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D L Eizirik

Publications and source records attributed to D L Eizirik.

At least 19 recordsLinked to original sources
PubMed2001

A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta and interferon-gamma, are putative mediators of immune-induced beta-cell death and, under in vitro conditions, cause beta-cell apoptosis. We have recently shown that interleukin-1 beta + interferon-gamma modifies the expression of >200 genes in beta-cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta-cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta-cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta-cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta-cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta-cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta-cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta-cell dysfunction and death.

Adenoviridae↗
PubMed2001

Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.

BACKGROUND: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). METHODS AND RESULTS: The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. CONCLUSIONS: The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.

Animals↗
PubMed2001

Rat hepatic natural killer cells (pit cells) express mRNA and protein similar to in vitro interleukin-2 activated spleen natural killer cells.

Pit cells are liver-specific natural killer (NK) cells that can be divided into high- (HD) and low-density (LD) subpopulations. The characteristics of pit cells were further investigated in this report. LD and HD pit cells express the specific NK-activation markers gp42, CD25, and ANK44 antigen. LD cells and IL-2-activated NK cells have a high mRNA expression of perforin, granzymes, interferon-gamma, and tumor necrosis factor-alpha. LD pit cells, unlike spleen NK cells, have a weak response to IL-2 with regard to proliferation, cytotoxicity, and production of NK-related molecules. The characteristics of HD cells are intermediate between LD and spleen NK cells. These results show that pit cells, especially LD cells, possess characteristics similar to IL-2-activated NK cells. This is the first evidence on a molecular level that pit cells could be considered in vivo activated NK cells.

Animals↗
PubMed2001

Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 beta-exposed human and rat islet cells.

AIMS/HYPOTHESIS: Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes and T lymphocytes, and could thus contribute to mononuclear cell infiltration in Type I (insulin-dependent) diabetes mellitus. Cytokines induce MCP-1 mRNA expression in pancreatic rat beta cells. To investigate this issue, we analysed the signal transduction for IL-1 beta-induced MCP-1 expression in rat beta cells and in vitro MCP-1 mRNA expression and protein release by human islets as well as in vivo islet MCP-1 mRNA expression in prediabetic non-obese diabetic mice. METHODS: Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1 beta (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6-72 h with the cytokines IL-1 beta, IFN-gamma or the inducible nitric oxide synthase (iNOS) inhibitor NG-methyl-L-arginine or both. We measured MCP-1 mRNA by RT-PCR and protein by ELISA. The MCP-1 mRNA expression in islets from male and female non-obese diabetic mice (2-12 weeks of age) was measured by real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Interleukin-1 beta induced MCP-1 mRNA expression in rat beta cells, with a maximum induction after 6 h. A combination of p38 and ERK1/2 inhibitors decreased MCP-1 expression by 70%. IL-1 beta induced both MCP-1 mRNA expression and a threefold increase in medium MCP-1 protein accumulation in human islet cells. This effect was not prevented by iNOS blockers. In vivo there was an age-related increase in MCP-1 mRNA expression in islets from male and female non-obese diabetic mice, reaching a peak at 8 weeks. CONCLUSIONS/INTERPRETATION: In rat and human islet cells MCP-1 mRNA is induced by IL-1 beta. Both ERK1/2 and p38 MAPK, but not nitric oxide, contribute to MCP-1 expression. In non-obese diabetic mice MCP-1 mRNA expression increases with age, peaking at the early phases of insulitis. The production of MCP-1 by pancreatic beta cells could contribute to the recruitment of mononuclear cells into pancreatic islets in early Type I diabetes.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Dual role of interferon-gamma signalling pathway in sensitivity of pancreatic beta cells to immune destruction.

AIMS/HYPOTHESIS: Disruption of the interferon-gamma (IFN-gamma) signalling pathway at the level of interferon regulatory factor-1 (IRF-1) protects islets against cytokine-induced nitric oxide production and cell death in vitro. The aim of this study was to investigate the effects of a global disruption of IFN-gamma signalling, or a selective disruption of IRF-1, on beta-cell sensitivity to in vivo immune destruction. METHODS: In a first set of experiments, IFN-gamma receptor knockout mice (IFN-gammaR-/-) and interferon regulatory factor-1 knockout mice (IRF-1-/-) were rendered diabetic by injections of 50 mg streptozotocin i. p. on 5 consecutive days (MLDSTZ). RESULTS: Whereas no difference in sensitivity to MLDSTZ-induced diabetes could be observed between IFN-gammaR-/- mice and their 129/Sv/Ev controls (50% vs 55%, NS), there was an increased incidence of diabetes in IRF-1-/- mice (100% vs 67% in C57B1/6 mice, p < 0.05). A similar increased sensitivity to immune destruction of IRF-1-/- islets was observed when these islets were used as allografts. Islet graft survival rate of IFN-gammaR-/- and 129/Sv/Ev islets, when transplanted in alloxan-diabetic BALB/c recipients, was comparable (12.0 +/- 1.9 days vs 12.9 +/- 2.3 days, NS). Allograft rejection, however, of IRF-1-/- islets by BALB/c recipients occurred more rapidly than following transplantation to their C57B1/6 controls (9.1 +/- 2.0 days vs 13.1 +/- 1.5 days, p < 0.003). CONCLUSIONS/INTERPRETATION: These data indicate that IFN-gamma signal transduction at the beta-cell level is not essential for immune beta-cell destruction in vivo. Moreover, disruption of the IRF-1 gene in pancreatic islets increases susceptibility to beta-cell killing, suggesting that IRF-1 might be necessary for the expression of putative beta-cell "defence and/or repair" genes.

Animals↗
PubMed2001

A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis.

Apoptosis is likely to be the main form of beta-cell death in immune-mediated diabetes mellitus in rodents and possibly in humans. Clarification of the regulation of beta-cell death could indicate novel sites for therapeutic intervention in Type I (insulin-dependent) diabetes mellitus. We review the molecular effectors and signal transduction of immune-mediated beta-cell apoptosis. Data obtained on non-obese diabetic (NOD) mice suggest that macrophages and CD4+ T-cells are the main cellular effectors, whereas CD8+ T-cells are more important initiators of the immune process leading to beta-cell death. Perforin could be the effector molecule utilized by CD8+ T-cell initiation, whereas CD4+ mediated beta-cell destruction is mostly dependent on Fas/FasL and the cytokines IFNgamma and TNF-alpha. The macrophage cytokine IL-1beta in combination with IFN-gamma and TNF-alpha, plays an important role for beta-cell dysfunction and death. Signal transduction by these cytokines involves: (i) binding to specific receptors, (ii) signal transduction by cytosolic kinases (especially the so-called mitogen- and stress-activated protein kinases) and/or phosphatases, (iii) mobilization of diverse transcription factors - with nuclear factor kappaB (NF-kappaB), AP-1 and STAT-1 probably playing key roles for beta-cell apoptosis; (iv) up-regulation or down-regulation of gene transcription. Recent data obtained by microarray and proteomic analysis suggest that the process of beta-cell apoptosis depends on the parallel and/or sequential up-regulation and down-regulation of considerable numbers of genes, which can be grouped in gene modules or patterns according to their functions. A detailed characterization of these "gene modules", and of the signalling pathways and transcription factors regulating them could allow us to understand the ultimate mechanisms leading to beta-cell apoptosis.

Animals↗
PubMed2001

Double-stranded ribonucleic acid (RNA) induces beta-cell Fas messenger RNA expression and increases cytokine-induced beta-cell apoptosis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.

Animals↗
PubMed2001

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

Adenoviridae↗
PubMed2001

beta-cell apoptosis and defense mechanisms: lessons from type 1 diabetes.

Increased evidence suggests that apoptosis is the main mode of beta-cell death in early type 1 diabetes. Cytokines mediate beta-cell apoptosis, and in this article, we discuss some of the cytokine-modified genes that may contribute to beta-cell survival or death. The gene encoding for the inducible form of nitric oxide synthase is induced by interleukin (IL)-1beta or IL-1beta plus gamma-interferon in rodent and human islets, respectively. This leads to nitric oxide (NO) formation, which contributes to a major extent to beta-cell necrosis and to a minor extent to the process of beta-cell apoptosis. The main mode of cell death induced by cytokines in human beta-cells is apoptosis, whereas cytokines lead to both necrosis and apoptosis in rat and mouse beta-cells. It is suggested that the necrotic component in rodent islets is due to NO-induced mitochondrial impairment and consequent decreased ATP production. Human islets, possessing better antioxidant defenses, are able to preserve glucose oxidation and ATP production, and can thus complete the apoptotic program after the death signal delivered by cytokines. We propose that this death signal results from cytokine-induced parallel and/or sequential changes in the expression of multiple proapoptotic and prosurvival genes. The identity of these "gene modules" and of the transcription factors regulating them remains to be established.

Animals↗
PubMed2001

Identification of novel cytokine-induced genes in pancreatic beta-cells by high-density oligonucleotide arrays.

Type 1 diabetes is an autoimmune disease resulting from the selective destruction of insulin-producing beta-cells. Cytokines may contribute to pancreatic beta-cell death in type 1 diabetes. beta-cell exposure to interleukin (IL)-1beta induces functional impairment, whereas beta-cell culture for 6-9 days in the presence of IL-1beta and interferon (INF)-gamma leads to apoptosis. To clarify the mechanisms involved in these effects of cytokines, we studied the general pattern of cytokine-induced gene expression in beta-cells. Primary rat beta-cells were fluorescence-activated cell sorter-purified and exposed for 6 or 24 h to control condition, IL-1beta + INF-gamma, or IL-1beta alone (24 h only). Gene expression profile was analyzed in duplicate by oligonucleotide arrays. Nearly 3,000 transcripts were detected in controls and cytokine-treated beta-cells. Of these, 96 and 147 displayed changes in expression after 6 and 24 h, respectively, of exposure to IL-1beta + INF-gamma, whereas 105 transcripts were modified after a 24-h exposure to IL-1beta. The cytokine-responsive genes were clustered according to their biological functions. The major clusters observed were metabolism, signal transduction, transcription factors, protein synthesis/ processing, hormones, and related receptors. These modifications in gene expression may explain some of the cytokine effects in beta-cells, such as decreased protein biosynthesis and insulin release. In addition, there was induction of diverse cytokines and chemokines; this suggests that beta-cells may contribute to mononuclear cell homing during insulitis. Several of the cytokine-induced genes are potentially regulated by the transcription factor NF-kappaB. Clarification of the function of the identified cytokine-induced gene patterns may unveil some of the mechanisms involved in beta-cell damage and repair in type 1 diabetes.

Animals↗
PubMed2001

Cytokine induction of Fas gene expression in insulin-producing cells requires the transcription factors NF-kappaB and C/EBP.

Fas-mediated cell death may play a role in the autoimmune destruction of pancreatic beta-cells in type 1 diabetes. beta-Cells do not express Fas under physiological conditions, but Fas mRNA and protein are induced in cytokine-exposed mouse and human islets, rendering the beta-cells susceptible to Fas ligand-induced apoptosis. The aim of the present study was to investigate the molecular regulation of Fas by cytokines in rat beta-cells and in insulin-producing RINm5F cells. Fas mRNA expression was increased 15-fold in fluorescence-activated cell sorting-purified rat beta-cells exposed to interleukin (IL)-1beta, whereas gamma-interferon had no effect. Transfection experiments of rat Fas promoter-luciferase reporter constructs into purified rat beta-cells and RINm5F insulinoma cells identified an IL-1beta-responsive region between nucleotides -223 and -54. Inactivation of two adjacent NF-kappaB and C/EBP sites in this region abolished IL-1beta-induced Fas promoter activity in RINm5F cells. Binding of NF-kappaB and C/EBP factors to their respective sites was confirmed by gel shift assays. In cotransfection experiments, NF-kappaB p65 transactivated the Fas promoter. NF-kappaB p50 and C/EBPbeta overexpression had no effect by themselves on the Fas promoter activity, but when cotransfected with p65, each factor inhibited transactivation by p65. These results suggest a critical role for NF-kappaB and C/EBP factors in cytokine-regulation of Fas expression in insulin-producing cells.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation.

Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause apoptosis of triglyceride-laden pancreatic beta-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent cell death. This "lipotoxicity" hypothesis could explain development of type 2 diabetes in obesity. The present study examines whether prolonged exposure to FFA affects survival of isolated normal rat beta-cells and whether the outcome is related to the occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5-100 nmol/l of unbound oleate and palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation was observed between the percentage of dead beta-cells on day 8 and their cellular triglyceride content on day 2. For equimolar concentrations of the tested FFA, oleate caused the lowest beta-cell toxicity and the highest cytoplasmic triglyceride accumulation. On the other hand, oleate exerted the highest toxicity in islet non-beta-cells, where no FFA-induced triglyceride accumulation was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both in islet beta-cells and non-beta-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent mechanism. The ability of normal beta-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo.

Animals↗
PubMed2000

Interleukin-1beta induces ornithine decarboxylase activity in insulin-producing cells.

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.

Animals↗
PubMed2000

Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression.

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.

Animals↗
PubMed2000

NF-kappaB is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells.

Reactive oxygen species play an important role in the cytotoxic effect of inflammatory cytokines on pancreatic beta-cells in type 1 diabetes mellitus. The antioxidant enzyme manganese superoxide dismutase (MnSOD) is part of the cellular defenses against these deleterious radicals. MnSOD gene expression is induced by cytokines in insulin-producing cells, but the transcriptional regulation of MnSOD expression in these cells is not well understood. In this report, we investigated the transcriptional regulation by cytokines of the rat MnSOD gene in insulin-producing cells. By transient transfections with promoter-luciferase reporter constructs, we identified two interleukin (IL)-1beta-responsive elements, conferring each an additive 3-fold IL-1beta-induced transcriptional activity. The first is located in the promoter region, whereas the second is located in the second intron of the MnSOD gene. Interestingly, the intronic element is required for interferon-gamma-induced potentiation. Site-directed mutagenesis and band-shift assays showed that an NF-kappaB binding site in each region is necessary, but not sufficient, for transcriptional induction by IL-1beta. Our results suggest that NF-kappaB may cooperate with CCAAT/enhancer-binding protein factors in the promoter region and with octamer and Ets factors in the intronic region.

Animals↗
PubMed2000

Interleukin-1beta regulates phospholipase D-1 expression in rat pancreatic beta-cells.

The cytokine interleukin (IL)-1beta induces a biphasic effect in rat pancreatic islets, with an early and transitory stimulation of insulin release followed by progressive functional suppression. To clarify the mechanisms involved in these effects, we have recently performed a differential display of messenger RNA (mRNA) by RT-PCR (DDRT-PCR) on rat beta-cells exposed for 6 or 24 h to IL-1beta. Among the different IL-1beta-induced genes, there was an early and transient increase in phospholipase D-1 (PLD1) expression. PLD1 can induce phosphatidic acid formation and subsequent activation of protein kinase C, a process which stimulates insulin release. In the present study, we characterized the regulation of PLD isoforms by IL-1beta in pancreatic beta-cells. By using different combinations of primers and RT-PCR, we observed that IL-1beta induces an early increase (2 and 6 h) in the expression of both alternatively spliced isoforms of PLD1 (PLD1alpha and 1b). Prolonged exposure to IL-1beta (12 and 24 h) caused a decrease of PLD1a mRNA expression compared with control beta-cells, and lead to a return of PLD1b mRNA to basal level. NG-methyl-L-arginine (LMA), a blocker of the inducible form of nitric oxide synthase (iNOS), prevented this late inhibitory effect of IL-1beta, suggesting that IL-1beta-induced decrease in PLD1a expression is NO-mediated. IL-1beta induced an early (2-6 h) and sustained (16-24 h) increase in PLD1a mRNA expression in insulin-producing RINm5F cells. This was paralleled by a cytokine-induced increase in PLD1 protein expression and enzyme activity. RINm5F cells, but not primary beta-cells, expressed PLD2, and the expression of this gene was not affected by IL-1beta. In conclusion, we have shown that the cytokine IL-1beta regulates PLD1 expression in primary and clonal beta-cells. The early induction of PLD1 probably contributes to the early stimulatory effects of IL-1beta on islet insulin release.

Alternative Splicing↗
PubMed2000

Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells.

Enteroviruses may be involved in the pathogenesis of insulin-dependent diabetes mellitus, either through direct beta-cell infection or as triggers of autoimmunity. In the present study we investigated the patterns of infection in adult human islet cell preparations (consisting of 56+/-14% beta-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells, but only CBVs caused cell death. One week after infection, the insulin response of the beta-cells to glucose or glucose plus theophylline was most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these cells. DNA fragmentation was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3% in controls; P<0.01). CAV-9 infection did not induce DNA fragmentation. One week after infection the majority of infected cells showed characteristics of secondary necrosis. Medium nitrite and inducible nitric oxide synthase messenger ribonucleic acid levels were not significantly up-regulated by CBV infection. These results suggest that several enteroviruses may infect human beta-cells. The infection may result in functional impairment or death of the beta-cell or may have no apparent immediate adverse effects, as shown here for CAV-9. Coxsackie B viruses cause functional impairment and beta-cell death characterized by nuclear pyknosis. Apoptosis appears to play a minor role during a productive CBV infection in beta-cells.

Adult↗