

& 2217-PSIRT3 and SIRT6 Suppression Impairs Pancreatic β-Cell Function and May Contribute to Onset of Type 2 DiabetesPAUL W. CATON, SARAH J. RICHARDSON, JULIUS KIESWICH, MARCO BUGLI-ANI, PIERO MARCHETTI, NOEL MORGAN, MOHAMMAD M. YAQOOB, MARK J. HOLNESS, MARY C. SUGDEN, London, United Kingdom, Plymouth, United Kingdom, Pisa, Italy

Nicotinamide mononucleotide (NMN) improves pancreatic β-cell function by increasing β-cell NAD+ levels, though the mechanism responsible has yet to be fully described. SIRT3 and SIRT6 are, respectively, mitochondrial and nuclear NAD+ dependent enzymes that control reactive oxygen species (ROS) production and exert anti-infl ammatory effects. Since chronic infl ammation and mitochondrial dysfunction mediate β-cell impairment and onset of type 2 diabetes (T2DM), we examined the role of SIRT3 and SIRT6 in maintenance of β-cell function and NMN action.

We examined SIRT3 and SIRT6 in islets isolated from T2DM and non-diabetic human donors (Ave. plasma glucose; 10.4 vs. 7.9 mmol/L), islets isolated from control and high-fat fed mice (HFD; 40% fat; 16 wks) and in INS1 cells transfected with siSIRT3 (150 nM; 72 h).

SIRT3 and SIRT6 expression was decreased in human T2DM islets and in mouse islets incubated with IL1β/TNFα (5 ng/ml; 48 h) in association with decreased insulin secretion (3, 17 mmol/L glucose; 1h). In HFD-mice levels of islet SIRT6, but not SIRT3, were suppressed in parallel with lack of islet compensation to insulin resistance. SIRT1 activator or inhibitor studies showed that SIRT1, which reportedly resolves β-cell infl ammation, regulates expression of SIRT6 but not SIRT3 in β-cells, suggesting SIRT6 may partly mediate effects of SIRT1. Thus, we next examined specifi c effects of decreased SIRT3. Immunofl uorescence showed SIRT3 is expressed in β-cells of mouse and human islets. SIRT3 knockdown in INS1 cells lowered insulin secretion and expression of key β-cell genes and increased levels of ROS (CM-H2DCFDA fl uorescence), IL1β expression and apoptosis (Caspase 3 activity). SIRT3 knockdown also blocked the protective effects of NMN (100 µM; 48 h) against pro-infl ammatory cytokines.

Decreased SIRT3 and SIRT6 levels may contributory to impaired β-cell function in T2DM. Strategies to activate or increase SIRT3 or SIRT6 may represent an effective treatment approach for T2DM

Supported by: Diabetes UK; EFSD/Lilly Fellowship (to P.W.C.); DRWF (to S.J.R.)

2218-PThe Association of Serum Osteocalcin With Glucose Control, Pan-creatic Function and Insulin Sensitivity in Type Two Diabetes Pa-tientsXUEFEI RUI, HUI SHENG, CHUNPING PAN, JUNLEI SU, CHENYU ZHAN, SHANGYU CHAI, BIN SU, CHUNHUA QIAN, XIAOYUN CHENG, JIYING WANG, YUQI HAN, SHEN QU, Shanghai, China

This study is to investigate the association between serum osteocalcin and glucose control, pancreatic function and insulin sensitivity in the type 2 diabetes mellitus (T2DM) Chinese patients.

1019 T2DM patients were recruited (Male:432, Female:587, Average age: 65.96 ± 13.11 years). After measured BMI, the fasting plasma glucose(FPG), insulin(FINS), C peptide(F-CP), postprandial blood glucose (2hPBG) and C peptide (2hC-P), HbA1C and osteocalcin were determined. Homeostasis model assessment of β cell function (HOMA-β) and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated based on the above parameters.

The difference in osteocalcin levels between male and female are signifi cant [(13.51±12.37)ng/mL vs (16.22±10.40)ng/mL, P<0.001]. After adjustment for age and BMI, serum osteocalcin was negatively correlated with HbA1C, FPG and 2hPBG (P<0.05), and positively correlated with fasting C peptide (FC-P), 2h C peptide (2hC-P) and HOMA-β (P<0.05). In male, after adjustment for age and BMI, serum osteocalcin was negatively correlated with 2hPBG (P<0.05), and positively correlated with FC-P, 2hC-P and HOMA-β (P<0.05). In female, after adjustment for age and BMI, serum osteocalcin was negatively correlated with HbA1C, FPG (P<0.05). Multivariable liner regression analysis showed that serum osteocalcin was the independent related factor in infl uencing HOMA-β and HOMA-IR in male, and it was also the independent related factor in infl uencing HOMA-IR in all subjects (P<0.05).

In T2DM, osteocalcin was negatively associated with glucose, implying osteocalcin could lower blood glucose. And osteocalcin was positively asso-ciated with FC-P, 2hC-P and HOMA-β, supporting the idea that osteocalcin might improve glucose control through promoting insulin secretion of pan-creatic islet.

Supported by: NSFC (30900698); Doctoral Fund of Ministry of Education (20090072120020)

2219-PFoxO1 Contributes to Beta-Cell Compensation for Insulin Resis-tance and Oxidative StressTING ZHANG, DAE HYUN KIM, YOUSEF EL-GOHARY, RENNIAN WANG, SUZANNE BERTERA, RITA BOTTINO, SOJIN LEE, VIRTU NAVARRO CALABUIG, GEORGE GITTES, MASSIMO TRUCCO, HENRY DONG, Pittsburgh, PA, London, ON, Canada

Beta-cell compensation is an adaptive mechanism by which beta cells are exercised for increased insulin release to overcome insulin resistance for maintaining euglycemia in obesity. Failure in beta-cell compensation contributes to insulin insuffi ciency and overt diabetes with poorly understood mechanisms. FoxO1 is a forkhead transcription factor that plays a key role in mediating insulin action on target gene expression. To characterize how FoxO1 integrates insulin signaling to beta-cell compensation, we generated beta-cell specifi c FoxO1 transgenic (FoxO1-tg) mice, in which FoxO1 is expressed from the rat insulin II promoter (RIP), and determined the ability of islets with FoxO1 gain-of-function to compensate for fat-induced insulin resistance and streptozotocin (STZ)-elicited diabetes. RIP-FoxO1 transgenic production resulted in beta-cell mass expansion, accompanied by signifi cant upregulation of IRS2, Pdx1, MafA, FoxA2, Glut2 and insulin expression in islets. This effect accounted for enhanced glucose-stimulated insulin secretion, rendering FoxO1-tg mice refractory to fat-induced glucose intolerance. When fed a high fat, FoxO1-tg mice, as opposed to wild-type littermates, were able to maintain near normal glycemic control. When challenged with multiple low doses of STZ, FoxO1-tg mice were resistant to STZ-induced diabetes, correlating with FoxO1-mediated induction of anti-oxidative function in islets. We recapitulated this fi nding in cultured INS-1 cells, in which elevated FoxO1 production resulted in selective induction of superoxide dismutase and catalase, two key enzymes in anti-oxidation. We conclude that FoxO1 contributes to beta-cell compensation for insulin resistance and oxidative stress.

A570


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST


2220-PPancreatic Beta-Cell Specifi c Syntaxin-1A KO Causes Exocytotic Defects in Predocked and Newcomer Insulin GranulesTAO LIANG, TAIRAN QIN, LI XIE, YOUHOU KANG, LUCY OSBORNE, HERBERT Y. GAISANO, Toronto, ON, Canada

In type-2 diabetes (T2D), islet levels of exocytotic Syntaxin (Syn)-1A & cognate SNARE proteins are severely reduced. Syn-1A is known to mediate exocytosis insulin secretory granules (SG) as reported by previous in vivo studies employing global Syn-1A deletion or Syn-1A overexpression, which might have confounding effects. To assess Syn-1A function in beta−cells per se on glucose homeostasis, we generated beta-cell specifi c Syn-1A KO(beta-Syn-1A KO) mice. We utilized the Cre-loxP recombination strategy to obtain beta-cell-specifi c ablation of Syn-1A gene (RIP-Cre(+/+):Syn-1A (fl ox/fl ox)) and compared to littermate controls (RIP-Cre(-/-):Syn-1A(fl ox/fl ox)). Absence of Syn-1A in beta-Syn-1A KO islets was confi rmed by Western blotting. Beta-Syn-1A KO mice were viable with no effects on weight gain. Beta-Syn-1A KO mice had random & fasting hyperglycemia, with IPGTT showing sustained elevation of blood glucose levels with correspondingly reduced blood insulin levels compared to controls. Surprisingly, insulin tolerance test showed beta-Syn-1A KO mice exhibited increased insulin sensitivity, probably because of chronically reduced blood insulin levels. Islet perifusion assay on beta-Syn-1A KO islets showed impaired insulin secretion in both fi rst and second phase GSIS, with GLP-1 potentiating but not fully rescuing the secretion defi ciency. To unequivocally determine the beta-cell exocytotic defects, we employed total internal refl ection fl uorescence (TIRF) imaging to record and analyze SG motion and exocytosis. Previously docked and newcomer SGs in both fi rst and second phases were markedly reduced in Syn-1A KO beta-cells. Addition of GLP-1 recruited fewer SGs in beta-Syn-1A KO beta-cells. Our results indicate that fl uctuation in islet Syn-1A levels in T2D accounting for progressive impairment in GSIS and GLP-1 treatment/rescue induce differing infl uences on the kinetics of recruitment and fusion of different insulin SG pools.

Supported by: CIHR

2221-P

2222-PNTPDase3 Is Abundantly Expressed in Pancreatic Beta-Cells and Plays a Role in the Regulation of Insulin SecretionALEXANDER M. EFANOV, AUDRA L. KAUFFMAN, LISA S. BEAVERS, JAMES T. ALSTON, THOMAS B. FARB, JAMES V. FICORILLI, MARIALUISA C. MARCELO, MARTIN B. BRENNER, KRISTER B. BOKVIST, DAVID G. BARRETT, SAMREEN K. SYED, Indianapolis, IN

Extracellular ATP can act as a potent glucose-dependent insulin secreta-gogue through activation of P2-purinergic receptors and extracellular ATP may therefore have importance for the regulation of insulin secretion. Ectonucleotidases, a family of membrane-bound metabolizing enzymes, regulate extracellular ATP levels by degrading ATP to ADP and AMP. Ectonucleotidase activity will affect the relative proportion of the different nucleotides, which in turn will impact the level of stimulation exerted by extracellular ATP. We therefore investigated the expression and role of ectonucleotidases in pancreatic beta-cells. Of the ectonucleotidases studied only ENTPD3 (gene encoding NTPDase3) mRNA was detected at fairly abundant levels in human and rodent pancreatic islets as well as in insulinoma cells. ENTPD3 mRNA was also detected, although at lower levels, in pancreatic islets from diabetic animals and human diabetic patients. Detection of NTPDase3 in pancreatic tissues slices with a specifi c antibody demonstrated that the enzyme is situated in pancreatic beta-cells and not alpha-cells. ARL67156, a selective ectonucleotidase inhibitor, was utilized to evaluate the role of NTPDase3 in pancreatic beta-cell function. ARL67165 blocked degradation of extracellular ATP added to insulinoma cells. It also increased levels of endogenous ATP released from cells. Measurements of insulin secretion in insulinoma cells as well as rodent and human pancreatic islets demonstrated that ARL67156 potentiated glucose dependent insulin secretion and was additive to GLP-1 in enhancing insulin secretion. Thus, we have demonstrated that NTPDase3 is present in pancreatic beta-cells from multiple species and is involved in the regulation of insulin secretion.

2223-PRole of Altered Glucose Metabolism in the Glucotoxic Progression of KATP-Induced Neonatal DiabetesMARIA S. REMEDI, PAIGE COOPER, COLIN G. NICHOLS, St. Louis, MO

Glucotoxicity, leading to reduced pancreatic β-cell function and enhanced β-cell death are key events in the pathogenesis of type-2 diabetes. Defective insulin secretion is also crucial in monogenic diabetes. The normal β-cell responds to elevated blood glucose with an increase in [ATP]/[ADP] ratio which leads to ATP sensitive K+ (KATP) channel closure, membrane depolarization, Ca2+ infl ux through voltage-dependent Ca2+ channels, increase in intracellular [Ca2+]i which, in turn, triggers insulin secretion. Gain-of-function (GOF) KATP mutations cause Neonatal Diabetes Mellitus (NDM) and mild GOF polymorphisms are highly associated with type-2 diabetes. A novel mouse model of KATP-GOF induced NDM (by tamoxifen inducible expression of an ATP-insensitive mutant under Cre-recombinase control in β-cells) demonstrates not only development of profound diabetes that becomes progressively more severe over-time, but also dramatic secondary consequences of systemic diabetes: loss of insulin content and β-cell mass. Quantitative real-time PCR in KATP-GOF islets demonstrates a marked reduction in the insulin gene, but also an increase in the glucokinase gene (GK) (fi rst enzyme in glucose metabolism), refl ected in glucose hypermetabolism in these mice, which may contribute to the β-cell deterioration. If so, we hypothesize that glucotoxic effects in KATP-GOF will be abrogated (or worsened), by deceleration (or acceleration) of metabolism. To test this in vivo, we crossed our KATP-GOF mice with mildly glucose-intolerant heterozygous GK+/- knockout mice. Progression of diabetes, assessed by blood glucose levels over time, is slower in double transgenic GK+/-/KATP-GOF compared with KATP-GOF and glucose tolerance is enhanced in GK+/-/KATP-GOF mice 12 days after disease induction. These results demonstrate an increased metabolism in KATP-GOF β-cells, and that deceleration of metabolism delays the development of hypermetabolism-induced glucotoxicity in KATP-induced NDM.

WITHDRAWN

A571


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2224-PThe Effect of Insulin Resistance and Diabetes on Islet Cell Protein Dynamics: A Novel Combination of Dynamic and Quantitative Pro-teomicsCYRUS F. KHAMBATTA, Emeryville, CA

Insulin resistance (IR) and islet cell failure are the two fundamental processes underlying type 2 diabetes (T2D). Alterations in mitochondrial protein turnover have been implicated in the pathology of T2D, however the specifi c effects of IR and islet cell failure on the synthesis and degradation of the intracellular proteome have yet to be described. In this study, we measured protein synthesis and degradation rates of hundreds of islet cell proteins from insulin resistant Zucker rats (ZDF, n=9), diabetic Zucker rats (ZDF, n=9), and age-matched lean controls (ZLC, n=18). Using 2H2O labeling, we measured fractional synthesis rates (FSR) of proteins involved in the principal pathways of glucose stimulated insulin secretion (GSIS). Animals were labeled for 12 hours (n=3), 2 days (n=3), and 5 days (n=3) in order to measure the turnover of fast, medium and slow turnover proteins. We found that IR increased the FSR of 95% of all measured islet proteins, including those involved in the ER, glycolysis, cytoskeletal remodeling, mitochondria, and ribosomal protein synthesis. In diabetic animals, protein synthesis was increased in 51% of measured islet proteins, however there was a signifi cant and selective reduction of ribosomal protein synthesis. We combined FSR with label free quantitative proteomics to approximate changes in absolute synthesis and breakdown rates. IR increased absolute turnover rates in 82% of measured proteins, and islet cell failure selectively reduced absolute ribosomal protein synthesis. These results suggest that (a) the rapid rate of islet cell proliferation observed in IR is accompanied by increased fractional and absolute synthesis of proteins involved in GSIS, (b) ribosomal protein turnover is impaired in T2D, and (c) mitochondrial protein synthesis is increased in both IR and T2D, contrary to literature fi ndings.

2225-PThe ΔF508 Gene Mutation of Cystic Fibrosis Transmembrane Regu-lator Protein Leads to a Progressive Decline of Beta-Cell Function in Mice Carrying this MutationISMA BENTERKI, Montreal, QC, Canada

Cystic Fibrosis (CF) is the most common autosomal-recessive disorder in humans and is due to mutations in the gene encoding for the Cystic Fibrosis Transmembrane Regulator(CFTR). In CF patients, the most common mutation is the deletion of Phe508 (ΔF508), which leads to loss of function of CFTR channel. With increased life expectancy of CF patients, several secondary complications occur such as Cystic Fibrosis-Related Diabetes (CFRD) which affects 20 to 40% of CF patients. However, the mechanisms underlying CFRD are unknown. The objective of this study was to examine the impact of the ΔF508 CFTR mutation on glucose homeostasis in mice. To this aim, we compared cftr Δ F508/Δ F508 (Δ F508) male and female mice to wild-type (WT) littermates. ΔF508 male and female mice had lower body weight compared to WT mice. At 14 weeks of age, both male and female ΔF508 displayed improved oral glucose tolerance as compared to WT animals. This was associated with greater insulin sensitivity and improved beta-cell function in vivo, as assessed by hyperglycemic clamps. In contrast, 6-month old ΔF508 male mice were insulin resistant and had defective beta-cell function as compared to their WT littermates. In isolated islets from 14-week old mice, we observed a mild decrease in glucose-induced insulin secretion ex vivo but no difference in the sensitivity to proinfl ammatory cytokines. These results suggest that the ΔF508 mutation is associated with a subtle impairment in insulin secretion which becomes apparent when animals become insulin resistant with age.

Supported by: Cystic Fibrosis Canada

2226-PThe Role of the Androgen Receptor in β-Cell Function in Male MiceGUADALUPE NAVARRO, FRANCK MAUVAIS-JARVIS, Chicago, IL

In aging and castrated men, testosterone defi ciency predisposes to type 2 diabetes (T2D). Although the role of testosterone defi ciency in predisposing to visceral adiposity and insulin resistance is known, there is no information on the function of testosterone and the androgen receptor (AR) in male β-cells in physiology and diabetes. To address this issue, we generated a β-cell-specifi c AR knockout mouse (βARKO-/y) by crossing ARlox/y mice with RIP2-Cre transgenic mice. Male βARKO-/y mice showed a selective loss of AR protein expression in islet β-cells and a 50% decrease in AR expression in hypothalamus. Male βARKO-/y mice showed no alteration in food intake and body weight in aging. On a normal chow, male βARKO-/y mice displayed defi cient basal and glucose-stimulated insulin secretion (GSIS) and

developed glucose intolerance but remained normoglycemic in the fed and fasted state. However, following exposure to a high fat diet (HFD) while male βARKO-/y mice also developed alteration in GSIS and glucose intolerance, in contract, they became hypoinsulinemic and hyperglycemic in the fed and fasted states. In addition, cultured male βARKO-/y islets displayed decreased basal and glucose-stimulated insulin secretion during static incubation. Insulin sensitivity remained unaltered in male βARKO-/y mice. To assess the contribution of AR partial hypothalamic deletion to the male βARKO-/y diabetic phenotype, we generated a neuronal AR knockout mouse (NARKO-/y) using the Synapsin-Cre mouse. Male NARKO-/y mice showed no alteration in GSIS and glucose tolerance and remained normoglycemic and normoinsulinemic whether on a normal chow or HFD. Thus, the AR in β-cells is critical to insulin secretion in males and strategies aimed at selective AR delivery to β-cells without reproductive actions may prevent T2D in hypoandrogenic men.

Supported by: DK074970, DK007169; HD044405

2227-PSerotonin Regulates Glucose Stimulated Insulin Secretion from Pancreatic β-Cells during PregnancyMICA OHARA-IMAIZUMI, HAIL KIM, MASASHI YOSHIDA, YUKIKO TOYOFUKU, TADASHI OKAMURA, TOYOYOSHI UCHIDA, YOSHIO FUJITANI, MASAFUMI KAKEI, HIROTAKA WATADA, MICHAEL S. GERMAN, SHINYA NAGAMATSU, To-kyo, Japan, Daejeon, Republic of Korea, Saitama, Japan, San Francisco, CA

In response to the increased insulin resistance during pregnancy, pancreatic islets undergo not only an increase in β cell proliferation but also an increase in glucose stimulated insulin secretion (GSIS). Although several mechanisms for the increase of β cell proliferation during pregnancy have been reported, the mechanism of the increased GSIS has yet to be elucidated. Here we explored the role of 5-HT in the regulation of GSIS by analyzing Htr3a KO mice. Htr3a KO mice showed impaired glucose tolerance during pregnancy in spite of normal increase of β cell mass. GSIS was markedly increased in the pancreatic islets isolated from pregnant mice but not in the islets isolated from pregnant Htr3a KO mice. Activation of 5-HT3 receptor (Htr3) during pregnancy induced increased uptake of Ca2+ in response to glucose stimulation, which resulted in increased insulin exocytosis. Electrophysiological studies showed that activation of Htr3 induced a depolarizing shift of resting membrane potential in β cells, which decreased glucose threshold for insulin secretion. Thus, our data indicated that Htr3 signaling, in a paracrine/autocrine manner, plays an essential role in the increase of GSIS during pregnancy.

Supported by: JDRF

2228-PInsulin-Degrading Enzyme Is Necessary for Pancreatic Beta-Cell Glucose-Stimulated Insulin SecretionJOSE F. LOPEZ-ACOSTA, GERMAN PERDOMO, IRENE COZAR-CASTELLANO, Val-ladolid, Spain, Toledo, Spain

To fi nd new therapeutic targets for the treatment of diabetes is crucial for improving glycemic control and diabetes complications. Insulin-degrading enzyme (IDE) is a ubiquitous metalloprotease that degrades insulin. IDE is localized in a susceptibility locus for type 2 diabetes in humans. IDE-null mice are hyperinsulinemic and glucose intolerant. However, the role of IDE on pancreatic beta-cell function is completely unknown. Thus, we hypothesize that IDE is required for beta-cell glucose-stimulated insulin secretion.

To this end, we studied IDE expression levels in islets of hyperinsulinemic and hyperglycemic db/db mice. We measured IDE expression in INS-1E cells and primary rat islets, under physiological and pathophysiological levels of glucose and insulin. To understand the physiological role of IDE in the pancreatic beta-cell, we used siRNA-IDE in INS-1E cells and 1,10-Phenantroline (an inhibitor of IDE activity) in rat islets, performing glucose-stimulated insulin secretion (GSIS) to study beta-cell function. We also studied the expression of proteins involved in the secretory machinery of the beta-cell.

We showed that IDE expression was increased in islets of hyperinsulinemic db/db mice, correlating to plasma insulin levels. IDE expression was markedly downregulated by hyperglycemia in INS-1E cells and rat islets. In contrast, IDE was upregulated by hyperinsulinemia. Surprisingly, IDE loss-of-function reduced GSIS by 50%, whereas beta-cell insulin content remained unchanged. In parallel, secreted c-peptide levels were decreased by 50%. To study the molecular mechanisms underlying these effects, we measured glut2, glucokinase, sur1 and kir6.2 levels by RT-PCR, showing no changes in expression after IDE loss-of-function.

A572


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST


In conclusion, we unravel an unexpected role of IDE on beta-cell glucose-stimulated insulin secretion. These data suggest that IDE is a therapeutic target for diabetes treatment.

Supported by: Ministry of Economy and Competitivity (Spain)

2229-PReversing New-Onset Diabetes in NOD Mice by Combining Sitaglip-tin With GW9508, a GPR40 and GPR120 Dual AgonistA. ANSARULLAH, YAN LU, MARTHA HOLSTEIN, BRITTANY DERUYTER, ALEX RABINOVITCH, ZHIGUANG GUO, Sioux Falls, SD

GPR40 is abundantly expressed in β cells and intestinal L cells. GPR120 is expressed on the intestinal L cells and some immune cells. Activating GPR120 has an anti-infl ammatory effect and stimulates GLP-1 release. DPP-IV inhibitor also has an anti-infl ammatory effect and inhibits GLP-1 degradation. In this study, we investigated the effect of combining sitagliptin with GW9508, a GPR40 and GPR120 dual agonist, on reversing new-onset diabetes in NOD mice. New-onset diabetic NOD mice were orally treated either with sitagliptin at 30 mg/kg, or GW9508 at 5 mg/kg, or in combination twice a day, for up to 6 weeks. Nonfasting blood glucose level was determined. CD4+CD25+FoxP3+ T cells, Th17 T cells, and plasma GLP-1 were measured. Diabetes was reversed in 12.5% of mice treated with vehicle for GW9508 (DMSO, n=16), in 53.3% of mice treated with GW9508 (n=15), in 45.4% of mice treated with sitagliptin (n=11), and in 75.0% of mice treated with GW9508 and sitagliptin. The insulitis score was signifi cantly lower in GW9508 and sitagliptin treated mice. The percentage of CD4+CD25+FoxP3+ T cells in pancreatic lymph nodes was signifi cantly increased in sitagliptin alone treated mice and in GW9508 and sitagliptin combination treated mice (P<0.05) but not in GW9508 alone treated mice compared to control mice. The percentage of IL-17+ cells and IL-17+CD4+ T cells in pancreatic lymph nodes and the ratio of IL-17+CD4+ cells/ CD4+CD25+Foxp3+ T cells were signifi cantly decreased in GW9508 and sitagliptin treated mice (P<0.01). The percentage of insulin+/BrdU+ β cells and insulin+/Ki67+ β cells in islets were signifi cantly increased compared to prediabetic NOD mice (P<0.01). Our studies demonstrated that GW9508 and sitagliptin improve the diabetes reversal through modulating infl ammation and stimulating β cell regeneration in new-onset diabetic NOD mice. Combining a DPP-IV inhibitor with a GPR40/GPR120 agonist is a new therapeutic approach for treating type 1 diabetes.

2230-PTRP Channels are Involved in Restoring First-Phase Insulin Secre-tion by Islets Exposed to High PalmitateKYOKO MIURA, SHIMPEI OGAWA, HARUKA KAWANABE, YOSHIRO KITAHARA, Kawasaki, Japan

Delayed and excessive postprandial insulin secretion is a characteristic feature of obese and insulin resistant patients. β cell lipotoxicity is thought to play an important role in the development of such dysfunction. In this study, we tried to fi nd a new target to restore early-phase insulin secretion by β cells exposed to high palmitate (PA) using rat islets. After islets were exposed to 0.5 mM PA for 48 hrs, expression of Kir6.2 was decreased to 50 % of that in normal islets, UCP2 expression was increased to twice the normal level, and the ATP/ADP ratio was unchanged after 16.7 mM glucose stimulation. Consequently, glucose-stimulated insulin secretion (GSIS) was impaired. Nateglinide (NT) restored fi rst-phase insulin secretion by these islets with a peak at 2 min after stimulation of perifused islets, while the peak of insulin secretion induced by glimepiride (GP) was delayed until 6 min. The effect of NT was not blocked by diazoxide (DZ), while that of GP was blocked by DZ. These data indicated that NT restored fi rst-phase insulin secretion via a KATP channel independent pathway in islets exposed to PA. Then, we performed a microarray analysis to identify the molecular targets related to restoration of fi rst-phase insulin secretion by NT. In islets exposed to PA, TRP A1 and TRP C5 were increased to 4-fold of that in normal islets. Then we tested the effect of a TRP channel inhibitor (SKF9635(SKF)) on the insulinotropic action of NT. SKF blocked the effect of NT in islets exposed to PA, while it did not alter the action of NT on normal islets. In addition, the effect of GP was not blocked by SKF irrespective of whether the islets were exposed to PA. These fi ndings suggest that PA-induced lipotoxicity impairs the glucose-dependent pathway and GSIS, while TRP A1 and TRP C5 are promoted to regulate fi rst-phase insulin secretion. The compounds that interact with these channels may be effective for restoring early-phase insulin secretion and improving delayed and excessive postprandial insulin secretion.

2231-PActivation of Trpm2 Channel by Glucose Metabolism and GLP-1 in Pancreatic Beta-CellMASASHI YOSHIDA, SHIHO YAMATO, KATSUYA DEZAKI, MASANOBU KAWAKA-MI, TOSHIHIKO YADA, MASAFUMI KAKEI, Saitama, Japan

Here we report a novel mechanism of Glucagon-like peptide 1 (GLP-1) on back-ground non-selective cation channel current (NSCC) in rat pancreatic beta cells. Perforated whole-cell patch-clamp technique was used. The resting membrane conductance that was measured by ramp clamp from -100 mV to -50 mV at 5.6 mM glucose with 100 µM tolbutamide was 31.5 ± 7.6 pS/pF (n=7). The resting conductance was further increased to 62.2 ± 15.7 pS/pF (n=7, P<0.05 with paired test) after addition of 10-10 M exendin-4 that is a GLP-1 receptor agonist. Exendin-4 >10-11M dose-dependently increased inward current. The exendin-4 sensitive current obtained by subtracting background current in the absence of exendin-4 from the current recorded during exposure to exendin-4 at 10-10 M reversed around -20 mV, indicating that exendin-4 activates NSCC. The NSCC sensitive to exendin-4 was inhibited by 2-APB, a specifi c blocker of Transmembrane receptor potential melastatin 2 (Trpm2) channel. In the presence of 10-7 M exendin-9-39, GLP-1 receptor antagonist, exendin-4 at 10-10 M did not increase NSCC. This indicates that exendin-4 binding to GLP-1 receptor is invoved in activation of NSCC. Dibutyryl-cAMP at 1 mM activated NSCC. In the presence of 10 µM H89, protein-kinase A inhibitor, 10-10 M exendin-4 increased NSCC. PKA activator, 6-Benz cAMP and 6-Phen cAMP did not increase the NSCC. 8-pCPT-2-O-Me-cAMP at 10 µM, EPAC2 activator, increased NSCC to an extent similar to that on exposure to exendin-4. Native GLP-1 and liraglutide, GLP-1 analogue, increased the NSCC that was also inhibited by 2-APB. Increase of glucose concentration from 2.8 mM to 16.6 mM elicited NSCC that was blocked by 2-APB. We conclude that Trpm2-channel actvation is mediated by cAMP-EPAC2 pathway that may be a mechanism of GLP-1 mediated potentiation of glucose-stimuated insulin secreion. Glucose metabolism also activates Trpm2 channels to facilliate insulin secretion.

Supported by: Japan Society for the Promotion of Science

2232-PA New Cross-Talk between Exocrine and Endocrine Pancreatic Cells in Cystic Fibrosis: Infl uence of CFTRΔF508 Mutation and LPS-Generated Microparticles on Insulin CellsANDREI A. CONSTANTINESCU, CÉLINE GLEIZES, FATIHA ZOBAIRI, IOAN L. MI-TREA, GHEORGHE STOIAN, GILLES PRÉVOST, FLORENCE TOTI, LAURENCE KES-SLER, Strasbourg, France, Bucharest, Romania

In cystic fi brosis (CF), diabetes is detected as the result of exocrine pancreas alteration followed by endocrine dysfunction at later stage. Microparticles (MPs) are plasma membrane fragments shed from stimulated or damaged cells that act as cellular effectors. Our aim was to study the contribution of MPs generated by chronic infection to pancreatic exocrine and endocrine cell cross-talk and its signifi cance in the development of diabetes in CF patients.

Normal human exocrine pancreatic cells (PANC-1) or with CFTRΔF508 mutation (CFPAC-1) were stimulated by LPS from P.aeruginosa to produce MPs. RIN-m5F rat β cells were targeted by 10 nM MPs (18h) or submitted to 25 µM CFTR(Inh)-172 thereby limiting CFTR expression at cell surface. NFkB activation was detected in HEK-Blue reporter cells. Secreted insulin, IL-6 and IL-8 were measured in supernatant by ELISA. Cell apoptosis was quantifi ed by cytometry and lysosomal activity by Neutral Red.

CFTR-172 inhibitor induced a 9 fold increase in apoptosis (n=4; p<0.0001) and a 70% (n=6; p<0.0001) decrease in insulin secretion by RIN-m5F. LPS had no effect on endocrine cell apoptosis or insulin secretion but decreased the lysosomal activity by 18% (n=5; p=0.0007). Addition of MPs from LPS-treated CFPAC-1 generated a 40% (n=6; p<0.0001) decrease of insulin secretion, and a 27% (n=5; p=0.0001) reduction in lysosomal activity in the presence of LPS. MPs from LPS-treated PANC-1 cells did not alter insulin secretion or lysosomal activity. All types of MPs induced RIN-m5F apoptosis. MPs from CFPAC-1 induced a dose-dependent activation of NFkB, while MPs from PANC-1 did not. IL-6 and IL-8 were detected only in supernatant from cells submitted to MPs.

Our results suggest that inhibition of CFTR at the plasma membrane surface is associated with a decrease in insulin secretion while infections might contribute to CF-related diabetes through MPs exchanges between exocrine and endocrine cells.

Supported by: Association Vaincre la Mucoviscidose

A573


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2233-PZinc Regulates Insulin Secretion through Interaction With Histidine Residues of SUR1HISAMITSU ISHIHARA, YUICHIRO OTSUKA, SUGURU YAMAGUCHI, ASAMI FU-RUKAWA, MITSUHIRO NAKAZAKI, Tokyo, Japan

Zinc is accumulated in insulin-containing granules of beta-cells and is co-secreted with insulin. It has been suggested that zinc ions regulate sulfonylurea receptor type 1 (SUR1) through interaction with histidine residues of the SUR1. In order to examine roles of zinc in regulation of SUR1 function and of insulin secretion, we have expressed in MIN6 cells wild-type hamster SUR1 or its mutants in which histidine residues were substituted to alanine. First, to express SUR1 proteins in a regulated manner, MIN6 cells have been stably transfected with a tetracycline-sensitive transcription activator, and subsequently with a tetracycline-sensitive transcription silencer, generating MIN6TET3GTS. Then, MIN6TET3GTS cells were stabely transfected with wild-type or mutant SUR1 cDNAs driven by a tetracycline responsive promoter. Selected clones were treated with 1 µM doxycycline and insulin secretion was measured by 1-hour static incubation. MIN6 cells overexpreessing wild-type SUR1 secreted insulin in response to 100 nM glimepride in the presence of 5 mM glucose, which was suppressed by 30 µM zinc by 47 ± 11%. In MIN6 cells expressing SUR1 with a substitution of His326 to Ala, insulin secretion in response to 100 nM glimepride was not suppressed by 30 µM zinc. In MIN6 cells expressing SUR1 with a substitution of His332 to Ala, insulin secretion in response to 30 nM glimepride was enhanced with 30 µM zinc (by 45 ± 7%). These results indicated that zinc modulates insulin secretion through interaction with SUR1 and suggested autocrine or paracrine regulation of zinc in islet secretory function.

2234-PHuman Exfoliated Deciduous Teeth Conditioned Medium Improved Diabetic Control in Streptozotocin-Induced Diabetic Mice by Pro-tection of Pancreatic Beta-Cells Function and its SurvivalTAKAKO IZUMOTO, SHIN TSUNEKAWA, AKIHITO YAMAMOTO, EITA UENISNI, KOTA ISHIKAWA, ATSUSHI IIDA, HIDETADA OGATA, ATSUSHI HUJIYA, TETSUJI OKAWA, YUSUKE SEINO, YOJI HAMADA, MINORU UEDA, YUTAKA OISO, Na-goya, Japan

Stem cell transplantation has shown an effect on a regeneration of pancreatic beta-cells and improved diabetic control. The mechanism of anti-diabetic effects was considered to be a paracrine action rather than differentiation of transplanted cells. Meanwhile, recent studies showed that treatment with human exfoliated deciduous teeth conditioned medium (SHED-CM) that required no immunosuppressive agents had the effect of functional recovery on spinal cord injury and cerebral infarction in rodent models. Therefore, we investigated the effect of SHED-CM on pancreatic beta-cells fuction and its survival. In in vivo study, streptozotocin (STZ) (50mg/kg/day) was administrated to C57B/6J mice for 5 days, then DMEM (2ml/day), SHED-CM (2ml/day), or exendine-4 (Ex4) (48nmol/day) was injected intravenously for 14 days. SHED-CM and Ex4 improved the elevated casual blood glucose and the glucose tolerance during IPGTT signifi cantly compared to DMEM. SHED-CM and Ex4 increased the level of plasma insulin during IPGTT as well. It is noteworthy that the effects of SHED-CM on glycemic control and insulin secretion were greater than that of Ex4. Compared to DMEM, SHED-CM and Ex4 increased pancreatic insulin content signifi cantly by 86% and 35%, respectively. Pathological study revealed that SHED-CM ameliorated the morphological change of pancreatic islets and signifi cantly increased the area of insulin-positive cells. Furthermore, in vitro investigations were performed in MIN6 cells. Pre-incubation with SHED-CM for 6 hours enhanced glucose stimulated insulin secretion more than that with Ex4. Examination of DAPI staining exhibited that SHED-CM also alleviated STZ induced cell death. These date suggest that SHED-CM has the protective effects on pancreatic beta-cells in STZ-Induced Diabetic Mice and has the potential as a novel therapy for diabetes.

2235-PInvolvement of c-Jun NH2-Terminal Kinase (JNK) Pathway in FFA-Induced Beta-Cell DysfunctionKHAJAG KOULAJIAN, LORETTA LAM, ELENA BURDETT, ADRIA GIACCA, Toronto, ON, Canada

It has been reported that JNK is activated by palmitate in vitro in β-cells and is involved in palmitate-induced inhibition of insulin gene transcription. However, it is still unclear whether oleate also activates JNK. Furthermore, it is uncertain whether JNK is causal in the decrease of glucose stimulated insulin secretion (GSIS) induced by oleate or palmitate. Therefore, the objective of this study was to investigate the causal role of JNK activation

in free fatty acid (FFA)-induced β-cell dysfunction. JNK inhibition was achieved either by using 1) the JNK specifi c inhibitor SP600125, or 2) JNK-1 null mice (JNK-1 is the main isoform associated with obesity and type 2 diabetes). 1) Isolated rat pancreatic islets were incubated for 48h in oleate or palmitate (0.4 mM in 0.5% FFA-free BSA) with or without the JNK inhibitor SP600125. Oleate impaired GSIS and decreased islet insulin content. The pharmacological inhibition of JNK did not prevent the impairing effects of oleate on insulin content or GSIS. However, when palmitate was used, pharmacological inhibition of JNK prevented the decrease in insulin content but not GSIS. Palmitate, but not oleate, induced an increase in phosphorylated JNK (marker of activation) which was prevented by the JNK inhibitor. 2) Isolated islets of JNK-1 null mice and their littermate controls were cultured for 48h in oleate or palmitate (same conditions as rat islets) In mouse islets, the genetic inhibition of JNK-1 did not prevent the impairing effects of oleate on islet insulin content or GSIS, but prevented completely both the decrease in islet insulin content and the decrease in GSIS caused by palmitate. These data suggest that the impairing effect of oleate on β-cell function is not dependent on JNK. However, JNK pathway is involved in palmitate-induced β-cell dysfunction. The difference in GSIS between rat and mouse islets exposed to palmitate with JNK inhibition remains to be clarifi ed.

2236-PMarked Upregulation of Proinsulin Biosynthesis in Obese db/db Mouse IsletsCRISTINA ALARCON, BRANDON B. BOLAND, PATRICK C. MOORE, YUJI UCHI-ZONO, CHRISTOPHER J. RHODES, Chicago, IL

Dysfunctional pancreatic β-cells mark the onset of obesity-linked type 2 diabetes. However, it is unknown whether the dysfunction resides at the level of insulin production. We examined proinsulin synthesis in two closely related strains of db/db mice: C57BL/6J db/db mice (6J-db) which are obese, insulin resistant, hyperinsulinemic and glucose intolerant but have apparent compensatory β-cell mass; and C57BLKS/J db/db mice (KS-db) which are also obese, hyperinsulinemic and insulin resistant, but are diabetic due to β-cell loss. Immunofl uorescence analysis indicated a 3-fold increase in β-cell numbers in 6J-db mice, and a >30% decrease of β-cells in KS-db mice. But islet studies showed 6J-db islets had similar insulin content as control mice, despite the increased β-cell numbers. Glucose-stimulated insulin secretion (GSIS) was lost and the secreted proinsulin:insulin ratio increased (2-fold). Insulin content in KS-db islets was decreased (3-fold) in line with β-cell loss; but GSIS was apparent when normalized to insulin content. Glucose-regulated proinsulin synthesis was increased in both 6J-db (12-fold) and KS-db (7-fold) islets over that at basal glucose in control mice. Electron microscopy (EM) analysis of freshly isolated islets revealed marked degranulation of β-cells and massive upregulation of the RER and Golgi apparatus complementary to increased insulin production. There was little indication of ER-stress. Intriguingly, EM analysis of 6J-db and KS-db islets after 12 h incubation at normal 5.6 mmol/L glucose, indicated a near complete re-granulation of the β-cells. We conclude that β-cells exert themselves hard for a marked upregulation of insulin production in obese-diabetes and newly synthesized (pro)insulin is rapidly secreted to meet the hyperinsulinemic demand of insulin resistance. When glucose levels are normalized in vitro the β-cells appear to recover. This underlines the dynamic functional plasticity of β-cells and that ‘β-cell rest’ could be a useful therapeutic strategy.


2237-PPWD/PhJ Mice Have Primary Hyperinsulinemia Due to Enhanced Nutrient-Stimulated Insulin SecretionMAGGIE M. HO, XIAOKE HU, KWAN YI CHU, SUBASHINI KARUNAKARAN, JAMES D. JOHNSON, SUSANNE M. CLEE, Vancouver, BC, Canada

The PWD/PhJ (PWD) wild-derived inbred mouse strain is genetically very different to the commonly used strains. We have shown that in comparison to C57BL/6J (B6) mice, PWD mice have fasting hyperinsulinemia in the absence of obesity and whole-body insulin resistance. We examined potential mechanisms for their hyperinsulinemia. β-cell mass, % insulin staining area, and islet sizes are similar to B6 mice. PWD mice have markedly elevated glucose-stimulated insulin secretion compared to B6 mice. Perifusions revealed PWD mice have >3-fold increased glucose (20 mM)-stimulated insulin secretion (P=0.008) with no obvious distinction between fi rst and second phase secretion. Basal secretion (3 mM glucose) was similar to or lower than B6 mice. Potassium (30 mM)-stimulated secretion did not differ between the strains, either in amount or pattern.

A574


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST


These results were confi rmed in static incubations, where total glucose (16.7 mM)-stimulated insulin release was 2.3-fold higher in PWD mice (P=0.002) despite having >2-fold lower islet insulin content (P<0.0001) compared to B6 mice. Insulin secretion in response to other nutrients (e.g. sub-maximal glucose, palmitate, α-ketoisocaproate) was also signifi cantly increased. Diazoxide completely inhibited glucose-stimulated insulin secretion in PWD mice. The addition of 30 mM potassium to 16.7mM glucose overcame the strain difference. Combined, these data suggest hyperinsulinemia in PWD mice results from increased insulin secretion due to increased generation of metabolite-derived signals. Discovery of the molecular genetic mechanisms underlying the increased nutrient-stimulated insulin secretion in PWD mice may suggest novel ways to augment islet function in the treatment of type 2 diabetes or in islet transplants for type 1 diabetes.

2238-PUndercarboxylated Osteocalcin (ucOC) Is Correlated With Insulin Secretion Evaluated by Glucagon Loading Test in Patients With Type 2 DiabetesYUICHI TAKASHI, YOKO MATSUZAWA, JUN SAITO, MASAO OMURA, TETSUO NISHIKAWA, Yokohama, Japan

Undercarboxylated osteocalcin (ucOC) has been shown to regulate insulin secretion and insulin sensitivity in rodents. However, data on the correlation between ucOC and glucose metabolism in humans is limited and controversial. Thus we examined the effect of ucOC in the insulin secretion ability evaluated by glucagon loading test. We recruited 86 patients with type 2 diabetes under written informed consent, and performed glucagon loading test on the 3rd morning of hospitalization. The average ofage, duration of diabetes, BMI, and HbA1c (NGSP) of the subjects were 62.1y.o., 12.2years, 25.4kg/m2 and 9.2%, respectively. ucOC was 2.90ng/ml in average, and it was not signifi cantly correlated with age, duration of diabetes, BMI, HbA1c, and fasting plasma glucose. In addition, ucOC showed no signifi cant correlation with C-peptide (CPR) before and 6 minutes after glucagon loading, or ΔCPR (CPR6min - CPR0min). However, when we focused on the patients with HbA1c less than 8.0% (n=25), ucOC was shown to correlate positively with CPR after glucagon loading (r=0.44, p=0.027), and also with ΔCPR (r=0.47, p=0.018). This is the fi rst report which assessed the relationship of ucOC and glucagon loading test in type 2 diabetic patients. ucOC is suggested to be a useful marker to evaluate the insulin secretion ability such as post-prandial responsiveness of insulin production in those patients, but we have to pay careful attention to severe hyperglycemia which may deteriorate the function of pancreatic beta cell.

2239-PMechanisms of Fuel Surfeit Detoxifi cation in Pancreatic β-CellsYVES MUGABO, SHANGANG ZHAO, JULIEN LAMONTAGNE, MARCO PINEDA, ERIK JOLY, MURTHY MADIRAJU, MARC PRENTKI, Montreal, QC, Canada

Elevated glucose and free fatty acids (FFA) cause pancreatic β-cell dysfunction, depletion of insulin stores and apoptosis (glucolipotoxicity). If much knowledge has been gained on the biochemical basis of gluco-lipotoxicity little is known as to how the β-cell detoxifi es fuel excess. We tested the hypothesis that β-cells averts metabolic stress caused by fuel surfeit through glucose metabolism via the glycerolipid/FFA cycle and the subsequent release of lipolysis-derived glycerol and long-chain FFA.

Isolated rat islets exposed for 1 h to elevated glucose concentrations showed enhanced glycerolipid/FFA cycling and converted signifi cant amount of glucose carbon to FFA and exported a large fraction of them out of the cell. The saturated stearic and palmitic acids were the predominant FFA species released into the medium. Glycerol release, an index of lipolysis, and glucose utilization did not show saturation even at 16 mmol/l glucose, unlike glucose oxidation, ATP production and insulin secretion. Incorporation of glucose carbons to “inert” triglycerides and cholesterol esters were also elevated with increasing glucose concentration.

The results suggest that in islets as external glucose concentration is increased, the pathways involved in signaling for secretion and energy production reach saturation earlier than detoxifi cation processes, and that at high glucose islets convert a signifi cant portion of the sugar into glycerol and long chain FFA, which are exported outside the β-cell. The identifi cation of fuel surfeit detoxifi cation mechanisms by a targeted metabolomic approach that we are currently performing should reveal additional pathways of β-cell fuel detoxifi cation. Targeting fuel detoxifi cation processes may provide a novel approach to preserve β-cell mass and function.

Supported by: IRSC; FRQS; Diabète Québec

2240-PAlterations in Islet Morphology as a Function of Insulin Sensitivity in HumansTERESA MEZZA, GIOVANNA MUSCOGIURI, GIAN PIO SORICE, JIANG HU, GENN-ARO CLEMENTE, ANDREA GIACCARI, ROHIT N. KULKARNI, Boston, MA, Rome, Italy

Type 2 diabetes develops when insulin secretion fails to cope with worsening insulin resistance. It is becoming evident that insulin resistance can also impact non-classical tissues including the pancreatic β-cells. For example, obese insulin resistant patients exhibit an enhanced β-cell mass as an adaptive response although the underlying mechanism(s) is not fully understood. To explore the alterations that occur in islet morphology, as part of the adaptive mechanism, we performed hyperinsulinemic euglycemic clamps in 18 Caucasian non-diabetic patients, dividing them into insulin resistant (IR, n=9) or insulin sensitive (IS, n=9) (10 F/8 M, 51±15 yrs., BMI 27.9±5.3 kg/m2) groups according to glucose uptake; incretins were evaluated during a Mixed Meal Test. Subsequently, all patients underwent duodeno-pancreatectomy, and pancreases were collected for immunohistochemistry for glucagon, insulin and somatostatin+ cells to assess islet morphology. Apoptosis was evaluated by TUNEL, proliferation by Ki67, and ductal cells by CK19 immunostaining. Assessment of the entire group revealed a direct correlation between GU and islet size (r= -0.74; p<0.001), and between GU and % glucagon area expressed as a fraction of the total pancreas (%GLUCA, r=-0.65; p=0.003). IR group displayed a signifi cant reduction in β/α ratio and increased islet size (2456±332.2 vs 5156±944.8 µm2, p<0.01). While no differences were evident in proliferation, apoptosis or β cell area, the IR group displayed increased insulin+CK19+ cells (p<0.001), scattered islets (<8 cells) (p=0.04) and a larger β-cell nuclear area (p=0.03). Further, GLP1 levels correlated with %GLUA (r=0.63, p=0.04) and we detected GLP-1 immunoreactivity in α-cells. To our knowledge this is the fi rst report correlating in vivo insulin sensitivity indices and incretin parameters with islet morphology. Our data suggest that impaired insulin sensitivity and an increase in GLP-1 in α-cells contribute to enhance β-cell mass.

2241-PChronic Effects of Fatty Acids on Insulin Secretion, Oxygen Con-sumption, Intracellular Calcium Signaling and Gene Expression Profi le in Mouse and Human Pancreatic IsletsNICOLAI M. DOLIBA, QING LIU, PAN CHEN, CHANGHONG LI, CHENGYANG LIU, PATRICK SEALE, ALI NAJI, FRANZ M. MATSCHINSKY, Philadelphia, PA

Chronic hyperglycemia and hyperlipidemia are characteristics of type 2 diabetes and they are known to cause β-cell dysfunction termed “glucolipotoxicity”. The expression of PGC-1α is elevated in islets from different animal models of diabetes but its mechanistic role in β-cell glucolipotoxicity remains unclear. This study was to evaluate the inter-connection between expressions of PGC-1α, mitochondrial energy metabo-lism, calcium signaling and insulin secretion (IS) in mouse and human islets. To duplicate glucolipotoxicity in vitro, islets were cultured for 3 days with 0.5 mM palmitic acid (PA) at 1% BSA and different concentrations of glucose: 10, 16 and 25 mM. The inhibitory effect of PA on IS was evident at 16 mM in mouse and human islets. Despite inhibition of IS by PA, the oxygen consumption rate (OCR) in response to glucose or FCCP was unchanged in PA treated mouse islets, suggesting that inhibition of IS by PA occurred at steps downstream of ATP production. Gene expression of PGC-1α, PPAR-γ, CPT-1A, Cyt c and Cox5b were increased and expression of GCK was decreased after mouse and human islets exposure to 16 mM glucose and 0.5 mM PA. Increasing the glucose to 25 mM in the presence of PA led to greater inhibition of IS and the gene expression profi le exhibited the following changes: decreased expression of PGC-1α, slightly increased expression of CPT-1A with no changes in expression of PPAR-γ, Cyt c, Cox5b or GCK. These changes were associated with increased basal OCR and decreased stimulation of OCR by glucose but normal response to FCCP. The inhibition of IS was correlated with impaired glucose-stimulated calcium infl ux. We conclude that the phenotypic manifestation of glucolipotoxicity depends characteristically on the glucose concentration: at 16 mM glucose islets exhibit an adaptive response as evidenced by increased expression of PGC-1α, PPAR-γ, CPT-1A and Cox5b but this adaptation collapses at 25 mM glucose.

Supported by: NIH

A575


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


and insulin secretion. We previously showed that murine islets cultured in a microfl uidic device maintained ECs better than untreated islets due to improved access of albumin. We further explored the independent effects of a hypoxia mimetic, cobalt chloride (CoCl2) and exogenous VEGF on islet ECs. There was no difference in immunofl uorescence staining of PECAM-1 at 24hrs, however at 48 and 72 hrs there were signifi cant increases in islet EC area and connections in CoCl2 treated islets compared to control and VEGF treated islets. CoCl2 treatment signifi cantly increased VEGF-A secretion from islets, and in the Min6 cell line induced a signifi cant increase in VEGF-A, Tie-2 and Angiopoietin-1 mRNA levels but not in Tie-1 or Angiopoietin-2 mRNA levels. Additionally we found that glucose-stimulated insulin secretion and mitochondrial membrane potential in CoCl2 treated islets were comparable to controls. The glucose-stimulated NAD(P)H response was also similar between the two treatments as measured using two-photon microscopy. Interestingly, CoCl2 treated islets had an enhanced cytoplasmic NAD(P)H response compared to controls. We are currently examining whether this is due to NADH or NADPH. Our results suggest pre-treatment with CoCl2 in microfl uidic fl ow improves islet vasculature better than exogenous VEGF treatment by inducing endogenous growth factor secretion and access to islets; while preserving islet function and metabolism, perhaps by harnessing glycolysis. Future studies will probe islet metabolism and revascularization post transplantation in a diabetic mouse model.

2245-PAnalysis of Gene Expression Profi ling in Multiple Pancreatic Alpha and Beta-Cell Lines and Isolated Islets from Two Diabetic Mouse ModelsJIE WANG, YING CHEN, WEI TANG, CHAO LIU, KWAME OSEI, DONALD F. STEIN-ER, Chicago, IL, Jiangyin, China, Nanjing, China, Columbus, OH

Islet α and β cells derived from the same progenitors play opposing roles in the control of glucose homeostasis. Disturbances in their function are critical in the diabetes pathogenesis, but signature genes for these 2 cell-types’ unique features under (patho)physiological conditions are still not fully understood. In this follow-up study, we have applied Affymetrix Mouse Expression Set 430 microarrays and further analyzed gene expression profi ling in 2 α-cell lines (TC1.6 and TC-DeltaPC2) vs. 3 β-cell lines (MIN6, Ins2+/Akita, and Ins2+/+) and in islets from 4- and 12-wks-old Akita and HIMP1-Tg-L2 diabetes model mice vs. their littermates. The results show that the genes Gcg, Pou3f4, Arx, HIMP1, Cart, or Gpr81 in the 2 α-cell lines are expressed 16- to 120-fold higher compared to their untraceable transcripts in the 3 β-cell lines. By contrast, aside from β-cell marker genes defi ned previously, various genes such as Nnat, ucn3, Trpm5, Dpp4, Hopx, Enho, Hadh, and Bace2 in the 3 β-cell lines are expressed 10- to 100-fold higher compared to their faint transcript signals in the 2 α-cell lines. Intriguingly, the increased Cart transcripts and the reduced Nnat, ucn3, and Trpm5 transcripts showed in both Akita and HIMP1-Tg-L2 islets with age compared to controls, in spite of the currently undefi ned mechanisms underlying these changes. The reduced transcripts of various heat shock proteins and the increased transcripts of some oxidative stress induced genes were also evident in both Akita and HIMP1-Tg-L2 islets, suggesting these changes may link to diabetes caused by various primary factors. The gene expression profi ling in Ins2+/Akita vs. Ins2+/+ β-cells are largely compatible to that in the 12-wks-old Akita islets vs. controls, irrespective of small portion of discrepancies. In summary, our approach exposed novel signature genes for the (a)typical features of islet α- and β-cells and would be new targets for protection of islet cell function in diabetes.

2246-PRole of Heat Shock Protein 90 (Hsp90) in Insulin SecretionNATALIA A. TAMARINA, LOUIS H. PHILIPSON, Chicago, IL

In the face of high demand for insulin production, pancreatic β-cells are prone to chronic proteotoxic stress, which is triggered by the appearance of non-native and damaged proteins and can lead to defective insulin secretion and diabetes. Hsp90 proteins are key regulators of stress response pathways and normal cellular proteostasis. HSP 90 is also a regulator of constitutive secretory function in yeast. We hypothesize that HSP90 chaperone machinery is involved in modulating β-cell stress responses and insulin secretion.

Mouse insulinoma cells MIN6 were treated with the Hsp90 blocker 17-allylamino-17-dimethoxy-geldanomycin (17-AAG). 17-AAG treated cells revealed a reversible inhibition of insulin secretion. To study the effect of 17AAG on the distribution of newly synthesized insulin granules, MIN6 cells were transduced with an adenoviral vector expressing GFP-tagged

2242-PRole of Osteocalcin in Physiology of Human IsletsVICTORIA MEISSNER, YONG WANG, BARBARA BARBARO, GERARD KARSENTY, JOSE OBERHOLZER, KIRSTIE K. DANIELSON, Chicago, IL, New York, NY

While insulin stimulates osteoblast differentiation and proliferation, mouse models demonstrate that the osteoblast-derived hormone uncarboxylated osteocalcin (unOC) promotes beta-cell proliferation and insulin secretion. Our goal was to explore the potential clinical signifi cance of employing unOC as adjuvant therapy for improving islet transplant by investigating these fi ndings in humans. To assess acute effects of unOC on islets, we used microfl uidic technology to perfuse human islets with four concentrations of unOC (3.4 pg/ml, 34 pg/ml, 340 pg/ml, 1.2 ng/ml) in low glucose (2.8 mM); the technology provides dynamic data on islet mitochondrial potential, calcium infl ux, and insulin secretion. Of the four unOC concentrations, 340 pg/ml induced stronger and more consistent mitochondria depolarization and surges of intracellular calcium compared to 3.4 and 34 pg/ml, while nanomolar concentrations (1.2 ng/ml) induced no mitochondrial or calcium response. These fi ndings suggest that human islets are directly affected by unOC in low glucose, and respond better to pg/ml unOC concentrations similar to mouse models. Analysis of insulin indicated that 340 pg/ml unOC also induced greater insulin secretion compared to control without unOC. Next, to study the effect of unOC on islet insulin secretion across increasing glucose concentrations we used static culture. Islets were cultured either in 2.8, 8, 18 or 25 mM glucose, and treated with either 3.4 pg/ml, 1.2 ng/ml, 1.2 ug/ml, or no unOC; samples were taken at 1, 2.5 and 4 hours to measure insulin. When islets were cultured at low 2.8 mM glucose, the lowest concentration of unOC (3.4 pg/ml) induced a higher insulin secretion at all time points, while the highest unOC (1.2 ug/ml) had a lower insulin secretion, both compared to control. Interestingly this phenomenon reversed when islets were cultured at high glucose (25 mM). In summary, human unOC appears to regulate human islets in a glucose-dependent manner. Research is needed to determine potential application in islet transplant.

2243-PCOPII Dependent ER Exit of Proinsulin in Pancreatic Beta-CellsXUEQUN CHEN, JINGYE FANG, KEZHONG ZHANG, MING LIU, Detroit, MI, Ann Arbor, MI

Pancreatic beta cells have well developed ER to adapt to the massive production and processing of proinsulin. COPII vesicle is a conserved mechanism for ER export of cargo proteins from yeast to mammals. However, its role in beta cells has not been investigated. Our goal is to understand the molecular mechanism by which proinsulin, the dominant cargo protein in beta cells, exits ER. As a reporter to monitor ER export in both MIN6 cells and isolated mouse islets, recombinant adenovirus was created to express human preproinsulin fused with mCherry. Recombinant adenoviruses expressing wild type (WT) Sar1 and its dominant negative (DN) mutants T39N and H79G were also created to study COPII dependent ER exit.

At fi rst, the normal expression and processing of proinsulin-mCherry fusion protein and its correct targeting to insulin granules were confi rmed by Western blotting (WB) and confocal imaging. In addition, the viral titer dependent overexpression of WT and DN Sar1 was also verifi ed by WB. Next, it was demonstrated by WB, confocal and total internal refl ection fl uorescence imaging that DN but not WT Sar1 blocked ER exit of proinsulin in MIN6 cells. In addition, in vitro budding assay, using permeabilized MIN6 cells, purifi ed Sar1 protein and rat liver cytosol, demonstrated that proinsulin was packaged into COPII vesicles. Moreover, blockage of ER exit by DN Sar1 strongly induced all ER stress markers tested by both real time PCR and WB including Bip, CHOP, XBP-1, Hrd1 and phospho EIF2α. Under these conditions, steady state level of proinsulin was dramatically reduced without changing the synthesis rate of proinsulin, presumably by increasing its degradation. This possibility is being examined by proteasome inhibitor MG-132. These results indicated that ER exit of proinsulin is COPII dependent and plays a vital role for ER homeostasis and normal function of beta cells.

2244-PDetermining the Effect of a Hypoxia Mimetic and Microfl uidic Flow on Islet Vasculature and FunctionKRISHANA S. SANKAR, SVETLANA SALTAMENTOVA, JONATHAN V. ROCHE-LEAU, Toronto, ON, Canada

Islet transplantation is less invasive and cheaper than pancreas trans-plantation for the treatment of Type 1 diabetes; however, poor revasculariza-tion continues to be a major issue that leads to reduced transplant function. Our research aims to enhance the effi ciency of transplantation by priming islets for revascularization while maintaining glucose-stimulated metabolism

A576


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST


disappeared after 3 d of HG and was also partially restored by subsequent metformin treatment. Additionally, metformin increased AMP activated kinase (AMPK) phosphorylation while suppressing CD36 expression in INS-1 cells. Compound C, an AMPK inhibitor, failed to block the suppressive effect of metformin on CD36 expression. Moreover, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK agonist, could not suppress CD36 expression. Treatment with hydrogen peroxide elevated the expression of CD36 that was suppressed by N-acetyl cysteine in INS-1 cells.

Our results suggest that metformin suppresses CD36 expression elevated by HG via a AMPK independent mechanism. This effect may be associated with ability of metformin to prevent glucotoxicity in INS-1 cells.

2249-PInsulinotropic Action of Apolipoprotein A-I through ApoA-I/Cdc42/cAMP/PKA Pathway in MIN6 CellsKOKI MATSUMURA, YUKI KIMURA, HIROSHI MURAKAMI, MAKI YAMASHITA, KOTA MATSUKI, JUTARO TANABE, JUN MATSUI, NAOKI TAMASAWA, Hirosaki, Japan

We confi rmed that insulin secretion is induced by ApoA-I and then revealed the mechanisms for this activity in MIN6 cells.

(1)MIN6 cells were cultured in DMEM containing 25 mM glucose and 10% FBS.Glucose-stimulated insulin secretion (GSIS) was assessed by exposure to medium containing 0.5, 5.5 or 25 mM glucose for 1 h each. Insulin concentrations(ng/mg protein/mL) were measured by ELISA. (2) The effects of ApoA-I in GSIS,and their dose dependency (ApoA-I; 5, 10, 25 and 50 µg/ml), were determined under the same conditions with MIN6 cells.(3)The infl uence of SQ22536 (cAMP inhibitor) and Rs-cAMPS (cAMP-dependent protein kinase inhibitor) on ApoA-I action was studied in order to investigate the action mechanisms. (4) Small interference RNA (siRNA) transfection. MIN6 cells were transfected with the siRNA of ABCA1,Cdc42 and control, in the absence or presence of ApoA-I using TaqMan® MicroRNA Assays. After the incubation, insulin levels in the medium as well as mRNA and protein levels of ABCA1 and Cdc42 were determined with RT-PCR and western blotting.

(1)MIN6 cells secreted insulin in a glucose-dependent manner. (2) Addition of ApoA-I further increased insulin secretion by 4- to 8-fold, depending on the glucose concentration. (3)Both SQ22536 and Rp-cAMPS strongly inhibited the effects of ApoA-I on insulin secretion.(4) On 25mM glucose, siRNA of ABCA1 and Cdc42 signifi cantly decreased the effects of ApoA-I on insulin secretion compared with negative control group. Manifestation of ABCA1 and Cdc42 protein was less than that of negative control group as well.

For lipid effl ux in macrophages, ApoA-I activate the small G protein Cdc42 via ATP-binding cassette transporter A1 (ABCA1), and then Cdc42 activates adenylate cyclase and protein kinase A(PKA).We demonstrated in this study that ApoA-I enhances glucose-stimulated insulin release through Cdc42/cAMP/PKA pathway, via K(ATP)- independent insulinotropic action.

2250-PPI3K Mediates Islet Neogenesis-Associated Protein Effect upon β-Cell FunctionBÁRBARA MAIZTEGUI, CAROLINA L. ROMÁN, MARÍA I. BORELLI, JUAN J. GA-GLIARDINO, La Plata, Argentina

Islet neogenesis associated protein pentadecapeptide (INGAP-PP) increases insulin secretion (IS) and β-cell mass in normal rats; however, the mechanism by which it produces its effects is not clear. Thus, we studied INGAP-PP effect on IS, glucose (G) metabolism, glucokinase (GK) activity and protein expression and the PI3K pathway in cultured normal rat islets. Islets were isolated by collagenase digestion and cultured for 4 days in RPMI with 2 g/L NaHCO3, 5% FBS, 1% penicillin/streptomycin and 10 mM G, with or w/o 10 µg/ml INGAP-PP. Islets were preincubated in 3 mM G for 45 min and then incubated with 3, 8 and 16 mM G to measure IS, G metabolism (14CO2 and 3H2O production), GK activity (G-6-P production), GK, IR and PI3K protein expression (Western Blot [WB]) and PI3K/IRS-1 complex (immuneprecipitation and WB). We also studied the effect of Wortmannin (W), a PI3K inhibitor (150 and 300 nM) on G-induced IS in both groups. Results: (C vs. INGAP-PP; *p<0.05): IS (ng/islet/h): 3 mM G: 0.3 ± 0.03 vs. 0.3 ± 0.04; 8 mM G: 1.6 ± 0.2 vs. 2.4 ± 0.3*; 16 mM G: 2.4 ± 0.2 vs. 4.7 ± 0.3*; 16 mM +W 150 nM: 1.7 ± 0.3 vs. 1.8 ± 0.3; 16 mM G + W 300 nM: 0.9 ± 0.1 vs. 0.9 ± 0.3 (% inhibition with respect to 16 mM G: C, 64 ± 6 and INGAP-PP, 81 ± 6*). Glucose oxidation 14CO2 (pmol/islet/120 min): 3 mM G: 0.3 ± 0.04 vs. 0.5 ± 0.07; 8 mM G: 0.4 ± 0.06 vs. 0.7 ± 0.1*; 16 mM G: 0.6 ± 0.1 vs. 1.4 ± 0.1*. Glucose utilization 3H2O (pmol/islet/120 min): 3 mM G: 1.3 ± 0.2 vs. 1.8 ± 0.3; 8 mM G: 1.9 ± 0.3 vs. 3.2 ± 0.4*; 16 mM G: 3.3 ± 0.4 vs. 5.8 ±

proinsulin (Ins-Cpep-GFP) and studied by live confocal imaging. While in control cells newly synthesized insulin granules rapidly appear at the plasma membrane, in 17AAG treated cells granules congregated in the area of the Golgi network. Co-expression of a Golgi-specifi c fl uorescent protein with Ins-Cpep-GFP showed a continued attachment of granules to the Golgi, revealing an apparent block to normal traffi cking. ShRNA-expressing plasmids for Hsp90alpha or Hsp90beta genes were used to knock down gene expression in stably transfected clones of MIN6 cells. As shown with immunofl uorescence staining of shRNA-expressing transfected clones for insulin, the Hsp90alpha knockdown in MIN6 cells reproduces the insulin traffi cking defect observed with 17-AAG treatments. Hsp90beta knockdown on the other hand does not show same defect. The data suggest that some Hsp90alpha client proteins are necessary for traffi cking of newly synthesized granules to the plasma membrane. Thus, Hsp90alpha can regulate insulin secretion, possibly connecting with cellular stress or environmental factors that may impact carbohydrate metabolism and Type 2 Diabetes.

2247-POver-Nutrition Increased DNA Methylation of Insulin Gene Pro-moter in Pancreatic Beta-CellsKOTA ISHIKAWA, SHIN TSUNEKAWA, YUSUKE SEINO, EITA UENISHI, TAKAKO IZUMOTO, ATSUSHI IDA, HIDETADA OGATA, ATSUSHI FUJIYA, TETSUJI OKA-WA, YOJI HAMADA, YUTAKA OISO, Nagoya, Japan

Type 2 Diabetes (T2D) is a disease of insulin insuffi ciency caused by failure of pancreatic beta-cell function and mass. Recent studies showed that elevated DNA methylation (DNAmet) of insulin gene (Ins) promoter, especially the site of cAMP response element (CRE), was associated with Ins expression negatively in subjects with T2D. IRS2 that is essential for beta cell survival also contains CRE and CpG islands in the promoter. Therefore, we hypothesized that DNAmet of Ins and IRS2 affected the progression of diabetes. The aim of this study was to investigate the effect of over-nutrition on DNAmet and gene expression of Ins and IRS2 in pancreatic beta-cells. Rat insulinoma cells (INS1) were cultured at either 11.2 mmol/L normal (NG) or 22.4 mmol/L high glucose (HG) condition for 14 days in the presence or absence of 0.4 mmol/L palmitate (P). Among these conditions, incubation at HG and HG with P signifi cantly decreased the expression of Ins compared with that at NG. Similarly HG and HG with P markedly increased DNAmet of CRE in Ins promoter time-dependently, and P did not have an infl uence on the DNAmet at NG condition. (N: 4%, N with P: 4.6%, HG: 15.3%, HG with P: 16.3%) Meanwhile, all conditions affected neither DNAmet of CRE nor gene expression of IRS2. Methylation of the CpG sites in the promoter signifi cantly suppressed Ins promoter driven luciferase activity. In contrast, addition of methylation inhibitor, 5-Aza-2,-deoxycytidine, decreased DNAmet of CRE and signifi cantly improved Ins expression that was reduced by HG. When comparing the effectiveness of various drugs, such as Metformin, Exendin-4, Bezafi brate or N-acetyl-cysteine, only Metformin ameliorated the decline of Ins expression and the elevated DNAmet of CRE. These data suggested that elevated DNAmet of Ins promoter that was induced by over-nutrition, especially glucotoxicity, might be implicated in progression of T2D. Metformin may have the direct anti-diabetic effect on pancreatic beta-cells as a potent inhibitor of DNAmet.

2248-PPrevention of Glucotoxicity by Metformin in Insulin Producing INS-1 Cells: The Involvement of Fatty Acid Translocase Cluster Deter-minant 36 (CD36)JUN SUNG MOON, YONG-WOON KIM, SO-YOUNG PARK, JONG-YEON KIM, JI SUNG YOON, HYOUNG WOO LEE, KYU CHANG WON, Daegu, Republic of Korea

Although metformin regulates blood glucose levels primarily by improving insulin sensitivity, there is evidence that it also preserves insulin secretion by directly protecting pancreatic β-cells from glucotoxicity or lipotoxicity. However, the exact underlying mechanism has not been clarifi ed. In the present study, we hypothesized that the benefi cial effect of metformin on β-cells may be mediated by altering fatty acid translocase cluster determinant 36 (CD36), a fatty acids transporter. To test this hypothesis, we evaluated the ability of metformin to prevent glucotoxicity and the associated changes of CD36 expression in INS-1 cells.

Twelve hours of exposure to high glucose (HG; 30 mM) concentrations increased CD36 expression, palmitate uptake, and oxidative stress in the INS-1 cells. These effects were suppressed by treatment with metformin. Insulin expression was decreased by HG treatment and partially restored by metformin. Glucose-stimulated insulin secretion (GSIS) completely

A577


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


DPP-4I(KD) group and C57BL/6J served as normal control(CN) group. During 8 weeks intervention period, general features were collected weekly, FINS detection, OGTT and IPITT were performed at 7w and 15w respectively. 2. Analyzed morphology, proliferation and apoptosis in islet beta-cell;3.Evaluated the expression of PDX-1 and MafA. Result: 1. 7 week-old kkay mice will be proper models for the study after 2 weeks of HFD. At 15 weeks old, general conditions, glucose tolerance, insulin sensitivity and Islet morphology were improved in KE group and KD group. 2. Propotion of ki67 positive cells in intervention groups were higher than other groups(P<0.01). Apoptosis in islets showed no signifi cant differences among these groups. 3. Expression of MafA were increased in intervention groups. There were no signifi cant difference in the expression of PDX-1. Conclusion: 1.it is a proper kkay mice model of hyperinsulinemic-prediabetes by early administrating HFD; 2.early intervention of life style and administration of MK0626 could delay the onset of diabetes. 3. There were no obvious changes in the expression of PDX-1 at early stage of diabetes in kkay mice. MafA may play a crucial role in the development of diabetes in this model.

2254-PDissecting the Factors Affecting the Functional Properties of Iso-lated Human IsletsMARA SULEIMAN, MARCO BUGLIANI, FAROOQ SYED, FRANCESCO OLIMPICO, LUCIANA MARIOTTI, SILVIA FORNACIARI, DANIELE FOCOSI, FABRIZIO SCAT-ENA, UGO BOGGI, FRANCO FILIPPONI, PIERO MARCHETTI, DECIO L. EIZIRIK, MIRIAM CNOP, LORELLA MARSELLI, Pisa, Italy, Brussels, Belgium

Human pancreatic islets (HPI) are being used for clinical and basic research studies. However, little information is available on variables affecting the functional properties of HPI following isolation.

We prepared HPI from 350 non-diabetic donors (age, 60±17 yrs; M/F, 184/166; BMI, 25.2±3.9 kg/m2; pancreas cold ischemia time: 15±7 hrs) and assessed insulin release (IR, µU/islet/min) in response to 3.3 mM glucose (G), 16.7 mM G, 100 µM glyburide (gly) and 20 mM arginine (arg); islet insulin positive area (IIPA) before isolation (n: 51) as well as beta cell proportion in islet preparations (n: 40) were evaluated by immunocytochemistry (ICC), and islet cell viability was assessed by nuclear dyes (n: 40). Correlation analyses were performed between clinical parameters, islet preparation characteristics, IIPA and IR.

Donor characteristics were not associated with any of the considered islet features. Islet purity by dithizone staining was 65±23%, correlated (p=0.04) with the proportion of beta cells as quantifi ed by ICC and did not affect islet cell viability (that was 95.4±3.7%). IIPA was 72±12%. IR was assessed at 4±3 days from isolation; it was 0.037±0.015 at 3.3 mM G and increased to 0.104±0.073 with 16.7 mM G (p<0.01 vs 3.3 G), 0.103±0.07 with gly (p<0.01) and 0.088±0.059 with arg (p<0.01). The respective stimulation index values were: 2.8±1.7, 2.9±1.7 and 2.4±1.3. IR was not affected by IIPA. Basal and stimulated IR was higher at increasing islet preparation purity (all p<0.01); in addition, there were inverse relationships between IR and the number of days from isolation to stimulation experiments (all p<0.01). These correlations were confi rmed after multiple regression.

In conclusion, islet purity and the time from isolation to the day of functional experiments are major factors affecting ex-vivo HPI insulin secretion capability. It remains to be determined whether this may be the case when the molecular properties of HPI are evaluated or in the clinical islet transplantation setting.

2255-PBeta Cell G-Protein Coupled Estrogen Receptor (GPER) Modulates the Genetic Effect of ZnT-8 on Glucose Induced Insulin Secretion by Cyclosporin AEUN SEOK KANG, HYE JIN WANG, EUN YEONG CHOE, OBIN KWON, HANNA SEOK, YONGIN CHO, YU JUNG YUN, YOUN JEONG CHOI, KYU HA HUH, MYUNG SOO KIM, YU SEUN KIM, SOON IL KIM, BYUNG-WAN LEE, CHUL WOO AHN, BONG SOO CHA, HYUN CHUL LEE, CHUL HOON KIM, Seoul, Republic of Korea

Cyclosporin A (CsA) is widely used in organ transplanted patients to prevent rejection but it has diabetogenic effect by inhibition of insulin secretion from pancreatic beta cell. It is reported that the genetic variations in rs13266634 of zinc transporter-8 (ZnT8) show different susceptibility to CsA in glucose stimulated insulin secretion. In this study, we studied the effect of G protein-coupled estrogen receptor (GPER) agonist, G1, on insulin secretion in beta cell according to ZnT8 genotype under CsA treatment. INS1E and MIN6 Cells were transfected with ZnT8 cDNA (Wild R325 or Mutant W325). Cells were treated with CsA with/without G1. We measured insulin secretion in cells transfected with ZnT8-R325 or ZnT8-W325. Amount of insulin secretion in ZnT8-R325 expressing cells was reduced

0.6*. GK activity (pmol/islet/h): 2.4 ± 0.6 vs. 4.8 ± 0.3*. Protein expression (INGAP-PP % above control): GK 91.7 ± 3.8*; IR 75 ± 1.5*; PI3K 155 ± 15*; PI3K-IRS-1 complex 76 ± 5*. Our data suggest that the positive modulatory effect of INGAP-PP upon G-induced IS could be partly ascribed to its effect upon GK activity and G metabolism mediated via PI3K.

Supported by: CONICET (PIP1211)

2251-PInhibition of Pancreatic Beta-Cell CaMKII Reduces Glucose-Stim-ulated Calcium Infl ux and Insulin Secretion, which Manifests With Impaired Glucose TolerancePRASANNA K. DADI, NICHOLAS C. VIERRA, ROGER J. COLBRAN, DAVID A. JA-COBSON, Nashville, TN

Pancreatic β-cell Ca2+ entry via voltage dependent Ca2+ channels (VDCCs) is coupled to insulin secretion. While CaMKII can modulate insulin secretion, the underlying mechanism(s) and whether CaMKII regulates glucose homeostasis remains unknown. Here we report that mice with conditional β-cell CaMKII inhibition show signifi cantly impaired glucose tolerance due to decreased glucose stimulated insulin secretion. Inhibition of β-cell CaMKII results in reduced VDCC facilitation, causing diminished glucose-stimulated action potential fi ring frequency. Glucose-stimulated Ca2+ entry is also reduced in islets with β-cell CaMKII inhibition as well as in β-cells treated with a peptide inhibitor of CaMKII, but interestingly this is mediated by both a reduction in β-cell VDCC Ca2+ entry as well as decreased endoplasmic reticulum Ca2+ levels. These results reveal that CaMKII activation provides an important positive feedback mechanism to further elevate β-cell Ca2+ levels, exposing how CaMKII enhances glucose-stimulated insulin secretion.

Supported by: DK096122, DK081666, DK020593

2252-PLong-Term Culture of Pancreatic Tissue Slices for the Study of En-docrine and Exocrine Cells In SituANJA MARCINIAK, CLAUDIA SELCK, STEPHAN SPEIER, Dresden, Germany

Pancreatic tissue slices represent an excellent approach to study endocrine and exocrine cell physiology in situ as they maintain pancreas morphology and lack any enzymatic or extensive mechanical injury during preparation. However, in culture the exocrine tissue of acute slices undergoes rapid degeneration which prevents long-term studies of exocrine cells and hampers the investigation of endocrine cells in an in situ environment. Therefore, we aimed to establish an organotypic culture system for pancreatic tissue slices which preserves cell morphology and function, enabling long-term in situ studies of exocrine and endocrine cells.

Adult mouse pancreatic tissue slices of 150 µm thickness were cultured under various conditions using the interface-membrane technique. Slice surface area, cell morphology and viability were assessed on tissue and single cell level using fl uorescent reporter mice and indicators in combination with stereomicroscopy and laser scanning microscopy. Further more, stimulated secretion from endocrine (insulin) and exocrine (amylase) cells were measured. Our experiments revealed optimized culture conditions which signifi cantly prolonged the culture period of viable and functional tissue slices. Slice morphology was well-preserved with 90.3±6.4 % of the initial surface area still present after 7 days. Detailed analysis of islets of Langerhans and acini demonstrated sustained structural integrity and only minimal cell death was detected. Importantly, beta cells remained glucose responsive with a comparable stimulation index for insulin release in fresh and 7 day cultured slices (2.3±0.5 vs. 2.0±0.4). Additionally, over the entire culture period acinar cells showed a typical caerulein concentration dependent stimulated amylase secretion. Thus, we present a long-term organotypic culture system for pancreatic tissue slices allowing longitudinal studies simultaneously on endocrine and exocrine cells under near-physiological conditions.

2253-PEffect of DPP-4 Inhibitor MK0626 and Exercise on the Protection of Islet Function in Early Pre-Diabetic kkay MiceYUPENG LI, HUI TIAN, JING XIAO, YU PEI, XIAOFEI HAN, HUA SHU, Tianjin, China, Beijing, China

Objective: To study the changes of islet beta-cell morphology and function after lifestyle intervention and administration of dpp-4 inhibitor (MK0626) in HFD induced pre-diabetes kkay mice. Method: 1. Establish models of prediabetes-hyperinsulinism by administrating HFD to kkay mice. 2. 5 weeks old male HFD induced kkay mice were divided randomly into: kkay normal diet(KN) group, kkay high fat diet(KH) group, kkay exercise(KE) group, kkay

A578


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST


When comparing cells exposed to 11 mmol/L glucose to those at 2.8 mmol/L, GLP-1 effect on GIIS was blunted in K and Q in respect to NEO cells. GLP-1 signifi cantly increased cell proliferation in NEO (33+8%, p<0.01 in comparison to BSA treated cells) but its effect was reduced in K (17+6%, p=ns) and abolished in Q cells (-10+2%, p=ns). Similarly, GLP-1 signifi cantly increased viability in NEO (50+19%, p<0.01 in comparison to BSA treated cells) but not in K (18+5%, p=ns) and Q cells (7%+3%, p=ns). GLP-1 signifi cantly reduced apoptosis in NEO (54+9%, p<0.05) and in K cells (56+16%, p<0.05) but had no signifi cant effect in Q cells (29+5%, p=ns). These results were confi rmed with fl ow citometry (-57+10%, p <0.05 in NEO; -38+16%, p< 0.05 in K; -18+7% in Q, p=ns). Finally, GLP-1 increased Akt and ERK 1/2 phosphorylation in NEO (p<0.05 for both vs. staurosporine incubated cells) and in K cells (p<0.05 for both) while it had no effect in Q cells (p=ns for both). GLP-1 decreased Bax/Bcl-2 ratio in NEO (p<0.05) and in K cells (p<0.05) but not in Q cells (p=ns). In conclusions, our fi ndings indicate that overexpression of ENPP1, especially with the Q121 variant, reduces the well known secretagogue, proliferative and anti-apoptotic effects of GLP-1 on beta cells likely playing a pathogenetic role for defective insulin secretion.

2258-PDownregulation of Vasoactive Intestinal Peptide Expression in Type 2 DiabetesFATMA ZEHRA HAPIL, HALE TASYUREK, NIL GUZEL, AHTER D. SANLIOGLU, MUSTAFA KEMAL BALCI, SALIH SANLIOGLU, Antalya, Turkey

Chronic insulin resistance and a progressive decline in βετα cell function are the two hallmarks of Type 2 Diabetes (T2D). Because beta cell mass and function inevitably decline in T2D, therapeutic agents with the potential to stimulate postprandial insulin secretion and preserve beta cells are crucial for the management of T2D. Vasoactive Intestinal Peptide (VIP) is an islet endocrine neuro-peptide which functions as an anti-infl ammatory factor, induces immune tolerance and stimulates post-prandial insulin secretion. Because of its diverse functions, we suspected that VIP expression pattern might have been altered during the course of T2D. To test this hypothesis, we fi rst generated a Diet Induced Obesity (DIO) animal model via feeding C57BL/6J mice with diets enriched in fat (HFD). Streptozotocin was then administered to induce beta cell loss resulting in hyperglycemia. Insulin, glucagon and VIP content of the pancreatic islets were revealed by immunohistochemistry. Mice fed with HFD acquired considerable amount of abdominal fat but consumed less water compared to mice fed with normal diet (ND). Intraperitoneal Glucose Tolerance Tests (IPGTT) revealed impaired beta cell function in obese animals. Additionally, insulin resistance in HFD fed animals was confi rmed through administration of a fast acting insulin analogue (Insulin Lispro, Humalog, Elli Lilly). Intriguingly, diabetic obese animals displayed reduced levels of VIP expression in pancreatic islets compared to obese and/or control animals. Therefore, downregulation of VIP expression is correlated with the induction of diabetes in an experimental animal model of T2D. In conclusion, VIP mediated gene therapy approaches might be useful in the setting of T2D.

Supported by: Akdeniz University; TUBITAK (111S157)

2259-PEffects of Artemisia Dracunculus L. Extract (PMI-5011) on Insulin Secretion and Insulin Signaling in INS-1 CellsZHONG Q. WANG, XIAN H. ZHANG, YONGMEI YU, DAVID RIBNICKY, WILLIAM T. CEFALU, Baton Rouge, LA, New Brunswick, NJ

Botanical extracts have been proposed as alternative treatments for diabetes, yet specifi c mechanisms for many botanicals have not been determined. However, extracts of Artemisia Dracunculus L. (termed PMI-5011), have been shown to improve glucose metabolism and enhance insulin signaling in vitro and in vivo studies. We sought to evaluate the effects of PMI-5011 on insulin secretion and insulin signaling in cultured β-cells (INS-1).

INS-1 cells were treated with PMI-5011 at doses of 0, 2.5, 5, 10 and 25 µg/ml in normal (100 mg/dl) and high glucose (300 mg/dl) for 60 min. Medium insulin levels (assessed by ELISA) were 1.07 ± 0.01 (P<0.05) , 1.25 ± 0.02 (P<0.01), 1.37 ± 0.02 (P<0.001) and 0.84 ± 0.06 fold of control in normal glucose condition; 1.13 ± 0.04 (P<0.05), 1.05 ± 0.03, 1.08 ± 0.05, and 0.62 ± 0.04 in high glucose condition, respectively. Cells were then treated with various doses of PMI-5011 overnight and IRS-1, IRS-2, PI 3 K and GLUT2 in the lysates were measured using western blotting assay. Data showed that PMI-5011 greatly increased IRS-2 and GLUT-2 abundance, modestly increased IRS-1 and PI-3K protein content at doses from 1 to 5 µg/ml, but high dose of PMI-5011 (25 µg/ml) decreased protein content at normal

by CsA but not affected in ZnT8-W325 expressing cells. G1 attenuated the inhibitory effect of CsA on glucose stimulated insulin secretion in beta cells. However this difference according to the genotype disappeared when cells were treated with G1. The result suggests that 1) ZnT8-W325 genotype shows a resistance against CsA induced attenuation in glucose stimulated insulin secretion compared with ZnT8-R325 genotype. 2) GPER agonist, G1, attenuates the effect of CsA on glucose stimulated insulin secretion. 3) G1 modulates the genetic effect of ZnT-8 on the effect of CsA on glucose stimulated insulin secretion.

Supported by: Ministry of Education, Science and Technology (NRF-2010-013-E0008), (NRF-2012000891)

2256-PLoss of Cytochrome C Signaling Associated With Impaired Insulin Secretion by Islets Cultured in Low GlucoseAUSTIN ROUNTREE, MARK LISOWSKI, JARED RADTKE, FRANCIS KIM, CHRIS-TIANE S. HAMPE, IAN R. SWEET, Seattle, WA

Objective: The aim of the study was to assess the relative control of insulin secretion rate by calcium infl ux and signaling from cytochrome c.

Research design and methods: The experimental approach was to study islets cultured at low glucose, conditions known to impair glucose response of insulin secretion, in part through the loss of glucokinase. The relative contributions of mitochondrial signaling and calcium infl ux to the loss of secretory function, and the subsequent recovery by a glucokinase activator were determined. Oxygen consumption rate, ATP, reduction and translocation of cytochrome c, calcium and insulin secretion rate were measured in response to either glucose or a mitochondrial fuel (α-ketoisocaproate).

Results: Culture in 3 mM glucose for 2 days diminished glucose-stimulated oxygen consumption relative to islets cultured at 11 mM glucose, and abolished response of cytochrome c reduction and translocation, and insulin secretion. Surprisingly, glucose-stimulated calcium infl ux remained robust and was similar for islets cultured at 3 or 11 mM glucose. Responses to 10 mM α-ketoisocaproate by islets cultured at both concentration of glucose were similar, demonstrating that loss of mitochondrial and secretory capacity did not cause the impaired glucose-response of insulin secretory observed in the islets cultured in 3 mM glucose. A glucokinase activator acutely restored cytochrome c reduction and translocation, and insulin secretion, independent of effects on calcium infl ux.

Conclusions: In summary, cytochrome c reduction and translocation are essential signals in the stimulation of insulin secretion, the loss of which can result in impaired insulin secretion even when calcium response is intact.

2257-PENPP1 Infl uences the Effect of GLP-1 on Insulin Secretion, Prolif-eration and Apoptosis of INS-1E Pancreatic Beta CellsNUNZIA CAPORARRELLO, CRISTINA PARRINO, MARIANNA R. STANCAMPI-ANO, RICCARDO VIGNERI, LUCIA FRITTITTA, Catania, Italy

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) play a signifi cant role on insulin signaling, insulin secretion and glucose metabo-lism. In INS-1E cells transfected with either the K121 (K) or the Q121 (Q) of ENPP1 or neomycin-resistance gene (NEO) we investigated the effect of 100 nmol/l glucagon-like peptide-1 (GLP-1) on 1) glucose-induced insulin secretion (GIIS); 2) cell proliferation (BrdU incorporation assay) and viability (MTS assay); 3) staurosporine induced apoptosis (caspase-3/7 activity and fl ow citometry); and 4) pathways involved in apoptosis (e.g. phosphorylation of Akt and ERK1/2 and, the ratio of Bax/Bcl-2 by western blot analysis).

A579


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS

ISLET BIOLOGY—SIGNAL TRANSDUCTION

MODY carriers when compared to controls (copy number mean 3837±1162; median=1030, IQR=458-3318 vs. mean 81.3±48.2; median=14, IQR=0-126; p=0.00001). miR-103 correlated negatively with triglycerides (p=0.02) and systolic blood pressure (p=0.01), but did not correlate with HbA1c, age, or type of mutation (transactivation vs. dimerization domain).

In conclusion, we have identifi ed miR-103 to be negatively controlled by HNF1A and strongly upregulated in the serum of HNF1A-MODY carriers. The pathophysiology of HNF1A-MODY may be associated with the overexpression of miR-103 which has been previously linked to diabetes and obesity.

& 2262-PHydrogen Peroxide Is a Metabolic Secondary Signal in Pancreatic Beta-CellsALYSSA K. ARATA, RAMILA SHAH, PATRICK MOORE, CHRISTOPHER J. RHODES, Chicago, IL

Islet β-cells are thought to be susceptible to oxidative damage, in part due to low levels of antioxidant enzymes. We examined which of these enzymes were lacking in normal isolated rat islets relative to hepatocytes at the mRNA, protein, and enzyme activity levels. Essentially, we found that the levels of superoxide dismutases (SOD1 and SOD2), which convert superoxide into H2O2, were comparable to that in liver. However, glutathione peroxidase (GPX1) and catalase (CAT), which dispose of H2O2 to water, were lacking in islets (GPX1 activity was <20% and CAT activity was negligible) relative to hepatocytes (p≤0.01). As such, normal β-cells are wired for H2O2 production from oxidative metabolism-generated superoxide.

We postulate that H2O2 may act as a metabolic secondary signal in β-cells. Using Amplex Red fl uorometry, we found endogenous H2O2 production in isolated islets to rapidly increase within 2 min of raising glucose concentrations from basal 2.8 mM to stimulatory 16.7 mM (4-fold; p≤0.04) and remain elevated until reintroduction of basal glucose returned H2O2 to resting levels. Adenoviral-mediated expression of CAT (AdV-CAT) in islets prevented a glucose-induced increase in H2O2 (>90%; p≤0.04). Elimination of H2O2 by AdV-CAT signifi cantly inhibited glucose-induced insulin secretion, especially its amplifi cation under hypercalcemic clamp conditions (p≤0.05). Moreover, glucose-induced activation of mammalian target of rapamycin (mTOR), specifi cally mTOR complex-1 (mTORC1) and not mTORC2, was signifi cantly inhibited in AdV-CAT islets (p≤0.03). Conversely, enhancing H2O2 production with AdV-SOD2 increased glucose activation of mTORC1 (p≤0.008). In addition, glucose-induced H2O2 production in islets was capable of inhibiting protein tyrosine phosphatases (PTPs), particularly PTPN11. Thus, the antioxidant system of islet β-cells is coordinated to generate H2O2 as a normal metabolic secondary signal that amplifi es specifi c cellular functions including regulated insulin secretion.


& 2263-PRegulation of Pancreatic Islet Beta-Cell Functions by Nrf2 In Vivo and In VitroXIAOQING HE, QIANG MA, Morgantown, WV

Pancreatic islet beta cells secrete insulin in response to blood glucose to maintain body’s metabolic fuel homeostasis. On the other hand, beta cells are susceptible to a range of toxic signals such as high glucose, free fatty acids, oxidative stress, and infl ammation, which are major contributing factors to the development of diabetes. The nuclear factor erythroid 2-related factor 2 (Nrf2) is an emerging regulator of cellular defense against oxidative and infl ammatory insults. We have previously found that mice with targeted knockout (KO) of Nrf2 were sensitive to streptozotocin (STZ) induced diabetic lesions. In the present study, islet beta cells from Nrf2 wild-type (WT) and KO mice were analyzed in vivo and in vitro. Treatment with STZ induced diabetic conditions that were signifi cantly more severe in Nrf2 KO than WT mice (blood glucose of 558±38 mg/dl for KO and 380±17 for WT). In addition to reducing pancreatic islet size, STZ induced islet oxidative stress and infl ammation that were signifi cantly more apparent in KO than WT. Characterization of Nrf2 KO mice revealed that loss of Nrf2 induced a sub-diabetic condition with signifi cantly reduced glucose tolerance and reduced levels of serum insulin. To examine if Nrf2 directly affects islet functions, pancreatic islets were isolated. Islet cells from KO mice showed increased oxidative stress and apoptosis, and reduced survival in the presence of high glucose. Furthermore, Nrf2 KO islets exhibited signifi cantly decreased insulin secretion upon stimulation with glucose in comparison with WT. Taken together the fi ndings revealed that Nrf2 directly regulates islet beta cell functions by modulating glucose stimulated insulin secretion and by boosting protection against oxidative stress and STZ toxicity.

Supported by: NIOSH

glucose condition. Although, PMI-5011 greatly increased GLUT2 abundance at low doses (1 and 2.5 µg/ml), the effects of PMI-5011 on insulin signaling were reduced at high concentration of glucose. IRS-2, PI-3 and GLUT2 content decreased with increase of PMI-5011 concentrations. Furthermore, silent IRS-1 using IRS-1 siRNA transfections resulted in reduction of insulin secretion, IR β and GLUT2 abundance in INS-1 cells as well as blocked the effects of PMI-5011 on insulin release.

This study suggests that PMI-5011 increases insulin secretion in cultured pancreatic β-cell by enhancing insulin signaling pathway.

2260-PHigh Fidelity Taq DNA Polymerase Leads to Erroneous Methylation Analysis of the Human Insulin GeneALYSSA R. GOLDBACH, LAUREN A. KENNA, EITAN M. AKIRAV, Mineola, NY

Differential DNA methylation of CpG dinucleotides (CpG-DNs) can maintain or repress gene transcription in a cell specifi c manner. Insulin-producing β-cells show reduced methylation of the insulin gene. Identifi cation of differentially methylated CpG-DNs is used for the study of insulin gene regulation and for the detection of β-cell death in the blood of diabetic patients. Gene methylation can be detected by bisulfi te treatment of genomic DNA resulting in the conversion of unmethylated cytosines (C) to thymidines (T), while methylated Cs remain intact. Bisulfi te treated DNA is then amplifi ed in a PCR reaction using Taq DNA polymerase (Taq-pol) followed by sequencing of the PCR amplicon. Native Taq-pol lacks proofreading capacity, presenting with a higher probability of DNA mutations during polymerization. Recently engineered Taq-pols offer DNA proofreading and are known as high fi delity (HF) Taq-pols. We tested whether HF Taq-pols can be used for the amplifi cation and analysis of methylated or demethylated human insulin DNA treated with bisulfi te. Synthetically methylated or demethylated human insulin DNA was treated with bisulfi te followed by a PCR reaction using HF and low fi delity (LF) Taq-pols. Amplicons were gel purifi ed, cloned and sequenced. Surprisingly, HF Taq-pol showed higher rates of nucleotide substitution and deletion of demethylated insulin DNA when compared with low fi delity (LF) Taq-pol. These errors reduced the ability to detect demethylated CpG-DNs, impairing the correct analysis of DNA methylation. Methylated insulin DNA, in which CpG-DNs are spared following bisulfi te treatment, was less prone to mutation by HF Taq-pols, suggesting that unconverted CpG-DNs can be detected by HF Taq-pol. In conclusion, our data show an increase in the rate of mutations of bisulfi te treated insulin DNA when using HF Taq-pols, highlighting the importance of using LF Taq-pols for the analysis of gene methylation in diabetes.

Supported by: JDRF (17-2012-588)


Guided Audio Tour: Beta Cell Signal Transduction (Posters: 2261-P to 2268-P), see page 15.

& 2261-PIdentifi cation of Circulating MicroRNAs inHNF1A-MODY CarriersSIOBHAN BACON, CAROLINE BONNER, KRISTINE C. NYHAN, MA P. KYITHAR, JASMIN SCHMID, SEAN KILBRIDE, JOCHEN H.M. PREHN, MARIA M. BYRNE, Dublin, Ireland

HNF1A-MODY is caused by mutations in the TCF1/hepatocyte nuclear factor 1-alpha (hnf1a) gene, the most common being Pro291fsinsC. Micro-RNAs (miRNAs) have emerged as potent regulators of gene function in numerous diseases including diabetes. miRNAs are typically 19-25 nucleotide non-coding RNA molecules, which suppress gene expression by repression of protein production or mRNA degradation. To date, there have not been any studies investigating the potential involvement of miRNAs in the pathogenesis of HNF1A-MODY.

In this study, a miRNA array assay was carried out in INS-1 cells inducibly expressing Pro291fsinsC-HNF1A, which acts as a dominant-negative mutant in vitro. Among several other miRNAs, inducible expression of Pro291fsinsC-HNF1A caused a signifi cant upregulation of miR-103 expression. Elevated miR-103 levels upon induction of Pro291fsinsC-HNF1A was validated by real-time PCR. miRNAs have been shown to be secreted from cells, and present in exosomes in the serum of humans. We next determined serum levels of miR-103 in n=31 HNF1A-MODY carriers and n=10 MODY-negative family members by absolute real-time PCR analysis. Mutations in the HNF1A gene included L17H, P291fi nsC, S352fsdelG, F426X, P379T, and IVS7-6G>A. Strongly elevated levels of miR-103 were detected in the serum of HNF1A-

A580


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


secretion and islet β-cell function. We showed recently that GABA exerts regenerative and protective effect on the islet β-cells under both in vitro and in vivo conditions. Here, we illustrated the signaling pathways conveying the trophic effects of GABA in the islet β-cells. Western blot analysis showed that GABA time- and dose-dependently induced Akt, ERK, and CREB phosphorylation in clonal insulin-secreting INS-1 cells, which are key mediators for cell proliferation and survival. Thymidine incorporation assay revealed that GABA induced cell proliferation in INS-1 cells, which was inhibited by pharmacological inhibition using PI3K inhibitor LY294002, MAPK inhibitor PD98059, and PKA inhibitor H89, suggesting the role of these pathways in mediating GABA-induced β-cell proliferation. Western blot and fl ow cytometry analysis showed that infl ammatory cytokines i.e. a mixture of TNF-α (50 ng/ml), IFN-γ (50 ng/ml), and IL1-β (10 ng/ml) markedly induced apoptosis in INS-1 cells and isolated adult human islet β-cells, which was attenuated by GABA treatment. GABA also inhibited reactive oxygen species (ROS) production in INS-1 cells and human islets, suggesting that GABA protected β-cells from apoptosis by suppressing ROS levels. In isolated normal human islets, GABA signifi cantly induced Akt phosphorylation, which however, was not observed in type 2 diabetic islets (T2D). Furthermore, GABA-induced ERK and CREB phosphorylation were exaggerated in T2D islets, altogether suggesting an altered GABA signaling pathway in T2D. In summary, our results suggest that GABA exerts trophic effects by promoting β-cell proliferation via PI3K/Akt, MAPK/ERK, and PKA/CREB signaling pathways, which are altered in T2D, and that GABA promotes β-cell survival via inhibition of apoptosis, partially through inhibiting ROS production.

Supported by: Banting and Best Diabetes Center; JDRF; CIHR

& 2267-PATF3 and PDX-1 Exert Opposing Effects on Glucokinase Down-regulation and Pancreatic β-Cell Dysfunction by Chronic Ethanol ConsumptionWON HO KIM, KEON JAE PARK, JI YEON KIM, Chungbuk, Republic of Korea

Recently, we suggested that chronic alcohol consumption-induced pancreatic β-cell dysfunction was caused by glucokinase (GCK) nitration and its downregulation, and thus leading to the impaired glucose metabolism and insulin resistance; however, little is known about the exact mechanisms underlying chronic alcohol consumption-induced GCK downregulation. Here, we found that pancreatic β-cells of ethanol fed-mice exhibited a decrease in GCK mRNA and protein, and an increase in activating transcription factor 3 (ATF3), which were determined by ethanol metabolism pathway. We also identifi ed four putative ATF3 binding sites located between -404 and -287 of GCK promoter as the repressor region associated with ethanol-mediated GCK downregulation. Compared with wild-type pRGP-404/Luc-transfected cells, ATF3 inhibitory effects on GCK transcriptional activity were remarkably attenuated by site- or deletion-mutagenesis in the region containing ATF3-binding site, especially between -299 and -287. ChIP assay also shows that ATF3 inhibits GCK promoter activity via a direct recruitment to GCK promoter region spanning from -404 to -287. Furthermore, ethanol-induced ATF3 interferes the interaction of PDX-1 with p300, a histone acetytransferase, through deacetylation of PDX-1 as well as histone H3 and H4, resulting in a decrease of GCK transcriptional activity. Chromatin remodeling of GCK was dependent on ATF3-induced SIRT1. Collectively, we propose a mechanism for ethanol consumption-induced ATF3 plays as a negative regulator of GCK expression via its deacetylation and thus promotes pancreatic β-cell dysfunction. Also, ATF3-mediated GCK downregulation may offer therapeutic strategies to combat the impaired glucose metabolism, insulin insensitivity, and hyperglycemia by chronic ethanol consumption.

Supported by: Korea National Institute of Health

& 2268-PYES, a Src Family Kinase, Is the Upstream Activator of Cdc42 in β-CellsSTEPHANIE M. YODER, STACEY L. DINEEN, DEBBIE C. THURMOND, Indianapolis, IN

Second-phase insulin secretion requires the mobilization of insulin secretory granules from the cytosol to the plasma membrane. Cdc42, a small Rho family GTPase, is recognized as the proximal glucose-specifi c trigger critical to second-phase insulin secretion. Cdc42 is activated within 3 min and signals downstream to activate the p21-activated kinase (PAK1) within 5 min. PAK1 then signals to Raf-1/MEK/ERK to induce fi lamentous actin remodeling, which in turn mobilizes insulin granules to the plasma membrane, supporting second-phase insulin secretion. However, what

& 2264-PProlactin Increases Tryptophan Hydroxylase 1 (TPH1) Expression through JAK/STAT Pathway in Beta CellsHITOSHI IIDA, TAKESHI OGIHARA, KYOKO FUJIMAKI, MOTOYUKI TAMAKI, YUKIKO TOYOFUKU, HAIL KIM, YOSHIO FUJITANI, HIROTAKA WATADA, Tokyo, Japan, Daejeon, Republic of Korea

Lactogenic hormones increase beta cell mass to compensate for the insulin resistance during pregnancy. Previously, we have shown that serotonin play important role in beta cell proliferations, and that prolactin (Prl) induces expression of TPH1, a rate-limiting enzyme for serotonin synthesis. To investigate the precise mechanism of Prl-induced TPH1 expression, we performed 5’-RACE to isolate the beta cell specifi c promoter region and assessed the TPH1 promoter activity using luciferase assay in a mouse beta cell line, MIN6 cells. In MIN6 cells, Prl induced expression of TPH1, which was inhibited by the treatment with STAT5 inhibitor. 5’ RACE revealed an novel TPH1 transcription initiation site and we named this site as exon1b. The plasmid containing various lengths of 5’ fl anking region of exon1b showed basal luciferase activity, but not prolactin responsiveness. The survey of nucleotide sequence in 5’ fl anking region of this exon identifi ed two regions (GAS-A and GAS-B) which are similar to interferon gamma- activated sequence (GAS), binding site of STAT5, at about 9 and 6 kbp upstream of transcription start site. We generated plasmid containing 6X GAS-A or 6X GAS-B linked to heterologous promoter and luciferase gene. Prl activated luciferase activity of GAS-A containing plasmid by 6.3 fold, but not in GAS-B containing plasmid. The Prl responsiveness of GAS-A was inhibited by forced expression of dominant negative mutant of STAT5A as well as STAT5 inhibitor. We performed EMSA using GAS-A probe, and demonstrated that the binding activity of nuclear proteins was increased after treatment with Prl, and diminished by adding 100 times excess of cold probe including GAS consensus sequences and anti-STAT5 antibody. ChIP assay demonstrated that STAT5A bind to GAS-A region in MIN6 cells. Our fi ndings suggest that Prl induced TPH1 expression in beta cells is mediated by newly identifi ed promoter region and that this increases is mediated by the activation of STAT5.

& 2265-PDecoding Functionally Relevant MicroRNA Signaling in Glucose-Stimulated Beta-CellsSCOT J. MATKOVICH, TIFFANI C. GROSSENHEIDER, St. Louis, MO

MicroRNAs (miRs) are important contributors to mammalian cell biology and miRs such as miR-375 and -124a are required for appropriate beta-cell differentiation and glucose-stimulated insulin secretion (GSIS). However, miR roles in modulating beta-cell function in response to fl uctuations in serum glucose have not been comprehensively defi ned. Here, we have analyzed interlinked miR, mRNA, and RNA-induced silencing complex (RISC)-associated mRNA changes in response to glucose stimulation of rat INS-1 cells using unbiased and quantitative massively parallel sequencing technologies. Following overnight culture in 20 mM pyruvate / zero glucose and 16 h treatment with 1, 5.5, 11 or 25 mM glucose (n=6 per treatment), 56 miRs (of a total of 264 detected) were altered by at least 25% (FDR < 0.05) in at least one pairwise comparison of glucose concentrations. 745 RISC-associated mRNAs (of a total of ~12,000) were altered by at least 25%, with either no change or a directionally opposite change in the translationally competent mRNA compartment, and contained binding sites for at least one miR exhibiting a similar glucose-responsive abundance profi le. These mRNAs represent the most highly glucose-sensitive, miR-regulated species in INS-1 cells. Those suppressed (increased RISC abundance) in response to glucose included cyclins and activators of mitochondrial OXPHOS, while upregulated mRNAs included activators of fatty acid beta-oxidation (which decreases GSIS) and mediators of Ca2+ infl ux, intracellular Ca2+ release and ER stress. Specifi c matching of regulated miRs to regulated mRNAs using an in-house algorithm was confi rmed using miR mimics and anti-miRs with luciferase reporter assays and immunoblotting. The combined experimental and informatic approaches presented here demonstrate that multiple miRs control beta-cell GSIS, can be applied to intact islets and other tissues, and may fi nd broad utility as a method of defi ning biologically relevant miR contributions to cellular responses.

& 2266-PGABA Promotes β-Cell Proliferation and Survival via Mechanism Involving Activation of Akt, ERK and CREBDONG OK SON, INDRI PURWANA, XINYUN LIANG, AKSHAYA TADKASE, MAG-GIE ZHOU, SUZANNE WANG, XIAOMING LI, QINGHUA WANG, Toronto, ON, Canada

Gamma-aminobutyric acid (GABA), an important amino acid, is produced by pancreatic β-cells and plays a role in regulating insulin and glucagon

A581


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


islet results in impaired glucose tolerance due to reduced insulin secretion. Cellular analyses of islets lacking either Clock or Bmal1 reveal refractory responses to both cyclase activators and potassium channel agonists, indicating defective β-cell function at the very latest stage of stimulus-secretion coupling. Surprisingly, in isolated wild-type islets, insulin secretagogues exhibit strong circadian variation. Disruption of either Clock or Bmal1, the two components of the forward limb of the clock, resulted in similar metabolic phenotypes, consistent with overlapping molecular targets. Moreover, conditional ablation of the pancreatic clock in mid-adult life was suffi cient to induce impaired glucose-stimulated insulin secretion. These results demonstrate a primary role for the islet clock in mammalian glucose homeostasis and reveal intrinsic rhythmic properties in β-cell function.

Supported by: JDRF (1-2008-114); NIH (T32DK007169), (T32HL07909), (R01DK090625-01A1)

2271-PActivation of Nrf2 by Phenolic Antioxidant and Triterpenoid Sup-presses the Basal and High Glucose Induced Expression of Thiore-doxin Interacting ProteinQIANG MA, XIAOQING HE, Morgantown, WV

Thioredoxin interacting protein (TXNIP) is a multifunctional protein induced by high glucose and suppressed by insulin. Persistent elevation of TXNIP has been implicated in the development of beta cell death and insulin resistance in diabetes. The molecular mechanism by which TXNIP expression is regulated in diabetes remains largely unclear. We have previously identifi ed TXNIP as a target gene of nuclear factor erythroid 2-related factor 2 (Nrf2), a CNC-bZip transcription factor important for cellular resistance to oxidants. In the current study, we show that Nrf2 activators, such as the phenolic antioxidant tert-butyl hydroquinone (tBHQ) and the triterpenoid bardoxolone methyl (CDDO-Me), inhibited the basal and high glucose induced expression of TXNIP by activating the Nrf2 pathway. tBHQ and CDDO-Me inhibited TXNIP mRNA and protein expression in human INS-1 beta cells and mouse hepatoma cells in concentration- and time-dependent manners. Inhibition was transcriptional because inhibition by Nrf2 was observed for the expression of a heterologous reporter gene under the control of TXNIP gene promoter. Inhibition requires the presence of a functional Nrf2. An antioxidant response element (ARE) sequence located in the upstream region of the TXNIP gene was identifi ed as the cis-acting element for inhibition. Inhibition correlated with recruitment of Nrf2 to the ARE sequence and suppression of transcription of the gene through positive regulators such as MondoA. The study established Nrf2 as a critical, negative regulator of TXNIP gene expression in both the absence and presence of high glucose. The fi ndings suggest that small molecule Nrf2 activators may be utilized to suppress TXNIP expression in diabetes.

Supported by: NIOSH

2272-PEffects of Chronic Islet Neuropeptide Y Expression on Glucose HomeostasisYUI MACHIDA, DANIEL HALLINGER, CHRISTINE BRUINSMA, CIRIACO VILLA-FLOR, EDEN GARCIA, REXFORD S. AHIMA, YUMI IMAI, Norfolk, VA, Philadelphia, PA

Neuropeptide Y (NPY) is expressed in the hypothalamus and plays a critical role in the regulation of feeding and energy homeostasis. Interestingly, NPY is also expressed in pancreatic islets, and its expression in islets is reduced in obese mice. We previously reported that islet mass and insulin secretion were increased in NPY knockout mice, suggesting that NPY negatively regulates islet mass and function. To test the functional signifi cance of NPY reduction in islets in obesity, we created transgenic mice expressing NPY under rat insulin promoter (Tg mice) to address the consequence of chronic islet NPY expression that prevents downregulation seen in wild type (WT) mice. We established two lines of Tg mice that express NPY in islets at 2.5 (Tg1) and 28 (Tg2) the levels in WT (p<0.05). There was no difference in NPY levels in the brains of Tg1 or Tg2 mice compared with WT. The secretion of NPY in the conditioned medium was 1.6 fold higher from Tg1 islets, and 2.3 fold higher from Tg2 islets (p<0.05). There were no signifi cant changes in body weight, glucose tolerance or whole body insulin sensitivity when Tg1 and Tg2 mice were maintained on normal chow diet. Islet architecture and glucose-stimulated insulin secretion (GSIS) from isolated islets were unaltered in Tg mice on normal chow diet. When Tg2 mice were fed a high-fat diet (HF), there was no difference in body weight or insulin sensitivity compared with WT. However, glucose tolerance was signifi cantly enhanced in Tg2 mice compared with WT on HF (2 way ANOVA p<0.05, time 0 to 30

upstream signals initiate the glucose-specifi c activation of Cdc42 in β-cells remain largely unknown. The Src Family Kinases (SFKs) have previously been implicated in Cdc42 activation and actin remodeling in other cell types, as well as in the phosphorylation of Caveolin-1 and βPix, the crucial Cdc42 GDI and GEF proteins mediating second-phase insulin secretion. Accordingly, we hypothesized that one or more SFKs regulate the activation of Cdc42 in islet β-cells to trigger the downstream events of second-phase insulin secretion. To test this hypothesis, we pharmacologically blocked SFK activation in MIN6 β cells, which signifi cantly reduced glucose-induced Cdc42 activation, PAK1 phosphorylation, and late-phase insulin secretion. In both human islets and MIN6 cells, phosphorylation of the SFKs was induced by the protein tyrosine phosphatase inhibitor pervanadate (pV), was glucose-specifi c, and occurred within 1 min of glucose stimulation, thus temporally placing SFK phosphorylation upstream of Cdc42 activation. Immunodetection revealed YES, one of the nine known SFKs, as the chief family member involved in these glucose-specifi c signaling events. Taken together, these data establish YES as the most proximal glucose-specifi c signal leading to the activation of the Cdc42-signaling cascade, ultimately triggering actin remodeling, granule mobilization and second-phase insulin secretion.

Supported by: NIH (DK076614), (DK067912 to D.C.T.), (4686676 to S.M.Y.)

2269-PInsulin, cAMP, and Phorbol Ester-Activated Cell Signaling Path-ways Phosphorylate Tomosyn-2 to Regulate Insulin Secretion in Pancreatic β-CellsSUSHANT BHATNAGAR, LINDSAY SCHNEIDER, ALEXANDER S. HEBERT, JOSH-UA COON, ALAN ATTIE, Madison, WI

We previously identifi ed tomosyn-2 as a gene underlying a quantitative trait locus controlling glucose and insulin levels in mice. We showed that tomosyn-2 is an inhibitor of insulin secretion.Furthermore, we identifi ed a SNP (Ser912→Leu) in the protein-coding region of tomosyn-2 that affects its proteasomal degradation.Tomosyn-2 is relatively uncharacterized protein and contains a syntaxin-binding domain; syntaxin is a member of the SNARE complex that regulates insulin secretion. To understand the mechanism that reverses the inhibitory effect of tomosyn-2 on insulin secretion, we investigated the effect of insulin secretagogues on the phosphorylation of tomosyn-2. Using quantitative phosphoproteomics, we identifi ed novel insulin, glucose, cAMP, and phorbol ester (PMA) responsive phosphorylation sites in the regulatory domain of tomosyn-2 protein. Mutagenesis experiments are being performed to establish the role of these phosphorylation sites in regulating the binding activity of tomosyn-2 to the proteins of the SNARE complex. Preliminary results show that by treating pancreatic β-cell line, INS1 (832/13) with the cocktail containing insulin, 8-Br-cAMP, and PMA resulted in increased degradation of tomosyn-2 protein compared to the mock treated cells. Furthermore, by using mass spectrometry and molecular biology approaches we have identifi ed an E3 ligase, Hrd1, which binds and regulates the abundance of tomosyn-2. These results led us to hypothesize that phosphorylation of tomosyn-2 regulates short and long-term insulin secretion by modulatingits binding activity and protein abundance, respectively. Herein, we propose that alterations in tomosyn-2 phosphorylation will lead to inappropriate insulin secretion from the β-cells. This will result in increased susceptibility towards type 2 diabetes and can also lead to potentially life threatening hypoglycemia during fasting.

2270-PInterplay between Genotype, Temporal Phase, and Age in Circadi-an-Induced Diabetes Mellitus and Beta-Cell FailureMARK PERELIS, BILIANA MARCHEVA, YUMIKO KOBAYASHI, CHIAKI OMURA, LOUIS PHILIPSON, JOSEPH T. BASS, Chicago, IL

The molecular clock is composed of an autoregulatory transcription-translation feedback loop that maintains alignment between internal biochemical processes and the sleep-wake/fasting-feeding cycle. Circadian oscillators are deployed to maintain metabolic constancy throughout the entire 24-hr solar cycle, but how local tissue clocks exert these effects has been unclear. We have shown that cell-autonomous expression of Bmal1, a bHLH-PAS domain transcription factor, within endocrine pancreas is essential for glucose-stimulated insulin secretion (Marcheva, et al 2010). Yet, whether clock function in early development or later life is important in this process has not been completely elucidated. Here we show that targeted ablation of either Clock or Bmal1 selectively within the pancreatic

A582


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2275-PSuperiority of Alogliptin versus Pioglitazone for Activation of Islet PPAR-Gamma Signaling and Blood Glucose Control in Partial Pan-createctomy Diabetic RatsDHANANJAY GUPTA, BASANTHI SATISH, MINA PESHAVARIA, THOMAS JET-TON, JOHN L. LEAHY, Colchester, VT

We have proposed PPARγ is a key regulatory molecule in islet β-cells. Upstream is FoxO1, and its activation by many factors including incretin hormones GLP-1 and GIP increases PPARγ expression. Downstream are key target genes including Pdx-1 and GIP receptor (GIPR). Also we have shown defective regulation of this system in diabetic rodents. This study tested clinical agents that act in this pathway on glycemia and PPARγ target gene expression in partial pancreatectomy (Px) rats. 5-wk old Sprague Dawley rats underwent 60% Px, 90% Px, or sham-Px surgery. From day 3 to 20, they received chow containing vehicle, DPP-4 inhibitor alogliptin (ALO, 20 mg/kg daily), thiazolidinedione pioglitazone (PIO, 2.5 mg/kg daily), or both. OGTT was performed on day 20 and the next day islets isolated for mRNA and immunoblots. In shams, both ALO and PIO caused 35%-60% increases in islet Pdx-1 and GIPR expression. ALO but not PIO lowered maximal glucose during the OGTT (140±10 mg/dL sham-vehicle to 113±6 mg/dL sham-ALO), with no additional lowering when used together. Normoglycemia is preserved after a 60% Px with modest increased expression of PPARγ and its target genes-none of the drug therapy impacted this response. The 90% Px caused modest hyperglycemia and marked reductions in islet expression of PPARγ (48±3%), Pdx-1 (42±4%) and GIPR (68±4%) of shams. PIO had little to no impact on islet PPARγ, Pdx-1 and GIPR expression, and no improvement in blood glucose. In contrast, ALO normalized the PPARγ mRNA level, with Pdx-1 by immunoblot reaching 78±11% and GIPR 109±3% of vehicle fed shams. The OGTT maximal glucose value fell from 220±10 mg/dL in 90% Px-vehicle to 157±7 mg/dL in 90% Px-ALO. There was no additional effect when ALO and PIO were used together. In summary, the islet PPARγ activating effect of PIO is lost in diabetic rodents because of the depressed PPARγ expression. In contrast, ALO works upstream to restore PPARγ signaling in tandem with improved glycemia.

Supported by: Takeda Pharmaceuticals

2276-PTAK-875 Is a Partial Agonist of the Free Fatty Acid Receptor GPR40ARTURO MANCINI, ERIC CARPENTIER, MICHEL BOUVIER, VINCENT POITOUT, Montreal, QC, Canada

GPR40 is a β-cell 7 transmembrane receptor (7TMR) for medium-to-long chain fatty acids (FAs) that mediates FA potentiation of glucose-induced insulin secretion. Several GPR40 agonists are progressing through clinical development as type 2 diabetes therapies. The most clinically advanced agonist, TAK875, showed promising results in recent phase 2 trials. Yet, our knowledge of the mechanisms linking GPR40 activation to insulin secretion remains incomplete. The aim of this study was to examine how FA affect the signaling properties and pharmacological effi ciency of TAK875. HEK293 cells were co-transfected with GPR40 and the bioluminescence resonance energy transfer biosensors obelin (Ca2+ biosensor) or EPAC (cAMP biosensor). The signals generated by the biosensors were measured by spectrophotometry before and immediately following stimulation of cells with different GPR40 ligands [palmitate , oleate, linolenate or TAK875]. Obelin data obtained in HEK293 were confi rmed in the insulin secreting cell line MIN6. Our results show that FAs rapidly (~2.5-4 s) and potently (~20-30 fold) induced Ca2+ infl ux in a GPR40-dependent manner. Different FAs were equipotent in this regard. The Ca2+ response provoked by TAK875 was partial relative to palmitate (~51%) and oleate (58%). Further, TAK875 antagonized Ca2+ infl ux induced by saturating levels of palmitate while potentiating that induced by subsaturating palmitate concentrations. In conclusion, we show that TAK875 behaves as a partial GPR40 agonist (i.e., a mixed agonist/antagonist). These fi ndings may have important implications for the use of TAK-875 as an insulin secretagogue in type 2 diabetes.

Supported by: CDA

2277-PEffect of GLP-1 Analogue, Exendin-4 on Inhibition of Islet FibrosisJI-WON KIM, SEUNG-HYUN KO, SHIN-YOUNG PARK, YU-BAE AHN, KUN-HO YOON, Seoul, Republic of Korea

Objective: Pancreatic islet fi brosis might be to lead the beta-cell loss and dysfunction in type 2 diabetes. Pancreatic stellate cell (PSC) are known to be related to pancreatic fi brosis and infl ammation, and are the result of extracellular matrix protein synthesis. Therefore, we investigated the effect of GLP-1 analogues on activated PSCs in vitro, and pancreatic islets fi brosis in vivo.

after 1 mg/g intraperitoneal glucose challenge). These results demonstrate that a chronic increase in NPY expression in pancreatic islets is able to maintain glucose homeostasis in diet-induced obesity.

Supported by: NIH (K08-DK071536), (R03-DK071536 to Y.I.), (P01-DK049210 to R.S.A.)

2273-PTranscription Factor Ets1 Inhibits Insulin Secretion in Mouse Beta-Cells and Is Induced in the Islet of High-Fat Feeding MiceYAN LUO, LUO HAI, LI HU, FENGLI HE, YABIN LI, ZHIGUANG ZHOU, FENG LIU, YAN-SHAN DAI, Changsha, China

Activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucolipotoxicity and inhibit insulin gene expression in beta cells. However, the downstream effector of ERK1/2 pathway mediating this effect is little known. Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38. Thioredoxin-interacting protein (TXNIP) is a key molecule linking ER stress and infl ammation in beta cells. This study was aimed to understanding downstream effectors of ERK pathway that mediates glucolipotoxicity in islet beta cells. We found that Ets1 protein was localized in insulin-staining positive cells but not in glucagon-staining positive cells by using immunocytochemistry and confocal microscopy, indicating that Ets-1 was enriched in the beta cell in the mouse pancreas. Immuonocytochemistry with an Ets1 Thr38 phospho- specifi c antibody showed that active, phosphorylated Ets1 was induced and enriched in the nucleus of the islet cells from high-fat diet (HFD) fed mice, compared to that from control mice. Ets1 was induced in Min6 insulinoma cells in response to palmitate stimulation. P38 kinase is known to inhibit ERK1/2 activity. Inhibition of P38 kinase by treating Min6 cells with SB203580 increased ERK1/2 phosphorylation and induced Ets1 mRNA expression. Adenovirus-mediated expression of Ets1 in cultured mouse islets inhibited glucose-stimulated insulin secretion. TXNIP was identifi ed by cDNA microarray to be induced by Ets1 in Min6 cells. Overexpression of Ets1 resulted in increased TXNIP protein in cultured mouse islets; conversely, knockdown of Ets1 by siRNA led to reduced TXNIP protein in Min6 cells. Chromatin IP showed that Ets1 was associated with txnip promoter region containing Ets1 binding site in Min6 cells. Collectively, our data indicate that Ets1 was activated by ERK in islet of HFD fed mice and suggest that Ets1 mediates glucolipotoxicity by inhibition of insulin secretion via activating TXNIP expression in beta cells.

Supported by: NSFC (81270905)

2274-PSPARC Enhances Muscarinic Receptor-Stimulated Insulin Secre-tion in Mouse Islet Beta Cells through Down-Regulation of RGS-4FENGLI HE, LI HU, LUO HAI, YABIN LI, YAN LUO, ZHIGUANG ZHOU, FENG LIU, YAN-SHAN DAI, Changsha, China

Secreted protein acidic and cysteine-rich (SPARC, or Osteonectin) is a secreted extracellular matrix protein that is involved in the development, remodeling and tissue repair by modulating cell-matrix interactions. It has been reported that blood SPARC level is increased in early type 2 diabetic patients and excercise induces skeletal muscle to release SPARC. The role of SPARC in beta-cells is unknown. Hence, we investigated the regulation of SPARC in Min6 insulinoma cells and the function of SPARC in regulation of insulin secretion in mouse islet cells. SPARC protein was detected in beta cells in mouse pancreas by immunocytochemistry. SPARC protein was induced dose-dependently by high concentration of glucose and down-regulated by insulin in Min6 cells. SPARC overexpression, or purifi ed recombinant SPARC protein enhanced muscarinic agonist, oxotremorine-M, stimulated insulin secretion in islet cells. To study the mechanism of such effect, we conducted cDNA microarray in Min6 cells overexpressing SPARC by adenovirus and found that overexpression of SPARC in Min6 cells down-regulated the expression of regulator of G-protein signaling-4 (RGS-4), a negative regulator of insulin secretion that inhibits muscarinic M3 receptor/G(q) protein coupling in beta cells. Western blot showed that SPARC overexpression in Min6 cells down-regulated RGS-4 protein levels, whereas knockdown of SPARC by siRNA up-regulated RGS-4 expression. Acetylcholine is released from pancreatic nerve endings and stimulates insulin secretion through M3 receptor in beta cells during food ingestion. Our data suggest that increased SPARC expression by high concentration of glucose would enhance beta cell M3 receptor signaling to stimulate insulin secretion. Collectively, these fi ndings indicate that SPARC is regulated by glucose and insulin in beta cells and that SPARC plays an important modulatory role in regulating insulin secretion via downregulation of RGS-4 expression.

A583


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


activation in response to nitric oxide is delayed. There is a 3-fold increase in DNA damage as early as 15 minutes after DEA/NO treatment, yet γH2AX formation is fi rst observed 120 minutes after treatment. In contrast, ATM activation in response to hydrogen peroxide is rapid and correlates with the induction of DNA damage (15 minutes). Our data suggests that while ATM is required in β-cells for accurate DNA repair, it may also act in a signaling capacity to modulate additional cell fate decisions.


2280-PMolecular Characterization of Rough Endoplasmic Reticulum Sub-proteome in Pancreatic Beta-CellsXUEQUN CHEN, JIN-SOOK LEE, PATRACIA SKALLOS, JINGYE FANG, Detroit, MI

Pancreatic beta cell has well developed endoplasmic reticulum (ER) for massive production and processing of proinsulin. Perturbation of ER homeostasis contributes to both type 1 and type 2 diabetes. Our goal is to systematically characterize the molecular machinery responsible for proinsulin biogenesis and maintenance of ER homeostasis. MIN6 cell was used to purify rough ER. Two different purifi cation schemes, differential ultracentrifugation and step sucrose gradient, were compared. The ER proteins were separated on 1D SDS-PAGE. Thirty fi ve gel slices were excised from each lane followed by in-gel digestion and LC-MS/MS analyses. A total of 1696 proteins were identifi ed in three experiments, among which 1279 proteins were found in at least two separate experiments. This represents a comprehensive coverage of all known and many novel players in beta cell ER. The main functional categories included protein translation (25% of total), protein folding (12%), ion homeostasis (8%); ER export (24%) and protein degradation (8%). Representative proteins are being confi rmed by immunostaining including ERO1, EDEM3, ARMET, CDKAL1 and TANGO1. To identify beta cell specifi c ER proteins, tissue specifi c gene expression and Blast analysis is being performed between our data and published ER proteomic data. Furthermore, novel targeted proteomics approach using multiple reaction monitoring technology has been developed to quantify the copy numbers per cell of unique ER proteins in both MIN6 and mouse islets. This study represents the fi rst comprehensive characterization of the molecular machinery for proinsulin biogenesis in the beta cell ER. A quantitative molecular model is being developed and will serve as a foundation to characterize the alteration of ER homeostasis under diabetes causing conditions.

2281-PPalmitate Acutely Activates mTORC1 and Promotes mRNA Transla-tion, Whereas Chronic High Fat Feeding Results in Global Transla-tional Blockade in Islet β CellsMASAYUKI HATANAKA, BERNHARD MAIER, EMILY K. SIMS, CARMELLA EV-ANS-MOLINA, RAGHAVENDRA G. MIRMIRA, Indianapolis, IN

Lipotoxicity from high fat diets is believed to trigger oxidative and endoplasmic reticulum (ER) stress in the islet β cell, resulting in β cell dysfunction and eventual death. Because oxidative and ER stress are linked to alteration in mRNA translation, we sought to elucidate the effect of lipotoxicity on β cell mRNA translation by use of the technique of polyribosomal profi le (PRP) analysis. PRP analysis involves the separation of total mRNA species by their association with actively translating polyribosomes or inactively translating monoribosomes. PRP analysis was performed on MIN6 β cells and islets exposed to 0.5 mM palmitate and on islets from high fat diet-treated mice. Upon exposure of MIN6 cells to 0.5 mM palmitate for 60 min, there was an increase in polyribosome-associated RNA and the transcripts encoding Pdx1 and Insulin showed a shift toward the polyribosomes, consistent with an increased translation of these species. In accordance with these fi ndings, palmitate acutely induced the phosphorylation of 4E-BP1 and S6K, targets of mTORC1. Treatment with palmitate for longer periods caused eIF2a phosphorylation and a shift of RNA species towards monoribosomes, consistent with a block in mRNA translation. Similar fi ndings were observed in palmitate-treated mouse islets. Next, we studied islets from C57BL/6J mice placed on a 42% kcal fat diet for 16 weeks. Islets from high fat-fed mice showed evidence of general translational inhibition by PRP analysis, with partitioning of many transcripts (including Ins1/2, Pdx1, Glut2, Atf4, Chop) to monoribosome fractions—a picture consistent with general, non-ER stress-mediated suppression of translation. Taken together, our data suggest that lipotoxicity acutely promotes mRNA translation in β cells, during intermediate periods of time leads to ER stress-mediated suppression of translation, and during chronic periods leads to generalized suppression of total translation.

Supported by: Manpei Suzuki Diabetes Foundation; NIH (R01DK060581), (R01DK083583)

Methods: PSCs cultured in 5.5 mM, 27.7 mM glucose and 27.7 mM with 10 nM Ex-4 for 3 and 9 days. Western blot analyses for the expressions of α-SMA and ECM proteins, such as CTGF, α-collagen I and fi bronectin, were performed. Level of Transforming growth factor-β in culture media are measured. For in vivo study, saline (control), Ex-4 (2.5 nM/kg/twice a day) and Insulin were injected into OLETF rats for 12 weeks. We analyzed pancreatic islet morphology, quantifi ed beta-cells mass and the severity of islet fi brosis.

Results: GLP-1 receptor expression was strongly induced with high glucose stimulation in PSCs. The protein level of a-SMA was increase by high glucose however attenuated by Ex-4 treatment for 3 days. After 9 days culture of PSCs in high glucose, the expressions of CTGF and α-collagen I synthesis were increased and also attenuated by Ex-4 treatment. Islet fi brosis in OLETF rats was signifi cantly attenuated by Ex-4 treatment than control group of glucose matched insulin treated group.

Conclusion: The data suggesting the effects of Ex-4 on high glucose induced PSCs activation and the prevention of islet destruction by fi brosis in the animal model of type 2 diabetes.

2278-PPalmitate Induces ROS Production and β-Cell Dysfunction by Acti-vating NADPH Oxidase via Src SignalingYUICHI SATO, SHIMPEI FUJIMOTO, ERI MUKAI, HIROKI SATO, YUMIKO TAHARA, KASANE OGURA, GEN YAMANO, MASAHITO OGURA, KAZUAKI NAGASHIMA, YUTAKA SEINO, NOBUYA INAGAKI, Kyoto, Japan, Nankoku, Japan, Osaka, Japan

Chronic hyperlipidemia impairs pancreatic β-cell function, referred to as lipotoxicity. In β-cells, reactive oxygen species (ROS) is an important factor that impairs glucose-induced insulin secretion (GIIS). We have reported an important role of endogenous ROS overproduction by activation of Src, a non-receptor tyrosine kinase, in impaired GIIS from diabetic rat islets. In this study, we investigated the role of ROS production by Src signaling in palmitate-induced dysfunction of β-cells. Exposure to palmitate for 24 h dose-dependently impaired GIIS and increased ROS production in rat insulinoma INS-1D cells. Exposure to 10 µM PP2, a Src inhibitior, for 30 or 60 min recovered impaired GIIS and decreased ROS overproduction due to 0.6 mM palmitate exposure for 24 h (palmitate exposure). Palmitate exposure caused Src activation detected by Tyr418 phosphorylation and increased activity of NADPH oxidase (NOX) and protein levels of NOX2, a pathological ROS source in β-cells. Exposure to PP2 for 30 min decreased augmented NOX activity but did not affect increased protein level of NOX2 by palmitate exposure. Palmitate exposure increased protein level of p47phox, a regulatory protein of NOX2, in membrane fraction compared to control, which is signifi cantly reduced by acute PP2 exposure (~30% reduction). Transfection of siRNA of p47phox suppressed augmented p47phox protein level, decreased augmented ROS production, and increased impaired G IS by palmitate exposure. In addition, acute exposure to PP2 ameliorated impaired G IS and decreased ROS production in isolated islets of KK-Ay mice, an obese diabetic model with hyperlipidemia. In conclusion, activation of NOX via Src signaling plays an important role in ROS overproduction and impaired G IS caused by chronic exposure to palmitate, suggesting a lipotoxic mechanism of β-cell dysfunction of obese mice.

2279-PThe Role of ATM in the β-Cell Response to Nitric OxideBRYNDON J. OLESON, JOHN A. CORBETT, Milwaukee, WI

Type-1 diabetes mellitus is an autoimmune disease characterized by selective destruction of the pancreatic β-cells. During islet infl ammation there is a release of pro-infl ammatory cytokines such as interleukin-1 (IL-1) and interferon-γ (IFNγ) that are believed to participate in the loss of functional β-cell mass. While nitric oxide (NO) mediates the destructive effects of cytokines, including DNA damage and ultimate β-cell death, it also activates protective pathways that promote β-cell recovery. The mechanisms regulating this recovery pathway have yet to be fully determined. Ataxia telangiectasia mutated (ATM) is a key component in the maintenance of genomic integrity, coordinating the DNA damage response and cell fate decisions following genotoxic stress. One target of ATM is histone H2AX, and phosphorylated H2AX (γH2AX) is one of the earliest and most sensitive markers of DNA double-strand breaks. In this study, we investigated the role of ATM in the repair of β-cell DNA following nitric oxide-induced damage. Exposure of INS 832/13 insulinoma cells and rat islets to cytokines IL-1 and IFNγ for 12 and 24 hours stimulates formation of γH2AX in both an ATM- and NO-dependent manner. γH2AX formation is also induced by treatment with the NO-donor DEA/NO. While the ATM inhibitor KU-55933 attenuates the repair of damaged DNA in β-cells, ATM

A584


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


and proliferation. Insulin secretion capability of Min6 was assessed in lipotoxic condition. The expression of effectors involved in β-cell function in addition to PI3K/Akt/FoxO1 was detected by real-time PCR and western blotting. The results revealed that stimulating Min6 cells with liraglutide caused β-cell proliferation to be substantially enhanced. On the contrary, palmitate signifi cantly increased lipoapoptosis levels in Min6 cells, which was inhibited by the addition of liraglutide. Moreover, liraglutide could effectively nullify the impairment of insulin secretion induced by palmitate. Furthermore, LY294002, a PI3K inhibitor, could signifi cantly abrogate liraglutide induced effects. Western blot revealed that the phosphorylation of Akt and FoxO1 was markedly decreased when treated with palmitate but was elevated following exposure to liraglutide. Moreover, the mRNA levels of Bax and p27 declined upon treatment with liraglutide but increased when incubated with palmitate. In addition, the inhibition of Pdx-1, an important effector for β-cell function, by palmitate was canceled with the addition of liraglutide. Therefore, we concluded that liraglutide markedly improved β-cell function under lipid stress and that the protective action of liraglutide was mediated by activation of PI3K/Akt, which resulted in inactivation of FoxO1 along with the down-regulation of Bax and p27 and up-regulation of Pdx-1 expression.

Supported by: NSFC (81100581 to S.S.)

2285-PFunctional Compartmentalization of the cAMP-Signal Pathways in Pancreatic β−CellsRAWAN DAMOUNI, LIAT MUKTADER, JULIA PISKAROV, DANIELLE MELLOUL, RAFAEL NESHER, Jerusalem, Israel

cAMP pathways are crucial for the maintenance of pancreatic β−cell phenotype, mediating metabolic, hormonal, neural and ionic stimuli. They affect variety of genes, ion balance, enzymes as well as exocytotic processes. This multifaceted control of unrelated processes must be accomplished through functional compartmentalization. We followed the expression of nine unrelated genes after RNAi-mediated specifi c knockdown of six cAMP-dependent PDE isoenzymes or the catalytic subunits of PKA in INS-1E cells, stimulated with glucose, Liraglutide (Lrgt) or with cytokines. Cells treated with glucose, Lrgt, cytokines and the combination of two stimuli displayed different response to downregulation of specifi c PDE isoforms or to one of the catalytic subunits of PKA. Thus, cAMP originating from one or more adenylate cyclase isoenzymes may activate specifi c cAMP-dependent kinase, resulting in the phosphorylation of target cytosolic or nuclear effectors. Compartmented cAMP may be hydrolyzed by specifi c isoform of PDE. PDEs, PKAs, Ecaps and phosphatases may be spatially anchored to an AKAP in close proximity to the affector protein. The relevance for novel approaches to treat diabetes mellitus is discussed.

Supported by: Israel Academy of Sciences and Humanities

2286-PNeuronostatin Exerts Direct Effects on Pancreatic Alpha CellsMOLLISA ELRICK, GINA YOSTEN, WILLIS SAMSON, St. Louis, MO

Diabetes mellitus Type 2 (T2DM) is a life threatening disease that affects over 340 million people worldwide (World Health Organization, 2012). Left untreated, T2DM has long term, devastating consequences including cardiovascular disease and renal failure. Current therapies slow disease

2282-PInfl ammatory Cytokines Induce Expression and Activity of the Ubiquitin-Proteasome System (UPS) in INS-1E Cells and Human IsletsMARCO BUGLIANI, MORTEN LUNDH, FAROOQ SYED, DANNY H.C. CHOU, MARA SULEIMAN, BRIDGET K. WAGNER, THOMAS MANDRUP-POULSEN, PIERO MAR-CHETTI, Pisa, Italy, Copenhagen, Denmark, Boston, MA, Cambridge, MA

The UPS is the main cellular protein-degradation pathway and has been proposed to regulate beta cell function and survival.

Here we studied the expression and activity of the UPS in INS-1E cells and human islets isolated from 9 multi-organ donors, aged 67±14 years, with BMI of 25.6±1.9 kg/m2, exposed for up to 24h to different combinations and concentrations of the proinfl ammatory cytokines IL-1beta, IFN-gamma and TNF-alpha, which was then correlated with beta cell survival.

In both INS-1E cells and human islets, IL-1β+IFN-γ or IL-1β+IFN-γ+TNF-α, dose-dependently increased chymotrypsin-, trypsin- and caspase-like proteasome activities by 20-40% (p<0.05 vs control cells) after 1 h, plateauing by 12-24h. The increase in UPS activity correlated with early and persistently increased gene expression of the proteasomal subunits PSMA2, PSMA7, PSME1, PSMB8, PSMB9 and PSMB10, as suggested by microarray analysis and confi rmed by qPCR. Putative inhibition of deubiquitination or block of lysine deacetylation by the small-molecule inhibitors BRD0476 and MS275, respectively, partially prevented cytokine-induced beta cell death and downregulated PSMA7, PSME1 and/or PSMB10, without affecting cytokine-induced proteasomal activity. In conclusion, proinfl ammatory cytokines induced UPS expression and activity, which was related with beta cell apoptosis.

Supported by: NAIMIT, Contract n° Health (F2-2009-241447)

2283-PPharmacological Activation of α7-Nicotinic Acetylcholine Recep-tor (α7AChR) Attenuates Cytokine-Mediated Islet Infl ammationDHANANJAY GUPTA, BASANTHI SATISH, JACK L. LEAHY, THOMAS JETTON, Colchester, VT

To preserve islet survival, it is critical to identify counter-infl ammatory pathways that curtail cytokine responsiveness. α7AChR is a ligand-gated ion channel receptor that is an integral part of the vagus nerve-mediated “cholinergic anti-infl ammatory” signaling response. We aimed to determine if α7AChR signaling is active in islet β-cells and if its molecular effectors attenuate islet infl ammation in vitro and in the MLDS model. We confi rmed that α7AChR is expressed in β-cells by WB and qPCR. PNU (282987), a high affi nity agonist of α7AChR, induced sustained phosphorylation of STAT3 (T-705) in INS-1 β-cells and isolated islets. We evaluated the impact of α7AChR activation on islet infl ammation by pretreatment of islets +/- PNU (1h, 100µM) by 5 h treatment with cytokine cocktail (IL-1B, TNF-α, IFNγ). Whereas in the cytokine alone treated islets, the anti-infl ammatory pSTAT3 signal upstream of NFκB was reduced to 21% of control (no cytokine) islets, PNU treatment restored pSTAT3 levels to 91% of the control. The pro-infl ammatory marker, phosphorylated NFκB, was reduced in PNU-treated islets (1.5-fold vs. 2.4-fold in cytokine only). Pro-infl ammatory iNOS protein was induced 9.6+1.7-fold over control in cytokine treated islets alone, and upon PNU pretreatment, the iNOS induction signal was reduced to 3.7+0.5-fold. To evaluate in vivo impact of α7AChR activation on glucose homeostasis, MLDS (50 mg/kg, 5 d) B6 mice were treated with or without SQ injections of PNU (10 mg/kg, 3 w). This resulted in markedly improved fed blood glucose (176 mg/dL vs 371mg/dL), signifi cantly improved glucose tolerance (p<0.05) compared to vehicle treated MLDS mice. In summary, we identifi ed a novel mechanism to counter islet infl ammation in β-cells based on α7AChR mediated activation of pSTAT3 and reduced activation of NFκB and iNOS. As such, we propose in vivo activation of α7AChR signaling holds potential to preserve islet function in T1DM.

2284-PThe PI3K/Akt/FoxO1 Pathway Mediates the Protective Action of Liraglutidein β-Cells Under Lipotoxic StressSHIYING SHAO, Wuhan, China

Liraglutide, a modifi ed form of glucagon-like peptide-1, has been found to improve β-cell function. However, the effect of liraglutide on β cells under lipotoxic stress and the underlying molecular mechanisms remain unclear. In the present study, we investigated the role of PI3K/Akt/FoxO1 signaling in liraglutide involved β-cell protection. Min6 mouse insulinoma cells were exposed to fatty acid palmitate with/without liraglutide. Flow cytometric analysis and MTT assay were performed to evaluate β-cell apoptosis

A585


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


NF-κB, p38 MAPK, p53 & STAT3) in human islet progenitor cells (HIPCs) and the rodent β-cell line INS-1. Viral RNA was determined by quantitative RT-PCR and phosphoprotein levels of apoptotic factors by Bioplex in the infected cells at 4 hrs, 12 hrs, day 1, 2, 3, 4 and 5 post inoculation.

Both CVB4 and CVB5 replicated in INS-1 at a multiplicity of infection (MOI) of 1 and 10 cells while only CVB5 replicated in HIPC (MOI 10). CVB5 infection of HIPCs induced JNK, p38 MAPK, ERK1/2 and c-Jun on day 1 post-infection (p<0.05). These factors were also induced in INS-1 cells infected with CVB4 and CVB5 on day 1-2 except for ERK1/2 which increased 4 hours post-infection (p<0.05). NF-κB and STAT3 phosphoprotein increased in INS-1 cells infected with CVB4 and CVB5 on day 1-3 post-infection (p<0.05). However there was no increase in these two phosphoproteins in the HIPC infected with CVB5. Akt which is an anti-apoptotic factor decreased in INS-1 but increased slightly in the HIPC. There were no changes in p53 phosphoprotein levels in INS-1 but it decreased in the HIPC.

In summary, we have shown for the fi rst time that CVB4 and CVB5 induce multiple apoptotic pathways in HIPC and INS-1 cells.

Supported by: NHMRC (Australia)

2289-PModulating Estrogen Receptor Activity and SERM Action in Pan-creatic β-Cells With Nuclear Coregulator ExpressionELLEN N. DOVER, WAYNE D. GRAHAM, PETER A. ANTINOZZI, Winston-Salem, NC

Modulation of estrogen receptor (ER) activity in pancreatic β-cells has the therapeutic potential to infl uence survival, proliferation, and differentiation pathways important in diabetes progression. Selective estrogen receptor modulators (SERMs) are a class of estrogenic compounds which have the unique property of tissue specifi city, where a given compound can act as an agonist in one cell type and antagonist in another. This cell-type specifi city is largely driven by the complement of ER isoforms and coregulator proteins present in the cell. To characterize SERM specifi city in pancreatic β-cells, we evaluated an estrogen responsive transcription assay with a combinatorial strategy. Assay parameters included a panel of SERMs tested against various combinations of transiently expressed ER isoforms and nuclear receptor coregulators. Transcriptional activity was assessed with an estrogen response element (ERE) driven luciferase assay in the INS1E β-cell line. Transient ERα and ERβ expression increased ERE-dependent expression 12.7 ± 2.6 fold and 2.7 ± 0.2 fold, respectively, over the vector control. To evaluate the impact of specifi c nuclear receptor coregulators on ER isoforms a co-expression strategy was utilized. In summary, nuclear receptor coactivator 5, NCOA5, preferentially enhanced ERα activity by 1.5 ± 0.2 fold with no observable effect on ERβ-mediated activity. Further analysis with pharmacological intervention demonstrated that NCOA5 potentiation of ERα activity was inhibited by both MPP (an ERα-selective antagonist) and Y134 (a raloxifene-derived SERM). These studies provide a strategy for the assessment of SERM and coregulator interactions and further emphasize the importance of tissue-specifi c expression of coregulators in modulating estrogen signaling in pancreatic beta cells.

Supported by: NIH/NIDDK (DK080151)

2290-PThe Role of MAPK Pathway in Palmitate-Induced Stimulation of Insulin Secretion and Apoptotic Beta-Cell DeathGABRIELE KAISER, CHARLOTT-AMÉLIE TEUTSCH, REBECCA DÖLKER, FELICIA GERST, SUSANNE ULLRICH, HANS-ULRICH HÄRING, Tübingen, Germany

Mitogen-activated protein kinases (MAPK) are involved in a variety of physiological processes, such as differentiation, proliferation and apoptosis. In insulin secreting cells the MEK1-ERK1/2 signaling pathway is activated by glucose and long chain free fatty acids (FFA) but its role is not well understood. FFA acutely stimulate glucose-induced insulin secretion (GIIS) whereas chronic exposure to high concentrations of unsaturated FFA and glucose causes apoptotic beta-cell death. The insulinotropic effect of FFA depends on the activation of the Gq-coupled free fatty acid receptor 1 (FFAR1/GPR40) which results in the stimulation of phospholipase C leading to IP3-dependent increase of cytosolic Ca2+ and diacylglycerol-induced protein kinase C (PKC) activation. On one hand, activation of PKCs (also by phorbol myristate acetate (PMA)) stimulates insulin secretion, while on the other hand, PKCs, especially PKCdelta, mediate palmitate-induced apoptosis. In this study we analyzed the mechanism and role of ERK1/2 activation by FFA in insulin secreting cells.

In INS-1E cells stimulation with palmitate (600 µM in the presence of 10 % FCS or 50 µM coupled to 1.2 % FCS) for 1 h and 24 h triggered phosphorylation

progression but do not provide a cure. How T2DM develops is still unclear and in particular little is know about the mechanisms underlying increased glucagon activity in the prediabetic state. Neuronostatin (NST), a product of the somatostatin prohormone, is produced by delta cells of the pancreas and was shown to enhance early gene expression in alpha cells of the islet. In isolated rat islets, NST inhibited glucose-stimulated insulin secretion via an indirect action on the alpha cell. Neuronostatin infusion in conscious, unrestrained male rats delayed glucose clearance and suppressed the insulin response to a glucose challenge. We hypothesized that acting at the level of the alpha cell, neuronostatin initiated a communication between that cell and the beta cell, resulting in supersession of glucose stimulated insulin release. In order to evaluate the signaling mechanisms underlying this potential action of neuronostatin, we exposed the immortalized murine alpha cell line, alphaTC1-9, to NST in low glucose conditions. We observed increased expression of both glucagon and cJun as measured by RT-PCR. Furthermore, alphaTC1-9 cells actively transcribe GPR107, which we have identifi ed to be the receptor that transmits neuronostatin’s signal in vitro and in vivo. Transfection of alphaTC1-9 cells with siRNA targeting GPR107 achieved a 70% knockdown of gene expression. Experiments are currently underway to examine if compromise of GPR107 in those cells alters responsiveness to NST. Endogenous NST from neighboring delta cells, acting through GPR107, may be an important factor in the onset of T2DM by virtue of its ability to stimulate alpha cell glucagon production and release, thus promoting disease progression.

Supported by: HL-066023

2287-PStudy of Immunomodulatory Role of Exendin-4 During the Insulitis Stage in an Autoimmnune Diabetes Animal ModelCARMEN SEGUNDO, FRANCISCO M. VISIEDO, ALBERTO CEBADA-ALEU, LAURA QUINTANA, MANUEL AGUILAR-DIOSDADO, Cádiz, Spain

Type 1 diabetes is an autoimmune pathology characterized by a specifi c loss of beta-cell mass. In a pre-onset phase, immune cells infi ltrate the pancreatic islets creating an infl ammatory stage named insulitis. GLP-1 receptor agonists have been used in type 2 diabetes treatment on the basis of their capacity to improve beta cell glucose induced insulin secretion. In addition, other benefi cial effects on beta cell survival have been reported and, thus, new therapeutic approaches have been explored. In this sense, our group has obtained important results which suggest an immunomodulatory effect of exendin-4 used in the fi rst stages of the insulitis process in type 1 diabetes. Biobreeding rats (BB) were intra-peritoneally treated from 4 to 9 weeks of age with exendin-4 at 40 microg/kg/day or vehicle. IPGTTs were performed at 7 and 9 weeks. Beta cell mass and infi ltrate score were studied comparing treated with control animals. In addition, T regulatory lymphocyte population was characterized in lymphoid cells isolated from spleen, thymus, pancreas associated lymph node and pancreatic infi ltration. Our results show an effect of exendin-4 in maintaining beta cell mass along the insulitis stage, whereas vehicle treated animals underwent a loss of beta cell mass in the same period. Exendin-4 also induces a decrement of infi ltration score in comparison with vehicle treated animals. In relation with T regulatory cell characterization, an increment of CD4+CD25+Foxp3+ lymphocytes was observed in exendin-4 treated animal infi ltrate. T regulatory population was also incremented in thymus of these animals and this result was accompanied with a loss of relative weight of organ. In conclusion, these results indicate that exendin-4 treatment inhibited beta cell mass loss during insulitis stage and reduced islet infi ltration grade. This effect may be mediated by an increment in T regulatory population in thymus, territory of T cell maturation, and in infi ltrated pancreas.

Supported by: Amylin Pharmaceuticals, LLC.

2288-PEnterovirus Infection Induces Signal Transduction Factors in Hu-man Progenitor Cells and INS-1 CellsSANDHYA R. NAIR, AMMIRA AL-SHABEEB, WILLIAM D. RAWLINSON, MARIA E. CRAIG, Sydney, Australia

Despite evidence supporting an association between enterovirus (EV) infection and type 1 diabetes, the etiological mechanism(s) for EV-induced beta cell destruction are not well understood. We and others have shown that infection with multiple EVs particularly Coxsackievirus B (CVBs) can induce β-cell production of proinfl ammatory cytokines and chemokines. In this study we examined pathways of β-cell death following EV infection.

We investigated the effects of CVB4 & CVB5 (GenBank accession numbers GQ126859 & GQ126860) on viral replication and expression of factors representing multiple apoptotic pathways (Akt, c-Jun, ERK1/2, JNK,

A586


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


protective effects on beta cell against oxidative stress, and MAPK and PI3K pathways but not AMPK pathway would be involved in these ghrelin’s protective effects.

Supported by: JSPS KAKENHI (22590991); Smoking Research Foundation

2293-PKlotho Coordinates With Transcriptional Factor, Mafs in the Differ-entiation and Establishment of Cell Integrity of Pancreatic/Adipose CellsMARIKO TSUCHIYA, MISAKA RYOICHI, KOSAKU NITTA, KEN TSUCHIYA, Tokyo, Japan

Klotho is known to obligate co-receptor of FGF23 and to also be involved in insulin/IGF1 and WNT signaling, suggesting that Klotho plays important roles in the differentiation of the pancreatic cell lineage and in glucose metabolism. In the present study we investigated the biological role of Klotho in cultured cells in coordination with maf transcriptional factors. We explored the relationship between klotho and mafs in cell differentiation and proliferation, which were quantifi ed by using CL-Quant software to analyze time-lapse images in a Nikon Biostation CT in combination with a high-throughput cell migration assay, and in the formation of cell polarity by measuring the expression of Na/K ATPase/PKC-ζ, a marker of cell signaling (β-catenine) in 3D culture. Previously, we showed that suppression of mafA mRNA with siRNA prevented adipose cell differentiation by 3T3-L1 cells, which suspension was restored and differentiated to form lipid droplet by addition of recombinant Klotho protein. On the other hand, suppression of mafB expression with siRNA in AsPc1 cells, a cell line from a pancreatic cancer, resulting in cell proliferation accompanied by induction of cell adhesion molecules and translocation of Na/K ATPase to the cell membrane. Up-regulation of Klotho expression affected localization of Na/K-ATPase/PKC- ζ and cell migration/proliferation, especially, Klotho exerts its effects in developing or (re-) differentiating cell conditions. In contrast, down-regulation of Klotho attenuated stabilization of ATPase and sequential cell proliferation. Taken together, it is likely that there is a cross-link between Klotho and mafs, one of possible mechanism is mediating translocation of Na/K ATPase, resulting in Ca+ channel stabilization/alternation of Ca+ concentration and signaling pathway including insulin, IGF-1, and Wnt that function in the differentiation and establishment of the cell integrity.

Supported by: MEXT (Japan) (23592021)

2294-PFibroblast Growth Factor Receptor 5 (FGFR5) Activation of Mi-togen-Activated Protein Kinase (MAPK) in Beta-Cells Requires FGFR1 ActivityPAMUDITHA N. SILVA, JONTHAN V. ROCHELEAU, DAWN M. KILKENNY, Toronto, ON, Canada

The survival and proliferation of pancreatic beta-cells is affected by extracellular stimuli. We have previously shown that fi broblast growth factor receptor 1 (FGFR1; R1) expression levels in beta-cells changes in response to various extracellular matrices, consequently regulating intracellular signaling. FGFR5 (R5), a newly described member of the FGFR family, is a close homologue of R1 that lacks canonical intracellular tyrosine kinase domains associated with classical intracellular signaling. We previously identifi ed R5 expression in murine pancreatic beta cells and beta-TC3 cells, and now confi rm expression in insulin-positive human islet cells through Western immunoblotting and immunofl uorescence. We also previously observed that R5 overexpression increases baseline p44/42 MAPK (ERK1/2) phosphorylation levels. We hypothesize that in the absence of functional kinase domains, R5-associated activity is due to secondary activation of R1. Preliminary data has shown that inhibition of R1 expression using siRNA causes an increase in ERK1/2 activity in control cells. Conversely, R1 siRNA reduced ERK1/2 activity in R5-overexpressing cells suggesting dependence upon R1 signaling. Interestingly, FGF2-induced ERK1/2 phosphorylation was enhanced in R5 cells with reduced R1 expression suggesting a direct R5-dependent mechanism of ligand-induced signaling in these cells. We observed no signifi cant changes in R5-cell proliferation in association with enhanced ERK1/2 activity but detected an approximate two-fold increase in cellular adhesion to collagen type IV and laminin substrates (major components of islet extracellular matrix) compared to controls. Overall, these data suggest that a synergistic relationship in basal signaling activity exists between R5 and R1, and despite the lack of intrinsic kinase activity, R5 can induce cellular responses to FGF ligands similar to R1.

Supported by: Banting and Best Diabetes Center (to P.N.S); NSERC (to J.V.R.); University of Toronto (to D.M.K.)

of ERK1/2 as evaluated by Western blotting. The effect on ERK1/2 was inhibited by the MEK1 inhibitor PD98059 (10 µM). PMA (100 nM) transiently phosphorylated ERK1/2 after 1 h but not 24 h. The phosphorylation of ERK1/2 was not transmitted by FFAR1/GPR40, as palmitate phosphorylated ERK1/2 in islets of WT and GPR40 knockout mice to the same extent. In accordance, PD98059 did not inhibit palmitate-induced potentiation of GIIS in static incubations of INS-1E cells. PD98059 itself did not induce apoptosis but augmented palmitate-induced beta-cell death.

These data suggest that ERK1/2 activation is not directly involved in FFA-induced potentiation of GIIS, but rather counteracts the pro-apoptotic effect of palmitate.

2291-PIdentifi cation of cis- and Trans-Regulatory Elements Controlling Expression of the Glycemia-Protective Enzyme Angiotensin-Con-verting Enzyme 2 (ACE2) in Pancreatic Beta-Cells by Analysis of Evolutionarily Conserved MotifsKIM B. PEDERSEN, KAVALJIT H. CHHABRA, ERIC LAZARTIGUES, New Orleans, LA

Clinical trials suggest that renin-angiotensin system (RAS) blockade hinders development of type 2 diabetes. Angiotensin-converting enzyme 2 (ACE2) counteracts the RAS, and our laboratory has reported that pancreatic ACE2 gene therapy improves beta-cell function and glycemia in diabetic mice. We aim to understand the regulation of ACE2 in the beta-cell and have previously found that transcription factors HNF1α and HNF1β induce ACE2 expression in insulinoma cells. Since there is a high degree of sequence similarity in the proximal 500 bp promoter regions upstream of the translation start site from different mammalian species, we hypothesized that ACE2 expression in beta-cells is controlled by evolutionarily conserved motifs of the ACE2 promoter. We analyzed conserved motifs by TRANSFAC software, in vitro mutagenesis of luciferase reporters controlled by the human -454/-1 ACE2 promoter sequence, and gel shift assays. Three predicted HNF1-binding motifs indeed bind HNF1α and HNF1β. Mutation of each of these motifs (at -244/-249, -314/-319, and -335/-340) signifi cantly (p < 0.001) decreases the promoter response to HNF1β in 832/13 insulinoma cells. Systematic mutation of additional 12 conserved motifs of the human ACE2 promoter gave 3 mutations (at -83/-88, -148/-153, and -178/-185) that decrease and 2 mutations (at -39/-44 and -282/-287) that increase promoter activity signifi cantly (p < 0.001). The mutations disrupt binding of several transcription factors such as COUP-TFII at -83/-88 and FOXA1/2 and FOXA3 at -148/-153. We fi nally observed with specifi c qRT-PCR assays that the high expression of ACE2 in islets from wild-type mice is due primarily to transcription from the proximal promoter region. We conclude that ACE2 expression in beta-cells is controlled from conserved transcription factor binding sites in the proximal promoter region.

Supported by: AHA (12EIA8030004)

2292-PGhrelin Protect Beta Cells From Oxidative Stress via Both MAPK and PI3K PathwaysMASAHIRO NISHI, YING WANG, TATSUYA ISHIBASHI, ASAKO DOI, HIROTO FURUTA, HIDEYUKI SASAKI, KISHIO NANJO, TAKASHI AKAMIZU, Wakayama, Japan

Ghrelin, the only peripheral orexigenic hormone, has been reported to show various effects. We have reported that ghrelin could inhibit insulin secretion via induction of IA-2beta (PNAS 103:885,2006) or AMPK/UCP2 pathway (FEBS Lett 584:1503,2010). Since UCP2 has protective effects against oxidative stress, we have investigated the protective effects on cell viability, proliferation and apoptosis of ghrelin against oxidative stress using hydrogen peroxide (H2O2) treated beta cell line (MIN6 cells). MIN6 cells were stimulated by ghrelin and/or H2O2 under different conditions and cell viability, proliferation or apoptosis was assessed by Cell counting kit, Cell proliferation ELISA BrdU kit or Cell death ELISA kit, respectively. Activation of AMPK, MAPK, or Akt was assessed by Western blot using phosphor-specifi c antibodies. H2O2 (100uM) decreased viability of MIN6 cells. Ghrelin, dose-dependently, ameliorated decreased viability induced by H2O2. Similar results were obtained for cell proliferation. H2O2 also induced apoptosis in MIN6 cells and ghrelin ameliorated these H2O2 effects dose-dependently. However, AMPK activator, AICAR (0.4mM), did not show protective effects against H2O2, otherwise exaggerated effects of H2O2. Ghrelin phosphorylated and activated MAPK(erk1/2) and Akt. Furthermore, MAPK inhibitor, PD98059 (20uM), or PI3K inhibitor, Wortmannin (200nM), blocked ghrelin’s protective effects against H2O2. Ghrelin would have

A587


Isle

t Bio

logy

/In

sulin

Sec

retio

n

POST

ERS


2295-PProinfl ammatory Cytokines Induce ER Stress With Concomitant Alter-ations in mRNA Ribosomal Occupancy in Islet β CellsANDREW T. TEMPLIN, BERNHARD MAIER, RAGHAVENDRA G. MIRMIRA, India-napolis, INIslet β cells are susceptible to a variety of stresses in the setting of diabetes, including high insulin demand, glucolipotoxicity, and infl ammation. Recent evidence suggests these stressors converge upon the endoplasmic reticulum (ER), leading to ER stress and an adaptive global translation initiation block. However, little is known about the actual β cell mRNA translational response in these conditions, and to what extent specifi c mRNAs associate with inactively translating ribosomes (monosomes) vs. actively translating ribosomes (poly-somes). To identify changes to mRNA translation, we exposed MIN6 β cells and mouse islets to a timecourse of cytokines (IL-1β, TNF-α, and IFN-γ), then performed polyribosomal profi ling (PRP) analysis by sucrose gradient sedimen-tation of cell lysates. Exposure to cytokines >24 h revealed progressive evidence of polyribosome depletion in MIN6 β cells, suggesting global translational initiation block consistent with ER stress. In accordance with activation of ER stress, increases in the mRNAs for Atf4, Chop, and spliced Xbp1 were observed at 24 h. Next, to determine if cytokines caused differential partitioning of β cell transcripts to monosomes vs. polysomes, gradient fractions from the PRP were collected and analyzed by qRTPCR. PRP of MIN6 β cells following pro-longed cytokine exposure revealed changes to ribosomal occupancy of specifi c transcripts analogous to those experienced during ER stress. Notably, while mRNA for many transcripts are partitioned to monosomes (repressed) under stress, transcripts for ATF4 and CHOP were partitioned to polysomes (activated). Ribosomal occupancy of other transcripts (insulin, Pdx-1) was not affected. These data provide evidence that cytokines cause translational changes in β cells comparable to ER stress, and which favor translation of specifi c mRNAs. Differential translation by such mechanisms may play key roles in the adverse effects of cytokines on islet β cell function.

Supported By: AHA (11PRE7230010 to A.T.T.); NIH (RO1DK060581 to R.G.M.)

2296-PPdx-1 Loss Contributes to β-Cell SERCA2 Transcriptional Dysregu-lation in Type 2 Diabetes MellitusJUSTIN S. JOHNSON, TATSUYOSHI M. KONO, DAN MOSS, SCOTT COLVIN, PAT-RICK FUEGER, CARMELLA EVANS-MOLINA, Indianapolis, IN β cell dysfunction plays a central role in the pathophysiology of Type 2

diabetes mellitus (T2DM). Our previous data from in vitro and in vivo models of T2DM demonstrate loss of β cell sarcoendoplasmic reticulum Ca2+ ATPase (SERCA2) expression that correlates with insulin secretory defects and acti-vation of ER stress pathways. In silico analysis of the SERCA2 promoter revealed multiple putative pancreatic and duodenal homeobox protein 1 (Pdx-1) binding sites, and Pdx-1 heterozygotes’ islets are known to manifest ER stress. Here, we hypothesized that loss of Pdx-1 in T2DM leads to dysregu-lated SERCA2 expression with concomitant alterations in β cell ER homeo-stasis. To test this hypothesis, INS-1 832/13 cells were treated with 25mm glucose and 5 ng/ml of IL-1β (HG+IL1β) to mimic the infl ammatory milieu of T2DM. Under control conditions, Pdx-1 overexpression led to increased SERCA2 levels. While HG+IL1β led to a loss of both Pdx-1 and SERCA2, adenoviral overexpression of Pdx-1 was able to rescue SERCA2 mRNA and protein levels. In contrast, siRNA-mediated knockdown of Pdx-1 in INS-1 cells resulted in a 59% loss of SERCA2 expression. To determine if Pdx-1 was a transcriptional regulator of the SERCA2 gene, luciferase assays were per-formed in NIH-3T3 cells by co-transfection of human Pdx-1 with constructs containing variable lengths of the human SERCA2 promoter. Results showed that a region 259 bp upstream of the transcriptional start site was suffi cient to confer Pdx-1 transactivation. These results suggest that Pdx-1 directly regulates the SERCA2 gene, and that loss of SERCA2 expression in the dia-betic β cell may be initiated by a preceding loss of Pdx-1.

Supported by: NIH (T32GM077229 to J.S.J.), (K08DK080225), (R03DK089147), (R01DK093954)

ISLET BIOLOGY—APOPTOSIS 2161-P - Diabetes...A556 For author disclosure information, see page 829....

Documents

Transcript of ISLET BIOLOGY—APOPTOSIS 2161-P - Diabetes...A556 For author disclosure information, see page 829....