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Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 ( Prkaa1 ) and α2 ( Prkaa2 ). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the β-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism. more...

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP 's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP , which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM., (© 2018 Chatterjee Bhowmick and Jeremic.) more...

In mammalian pancreatic β cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed β cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic β cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic β cells., (© 2018 Tsuchiya et al.) more...

Apoptotic death of pancreatic β cells is a major cause of type 2 diabetes mellitus (T2D) progression. Two isoforms of pyruvate kinase, PKM1 and PKM2, have been reported to participate in cell death in several cell types; however, little is known about their causal pathways in pancreatic β-cell death. We examined whether the suppression of PKM1 or PKM2 affects endoplasmic reticulum (ER) stress-induced apoptosis in a pancreatic β-cell line, MIN6, and Beta-TC-6 and found that knockdown of PKM1, but not of PKM2, leads to the induction of ER stress-induced apoptosis in these cells. We also investigated the mechanism by which PKM1 inhibits ER stress-induced apoptosis. We confirmed that PKM1 interacts with A-Raf, an upstream regulator of the MEK/ERK pathway, and that this interaction contributes to MEK1 phosphorylation by A-Raf. PKM1 knockdown suppresses the phosphorylation of MEK, ERK, and caspase-9 (Thr125), which is phosphorylated by the MEK/ERK pathway, thereby inhibiting the cleavage and activation of caspase-9. Thus, PKM1 knockdown activates the caspase-9/caspase-3 pathway under ER stress conditions and leads to apoptosis., (Copyright © 2017 Elsevier Inc. All rights reserved.) more...

Pro-inflammatory cytokines play a crucial role in the destruction of pancreatic β-cells, thereby triggering the development of autoimmune diabetes mellitus. We recently developed a cell-permeable fusion protein, PEP-1-heme oxygenase-1 (PEP-1-HO-1) and investigated the anti-inflammatory effects in macrophage cells. In this study, we transduced PEP-1-HO-1 into INS-1 insulinoma cells and examined its protective effect against cytokine-induced cell death. PEP-1-HO-1 was successfully delivered into INS-1 cells in time- and dose-dependent manner and was maintained within the cells for at least 48 h. Pre-treatment with PEP-1-HO-1 increased the survival of INS-1 cells exposed to cytokine mixture (IL-1β, IFN-γ, and TNF-α) in a dose-dependent manner. PEP-1-HO-1 markedly decreased cytokine-induced production of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA). These protective effects of PEP-1-HO-1 against cytokines were correlated with the changes in the levels of signaling mediators of inflammation (iNOS and COX-2) and cell apoptosis/survival (Bcl-2, Bax, caspase-3, PARP, JNK, and Akt). These results showed that the transduced PEP-1-HO-1 efficiently prevented cytokine-induced cell death of INS-1 cells by alleviating oxidative/nitrosative stresses and inflammation. Further, these results suggested that PEP-1-mediated HO-1 transduction may be a potential therapeutic strategy to prevent β-cell destruction in patients with autoimmune diabetes mellitus., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

Objective: Exendin-4 (Ex-4) is an anti-diabetic drug that is a potent agonist of the glucagon-like peptide-1 (GLP-1) receptor. It has already been approved for the treatment of type 2 diabetes mellitus, but its underlying mechanisms of action are not fully understood. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the transport and release of neurotransmitters in neurons, is expressed in pancreatic islet cells and β-cells. This study aimed to investigate whether CASK is involved in the insulin secretagogue action induced by Ex-4 in INS-1 cells., Material/methods: A glucose-stimulated insulin secretion (GSIS) assay was performed with or without siRNA treatment against CASK. The expression level and location of CASK were evaluated by real-time PCR, western blotting and immunofluorescence. With the use of a protein kinase A (PKA) inhibitor or an exchange protein directly activated by cAMP-2 (Epac2) agonist, immunoblotting was performed to establish the signaling pathway through which Ex-4 alters CASK expression., Results: Knock-down of CASK significantly attenuated the Ex-4-enhanced insulin release, and we showed that Ex-4 could increase transcription of CASK mRNA and expression of CASK protein but did not change the cellular location of CASK. A PKA inhibitor reduced the ability of Ex-4 to stimulate CASK expression, but an Epac2 agonist had no effect suggesting that regulation was mediated by the cAMP/PKA pathway., Conclusion: Our study suggests that the stimulation of β-cell insulin secretion by Ex-4 is mediated, at least in part, by CASK via a novel signaling mechanism., (© 2013.) more...

Aims/hypothesis: Protein gene product 9.5 (PGP 9.5) is a marker for neuroendocrine cells but has not been used for pancreatic islet cells and pancreatic endocrine tumors (PETs). Antibodies for PGP 9.5 are now commercially available for immunocytochemical study, with which immunostaining may be able to differentiate between benign and malignant PETs., Results: All 4 kinds of normal islet cells were positively immunostained for PGP 9.5-moderately positive for β-cells and strongly positive for δ-cells, whereas ganglion cells were immunostained more strongly than islet cells. Nine of 12 insulinomas were moderately to strongly positive for PGP 9.5. Two glucagonomas, 3 of 6 pancreatic polypeptidomas (PPomas), 3 of 9 gastrinomas, and 2 of 4 non-functioning PETs were negative for PGP 9.5., Materials and Methods: Thirty-four PETs were immunocytochemically stained for PGP 9.5 using a rabbit polyclonal antibody together with immunostaining for 4 pancreatic hormones, chromogranin A (CgA), and gastrin. PETs consisted of 12 insulinomas, 2 glucagonomas, 1 somatostatinoma (SRIFoma), 6 PPomas, 9 gastrinomas, and 4 non-functioning PETs., Conclusion/interpretation: PGP 9.5 immunostaining was universally positive for 4 kinds of islet cells and was moderately to strongly positive for 9 of 12 (75%) insulinomas. All 22 non-β-cell PETs were negative or weakly positive for PGP 9.5, and thus negative or weakly positive PGP 9.5 immunostaining may be used as a marker for potential malignancy and poor prognosis for non-β-cell PETs. more...

Background: Insulinoma was a rare tumor and its pathogenesis was poorly understood. There had no study that focused on the role of mTOR signaling pathway in insulinoma tumorigenesis., Materials and Methods: Expression of p-mTOR and its downstream p-P70S6K in insulinoma and normal pancreatic tissue was evaluated by immunohistochemical staining and Western blotting. In vitro study, an insulinoma cell line (INS-1) was treated with inhibitors of mTOR (rapamycin) or dual PI3K/mTOR inhibitor (NVP-BEZ235), RT-PCR, and Western blotting were applied to evaluate their influence on the expression of mTOR and P70S6K. Cell proliferation was evaluated by MTT test, cell cycle and apoptosis were analyzed by flow cytometry, insulin secretion level was evaluated by GSIS method., Results: Positive expression of p-mTOR and p-P70S6K was much higher in insulinoma tumor specimens than the normal pancreatic islet (P < 0.05). mTOR inhibitors can induce decreased expression of mTOR and P70S6K, which resulting in inhibiting INS-1 cell proliferation, insulin secretion and inducing apoptosis. NVP-BEZ235 had better influence on inhibiting the cell proliferation and inducing apoptosis than rapamycin., Conclusion: mTOR/P70S6K signaling pathway is involved in tumorigenesis of insulinoma, NVP-BEZ235 and rapamycin offer a promising role as novel drugs in treatment of insulinoma., (Copyright © 2012 Wiley Periodicals, Inc.) more...

Group VIB Phospholipase A(2) (iPLA(2)γ) is distributed in membranous organelles in which β-oxidation occurs, that is, mitochondria and peroxisomes, and is expressed by insulin-secreting pancreatic islet β-cells and INS-1 insulinoma cells, which can be injured by inflammatory cytokines, for example, IL-1β and IFN-γ, and by oxidants, for example, streptozotocin (STZ) or t-butyl-hydroperoxide (TBHP), via processes pertinent to mechanisms of β-cell loss in types 1 and 2 diabetes mellitus. We find that incubating INS-1 cells with IL-1β and IFN-γ, with STZ, or with TBHP causes increased expression of iPLA(2)γ mRNA and protein. We prepared INS-1 knockdown (KD) cell lines with reduced iPLA(2)γ expression, and they proliferate more slowly than control INS-1 cells and undergo increased membrane peroxidation in response to cytokines or oxidants. Accumulation of oxidized phospholipid molecular species in STZ-treated INS-1 cells was demonstrated by LC/MS/MS scanning, and the levels in iPLA(2)γ-KD cells exceeded those in control cells. iPLA(2)γ-KD INS-1 cells also exhibited higher levels of apoptosis than control cells when incubated with STZ or with IL-1β and IFN-γ. These findings suggest that iPLA(2)γ promotes β-cell proliferation and that its expression is increased during inflammation or oxidative stress as a mechanism to mitigate membrane injury that may enhance β-cell survival. more...

The pro-inflammatory cytokine IL-1β leads to losses in functional β-cell mass in part by inducing the expression of genes that produce soluble mediators of inflammation, such as cyclooxygenase-2 (COX2). In the current study, we sought to understand what factors control the COX2 gene in response to IL-1β and how prostaglandins downstream of COX2 impact pro-inflammatory gene transcription in pancreatic β-cells. We analyzed COX2 gene expression in response to different maneuvers impacting NF-κB proteins. Also, we report alterations in the expression of COX2, EP-3 and EP-4 receptor genes by PGD(2) and PGE(2). Moreover, we examined whether PGD(2) and PGE(2) regulated NF-κB and interferon-gamma activated sequence (GAS) reporter gene activity. IL-1β-mediated induction of the COX2 gene requires the p65 and p50 subunits of NF-κB. In addition, PGD(2) and PGE(2) coordinately alter COX2 and EP receptor gene expression patterns and potentiate the cytokine-mediated transcriptional activity of promoters containing NF-κB or GAS response elements., (Copyright © 2011 Elsevier Inc. All rights reserved.) more...

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells. more...

The objective of the present study was to examine the effects of melatonin on transcripts of isoforms of calcium/calmodulin-dependent protein kinases in rat insulinoma beta-cells INS-1. Investigations show that calcium/calmodulin-dependent kinase IV and calcium/calmodulin-dependent kinase 2d are expressed in human and rat pancreatic islets and INS-1 cells. By application of either forskolin or 3-isobutyl-1-methylxanthine for 6 hours, calcium spiking was evoked and the release of insulin was increased. The expression of the calcium/calmodulin-dependent kinase IV and calcium/calmodulin-dependent kinase 2d transcripts was significantly increased due to forskolin or 3-isobutyl-1-methylxanthine. Acute melatonin treatment (6 h) in the presence of either forskolin or 3-isobutyl-1-methylxanthine caused a significant decrease in insulin release and induced significant downregulation of calcium/calmodulin-dependent kinase IV and calcium/calmodulin-dependent kinase 2d transcripts in INS-1 batch cultures. The attenuating effect of melatonin on transcripts could be almost completely reversed by preincubation with the melatonin receptor antagonist luzindole. Thus, the insulin-inhibiting effect of melatonin in INS-1 cells is associated with significant changes in transcripts of calcium-signaling components suggesting that melatonin influences gene expression of components, which are known to be involved in insulin secretion or insulin gene expression. more...

Pancreatic beta-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K(ATP)-dependent glucose-stimulated insulin secretion (GSIS), there are important K(ATP)-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel (13)C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling. more...

Beta-cell apoptosis induced by adipokines may result in beta-cell dysfunction in type 2 diabetes. Resistin, an adipokine-linked obesity with type 2 diabetes, impairs glucose-stimulated insulin secretion (GSIS) in beta-cells. Presently, the effects of resistin on rat insulinoma cells RINm5F were examined. Treatment of RINm5F with resistin induced cell damage. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) protected resistin-mediated cytotoxicity in RINm5F. Incubation with resistin up-regulated caspase-3 activity and induced the formation of a DNA ladder. TIMP-1 attenuated these effects. The molecular mechanism of TIMP-1 inhibition of resistin-mediated cytotoxicity appeared to involve Akt phosphorylation and activation of IkB-alpha phosphorylation. Resistin treatment suppressed Akt phosphorylation and activated IkB-alpha phosphorylation, which could be attenuated by TIMP-1. We conclude that resistin can induce beta-cell apoptosis and that resistin-related beta-cell apoptosis can be prevented by TIMP-1. more...

The effect of leucine on glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is quite controversial, and mechanism involved in the effect has not been elucidated yet. Consequently, we aimed to investigate effect of leucine on GSIS and its mechanism focusing on contribution of AMP-activated protein kinase (AMPK) and pancreatic/duodenal homeobox-1 (PDX-1). Rat insulinoma beta-cells (INS-1, RIN m5F, DN-PDX-1#28 and PDX-1#6) were cultured with or without leucine, AICAR (AMPK agonist) or compound C (AMPK antagonist) for 48 hrs. In contrast to control, AICAR treatment decreased GSIS at high glucose and insulin content, also impaired protein and mRNA expression of PDX-1 and its downstream targets, glucokinase (GCK) and glucose transporter 2 (GLUT2). Compound C treatment had the opposite effects. We observed that neither AICAR nor compound C could affect expression of GCK and GLUT2 when PDX-1 expression was absent. Chronic leucine exposure inhibited GSIS at high glucose and insulin content in a dose-dependent manner, concomitant with an increase in AMPK and a decrease in PDX-1, GCK and GLUT2. The inhibitory effects of leucine was potentiated by AICAR treatment and rescued by compound C treatment. Finally, the inhibition of PDX-1 could potentiate the impaired effects induced by leucine whereas overexpression of PDX-1 could protect the cell from impairment induced by leucine. The study indicated that chronic leucine might result in an increase in AMPK and then a decrease in PDX-l, in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content. more...

Glucose homeostasis depends upon the appropriate release of insulin from pancreatic islet beta-cells. Postpandrial changes in circulating nutrient concentrations are coupled with graded release of stored insulin pools by the proportional changes in mitochondrial metabolism. The corresponding increased synthesis rates of both ATP and of anaplerotic metabolites have been shown to be mediators for nutrient-stimulated insulin secretion. Anaplerosis leads to the export of malate or citrate from the mitochondria, both of which can be recycled through metabolic pathways to reenter the Kreb's cycle. These metabolic cycles have the net effect of either transferring mitochondrial reducing equivalents to the cytosol, or of efficiently providing pyruvate to facilitate responsive changes in the Kreb's cycle flux in proportion to increased availability of glutamate and anaplerotic flux through glutamate dehydrogenase. Here, we describe siRNA knock-down and isotopic labeling strategies to evaluate the role of cytosolic and mitochondrial isoforms of malic enzyme in facilitating malate-pyruvate cycling in the context of fuel-stimulated insulin secretion. more...

The aim of the present investigation was to characterize the role of the MAPK kinase kinase-1 (MEKK-1) in stress-induced cell death of insulin producing cells. We observed that transient overexpression of the wild type MEKK-1 protein in the insulin-producing cell lines RIN-5AH and betaTC-6 increased c-Jun N-terminal kinase (JNK) phosphorylation and augmented cell death induced by diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine (DETA/NO), streptozotocin (STZ), and hydrogen peroxide (H2O2). Furthermore, DETA/NO or STZ induced a rapid threonine phosphorylation of MEKK-1. Silencing of MEKK-1 gene expression in betaTC-6 and human dispersed islet cells, using in vitro-generated diced small interfering RNA, resulted in protection from DETA/NO, STZ, H2O2, and tunicamycin induced cell death. Moreover, in DETA/NO-treated cells diced small interfering RNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK but not p38 and ERK. Inhibition of JNK by treatment with SP600125 partially protected against DETA/NO- or STZ-induced cell death. In summary, our results support an essential role for MEKK-1 in JNK activation and stress-induced beta-cell death. Increased understanding of the signaling pathways that augment or diminish beta-cell MEKK-1 activity may aid in the generation of novel therapeutic strategies in the treatment of type 1 diabetes. more...

Inhibition of the lipid phosphatase SH2-domain containing inositol phosphatase 2 (SHIP2) in L6-C10 muscle cells, in 3T3-L1 adipocytes and in the liver of db/db mice has been shown to ameliorate insulin signal transduction and established SHIP2 as a negative regulator of insulin action. Here we show that SHIP2 inhibition in INS1E insulinoma cells increased Akt, glycogen synthase kinase 3 and extracellular signal-regulated kinases 1 and 2 phosphorylation. SHIP2 inhibition did not prevent palmitate-induced apoptosis, but increased cell proliferation. Our data raise the interesting possibility that SHIP2 inhibition exerts proliferative effects in beta-cells and further support the attractiveness of a specific inhibition of SHIP2 for the treatment of type 2 diabetes. more...

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress. more...

Endoplasmic reticulum stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cell, and contributes to the development of type 1 diabetes. The chaperone molecule, glucose regulated proteins 78 (GRP78), is required to maintain ER function during toxic insults. In this study, we investigated the effect of GRP78 on the beta-cell apoptosis. We first measured GRP78 protein expression in different phase of streptozotocin-affected beta-cell by immunoblotting analysis. An insulinoma cell line, NIT-1, transfected with GRP78 was established, named NIT-GRP78, and used to study apoptosis, which was induced by streptozotocin or inflammatory cytokines. Apoptosis of NIT-1 or NIT-GRP78 cells was detected by flow cytometry, the transcription of C/EBP homologous protein (CHOP) was monitored by real-time PCR, the concentration of nitric oxide and the activity of superoxide dismutase were measured by colorimetric method. We found that, in comparison to NIT-1 cells, NIT-GRP78 cells responded to the streptozotocin or cytokines treatments with decreased concentration of nitric oxide, but increased activity of superoxide dismutase. In addition, the level of CHOP was also decreased in the NIT-GRP78 cells, which may mediate the resistance of the GRP78 overexpressed NIT-1 cells from apoptosis. Finally, we found that NIT-GRP78 cells were also more resistant than NIT-1 cells to cytotoxic T lymphocyte (CTL) specific killing detected by flow cytometry through target cells expressing green fluorescent protein cultured with effector cells and finally stained with propidium iodide. The data suggest that modulating GRP78 expression could be useful in preventing pancreatic beta-cell from the immunological destruction in type 1 diabetes individuals. more...

Background: 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels in rat insulinoma cells, by the same mechanism as leptin., Results: BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of KATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic beta-cell KATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of KATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of KATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin., Conclusions: This study shows that BVT.12777, like leptin, activates KATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates KATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to KATP channel activation, and may bypass up-stream signalling components of the leptin pathway. more...

Histidine decarboxylase (HDC) is an enzyme for decarboxylating l-histidine to histamine and is expressed in various types of cells including neuroendocrine tumors. Recent findings have demonstrated a high percentage of HDC immunoreactivity in many neuroendocrine tumors, including carcinoid tumors, small cell carcinomas of the lung, pheochromocytomas, and medullary carcinomas of the thyroid. HDC immunostaining was applied to pancreatic islet cells and related tumors to explore possible expression of HDC as a wide spectrum marker for neuroendocrine differentiation. A total of 24 cases (22 pancreatic endocrine neoplasms, one small cell carcinoma of the pancreas, and one mixed exocrine-endocrine carcinoma) along with normal pancreatic tissue were immunostained with the anti-HDC antibody. In a normal pancreas, a double immunostaining revealed possible colocalization of HDC with glucagon- or insulin-positive cells in the islets. Seventeen of 22 pancreatic endocrine neoplasms (77%) were found to be positive for HDC, and no distinct relation to hormonal activity was observed. One small cell carcinoma was strongly positive to HDC. One non-functional tumor with mixed exocrine and endocrine components showed a diffuse positive immunostaining for HDC, and some neoplastic glucagon- or somatostatin (SRIF)-positive cells coexpressed HDC. In conclusion, we demonstrated that the majority of pancreatic endocrine tumors expressed HDC, and we suggest that HDC is a wider new marker for neuroendocrine differentiation. more...

Context: Endocrine pancreatic tumors (EPTs) are rare lesions with varying biological behavior. Establishing malignancy is a challenge for clinicians and pathologists., Objective: To establish the role of proliferative, apoptotic, angiogenic, and hormonal markers as predictors of malignancy in EPTs., Design: Paraffin-embedded EPT samples were studied for prognostic markers., Patients: Twenty-one consecutive patients with a diagnosis of EPT., Main Outcome Measures: The proliferative fraction (topoisomerase IIalpha), microvascular density (CD34), vascular endothelial growth factor expression, and estrogen receptor-beta (ERbeta) expression were studied by immunohistochemistry on all EPTs. Apoptosis was also assessed with terminal deoxynucleotidyl transferase nick-end labeling., Results: We identified 13 benign and 8 malignant tumors. Topoisomerase IIalpha was significantly increased in malignant tumors (P =.001), while there were no differences in apoptosis, microvascular density, or vascular endothelial growth factor expression in association with malignancy. No correlation could be identified between microvascular density and vascular endothelial growth factor expression, and ERbeta was not detected. A receiver operating characteristic curve for topoisomerase IIalpha disclosed that above a labeling index of 13, the test had 88% sensitivity and 100% specificity for predicting malignancy., Conclusion: Cellular proliferation measured with topoisomerase IIalpha is a simple prognostic marker for malignancy in EPTs, unlike apoptosis, angiogenesis, or the presence of ERbeta, which were not associated with malignant behavior. These findings designate a defined field for future research on endocrine pancreatic carcinogenesis and a possible target for chemotherapeutic agents. more...

Many cells express a group VIA 84 kDa phospholipase A(2) (iPLA(2)beta) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA(2)beta in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA(2)beta in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA(2)beta participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA(2)beta-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA(2)beta protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA(2)beta transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA(2)beta, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA(2)beta tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA(2)beta residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA(2)beta catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA(2)beta activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA(2)beta and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA(2)beta-immunoreactive protein suggests that a signal transduction role of iPLA(2)beta in the native beta-cell might be attributable to a 70 kDa isoform of iPLA(2)beta. more...

Pancreatic beta-cells have low activities of the antioxidant enzyme catalase. Nitric oxide interacts with the haem group of catalase inhibiting its activity. We have studied the activity of catalase in beta-cells under conditions mimicking prediabetes and in which nitric oxide is generated from cytokine treatment in vitro. We also studied whether there is regulation of catalase enzyme activity by nitric oxide at the protein or gene expression level. RINm5F insulin-producing cells, treated for 24 h with cytokines, showed increased medium nitrite production (17+/-2.2 vs 0.3+/-0.2 pmol/ micro g protein) and significantly decreased cellular catalase activity (42.4+/-4.5%) compared with control cells. A similar reduction was seen in catalase-overexpressing RIN-CAT cells and in rat or human pancreatic islets of Langerhans. Catalase activity was also suppressed by the long-acting nitric oxide donor diethylenetriamine/nitric oxide adduct (Deta-NO) and this inhibition was reversible. The inhibition of catalase activity by cytokines in RINm5F cells was significantly reversed by the addition of the nitric oxide synthase 2 (NOS2) inhibitors nitro monomethylarginine or N-(3-(aminomethyl)benzyl)acetamidine (1400W). Protein expression was found to be unchanged in cytokine- or Deta-NO-treated RINm5F cells, while mRNA expression was marginally increased. We have shown that inhibition of catalase activity by cytokines is nitric oxide dependent and propose that this inhibition may confer increased susceptibility to cytokine- or nitric oxide-induced cell killing. more...

The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, we found that the rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), produce solely hepoxilin A(3) (HXA(3)). Since HXA(3) synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, it was tempting to speculate that a HXA(3) synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption we incubated AA with HeLa cells overexpressing the rat leukocyte-type 12-LOX. Neither HXA(3) nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA(3) synthase and 12-LOX activities. Thus, we conclude, that cells containing rat leukocyte-type 12-LOX also possess an intrinsic HXA(3) synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA(3) is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as RINm5F. more...

Background: After transplantation, islet damage occurs through oxidative stress and host immune rejection mediated in part by macrophage activation. We investigated the influence of the overexpression of catalase (CAT) and Cu/Zn superoxide dismutase (Cu/Zn SOD) by rat insulinoma INS-1 beta cells exposed to oxidative stress on their viability and murine macrophage activation., Methods: INS-1 cells were infected with adenoviral vectors containing CAT (AdCAT) or Cu/Zn SOD (AdSOD) genes. After 72 hours, noninfected and infected INS-1 cells were exposed to oxidative stress and their viability was assessed using a colorimetric assay. Murine peritoneal exudate macrophages (mPEM) incubated with the supernatant of infected and stressed INS-1 cells were tested for chemotaxis and cytokine release (TNF-alpha, IL-alpha and IFN-gamma)., Results: After infection, AdCAT and AdSOD gene transfer protected INS-1 cells from the toxicity of different oxidative reagents. The exposure of non-infected INS-1 cells to oxidative stress stimulated mPEM chemotaxis. INS-1 cells infection with AdCAT or AdSOD reduced significantly mPEM chemotaxis from 2.41 +/- 0.31 to 1.61 +/- 0.17 and from 2.53 +/- 0.24 to 1.27 +/- 0.14 respectively (n = 5; p < 0.05). Cytokine release by mPEM was stimulated after exposure to stressed noninfected INS-1 cell supernatant. CAT and Cu/Zn SOD overexpression by infected INS-1 cells decreased significantly the release of TNF-alpha from 268.18 +/- 30.18 to 81.40 +/- 23.58 pg/ml and from 446.96 +/- 75.47 to 20.37 +/- 2.38 pg/ml respectively (n = 6; p < 0.001). The overexpression of these enzymes also reduced significantly the release of IL-1beta and IFN-gamma., Conclusions: CAT or Cu/Zn SOD gene transfer to INS-1 cells preserved them from oxidative damage and reduced the macrophage activation induced by these pancreatic cells. Therefore, protection of pancreatic beta cells against oxidative injury by antioxidant enzymes gene transfer is an effective approach to overcome the deleterious actions of macrophages in pancreatic islet transplantation. more...

Accumulating evidence suggests that the cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) manifests a signaling role in insulin-secreting (INS-1) beta-cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in iPLA(2)beta-overexpressing INS-1 cells (Ma Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem 276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity, and enzymatic activity analyses are used to examine distribution of iPLA(2)beta in stimulated INS-1 cells in greater detail. Overexpression of iPLA(2)beta in INS-1 cells leads to increased accumulation of iPLA(2)beta in the nuclear fraction. Increasing glucose concentrations alone results in modest increases in insulin secretion, relative to parental cells, and in nuclear accumulation of the iPLA(2)beta protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and in time-dependent nuclear accumulation of iPLA(2)beta fluorescence, which is reflected by increases in nuclear iPLA(2)beta protein content and specific enzymatic activity. The stimulated effects are significantly attenuated in the presence of cell-permeable inhibitors of protein phosphorylation and glycosylation. These findings suggest that conditions that amplify insulin secretion promote translocation of beta-cell iPLA(2)beta to the nuclei, where it may serve a crucial signaling role. more...

Peptide hormones are synthesized as bigger prohormones, which are processed posttranslationally into smaller active hormones. Proinsulin and proglucagon are processed into insulin and glucagon by prohormone convertase (PC) 1/3 and 2. The current study was performed to test a hypothesis that there may be some difference in immunoreactive PC levels between normal islet cells and islet cell tumors, as the latter contain more prohormones than the former. All islet cell tumors, including insulinomas, gastrinomas, glucagonomas, pancreatic polypeptide-omas (PP-omas), and nonfunctioning islet cell tumors, contain fewer PCs than normal islet cells. The smaller PC levels in islet cell tumors may be responsible for the higher levels of prohormones in islet cell tumors, and the smaller levels of PCs in islet cell tumors may be another distinguishing characteristic of islet cell tumors. more...

A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2beta) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet beta-cells, and other sources. Proposed iPLA2beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta-cells. To further examine iPLA2beta functions in beta-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2beta activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA2beta cDNA. The iPLA2beta-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2beta-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2beta-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2beta-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2beta plays a signaling role in beta-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells. more...

Reversible protein phosphorylation is an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of certain insulin secretagogues and intracellular second messengers, known to stimulate or inhibit insulin secretion, on the activities of cation-independent serine/threonine PPases were investigated in insulin-secreting RINm5F insulinoma cells. Raising cellular cAMP through adenylyl cyclase activation and phosphodiesterase inhibition in intact cells, evoked inhibitory effects on PPase activities. The addition of a nitric oxide donor, cyclic nucleotides, or proinflammatory prostaglandins to RINm5F cell homogenates at widely different concentrations did not affect type-1 or -2A PPase activities. Phosphatidyl serine seemingly activated PPase-1, while inactivating PPase-2A. A protein kinase C-activating phorbol ester produced the opposite results when added to RINm5F cell homogenates. These studies suggest that several known intracellular second messengers are without effect on beta-cell PPase activities. However, phosphatidyl serine and protein kinase C activation, whose activity is transiently increased by glucose, may promote insulin release through PPase inactivation, likely contributing to the increase in phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism, assisting in activation of the stimulus-secretion coupling in insulin-producing cells. more...

This study was designed to test the hypothesis that poly(ADP-ribose) polymerase (PARP) plays a role in the repair of damage to mitochondrial DNA (mtDNA). A rat insulinoma cell line was transfected with a PARP antisense vector that was under the control of a dexamethasone promoter. Transfected cells were selected for stable integration of the antisense vector. Several cell lines containing the antisense vector were isolated. For these studies, one of these lines (clone 5) was chosen for further evaluation. When cells were treated with dexamethasone for 72 h, PARP activity was diminished by 60%. Western blot analysis revealed a concomitant reduction in PARP protein. When clone 5 cells were exposed to the simple methylating agent methylnitrosourea (MNU) without previous treatment with dexamethasone, repair of lesions in mtDNA was found to be similar to that seen in wild-type cells or in wild-type cells treated with dexamethasone. However, when clone 5 cells were pretreated with dexamethasone for 72 h, repair of MNU-induced damage was significantly inhibited. To ascertain whether the PARP activity that was inhibited by the antisense treatment was the same as that found in the nucleus, repair studies were performed on fibroblasts derived from PARP knockout mice and their normal wild-type controls. Attenuated repair was also seen in the cells in which the gene for PARP was inactivated. These are the first studies to demonstrate that PARP can facilitate the repair of simple alkylation damage to mtDNA. more...

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form. On the other hand, hardly detectable levels of glucose-6-phosphatase activity and protein were present in the parent glucose insensitive RINm5F cell line. The mRNA of the glucose-6-phosphate transporter was also more abundant in the INS-1 cells. The results support the view that the glucose-6-phosphatase system of the beta-cell is associated with the regulation of insulin secretion., (Copyright 2000 Academic Press.) more...

A 76-year-old man with hypoglycemic coma was diagnosed as malignant insulinoma with multiple hepatic metastases. Embolization was done for two-thirds of the hepatic mass and it rapidly lowered the serum immunoreactive insulin. He was discharged without medication and has been free from hypoglycemia. After the embolization, the serum creatine kinase (CK) level increased transiently although there was no evidence of myocardial infarction. On electrophoresis, the CK activity showed an abnormal peak, suggesting mitochondrial CK. CK release after embolization has been reported in only a few cases with endocrine tumors, which might indicate some relationship between active energy metabolism and mitochondrial CK. more...

Aims: Telomerase activation is known to be a common event in human cancer and may be a useful marker for malignancy. In general, the histological features of pancreatic endocrine tumours cannot be used to determine their malignant potential. The aim of this study was to investigate the role of testing telomerase activity in pancreatic endocrine tumours., Methods: Prospectively collected fresh frozen tissue specimens from 10 patients with pancreatic endocrine tumours (nine insulinomas, one adrenocorticotrophin producing pancreatic endocrine tumour) were examined by a highly sensitive polymerase chain reaction (PCR) based telomerase repeat protocol (TRAP)., Results: Of the 10 pancreatic endocrine tumours, three had telomerase activity. The positive cases included two frankly malignant tumours with liver metastases and one pancreatic endocrine tumour occurring in the setting of multiple endocrine neoplasia type 1. The latter had an infiltrative border. Vascular and perineural tumour infiltration was noted. In the two malignant pancreatic endocrine tumours with liver metastases, telomerase activity was noted in the tumour and the adjacent morphologically non-neoplastic pancreas., Conclusion: To our knowledge, this is the first report of the role of telomerase activity in pancreatic endocrine tumours. Telomerase activity might be useful for distinguishing between benign and malignant pancreatic endocrine tumours. more...

Cytokines might play a role in the development of insulin-dependent diabetes. Interleukin 1beta (IL-1beta) has been shown to alter the functional state of insulin-producing beta cells. This effect appears mediated through induction of certain proteins and suppression of others. The present study demonstrates that the ornithine decarboxylase (ODC) activity of rat beta cells and insulin-producing rat insulinoma (RIN) cells increases more than three-fold within 2 h of IL-1beta exposure. Both basal and IL-1 beta induced ODC activities completely disappear following a 2 h block of protein translation. In Western blot analysis, a 51-kDa protein varies in parallel with the ODC-activity. Exposure to IL-1beta increases the 51-kDa band through an effect at the transcriptional level. The higher cellular ODC activity in IL-1beta treated RIN cells is associated with an increased cellular content of its enzymatic product putrescine, but not of putrescine-derived products such as polyamines and GABA. The 30-fold lower ODC activity in rat beta cells forms a technical obstacle to studies on the regulation and functional significance of this enzyme in normal cells. The present findings list ODC-activity as an early response to the effect of interleukin 1beta on the transcriptional activity in insulin-producing cells. Further work is needed to identify whether ODC activation contributes to the previously described functional changes in pancreatic beta cells., (Copyright 2000 Academic Press.) more...

Length of (CTG)n triplet repeats in myotonic dystrophy protein kinase gene (DMPK) was estimated in tumors, normal tissues of the same organs, muscles, and leukocytes from three myotonic dystrophy (DM) patients and a non-DM patient. Using cDNA 25 as a probe, a Southern blot analysis of EcoRI- and BglI-digested DNA from these tissues demonstrated the longest expansion of the repeats in the tumors of DM patients. In all tissues from a non-DM patient, the repeat length was confirmed to be stable by PCR analysis. Our data suggest that expanded (CTG)n repeat in tumor tissues may have increased the instability. This study emphasizes the importance of a long-term prospective study on the incidence of tumors in DM to clarify the pathological interrelation between the two entities., (Copyright 1999 John Wiley & Sons, Inc.) more...

Recent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly-->Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg(972) substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg(972) IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg(972) IRS-1 variant (RIN-Arg(972)) in RIN beta cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg(972) IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg(972) IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg(972) showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg(972) at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg(972) IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic beta cells. More importantly, the results suggest that the common Arg(972) IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant. more...

Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture. more...

Hypoglycemia with a low serum immunoreactive insulin (IRI) level and serum immunoreactive C-peptide (IRC) level was found in a 74-yr-old female. Although a fasting test induced hypoglycemia, the responses of IRI and IRC during the fasting test, and the results of a glucose tolerance test, glucagon test, and secretin test did not indicate the presence of an insulinoma. However, the serum proinsulin level before the fasting test was 130.5 pmol/L (N: 3.0-10.0 pmol/L), and this high level was maintained throughout the test. Soon after surgical enucleation of the tumor, the patient's blood glucose levels increased. Postoperatively, the hypoglycemic status resolved, and the serum proinsulin levels returned to normal (2.8 pmol/L). Histopathological studies revealed a typical insulinoma. Immunohistochemical studies by the recently developed method for vacuolar-type H+ (V-ATPase), which is responsible for acidification of the intracellular compartments in eukaryotic cells, showed that normal islets stained positive, but not the tumor. This finding indicates that the insulin-secretory granules in the insulinoma cells existed in a microenvironment in which V-ATPase activity had been lost. This suggests that the reduced activity of V-ATPase on the endomembrane of the insulin-secretory granules in insulinomas may result in loss of the acidic microenvironment and impaired conversion of proinsulin by converting enzymes. more...

Synapsin I is a synaptic vesicle-associated protein involved in neurotransmitter release. The functions of this protein are apparently regulated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We reported evidence for CaM kinase II and a synapsin I-like protein present in mouse insulinoma MIN6 cells (Matsumoto, K., Fukunaga, K., Miyazaki, J., Shichiri, M., and Miyamoto, E. (1995) Endocrinology 136, 3784-3793). Phosphorylation of the synapsin I-like protein in these cells correlated with the activation of CaM kinase II and insulin secretion. In the present study, we screened the MIN6 cDNA library with the full-length cDNA probe of rat brain synapsin Ia and obtained seven positive clones; the largest one was then sequenced. The largest open reading frame deduced from the cDNA sequence of 3695 base pairs encoded a polypeptide of 670 amino acids, which exhibited significant sequence similarity to rat synapsin Ib. The cDNA contained the same sequence as the first exon of the mouse synapsin I gene. These results indicate that synapsin Ib is present in MIN6 cells. Synapsin I was expressed in normal rat islets, as determined by reverse transcriptase-polymerase chain reaction analysis. Immunoblot analysis after subcellular fractionation of MIN6 cells demonstrated that synapsin Ib and delta subunit of CaM kinase II co-localized with insulin secretory granules. By analogy concerning regulation of neurotransmitter release, our results suggest that phosphorylation of synapsin I by CaM kinase II may induce the release of insulin from islet cells. more...

This study examined the effect of glucose and 12-O-tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKC alpha and beta activation in the membrane fractions, respectively. Treatment with 5 mM and 20 mM glucose for 5 min and 20 min resulted in 6-7-fold increases in insulin secretion, and treatment with 1 microM TPA for 5 min also resulted in 3-fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKC alpha and beta immunoreactivities after treatment with 5 mM, 20 mM glucose and 1 microM TPA. Translocations of PKC alpha after treatment with glucose and TPA were greater than those of PKC beta in membrane fractions. These results suggest that TPA independently provokes insulin secretion via PKC activation and that PKC alpha and beta activation may be involved in insulin secretion in human insulinoma cells. more...

Protein tyrosine phosphatases (PTPs) play important roles in cell growth and differentiation of normal and tumor cells. In this study, we analyzed the PTP profile in two pancreatic islet tumor cell lines. Transcripts were isolated from alphaTC-1 (glucagon-secreting) and betaTC-1 (insulin-secreting) cell lines for templates. A pair of degenerative primers, based on the conserved regions of known PTPs, was used to amplify the transcripts by polymerase chain reaction. A total of 1,620 clones was examined by restriction enzyme analysis and cDNA sequencing. Twenty-one PTPs were identified, including nine cytosolic PTPs (TcPTP, P19PTP, PTP1B, PTPMEG, PTP1C, SYP, PTPH1, PTPL1, and PTPD1), nine transmembrane PTPs (PTPdelta, PTPgamma, PTPkappa, DEP-1, IA-2, LAR, PTPalpha, PTPNE3, and PTPepsilon), and three new PTPs--PTPmu-like PTPkappa-like, and IA-2beta. An RNase protection assay demonstrated that some of these PTPs were expressed predominantly in glucagonoma (i.e., PTPdelta and IA-2) and others in insulinoma (i.e., PTP1C, PTPkappa, and PTPNE3) cells. In this report, we present the first profile of PTPs in alpha and beta tumor cell lines. more...

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion. more...

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from beta TC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 microM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 microM), glucagon-like peptide I-(7-36) amide (0.1 microM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from beta TC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, beta TC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that beta TC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells. more...

We have previously reported that pancreatic islet beta-cells and clonal HIT insulinoma cells express an ATP-stimulatable Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme and that activation of this enzyme appears to participate in glucose-stimulated insulin secretion. To further examine this hypothesis, glucose-responsitivity and expression of ASCI-PLA2 activity in various insulinoma cell lines were examined. Secretagogue-stimulated insulin secretion was observed with beta TC6-f7 and early passage (EP)-beta TC6 cells. In contrast, RIN-m5f, beta TC3, and late passage (LP)-beta TC6 cells exhibited little secretagogue-induced secretion. A haloenollactone suicide substrate (HELSS) which inhibits ASCI-PLA2 activity ablated secretagogue-induced insulin secretion from beta TC6-f7 and EP-beta TC6 cells. All insulinoma cell lines studied expressed both cytosolic and membrane-associated Ca(2+)-independent PLA2 activities which were inhibited by HELSS. The cytosolic enzymatic activity in the glucose-responsive beta TC6-f7 and EP-beta TC6 cells was activated by ATP and protected against thermal denaturation by ATP, but this was not the case in the glucose-unresponsive RIN-m5f, beta TC3, or LP-beta TC6 cells. Comparison of the distribution of Ca(2+)-independent PLA2 activity revealed that membrane-associated activity was higher than cytosolic activity in beta TC6-f7 and EP-beta TC6 cells but not in RIN-m5f, beta TC3, or LP-beta TC6 cells. Insensitivity of cytosolic activity to ATP may prevent association of the PLA2 activity with membrane substrates and contribute to attenuated glucose-responsitivity in the RIN-m5f, beta TC3, or LP-beta TC6 cells. HIT insulinoma cells were also found to undergo a decline in both glucose-responsitivity and membrane-associated Ca(2+)-independent PLA2 activity upon serial passage in culture, and this was associated with a reduction in membrane content of arachidonate-containing phospholipids. These and previous results suggest that the ATP-stimulatable PLA2 enzyme may participate in glucose-induced insulin secretion. more...

Interleukin-1 (IL-1) may be a mediator of beta-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor kappa B (NF-kappa B), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-kappa B activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1 beta, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 microM) prevented the increase in nitrite production by rat islets exposed to IL-1 beta for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1 beta for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1 beta, tumor necrosis factor-alpha and interferon-gamma). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-beta but failed to block IL-1 beta-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1 beta-induced NF-kappa B activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic beta-cells, an effect not associated with inhibition of NF-kappa B activation. more...

Prohormone convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amino acids. This mechanism was initially inferred from the series of several endocrine and neuroendocrine precursor proteins, including proinsulin and proglucagon. To determine the cellular and subcellular distribution of PC1/3 and PC2 in the rat and human pancreas, immunohistochemistry was performed using polyclonal antisera against mouse PC1/3 (ST-28) and mouse PC2 (ST-29). These studies showed light and electron microscopic co-localization of insulin, PC1/3 and PC2, and the coexistence of glucagon and PC2 in the pancreatic islets. This tendency of colocalization was also depicted in one case of human insulinoma and three cases of human glucagonomas, as well as in rat insulinomas. In two cases of human insulinomas, incomplete processing of proinsulin was suggested by the absence of PC2. At the subcellular level in the rat pancreatic islet, the colocalization of PC1/3 and insulin, and that of PC2 and glucagon, were observed in the same secretory granules by immunoelectron microscopy and image analysis. These studies suggest that PC1/3 and PC2 can function with the specificities in the processing of proinsulin and proglucagon into their active forms, respectively, in the normal and neoplastic pancreatic islets. more...

RINm5F insulinoma cells show a defective physiological insulin secretory response to glucose stimulation. The short chain carbonic acid sodium butyrate induced a growth arrest during a 72-h tissue culture period. In contrast to control RINm5F cells, 2 mM glucose increased insulin secretion by more than 70% in these sodium butyrate-treated cells (1 mM) without any further increase of the secretory rate between 2 and 20 mM glucose. This effect of sodium butyrate on insulin secretion was assessed in comparison with its effect on gene expression of the GLUT1 and GLUT2 glucose transporter, hexokinase type I and type II, glucokinase and insulin. Sodium butyrate at a 1 mM concentration decreased GLUT1 gene expression by nearly 50%, but did not induce gene expression of the low-affinity GLUT2 glucose transporter above the detection limit. Furthermore, sodium butyrate increased glucokinase gene expression by more than 50% and hexokinase type II gene expression by more than 100%, while insulin gene expression was increased only by 24%. Hexokinase type II enzyme activity was increased by more than 100% without a concomitant significant change of the glucokinase enzyme activity. Sodium butyrate (2 mM) caused effects comparable with those of 1 mM sodium butyrate. Thus the improved insulin secretory responsiveness of RINm5F insulinoma cells after sodium butyrate treatment at low non-physiological millimolar glucose concentrations can be interpreted as a result of an increased hexokinase-mediated metabolic flux rate through the glycolytic chain. more...

Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet beta-cells, and ATP has attracted interest as a messenger of glucose metabolism within beta-cells. Glucose-induced insulin secretion from islets and HIT insulinoma cells is accompanied by activation of an ATP-stimulatable Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme, the catalytic activity of which resides in a 40 kDa protein. An analogous PLA2 enzyme in myocardium was recently found to consist of a complex of a 40 kDa catalytic protein with a tetramer of an isoform of the glycolytic enzyme phosphofructokinase (PFK). Association of the PFK isoform with the myocardial PLA2 catalytic protein was found to confer ATP sensitivity onto the enzyme complex. Here we demonstrate that the majority of HIT cell and islet ASCI-PLA2 catalytic activity elutes from a gel filtration column in a region corresponding to 400 kDa, suggesting that the 40 kDa beta-cell ASCI-PLA2 catalytic protein exists as part of a larger molecular mass complex. Islet and HIT cell ASCI-PLA2 activities were immunoprecipitated by antibodies directed against PFK, and the immunoprecipitates contained 40 and 85 kDa proteins which correspond to the molecular masses of the PLA2 catalytic protein and of a PFK monomer, respectively. Islet and HIT cell ASCI-PLA2 activities were selectively and reversibly adsorbed to affinity matrices containing immobilized PFK but not to similar matrices containing immobilized transferrin or bovine serum albumin. Addition of free PFK prevented binding of HIT cell ASCI-PLA2 activity to immobilized PFK matrices and promoted desorption of activity previously bound to such matrices. These results suggest that beta-cell ASCI-PLA2, like the myocardial enzyme, exists as a complex comprised of a catalytic protein and a PFK-like protein and raise the possibility that the ASCI-PLA2 complex may represent a component of the beta-cell glucose sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved in glucose-induced insulin secretion. more...