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Type 1 diabetes (T1D) is an autoimmune disease leading to β-cell destruction. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and organ formation. They participate in the pathogenesis of several autoimmune diseases, but the nature of miRNAs contributing to β-cell death in T1D and their target genes remain to be clarified. We performed an miRNA expression profile on human islet preparations exposed to the cytokines IL-1β plus IFN-γ. Confirmation of miRNA and target gene modification in human β-cells was performed by real-time quantitative PCR. Single-stranded miRNAs inhibitors were used to block selected endogenous miRNAs. Cell death was measured by Hoechst/propidium iodide staining and activation of caspase-3. Fifty-seven miRNAs were detected as modulated by cytokines. Three of them, namely miR-23a-3p, miR-23b-3p, and miR-149-5p, were downregulated by cytokines and selected for further studies. These miRNAs were found to regulate the expression of the proapoptotic Bcl-2 proteins DP5 and PUMA and consequent human β-cell apoptosis. These results identify a novel cross talk between a key family of miRNAs and proapoptotic Bcl-2 proteins in human pancreatic β-cells, broadening our understanding of cytokine-induced β-cell apoptosis in early T1D. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Cytokines contribute to pancreatic β-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)-1β + interferon (IFN)-γ and tumor necrosis factor (TNF)-α + IFN-γ in primary rat β-cells. RESEARCH DESIGN AND METHODS--Fluorescence-activated cell sorter-purified rat β-cells were exposed to IL-1β + IFN-γ or TNF-α + IFN-γ 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 β-cells, with temporal differences in the number of genes modulated by IL-1β + IFNγ or TNF-α + IFN-γ. 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 β-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-α, 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 β-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in β-cells. It also shows that cytokines modify alternative splicing in β-cells, opening a new avenue of research for the field. Diabetes 59:358-374, 2010 [ABSTRACT FROM AUTHOR]

OBJECTIVE--Chronic 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 B-cells from lipotoxic ER stress by glucagon-like peptide (GLP)-1 agonists utilized in the treatment of type 2 diabetes. RESEARCH DESIGN AND METHODS--INS-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. RESULTS--Exendin-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. CONCLUSIONS--These 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. Diabetes 58:2851-2862, 2009 [ABSTRACT FROM AUTHOR]

OBJECTIVE--Exposure of β-cells to inflammatory cytokines leads to apoptotic cell death through the activation of gene networks under the control of specific transcription factors, such as interferon-γ-induced signal transducer and activator of transcription (STAT)-1. We previously demonstrated that β-cells lacking STAT-1 are resistant to cytokine-induced cell death in vitro. The aim of this study was to investigate the effect of STAT-1 elimination on immune-mediated β-cell destruction in vivo. RESEARCH DESIGN AND METHODS--Multiple low-dose streptozotocin (STZ) was given to C57BL/6 mice after syngeneic STAT-1[sup -/-] or wild-type islet transplantation. STAT-1[sup -/-] and wild-type islets were also transplanted in alloxan-diabetic BALB/c and spontaneously diabetic nonobese diabetic (NOD) mice. Additionally, mice were treated with interleukin (IL)-1 blockade (IL-1 receptor antagonist [IL-1ra]) and low-dose T-cell suppression (cyclosporine A [CsA]). RESULTS--When exposed to multiple low-dose STZ in an immune-competent host, STAT-1[sup -/-] islets were more resistant to destruction than wild-type islets (28 vs. 100% diabetes incidence, P ≤ 0.05). STAT-1 deletion also protected allogeneic islet grafts against primary nonfunction in autoimmune NOD mice (0 vs. 17% using wild-type islets). However, no difference in survival time was observed. Additionally, treating recipients with IL-1ra and CsA prolonged graft survival in chemically diabetic BALB/c mice, whereas no difference was seen between STAT-1[sup -/-] and C57BL/6 grafts. CONCLUSIONS--These data indicate that STAT-1 is a key player in immune-mediated early [β-cell dysfunction and death. When considering the many effector mechanisms contributing to β-cell death following islet transplantation, multiple combined interventions will be needed for prolongation of β-cell survival in the autoimmune context of type 1 diabetes. Diabetes 56: 2169-2173, 2007 [ABSTRACT FROM AUTHOR]

β-Cells under immune attack are destroyed by the aberrant activation of key intracellular signaling cascades. The aim of the present study was to evaluate the contribution of the signal transducer and activator of transcription (STAT)-1 pathway for β-cell apoptosis by studying the sensitivity of β-cells from STAT-1 knockout (-/-) mice to immune-mediated cell death in vitro and in vivo. Whole islets from STAT-1-/- mice were completely resistant to interferon (IFN)-γ (studied in combination with interleukin [IL]-1β)-mediated cell death (92 ± 4% viable cells in STAT-1-/- mice vs. 56 ± 3% viable cells in wild-type controls, P ≤ 0.001) and had preserved insulin release after exposure to IL-1β and IFN-γ. Moreover, analysis of cell death in cytokine-exposed purified β-cells confirmed that protection was due to absence of STAT-1 in the β-cells themselves. Deficiency of STAT-1 in islets completely prevented cytokine-induced upregulation of IL-15, interferon inducible protein 10, and inducible nitric oxide synthase transcription but did not interfere with monocyte chemoattractant protein 1 and macrophage inflammatory protein 3α expression. In vivo, STAT-1-/- mice were partially resistant to development of diabetes after multiple low-dose streptozotocin injections as reflected by mean blood glucose at 12 days after first injection (159 ± 28 vs. 283 ± 81 mg/dl in wild-type controls, P ≤ 0.05) and diabetes incidence at the end of the follow-up period (39 vs. 73% in wild-type controls, P ≤ 0.05). In conclusion, the present results indicate that STAT-1 is a crucial transcription factor in the process of IFN-γ-mediated β-cell death and the subsequent development of immune-mediated diabetes. Diabetes 54:2396-2403, 2005 [ABSTRACT FROM AUTHOR]