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The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the b-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual b-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat b-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced ap-optosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-kB (NF-kB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticu-lum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls b-cell sensitivity to cytokines possibly by affecting the NF-kB–inducible nitric oxide synthase– endoplasmic reticulum stress pathway., SCOPUS: ar.j, info:eu-repo/semantics/published

The loss of functional pancreatic β-cell mass is an important hallmark of both type 1 and type 2 diabetes. The RNA-binding protein NOVA1 is expressed in human and rodent pancreatic β-cells. Previous in vitro studies indicated that NOVA1 is necessary for glucose-stimulated insulin secretion and its deficiency-enhanced cytokine-induced apoptosis. Moreover, Bim, a proapoptotic protein, is differentially spliced and potentiates apoptosis in NOVA1-deficient β-cells in culture. We generated two novel mouse models by Cre-Lox technology lacking Nova1 (βNova1−/−) or Bim (βBim−/−) in β-cells. To test the impact of Nova1 or Bim deletion on β-cell function, mice were subjected to multiple low-dose streptozotocin (MLD-STZ)-induced diabetes or high-fat diet-induced insulin resistance. β-cell-specific Nova1 or Bim deficiency failed to affect diabetes development in response to MLD-STZ-induced β-cell dysfunction and death evidenced by unaltered blood glucose levels and pancreatic insulin content. In addition, body composition, glucose and insulin tolerance test, and pancreatic insulin content were indistinguishable between control and βNova1−/− or βBim−/− mice on a high fat diet. Thus, Nova1 or Bim deletion in β-cells does not impact on glucose homeostasis or diabetes development in mice. Together, these data argue against an in vivo role for the Nova1-Bim axis in β-cells.

The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D) suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the β-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycaemic control and residual β-cell function during first year after diagnosis. In INS-1E cells and rat and human islets, pro-inflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat b-cells, suggesting an anti-apoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced apoptosis which correlated with reduced nuclear content of S536-phosphorylated NFκB subunit p65, lower nitric oxide production and diminished C/EBP-homologues protein (CHOP) expression indicative of decreased endoplasmic reticulum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls β-cell sensitivity to cytokines possibly by affecting the NFκB-iNOS-ER stress pathway.

The prevalence of diabetes has reached 8.8% in worldwide population and is predicted to increase up to 10.4% by 2040. Thus, there is an urgent need for the development of means to treat or prevent this major disease. Due to its role in inflammatory responses, several studies demonstrated the importance of the transcription factor nuclear factor-κB (NF-κB) in both type 1 diabetes (T1D) and type 2 diabetes (T2D). The two major NF-κB pathways are the canonical and the non-canonical. The later pathway is activated by the NF-κB-inducing kinase (NIK) that triggers p100 processing into p52, which forms with RelB its main dimer. Cytokines mediating the activation of this pathway are present in the serum of T1D and T2D patients. Conversely, limited information is available regarding the role of the alternative pathway on diabetes development and β-cell fate. In the present review, we will briefly describe the involvement of NF-κB on diabetes pathology and discuss new studies indicating an important role for the non-canonical NF-κB activation in β-cell function and survival. The non-canonical NF-κB pathway is emerging as a novel potential target for the development of therapeutic strategies to treat or prevent diabetes.

Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.

Activation of the transcription factor nuclear factor kappa B (NFkB) contributes to β-cell death in type 1 diabetes (T1D). Genome-wide association studies have identified the gene TNF-induced protein 3 (TNFAIP3), encoding for the zinc finger protein A20, as a susceptibility locus for T1D. A20 restricts NF-κB signaling and has strong antiapoptotic activities in β-cells. Although the role of A20 on NF-κB inhibition is well characterized, its other antiapoptotic functions are largely unknown. By studying INS-1E cells and rat dispersed islet cells knocked down or overexpressing A20 and islets isolated from the β-cell-specific A20 knockout mice, we presently demonstrate that A20 has broader effects in β-cells that are not restricted to inhibition of NF-κB. These involves, suppression of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), activation of survival signaling via v-akt murine thymoma viral oncogene homolog (Akt) and consequently inhibition of the intrinsic apoptotic pathway. Finally, in a cohort of T1D children, we observed that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predicted lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating reduced residual β-cell function and impaired glycemic control. In conclusion, our results indicate a critical role for A20 in the regulation of β-cell survival and unveil novel mechanisms by which A20 controls β-cell fate. Moreover, we identify the single nucleotide polymorphism rs2327832 of TNFAIP3 as a possible prognostic marker for diabetes outcome in children with T1D.

Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca2+ extrusion—the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. Biological and morphological methods (Ca2+ imaging, Ca2+ uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. Pmca (isoform 2) heterozygous inactivation increased intracellular Ca2+ stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. The present data indicates that inhibition of Ca2+ extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca2+ extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

An important feature of type 2 diabetes is a decrease in β-cell mass. Therefore, it is essential to find new approaches to stimulate β-cell proliferation. We have previously shown that heterozygous inactivation of the Na+/Ca2+ exchanger (isoform 1; NCX1), a protein responsible for Ca2+ extrusion from cells, increases β-cell proliferation, mass, and function in mice. Here, we show that Ncx1 inactivation also increases β-cell proliferation in 2-year-old mice and that NCX1 inhibition in adult mice by four small molecules of the benzoxyphenyl family stimulates β-cell proliferation both in vitro and in vivo. NCX1 inhibition by small interfering RNA or small molecules activates the calcineurin/nuclear factor of activated T cells (NFAT) pathway and inhibits apoptosis induced by the immunosuppressors cyclosporine A (CsA) and tacrolimus in insulin-producing cell. Moreover, NCX1 inhibition increases the expression of β-cell-specific genes, such as Ins1, Ins2, and Pdx1, and inactivates/downregulates the tumor suppressors retinoblastoma protein (pRb) and miR-193a and the cell cycle inhibitor p53. Our data show that Na+/Ca2+ exchange is a druggable target to stimulate β-cell function and proliferation. Specific β-cell inhibition of Na+/Ca2+ exchange by phenoxybenzamyl derivatives may represent an innovative approach to promote β-cell regeneration in diabetes and improve the efficiency of pancreatic islet transplantation for the treatment of the disease., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

Cell-to-cell communication mediated by gap junctions made of Connexin36 (Cx36) contributes to pancreatic β-cell function. We have recently demonstrated that Cx36 also supports β-cell survival by a still unclear mechanism. Using specific Cx36 siRNAs or adenoviral vectors, we now show that Cx36 downregulation promotes apoptosis in INS-1E cells exposed to the pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) involved at the onset of type 1 diabetes, whereas Cx36 overexpression protects against this effect. Cx36 overexpression also protects INS-1E cells against endoplasmic reticulum (ER) stress-mediated apoptosis, and alleviates the cytokine-induced production of reactive oxygen species, the depletion of the ER Ca(2+) stores, the CHOP overexpression and the degradation of the anti-apoptotic protein Bcl-2 and Mcl-1. We further show that cytokines activate the AMP-dependent protein kinase (AMPK) in a NO-dependent and ER-stress-dependent manner and that AMPK inhibits Cx36 expression. Altogether, the data suggest that Cx36 is involved in Ca(2+) homeostasis within the ER and that Cx36 expression is downregulated following ER stress and subsequent AMPK activation. As a result, cytokine-induced Cx36 downregulation elicits a positive feedback loop that amplifies ER stress and AMPK activation, leading to further Cx36 downregulation. The data reveal that Cx36 plays a central role in the oxidative stress and ER stress induced by cytokines and the subsequent regulation of AMPK activity, which in turn controls Cx36 expression and mitochondria-dependent apoptosis of insulin-producing cells.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to β-cell death in type 1 diabetes (T1D). MCL-1 is an anti-apoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent β-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from T1D patients. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human β-cells, a process partially prevented by MCL-1 overexpression. By generating a β-cell specific Mcl-1 knockout mouse strain (βMcl-1KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. β-cells from βMcl-1KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover, βMcl-1KO mice displayed higher hyperglycaemia and lower pancreatic insulin content after multiple low dose streptozotocin treatment. We found that the kinase GSK3β, the E3 ligases MULE and βTrCP and the deubiquitinase USP9x, regulate cytokine-mediated MCL-1 protein turnover in rodent β-cells. Our results identify MCL-1 as a critical pro-survival protein for preventing β-cell death and clarify the mechanisms behind its downregulation by pro-inflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance β-cell survival and thereby delay or prevent disease progression., SCOPUS: ar.j, info:eu-repo/semantics/published

Protein synthesis is increased by several-fold in stimulated pancreatic beta cells. Synthesis and folding of (pro)insulin takes place in the endoplasmic reticulum (ER), and beta cells trigger the unfolded protein response (UPR) to upgrade the functional capacity of the ER. Prolonged or excessive UPR activation contributes to beta cell dysfunction and death in type 2 diabetes, but there is another side of the UPR that may be of particular relevance for autoimmune type 1 diabetes, namely, the cross-talk between the UPR and innate immunity/inflammation. Recent evidence, discussed in this review, indicates that both saturated fats and inflammatory mediators such as cytokines trigger the UPR in pancreatic beta cells. The UPR potentiates activation of nuclear factor κB, a key regulator of inflammation. Two branches of the UPR, namely IRE1/XBP1s and PERK/ATF4/CHOP, mediate the UPR-induced sensitisation of pancreatic beta cells to the proinflammatory effects of cytokines. This can contribute to the upregulation of local inflammatory mechanisms and the aggravation of insulitis. The dialogue between the UPR and inflammation may provide an explanation for the parallel increase in the prevalence of childhood obesity and type 1 diabetes.

Type 1 diabetes is a chronic autoimmune disease involving the progressive loss of beta cell mass. Cytokines released by immune cells are early contributors to beta cell apoptosis. Thus, an understanding of the signal transduction mechanisms induced by cytokines in beta cells is necessary for the rational design of novel therapies to prevent or to cure this disease. Cytokine-mediated beta cell apoptosis is a complex phenomenon that includes activation of the transcription factors signal transducer and activator of transcription 1 and nuclear factor κB (NFκB), c-Jun N-terminal kinase, endoplasmic reticulum (ER) stress and the intrinsic mitochondrial apoptotic pathway. NFκB has both a pro-inflammatory and a pro-apoptotic role in beta cells. One of the mechanisms by which NFκB contributes to beta cell apoptosis is via activation of ER stress. The role for ER stress in beta cell apoptosis is not completely clarified but involves production of C/EBP homologous protein and activation of the intrinsic mitochondrial apoptotic pathway. In this issue of Diabetologia, Roggli et al (DOI 10.1007/s00125-011-2399-7) report on a new player in this elaborate response, the RNA-binding protein ARE/poly(U)-binding factor 1. This commentary discusses these findings and their relevance to the field.

Ca2+ may trigger apoptosis in beta-cells. Hence, the control of intracellular Ca2+ may represent a potential approach to prevent beta-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma-membrane Ca2+-ATPase (PMCA) overexpression on Ca2+-regulated apoptosis in clonal beta-cells. Clonal beta-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial Ca2+ concentration [Ca2+] using a combination of aequorins with different Ca2+ affinities, and on the endoplasmic-reticulum (ER) and mitochondrial pathways of apoptosis. beta-cell stimulation generated microdomains of high [Ca2+] in the cytosol and subcellular heterogeneities in [Ca2+] among mitochondria. Overexpression of PMCA decreased [Ca2+] in the cytosol, the ER and the mitochondria. PMCA overexpression activated the IRE1alpha-XBP1s but inhibited the PERK-eiF2alpha-CHOP and the ATF6-BiP pathways of the ER unfolded protein response. Increased Bax/Bcl-2 expression ratio (proapoptotic/antiapoptotic Bcl-2 family members) was observed in PMCA overexpressing beta-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, loss of mitochondrial membrane potential and apoptosis. In conclusion, clonal beta-cell stimulation generates microdomains of high [Ca2+] in the cytosol and subcellular heterogeneities in [Ca2+] among mitochondria. PMCA overexpression depletes intracellular [Ca2+] stores and, despite a decrease in mitochondrial [Ca2+], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca2+ homeostasis that could decrease beta-cell apoptosis in diabetes., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Exposure of insulin-secreting β-cells to inflammatory cytokines or high concentrations of free fatty acids, factors involved in the pathogenesis of type 1 and type 2 diabetes, leads to endoplasmic reticulum (ER) stress, β-cell dysfunction, and eventually apoptotic β-cell death. The aim of this study was to investigate the impact of ER stress on β-cells at the protein level to evaluate the contribution of post-transcriptional and post-translational changes in ER stress-induced β-cell damage. INS-1E cells were exposed in vitro to the ER-stress inducer cyclopiazonic acid (CPA) at two concentrations, and protein changes were evaluated using 2D-DIGE. CPA, 25 μM, led to massive apoptosis, accompanied by a near complete protein translation shut-down. CPA, 6.25 μM, led to adaptation of the β-cells to ER stress. Identification of the differentially expressed proteins in the two conditions led to the discovery of a clear pattern of defense pathways, with post-translational modifications playing a crucial role. Key alterations included inhibition of insulin translation and post-translational modifications in ER chaperones HYOU1 and HSPA5. Also, a central role for 14-3-3 proteins is suggested. In conclusion, INS-1E cells are highly sensitive to ER stress, leading to important post-transcriptional and post-translational modifications that may contribute to β-cell dysfunction and death.

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.

The pathogenesis of type 1 diabetes has a strong genetic component. Genome-wide association scans recently identified novel susceptibility genes including the phosphatases PTPN22 and PTPN2. We hypothesized that PTPN2 plays a direct role in beta-cell demise and assessed PTPN2 expression in human islets and rat primary and clonal beta-cells, besides evaluating its role in cytokine-induced signaling and beta-cell apoptosis., info:eu-repo/semantics/published

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.

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of diabetes, contributing to pancreatic beta-cell loss and insulin resistance. Components of the unfolded protein response (UPR) play a dual role in beta-cells, acting as beneficial regulators under physiological conditions or as triggers of beta-cell dysfunction and apoptosis under situations of chronic stress. Novel findings suggest that "what makes a beta-cell a beta-cell", i.e., its enormous capacity to synthesize and secrete insulin, is also its Achilles heel, rendering it vulnerable to chronic high glucose and fatty acid exposure, agents that contribute to beta-cell failure in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response and which defenses are triggered by beta-cells against these challenges. ER stress may also link obesity and insulin resistance in type 2 diabetes. High fat feeding and obesity induce ER stress in liver, which suppresses insulin signaling via c-Jun N-terminal kinase activation. In vitro data suggest that ER stress may also contribute to cytokine-induced beta-cell death. Thus, the cytokines IL-1beta and interferon-gamma, putative mediators of beta-cell loss in type 1 diabetes, induce severe ER stress through, respectively, NO-mediated depletion of ER calcium and inhibition of ER chaperones, thus hampering beta-cell defenses and amplifying the proapoptotic pathways. A better understanding of the pathways regulating ER stress in beta-cells may be instrumental for the design of novel therapies to prevent beta-cell loss in 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.

SCOPUS: ar.j, info:eu-repo/semantics/published

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.

A tight control of endoplasmic reticulum homeostasis is crucial for beta cell function and survival. We recently described that IL-1beta plus IFN-gamma deplete endoplasmic reticulum Ca2+ stores in beta cells, leading to endoplasmic reticulum stress and apoptosis. IL-1beta alone induced endoplasmic reticulum stress but failed to induce beta cell death, while IFN-gamma alone neither caused endoplasmic reticulum stress nor induced beta cell death. This suggests that IFN-gamma aggravates endoplasmic reticulum stress induced by IL-1beta, eventually triggering apoptosis. Here we tested this hypothesis and the mechanisms involved, by investigating the effects of IFN-gamma on endoplasmic reticulum-stress-induced beta cell apoptosis caused by a specific blocker of the sarcoendoplasmic-reticulum pump Ca2+-ATPase (SERCA).INS-1E cells or beta cells were pretreated with IFN-gamma and then exposed to the SERCA blocker cyclopiazonic acid (CPA) for induction of endoplasmic reticulum stress. Cell death was evaluated by Hoechst 342 and propidium iodide staining. Expression of genes related to endoplasmic reticulum stress was determined by real-time RT-PCR, while activation of the endoplasmic reticulum stress response was determined by analysing X-box binding protein-1 (Xbp1) splicing and using a reporter construct containing five copies of the unfolded protein response element (UPRE).CPA induces endoplasmic reticulum stress and apoptosis in insulin-producing cells. Pretreatment with IFN-gamma decreased the basal level of spliced Xbp1 mRNA, the basal and CPA-induced activity of the UPRE reporter, and the mRNA expression of several endoplasmic reticulum chaperones (Bip, Grp94 and Orp 150) and Sec61a. Furthermore, CPA-induced Chop mRNA expression and beta cell apoptosis were potentiated in cells that had been pretreated with IFN-gamma.CPA-induced endoplasmic reticulum stress and apoptosis is enhanced in IFN-gamma-treated beta cells. These effects are mediated via downregulation of the expression of genes involved in beta cell defence against endoplasmic reticulum stress.

1,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is an immune modulator that prevents experimental autoimmune diseases. Receptors for 1,25-(OH)(2)D(3) are present in pancreatic beta-cells, the target of an autoimmune assault in nonobese diabetic (NOD) mice. The aim of this study was to investigate the in vivo and in vitro effects of 1,25-(OH)(2)D(3) on beta-cell gene expression and death and correlate these findings to in vivo diabetes development in NOD mice. When female NOD mice were treated with 1,25-(OH)(2)D(3) (5 microg/kg per 2 d), there was a decrease in islet cytokine and chemokine expression, which was accompanied by less insulitis. Complementing these findings, we observed that exposure to 1,25-(OH)(2)D(3) in three cell systems INS-1(E) cell line, fluorescence-activated cell sorting purified rat beta-cells, and NOD-severe combined immunodeficient islets) suppressed IP-10 and IL-15 expression in the beta-cell itself but did not prevent cytokine-induced beta-cell death. This 1,25-(OH)(2)D(3)-induced inhibition of chemokine expression in beta-cells was associated with a decreased diabetes incidence in some treatment windows targeting early insulitis. Thus, although a short and early intervention with 1,25-(OH)(2)D(3) (3-14 wk of age) reduced diabetes incidence (35 vs. 58%, P < or = 0.05), a late intervention (from 14 wk of age, when insulitis is present) failed to prevent disease. Of note, only early and long-term treatment (3-28 wk of age) prevented disease to a major extent (more than 30% decrease in diabetes incidence). We conclude that 1,25-(OH)(2)D(3) monotherapy is most effective in preventing diabetes in NOD mice when applied early. This beneficial effect of 1,25-(OH)(2)D(3) is associated with decreased chemokine and cytokine expression by the pancreatic islets.

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.

Apoptosis is probably the main form of beta-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to beta-cell destruction through nuclear factor-kappaB (NF-kappaB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are beta-cell toxic also via NF-kappaB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced beta-cell apoptosis and determine whether TNFalpha, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-kappaB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified beta-cells by oleate, palmitate, and/or cytokines (IL-1beta, interferon-gamma, TNFalpha). Palmitate and IL-1beta induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFalpha did not potentiate FFA toxicity in primary beta-cells. The NF-kappaB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1beta but not by FFAs. Cytokines activated NF-kappaB in INS-1E and beta-cells, but FFAs did not. Moreover, FFAs did not enhance NF-kappaB activation by TNFalpha. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced beta-cell death but the mechanisms involved are different. Whereas cytokines induce NF-kappaB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-kappaB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of beta-cell death in T1DM and T2DM.

The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

Pancreatic beta-cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine-induced MCP-1 gene expression in human and rat pancreatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluorescence-activated cell sorting-purified rat beta-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1beta-responsive enhancer region between -2,180 bp and -2,478 bp. Mutation of either of the two nuclear factor (NF)-kappaB sites present in this region abrogated IL-1beta-induced MCP-1 promoter activity. Binding of NF-kappaB to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/p50 heterodimers and p65 homodimers. In vivo binding of NF-kappaB was confirmed by chromatin immunoprecipitation assay. Blocking of NF-kappaB activation in cytokine-exposed primary beta-cells by an adenovirus overexpressing a nondegradable form of IkappaBalpha or by pyrrolidine dithiocarbamate decreased IL-1beta-induced MCP-1 mRNA expression. We conclude that NF-kappaB plays an important role for MCP-1 expression in beta-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.

Viral infections may trigger the autoimmune assault leading to type 1 diabetes mellitus. Double-stranded RNA (dsRNA) is produced by many viruses during their replicative cycle. The dsRNA, tested as synthetic poly(IC) (PIC), in synergism with the proinflammatory cytokines interferon-gamma (IFN-gamma) and/or IL-1 beta, results in nitric oxide production, Fas expression, beta-cell dysfunction, and death. Activation of the transcription nuclear factor-kappa B (NF-kappa B) is required for PIC-induced inducible nitric oxide synthase expression in beta-cells, and we hypothesized that this transcription factor may also participate in PIC-induced Fas expression and beta-cell apoptosis. This hypothesis, and the possibility that PIC induces expression of additional chemokines and cytokines (previously reported as NF-kappa B dependent) in pancreatic beta-cells, was investigated in the present study. We observed that the PIC-responsive region in the Fas promoter is located between nucleotides -223 and -54. Site-directed mutations at the NF-kappa B and CCAAT/enhancer binding protein-binding sites prevented PIC-induced Fas promoter activity. Increased Fas promoter activity was paralleled by enhanced susceptibility of PIC + cytokine-treated beta-cells to apoptosis induced by Fas ligand. beta-Cell infection with the NF-kappa B inhibitor AdI kappa B((SA)2) prevented both necrosis and apoptosis induced by PIC + IL-1 beta or PIC + IFN-gamma. Messenger RNAs for several chemokines and one cytokine were induced by PIC, alone or in combination with IFN-gamma, in pancreatic beta-cells. These included IP-10, interferon-gamma-inducible protein-10, IL-15, macrophage chemoattractant protein-1, fractalkine, and macrophage inflammatory protein-3 alpha. There was not, however, induction of IL-1 beta expression. We propose that dsRNA, generated during a viral infection, may contribute for beta-cell demise by both inducing expression of chemokines and IL-15, putative contributors for the build-up of insulitis, and by synergizing with locally produced cytokines to induce beta-cell apoptosis. Activation of the transcription factor NF-kappa B plays a central role in at least part of the deleterious effects of dsRNA in pancreatic beta-cells.

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.

As reported in an issue of Journal of Proteome Research, mass spectrometry has been used to identify numerous proteins in pancreatic islets. Our group studies beta cell gene expression, and we were interested in whether proteins described in this study could be found at the level of the transcriptome. Microarray analysis is a powerful technique for quantitative measurements of the expression of thousand genes in parallel. However, in crude tumor biopsies, only a subset of transcripts correlate with protein levels, and it is still unknown how frequently mRNA expression correlates with amount of protein in well-differentiated cells. To address this issue, we presently compared data from mouse primary islet proteins obtained by proteomic analysis with RNA data from FACS purified primary rat beta cells obtained by microarray analysis (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation).

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress has been implicated in the development of type 2 diabetes, via effects on obesity, insulin resistance and pancreatic beta cell health. C/EBP homologous protein (CHOP) is induced by ER stress and has a central role in apoptotic execution pathways triggered by ER stress. The aim of this study was to characterise the role of CHOP in obesity and insulin resistance. METHODS: Metabolic studies were performed in Chop ( -/- ) and wild-type C57Bl/6 mice, and included euglycaemic-hyperinsulinaemic clamps and indirect calorimetry. The inflammatory state of liver and adipose tissue was determined by quantitative RT-PCR, immunohistology and macrophage cultures. Viability and absence of ER stress in islets of Langerhans was determined by electron microscopy, islet culture and quantitative RT-PCR. RESULTS: Systemic deletion of Chop induced abdominal obesity and hepatic steatosis. Despite marked obesity, Chop ( -/- ) mice had preserved normal glucose tolerance and insulin sensitivity. This discrepancy was accompanied by lower levels of pro-inflammatory cytokines and less infiltration of immune cells into fat and liver. CONCLUSIONS/INTERPRETATION: These observations suggest that insulin resistance is not induced by fat accumulation per se, but rather by the inflammation induced by ectopic fat. CHOP may play a key role in the crosstalk between excessive fat deposition and induction of inflammation-mediated insulin resistance., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2a phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections., SCOPUS: ar.j, info:eu-repo/semantics/published

OBJECTIVE We have previously shown that overexpression of the Na-Ca exchanger (NCX1), a protein responsible for Ca(2+) extrusion from cells, increases β-cell programmed cell death (apoptosis) and reduces β-cell proliferation. To further characterize the role of NCX1 in β-cells under in vivo conditions, we developed and characterized mice deficient for NCX1. RESEARCH DESIGN AND METHODS Biologic and morphologic methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release, and point counting morphometry) were used to assess β-cell function in vitro. Blood glucose and insulin levels were measured to assess glucose metabolism and insulin sensitivity in vivo. Islets were transplanted under the kidney capsule to assess their performance to revert diabetes in alloxan-diabetic mice. RESULTS Heterozygous inactivation of Ncx1 in mice induced an increase in glucose-induced insulin release, with a major enhancement of its first and second phase. This was paralleled by an increase in β-cell proliferation and mass. The mutation also increased β-cell insulin content, proinsulin immunostaining, glucose-induced Ca(2+) uptake, and β-cell resistance to hypoxia. In addition, Ncx1(+/-) islets showed a two- to four-times higher rate of diabetes cure than Ncx1(+/+) islets when transplanted into diabetic animals. CONCLUSIONS Downregulation of the Na/Ca exchanger leads to an increase in β-cell function, proliferation, mass, and resistance to physiologic stress, namely to various changes in β-cell function that are opposite to the major abnormalities seen in type 2 diabetes. This provides a unique model for the prevention and treatment of β-cell dysfunction in type 2 diabetes and after islet transplantation., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca2+ depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) contributes to β-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca2+ depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor κB (NFκB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFκB activation, as assessed by IκB degradation and NFκB DNA binding. Inhibition of NFκB or the Bcl-2 family member Bax pathways protected β-cells against TG- but not SNAP-induced β-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFκB and Bax.

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.

Type 1 diabetes mellitus (T1D) is characterized by severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. The triggering of autoimmunity against the beta-cells is probably caused by environmental agent(s) acting in the context of a predisposing genetic background. Once activated, the immune cells invade the islets and mediate their deleterious effects on beta-cells via mechanisms such as Fas/FasL, perforin/granzyme, reactive oxygen and nitrogen species and pro-inflammatory cytokines. Binding of cytokines to their receptors on the beta-cells activates MAP-kinases and the transcription factors STAT-1 and NFkappa-B, provoking functional impairment, endoplasmic reticulum stress and ultimately apoptosis. This review discusses the potential mediators and mechanisms leading to beta-cell destruction in T1D., Journal Article, Research Support, Non-U.S. Gov't, Review, info:eu-repo/semantics/published

Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors—improving β-cell resistance against immune attack—is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 ± 4.1% at MOI 50 in whole islets to 80.0 ± 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive β-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of β-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.

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.