Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.

Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumour necrosis factor alpha (TNFalpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNFalpha neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.     

Activation of extracellular signal-regulated kinase (ERK)1/2 contributes to cytokine-induced apoptosis in purified rat pancreatic beta-cells

Cytokines may contribute to beta-cell apoptosis in the early stages of type 1 diabetes mellitus. It has been reported recently that interleukin-1 beta (IL-1 beta) induces activation of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2 in neonatal rat islets. Since these kinases may participate in cytokine-induced apoptosis, we evaluated whether cytokines induce activation of MAPKs in FACS-purified primary rat beta-cells, and whether blockers of p38 and/or ERK1/2 prevent beta-cell death. IL-1 beta, but not interferon-gamma (IFN-gamma), caused phosphorylation of the substrates Elk-1, ATF-2 and hsp25, and the phosphorylation of both Elk-1 and hsp25 were decreased by the p38 blocker SB203580 (p38i) and the MAPK/ERK blocker PD 098059 (MEKi). When added together, p38i and MEKi decreased IL-1 beta-induced nitrite production over 24 hours by 60%, but did not affect IL-1 beta-induced manganese superoxide dismutase (MnSOD) mRNA expression. To test the effects of MAPK inhibitors on beta-cell death by necrosis or apoptosis, these cells were exposed for 6 or 9 days to IL-1 beta + IFN-gamma. This treatment induced cell death, mostly by apoptosis. The MEKi, but not the p38i, significantly decreased cytokine-induced apoptosis, thus decreasing the total number of dead cells. This protection was only partial, suggesting that ERK1/2 activation is not the only mechanism by which cytokines induce beta-cell apoptosis. We conclude that IL-1 beta induces activation of both p38 and ERK1/2, and that ERK1/2 contributes to the pro-apoptotic effects of the cytokine in primary beta-cells.     

IGFs, basic FGF, and glucose modulate proliferation and apoptosis induced by IFNgamma but not by IL-1beta in rat INS-1E beta-cells.

We investigated the effects of glucose and beta-cell growth factors (IGF-I, IGF-II, bFGF) on growth and apoptosis in the presence and absence of apoptosis inducing cytokines (IFNgamma, Il-1beta, TNFalpha). Rat INS-1E beta-cell viability was measured by WST-1 viability assay and cell counting, apoptosis by FACS analysis of annexin-V-FITC and fluorescein-dUTP (TUNEL-staining)-positive cells. Glucose alone maintained INS-1E beta-cell viability at high physiological concentrations (6.2-12.5 mmol/l), addition of IGF-II alone or in combination with bFGF further increased these glucose effects. The cytokines IFNg and IL-1beta, but not TNFalpha strongly induced INS-1E beta-cell apoptosis. Interestingly, glucose alone induced apoptosis at extremely low or very high concentrations. In combination with IFNg, low glucose (1.6 mmol/l) increased apoptosis by 25.6% (1SD 5.0%) and high glucose (50 mmol/l) by 22.8% (1SD 2.8%) compared to 12.5 mmol/l glucose. In contrast, glucose failed to modulate IL-1beta-induced apoptosis. Most importantly, IGF-II and bFGF inhibited apoptosis induced by IFNg, but not by IL-1beta. Therefore, IGF signaling, supported by bFGF and optimal glucose levels, maintains beta-cell viability in vitro. Cytokines IFNg and IL-1beta differentially interfere with intracellular signaling cascades stimulated by IGFs and bFGF or glucose, respectively.     

The imidazoline RX871024 stimulates insulin secretion in pancreatic beta-cells from mice deficient in K(ATP) channel function.

Effects of the imidazoline compound RX871024 on cytosolic free Ca(2+) concentration ([Ca(2+)]i) and insulin secretion in pancreatic beta-cells from SUR1 deficient mice have been studied. In beta-cells from wild-type mice RX871024 increased [Ca(2+)]i by blocking ATP-dependent K(+)-current (K(ATP)) and inducing membrane depolarization. In beta-cells lacking a component of the K(ATP)-channel, SUR1 subunit, RX871024 failed to increase [Ca(2+)]i. However, insulin secretion in these cells was strongly stimulated by the imidazoline. Thus, a major component of the insulinotropic activity of RX871024 is stimulation of insulin exocytosis independently from changes in K(ATP)-current and [Ca(2+)]i. This means that effects of RX871024 on insulin exocytosis are partly mediated by interaction with proteins distinct from those composing the K(ATP)-channel.     

Imidazoline RX871024 raises diacylglycerol levels in rat pancreatic islets

Imidazoline compound RX871024 and carbamylcholine (CCh) stimulate insulin secretion in isolated rat pancreatic islets. Combination of CCh and RX871024 induces a synergetic effect on insulin secretion. RX871024 and CCh produce twofold increases in diacylglycerol (DAG) concentration. The combination of two compounds has an additive effect on DAG concentration. Effects of RX871024 on insulin secretion and DAG concentration are not dependent on the presence of D609, an inhibitor of phosphatidylcholine-specific phospholipase C. It is concluded that as in case with CCh the increase in DAG concentration induced by imidazoline RX871024 contributes to the insulinotropic activity of the compound.     

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.     

Low protein diet confers resistance to the inhibitory effects of interleukin 1beta on insulin secretion in pancreatic islets*

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1beta (IL-1beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1beta for 1 h or 24 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1beta-induced nitric oxide (NO) production, lower inhibition of D-[U(14)C]-glucose oxidation to (14)CO(2) and less pronounced effect of IL-1beta on alpha-ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K(+) the inhibitory effect of IL-1beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta-cells of the deleterious effect of IL-1beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca(2+) homeostasis.     

Requirements of calcium fluxes and ERK kinase activation for glucose- and interleukin-1β-induced β-cell apoptosis

Increasing evidence indicates that beta-cell apoptosis and impaired secretory function were partly mediated by interleukin (IL)-1beta and/or high-glucose-induced beta-cell production of IL-1beta. However, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this study, we investigated whether Ca(2+) and extracellular signal-regulated kinase (ERK) activation plays a role for IL-1beta action in rat islets. Exposure of rat islets for 4 days to 33.3 mM glucose and 140 ng/ml IL-1beta- induced beta-cell apoptosis and impaired glucose-stimulated insulin secretion. By Western blotting with phosphospecific antibodies, glucose and IL-1beta were shown to activate ERK. Ca(2+) channel blocker nimodipine or ERK inhibitor PD98059 prevented glucose- and IL-1beta-induced ERK activation, beta-cell apoptosis, and impaired function. Furthermore, treatment with Ca(2+) ionophore ionomycin, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced ERK activation in rat islet. On the other hand, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl] (BAPTA/AM) and an inhibitor of calmodulin (W7) diminished IL-1beta-induced phosphorylation of ERK. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, mibefradil, or PD98059. Together, these data suggest that glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent in rat islets.     

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

BACKGROUND INFORMATION: Pancreatic beta-cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL-1beta (interleukin 1beta) induces distinct changes of beta-cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL-1beta and known pathways for IL-1beta actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL-1beta were monitored by measurements of nitrite and PGE(2) (prostaglandin E(2)). RESULTS: IL-1beta caused an enhancement of islet intracellular insulin degradation and an increase in the lysosomal incorporation of beta-cell secretory granules. The effects of IL-1beta were abolished by aminoguanidine, a selective inhibitor of inducible NOS (nitric oxide synthase), or by rofecoxib, a specific inhibitor of COX-2 (cyclo-oxygenase 2). In the absence of IL-1beta, nitroarginine, which is a selective inhibitor of constitutive NOS, caused a decrease in intracellular degradation of insulin in parallel with a decreased production of NO and PGE(2) by the islets. CONCLUSIONS: The correlation between the enhanced intracellular insulin degradation and lysosomal changes caused by IL-1beta suggests that insulin degradation may be effected by crinophagy. Under physiological conditions, significant beta-cell degradation of insulin may depend on the activity of COX-2, possibly stimulated by endogenous NO.     

Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.     

Inhibition of fetal rat pancreatic beta-cell replication by interleukin-1 beta in vitro is not mediated through pertussis toxin-sensitive G-proteins, a decrease in cyclic AMP, or protease activation.

It has been proposed that the cytokine interleukin-1 beta (IL-1 beta), secreted by islet-infiltrating macrophages, may be involved in the pathogenesis of insulin-dependent diabetes mellitus by participation in beta-cell destruction. Addition of IL-1 beta to isolated pancreatic islets in vitro results in cytotoxic effects on beta-cell function, but there is little information on the intracellular events that convey the actions of the cytokine. In the present study, fetal rat pancreatic islets containing a high fraction of beta-cells were exposed in culture to IL-1 beta. It was found that IL-1 beta markedly decreased beta-cell DNA synthesis, insulin secretion and cyclic AMP content. In order to explore whether the decrease in cAMP resulted from IL-1 beta interaction with GTP-binding proteins coupled to adenylyl cyclase, islets were treated for 24 h with pertussis toxin prior to addition of cytokine. While this treatment restored the decrease in cAMP, the reduced DNA synthesis and insulin secretion persisted. Pertussis toxin treatment without the addition of IL-1 beta resulted in increases in cAMP, DNA synthesis and insulin secretion. Addition of the stimulatory cAMP analog Sp-cAMPS also increase DNA synthesis and insulin secretion, but failed to affect the decrease in these functions evoked by IL-1 beta. The protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone, recently shown to protect completely against IL-1 beta-induced suppression of insulin production and secretion, was found to markedly reduce DNA synthesis without affecting insulin secretion. When the protease inhibitor was combined with IL-1 beta, the suppressed secretion was counteracted while DNA synthesis inhibition was not. It is concluded that cAMP stimulates DNA synthesis and insulin secretion in beta-cells, but that the inhibitory effect of IL-1 beta on these functions cannot be ascribed to the decrease in cAMP evoked by the cytokine. However, the repressive effect of the cytokine on insulin secretion, but not DNA synthesis, may be prevented by protease inhibition.     

Pancreatic beta-cell damage mediated by beta-cell production of interleukin-1. A novel mechanism for virus-induced diabetes

Viral infection is one environmental factor that may initiate beta-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta-converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma-induced iNOS expression by primary beta-cells. In addition, poly(I-C) + IFN-gamma-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a nitric oxide-dependent fashion.