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Objective: DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PK R-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabe tes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome. Methods: Whole exome sequencing was performed and identified variants we re confirmed by Sanger sequencing. DNAJC3 was silenced by RNAi in INS-1E cells, primary rat β-cells, human islets, and induced pluripotent stem cellderived β-cells. β-cell function and apoptosis were assessed, and potential media tors of apoptosis examined. Results: The two patients presented with juvenile-onset diabetes, short stature, hypothyroidism, neurodegeneration, facial dysmorphism, hypoacusis, microcephaly and skeletal bone deformities. They were heterozygous compound and homozygous for novel loss-of-function mutations in DNAJC3. DNAJC3 silencing did not impair insulin content or secretion. Instead, the knockdown induced rat and human β-cell apoptosis and further sensitized cells to endoplasmic reticulum stress, triggering mitochondrial apoptosis via the pro-apoptototic Bcl-2 proteins BIM and PUMA. Conclusions: This report confirms previously described features and expands the clinical spectrum of syndromic DNAJC3 diabetes, one of the five monogenic forms of diabetes per taining to the PERK pathway of the endoplasmic reticulum stress response. DNAJC3 deficiency may lead to β-cell loss through BIM- and PUMA-dependent activation of the mitochondrial pathway of apoptosis. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

Objective: 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.Research Design and Methods: INS-1E or fluorescence-activated cell sorter-purified primary rat beta-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 beta-cells against FFAs via the induction of the ER chaperone BiP and the antiapoptotic protein JunB that mediate beta-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 beta-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 beta-cell ER stress in diabetes. [ABSTRACT FROM AUTHOR]

The UPR (unfolded protein response) or ER (endoplasmic reticulum) stress response was first described 20 years ago. The field of ER stress has expanded tremendously since, moving from basic biology in yeast to human neurodegenerative, inflammatory, cardiovascular and neoplastic diseases. The ER stress response has also been implicated in diabetes development, affecting both insulin production by pancreatic β-cells and insulin sensitivity in peripheral tissues. In the present mini-review, we focus on recent progress in the field of ER stress in pancreatic β-cells. Recent advances in the understanding of lipotoxic ER stress and β-cell recovery from ER stress are discussed. [ABSTRACT FROM AUTHOR]

beta-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 beta-cell apoptosis by studying the sensitivity of beta-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)-gamma (studied in combination with interleukin [IL]-1beta)-mediated cell death (92 +/- 4% viable cells in STAT-1-/- mice vs. 56 +/- 3% viable cells in wild-type controls, P < or = 0.001) and had preserved insulin release after exposure to IL-1beta and IFN-gamma. Moreover, analysis of cell death in cytokine-exposed purified beta-cells confirmed that protection was due to absence of STAT-1 in the beta-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 3alpha 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 < or = 0.05) and diabetes incidence at the end of the follow-up period (39 vs. 73% in wild-type controls, P < or = 0.05). In conclusion, the present results indicate that STAT-1 is a crucial transcription factor in the process of IFN-gamma-mediated beta-cell death and the subsequent development of immune-mediated diabetes. [ABSTRACT FROM AUTHOR]

Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. We investigated whether advantage could be taken of such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. Control rats or animals injected with streptozotocin (STZ) were infused with solutions of d-glucose mixed with a tracer amount of d-[U-C]glucose, and the radio-active glycogen content of both liver and pancreas was then measured. After 48 h of infusion, the radio-active glycogen content of the pancreas was 30 times lower in STZ rats than in control animals, coinciding with a 50 times lower insulin content. In the control rats, a sizable labeling of pancreatic glycogen was also recorded when d-[U-C]glucose was infused for only the last 4 h of unlabeled d-glucose infusion; such a labeling was not decreased when the animals were further infused for 1 h with only the unlabeled hexose. Moreover, a pronounced difference in the pancreatic gland and blood radioactive content of control rats was still observed when the hyperglycemic animals were killed only 40 min after the iv injection of d-[U-C]glucose. In STZ rats transplanted with islets and later infused with d-[U-C]glucose, the total radioactive content and radioactive glycogen content were both much higher in the transplanted islets than in the pancreatic gland. These results allow one to define the conditions under which the administration of either 2-deoxy-2-[F]fluoro- d-glucose or C-labeled d-glucose could conceivably be used to favor the selective labeling of the endocrine, as distinct from exocrine, pancreas. [ABSTRACT FROM AUTHOR]

The fate of 6-deoxy-6-[I[iodo- d-glucose (6-DIG), injected intravenously, was compared in control rats and animals that had received streptozotocin and were then treated with insulin or not. In the control rats, the measurement of plasma radioactivity suggested that, after an initial and rapid (up to min 10) distribution phenomenon ( K value: 12.2 × 10 min), the clearance of the iodinated hexose occurred mainly by glomerular filtration ( K value: 0.2 × 10 min). Three minutes after the injection of 6-DIG, the radioactive content of muscle, liver, and pancreas, relative to the paired value in blood, was lower in untreated diabetic rats than in control animals. In the case of muscle and liver, such a difference was no longer observed when the treatment of the diabetic rats by insulin resulted in restoration of normoglycemia. In the pancreas, however, the radioactive content, whether expressed relative to the paired blood or liver value, remained significantly lower in the insulin-treated diabetic rats than in the control animals. No significant difference between control and diabetic rats, in terms of pancreatic radioactivity, was observed 10 min after the injection of 6-DIG. These findings indicate that advantage can be taken from the vastly different time course for 6-DIG uptake by pancreatic acinar and islet cells, as recently documented in vitro, to label preferentially the endocrine moiety of the pancreatic gland shortly after 6-DIG injection. [ABSTRACT FROM AUTHOR]