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DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patientsThe first genome-scale DNA methylation study on pancreatic islets from type 2 diabetic patients identifies disease-associated DNA methylation pattern that translate into aberrant gene expression in novel factors relevant for β-cell function and survival.

Pancreatic b-cell dysfunction and death are central in the pathogenesis of type 2 diabetes (T2D). Saturated fatty acids cause b-cell failure and contribute to diabetes development in genetically predisposed individuals. Here we used RNA sequencing to map transcripts expressed in five palmitate-treated human islet preparations, observing 1,325 modified genes. Palmitate induced fatty acid metabolism and endoplasmic reticulum (ER) stress. Functional studies identified novel mediators of adaptive ER stress signaling. Palmitate modified genes regulating ubiquitin and proteasome function, autophagy, and apoptosis. Inhibition of autophagic flux and lysosome function contributed to lipotoxicity. Palmitate inhibited transcription factors controlling b-cell phenotype, including PAX4 and GATA6. Fifty-nine T2D candidate genes were expressed in human islets, and 11 were modified by palmitate. Palmitate modified expression of 17 splicing factors and shifted alternative splicing of 3,525 transcripts. Ingenuity Pathway Analysis of modified transcripts and genes confirmed that top changed functions related to cell death. Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of transcription factor binding sites in palmitate-modified transcripts revealed a role for PAX4, GATA, and the ER stress response regulators XBP1 and ATF6. This human islet transcriptome study identified novel mechanisms of palmitate-induced b-cell dysfunction and death. The data point to cross talk between metabolic stress and candidate genes at the b-cell level.

OBJECTIVE-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 ␤-cell demise and assessed PTPN2 expression in human islets and rat primary and clonal ␤-cells, besides evaluating its role in cytokine-induced signaling and ␤-cell apoptosis.RESEARCH DESIGN AND METHODS-PTPN2 mRNA and protein expression was evaluated by real-time PCR and Western blot. Small interfering (si)RNAs were used to inhibit the expression of PTPN2 and downstream STAT1 in ␤-cells, allowing the assessment of cell death after cytokine treatment.RESULTS-PTPN2 mRNA and protein are expressed in human islets and rat ␤-cells and upregulated by cytokines. Transfection with PTPN2 siRNAs inhibited basal-and cytokine-induced PTPN2 expression in rat ␤-cells and dispersed human islets cells. Decreased PTPN2 expression exacerbated interleukin (IL)-1␤ ϩ interferon (IFN)-␥-induced ␤-cell apoptosis and turned IFN-␥ alone into a proapoptotic signal. Inhibition of PTPN2 amplified IFN-␥-induced STAT1 phosphorylation, whereas double knockdown of both PTPN2 and STAT1 protected ␤-cells against cytokine-induced apoptosis, suggesting that STAT1 hyperactivation is responsible for the aggravation of cytokine-induced ␤-cell death in PTPN2-deficient cells.CONCLUSIONS-We identified a functional role for the type 1 diabetes candidate gene PTPN2 in modulating IFN-␥ signal transduction at the ␤-cell level. PTPN2 regulates cytokine-induced apoptosis and may thereby contribute to the pathogenesis of type 1 diabetes.

OBJECTIVEChronic exposure of pancreatic β-cells to saturated free fatty acids (FFAs) causes endoplasmic reticulum (ER) stress and apoptosis and may contribute to β-cell loss in type 2 diabetes. Here, we evaluated the molecular mechanisms involved in the protection of β-cells from lipotoxic ER stress by glucagon-like peptide (GLP)-1 agonists utilized in the treatment of type 2 diabetes.RESEARCH DESIGN AND METHODSINS-1E or fluorescence-activated cell sorter–purified primary rat β-cells were exposed to oleate or palmitate with or without the GLP-1 agonist exendin-4 or forskolin. Cyclopiazonic acid was used as a synthetic ER stressor, while the activating transcription factor 4–C/EBP homologous protein branch was selectively activated with salubrinal. The ER stress signaling pathways modulated by GLP-1 agonists were studied by real-time PCR and Western blot. Knockdown by RNA interference was used to identify mediators of the antiapoptotic GLP-1 effects in the ER stress response and downstream mitochondrial cell death mechanisms.RESULTSExendin-4 and forskolin protected β-cells against FFAs via the induction of the ER chaperone BiP and the antiapoptotic protein JunB that mediate β-cell survival under lipotoxic conditions. On the other hand, exendin-4 and forskolin protected against synthetic ER stressors by inactivating caspase 12 and upregulating Bcl-2 and X-chromosome–linked inhibitor of apoptosis protein that inhibit mitochondrial apoptosis.CONCLUSIONSThese observations suggest that GLP-1 agonists increase in a context-dependent way the β-cell defense mechanisms against different pathways involved in ER stress–induced apoptosis. The identification of the pathways modulated by GLP-1 agonists allows for targeted approaches to alleviate β-cell ER stress in diabetes.

Aims/hypothesis Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.Methods Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic b-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1b þ IFN-c and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in b-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 b-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-1b þ IFN-c induce b-cell death and the nature of the downstream signals by which ER stress 'convinces' b-cells to trigger apoptosis.

Mutations in human Gli-similar (GLIS) 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD) in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1β + interferon-γ) or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial) pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS.