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In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of "metabolic memory", just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Diets rich in saturated fats may contribute to the loss of pancreatic β-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes β-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated β-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to β-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient β-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic β-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to β-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to β-cell apoptosis. In conclusion, JunB modulates the β-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic β-cell death. These findings elucidate novel β-cell-protective signal transduction in type 2 diabetes.

Aims/hypothesis Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca 2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)–C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. Methods Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. Results Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. Conclusions/interpretation XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.

Insulin-secreting pancreatic β-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to β-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of β-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of β-cell apoptosis leading to diabetes.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Endoplasmic reticulum stress-mediated apoptosis may play an important role in the destruction of pancreatic beta-cells, thus contributing to the development of type 1 and type 2 diabetes. One of the regulators of endoplasmic reticulum stress-mediated cell death is the CCAAT/enhancer binding protein (C/EBP) homologous protein (Chop). We presently studied the molecular regulation of Chop expression in insulin-producing cells (INS-1E) in response to three pro-apoptotic and endoplasmic reticulum stress-inducing agents, namely the cytokines interleukin-1beta + interferon-gamma, the free fatty acid palmitate, and the sarcoendoplasmic reticulum pump Ca(2+) ATPase blocker cyclopiazonic acid (CPA). Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by CPA, cytokines, and palmitate. Whereas palmitate- and cytokine-induced Chop expression was mediated via a C/EBP-activating transcription factor (ATF) composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the endoplasmic reticulum stress response element. Cytokines, palmitate, and CPA induced eIF2alpha phosphorylation in INS-1E cells leading to activation of the transcription factor ATF4. Chop transcription in response to cytokines and palmitate depends on the binding of ATF4 and AP-1 to the Chop promoter, but distinct AP-1 dimers were formed by cytokines and palmitate. These results suggest a differential response of beta-cells to diverse endoplasmic reticulum stress inducers, leading to a differential regulation of Chop transcription.

Activation of the transcription factor nuclear factor (NF)-κB by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-κB, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-κB activation in beta cells have focused on the canonical pathway. The aim of this study was to investigate whether cytokines activate the non-canonical pathway in beta cells, how this pathway is regulated and the consequences of its activation on beta cell fate. NF-κB signalling was analysed by immunoblotting, promoter reporter assays and real-time RT-PCR, after knockdown or overexpression of key genes/proteins. INS-1E cells, FACS-purified rat beta cells and the human beta cell line EndoC-βH1 exposed to cytokines were used as models. IL-1β plus IFN-γ induced stabilisation of NF-κB-inducing kinase and increased the expression and cleavage of p100 protein, culminating in the nuclear translocation of p52, the hallmark of the non-canonical signalling. This activation relied on different crosstalks between the canonical and non-canonical pathways, some of which were beta cell specific. Importantly, cytokine-mediated activation of the non-canonical pathway controlled the expression of ‘late’ NF-κB-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes. The atypical activation of the non-canonical NF-κB pathway by proinflammatory cytokines constitutes a novel ‘feed-forward’ mechanism that contributes to the particularly pro-apoptotic effect of NF-κB in beta cells.

Cytokines, such as IL-1beta and TNF-alpha, contribute to pancreatic beta-cell death in type 1 diabetes mellitus. The transcription factor nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced beta-cell apoptosis. Paradoxically, NF-kappaB has mostly antiapoptotic effects in other cell types. The cellular actions of NF-kappaB depend on the cell type, the nature and duration of the stimulus, the periodicity, and the degree of activity of the particular dimers involved. To clarify the reasons behind the proapoptotic effects of NF-kappaB in pancreatic beta-cells, we compared the pattern of cytokine-induced NF-kappaB activation between rat insulin-producing cells (INS-1E cells) and fibroblasts (208F cells). NF-kappaB activation was induced in INS-1E cells and in 208F cells after exposure to cytokines, but apoptosis was induced only in INS-1E cells, with a more pronounced proapoptotic effect of IL-1beta than of TNF-alpha. NF-kappaB activation in IL-1beta-exposed INS-1E cells was earlier and more marked as compared with TNF-alpha-exposed INS-1E cells or IL-1beta-exposed 208F cells. Both cytokines induced a prolonged (up to 48 h) and stable NF-kappaB activation in INS-1E cells, whereas IL-1beta induced an oscillatory NF-kappaB activation in 208F cells. p65/p65 and p65/p50 were the predominant NF-kappaB dimers in IL-1beta-exposed INS-1E cells and 208F cells, respectively. IL-1beta induced a differential usage of cis-elements in the inducible nitric oxide synthase promoter region in the two cell-lines and an increase in ERK1/2 activity in INS-1E cells but not in 208F cells. Cytokine-induced expression of IkappaB isoforms and other NF-kappaB target genes (Fas, MCP-1, and inducible nitric oxide synthase) was severalfold higher in INS-1E cells than in 208F cells. These results suggest that cytokine-induced NF-kappaB activation in insulin-producing cells is more rapid, marked, and sustained than in fibroblasts, which correlates with a more pronounced activation of downstream genes and a proapoptotic outcome.

Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). In the present study we show that cytokine-induced apoptosis and necrosis in primary rat beta-cells and INS-1E cells largely depends on NO production. IL-1beta + IFN-gamma, via NO synthesis, markedly decreased SERCA2b protein expression and depleted ER Ca(2+) stores. Of note, beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol-requiring ER-to-nucleus signal kinase 1alpha (IRE1alpha) and PRK (RNA-dependent protein kinase)-like ER kinase (PERK)/activating transcription factor 4 (ATF4), but not ATF6. In contrast, the ER stress-inducing agent thapsigargin triggered these four pathways in parallel. In conclusion, our results suggest that the IL-1beta + IFN-gamma-induced decrease in SERCA2b expression, with subsequent depletion of ER Ca(2+) and activation of the ER stress pathway, is a potential contributory mechanism to beta-cell death.

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.

Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico “phenome–interactome analysis” on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17 . Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β–induced NF-κB activity and protection against IL-1β–mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Type 1 diabetes is an autoimmune disorder characterized by chronic inflammation and pancreatic β-cell loss. Here, we demonstrate that the proinflammatory cytokine interleukin-1β, combined with interferon-γ, induces the expression of the Bcl-2 homology 3 (BH3)-only activator PUMA (p53 up-regulated modulator of apoptosis) in β-cells. Transcriptional activation of PUMAis regulated by nuclear factor-κB and endoplasmic reticulum stress but is independent of p53. PUMA activation leads to mitochondrial Bax translocation, cytochrome c release, and caspase-3 cleavage resulting in β-cell demise. The antiapoptotic Bcl-XL protein is localized mainly at the mitochondria of the β-cells and antagonizesPUMAaction, but Bcl-XL is inactivated by the BH3-only sensitizer DP5/Hrk in cytokine-exposed β-cells. Moreover, a pharmacological mimic of the BH3-only sensitizer Bad, which inhibits Bcl-XL and Bcl-2, induces PUMA dependent β-cell death and potentiates cytokine-induced apoptosis. Our data support a hierarchical activation of BH3-only proteins controlling the intrinsic pathway of β-cell apoptosis in the context of inflammation and type 1 diabetes. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., SCOPUS: ar.j, info:eu-repo/semantics/published

OBJECTIVE: Cytokines contribute to pancreatic beta-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in beta-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes. CONCLUSIONS: The present study doubles the number of known genes expressed in primary beta-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in beta-cells. It also shows that cytokines modify alternative splicing in beta-cells, opening a new avenue of research for the field., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Chronic exposure of pancreatic beta-cells to saturated free fatty acids (FFAs) causes endoplasmic reticulum (ER) stress and apoptosis and may contribute to beta-cell loss in type 2 diabetes. Here, we evaluated the molecular mechanisms involved in the protection of beta-cells from lipotoxic ER stress by glucagon-like peptide (GLP)-1 agonists utilized in the treatment of type 2 diabetes., info:eu-repo/semantics/published

Free fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.

Udgivelsesdato: July AIMS/HYPOTHESIS: IL-1beta and TNF-alpha contribute to pancreatic beta cell death in type 1 diabetes. Both cytokines activate the transcription factor nuclear factor-kappaB (NF-kappaB), but recent observations suggest that NF-kappaB blockade prevents IL-1beta + IFN-gamma- but not TNF-alpha + IFN-gamma-induced beta cell apoptosis. The aim of the present study was to compare the effects of IL-1beta and TNF-alpha on cell death and the pattern of NF-kappaB activation and global gene expression in beta cells. METHODS: Cell viability was measured after exposure to IL-1beta or to TNF-alpha alone or in combination with IFN-gamma, and blockade of NF-kappaB activation or protein synthesis. INS-1E cells exposed to IL-1beta or TNF-alpha in time course experiments were used for IkappaB kinase (IKK) activation assay, detection of p65 NF-kappaB by immunocytochemistry, real-time RT-PCR and microarray analysis. RESULTS: Blocking NF-kappaB activation protected beta cells against IL-1beta + IFNgamma- or TNFalpha + IFNgamma-induced apoptosis. Blocking de novo protein synthesis did not increase TNF-alpha- or IL-1beta-induced beta cell death, in line with the observations that cytokines induced the expression of the anti-apoptotic genes A20, Iap-2 and Xiap to a similar extent. Microarray analysis of INS-1E cells treated with IL-1beta or TNF-alpha showed similar patterns of gene expression. IL-1beta, however, induced a higher rate of expression of NF-kappaB target genes putatively involved in beta cell dysfunction and death and a stronger activation of the IKK complex, leading to an earlier translocation of NF-kappaB to the nucleus. CONCLUSIONS/INTERPRETATION: NF-kappaB activation in beta cells has a pro-apoptotic role following exposure not only to IL-1beta but also to TNF-alpha. The more marked beta cell death induced by IL-1beta is explained at least in part by higher intensity NF-kappaB activation, leading to increased transcription of key target genes.

Free fatty acids cause pancreatic beta-cell apoptosis and may contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum stress. Reductions in eukaryotic translation initiation factor (eIF) 2alpha phosphorylation trigger beta-cell failure and diabetes. Salubrinal selectively inhibits eIF2alpha dephosphorylation, protects other cells against endoplasmic reticulum stress-mediated apoptosis, and has been proposed as a beta-cell protector. Unexpectedly, salubrinal induced apoptosis in primary beta-cells, and it potentiated the deleterious effects of oleate and palmitate. Salubrinal induced a marked eIF2alpha phosphorylation and potentiated the inhibitory effects of free fatty acids on protein synthesis and insulin release. The synergistic activation of the PERK-eIF2alpha branch of the endoplasmic reticulum stress response, but not of the IRE1 and activating transcription factor-6 pathways, led to a marked induction of activating transcription factor-4 and the pro-apoptotic transcription factor CHOP. Our findings demonstrate that excessive eIF2alpha phosphorylation is poorly tolerated by beta-cells and exacerbates free fatty acid-induced apoptosis. This modifies the present paradigm regarding the beneficial role of eIF2alpha phosphorylation in beta-cells and must be taken into consideration when designing therapies to protect beta-cells in type 2 diabetes.