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Progressive failure of insulin-producing β-cells is the central event leading to diabetes, but the signaling networks controlling β-cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining the function and survival of human β-cells. RNA sequencing analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling. In particular, SRp55-mediated splicing changes modulate the function of the proapoptotic proteins BIM and BAX, JNK signaling, and endoplasmic reticulum stress, explaining why SRp55 depletion triggers β-cell apoptosis. Furthermore, SRp55 depletion inhibits β-cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human β-cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55, that may cross talk with candidate genes for diabetes. [ABSTRACT FROM AUTHOR]

We describe a new syndrome characterized by early-onset diabetes associated with bone marrow failure, affecting mostly the erythrocytic lineage. Using whole-exome sequencing in a remotely consanguineous patient from a family with two affected siblings, we identified a single homozygous missense mutation (chr15.hg19:g.48,626,619A>G) located in the dUTPase (DUT) gene (National Center for Biotechnology Information Gene ID 1854), affecting both the mitochondrial (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) isoforms. We found the same homozygous mutation in an unrelated consanguineous patient with diabetes and bone marrow aplasia from a family with two affected siblings, whereas none of the >60,000 subjects from the Exome Aggregation Consortium (ExAC) was homozygous for this mutation. This replicated observation probability was highly significant, thus confirming the role of this DUT mutation in this syndrome. DUT is a key enzyme for maintaining DNA integrity by preventing misincorporation of uracil into DNA, which results in DNA toxicity and cell death. We showed that DUT silencing in human and rat pancreatic β-cells results in apoptosis via the intrinsic cell death pathway. Our findings support the importance of tight control of DNA metabolism for β-cell integrity and warrant close metabolic monitoring of patients treated by drugs affecting dUTP balance. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is a chronic autoimmune disease characterized by specific destruction of pancreatic [beta]-cells by the immune system. Linkage and genome-wide association studies have identified more than 50 loci across the human genome associated with risk of type 1 diabetes. Recently, basic leucine zipper transcription factor 2 (BACH2) has been associated with genetic risk to develop type 1 diabetes, in an effect ascribed to the immune system. We evaluated whether BACH2 may also play a role in immune-mediated pancreatic [beta]-cell apoptosis. BACH2 inhibition exacerbated cytokine-induced [beta]-cell apoptosis in human and rodent [beta]-cells by the mitochondrial pathway of cell death, whereas BACH2 overexpression had protective effects. BACH2 silencing and exposure to proinflammatory cytokines increased phosphorylation of the proapoptotic protein JNK1 by upregulation of mitogen-activated protein kinase kinase 7 (MKK7) and downregulation of PTPN2. JNK1 increased phosphorylation of the proapoptotic protein BIM, and both JNK1 and BIM knockdown protected [beta]-cells against cytokine-induced apoptosis in BACH2-silenced cells. The present findings suggest that the type 1 diabetes candidate gene BACH2 regulates proinflammatory cytokine-induced apoptotic pathways in pancreatic [beta]-cells by crosstalk with another candidate gene, PTPN2, and activation of JNK1 and BIM. This clarifies an unexpected and relevant mechanism by which BACH2 may contribute to diabetes. [ABSTRACT FROM AUTHOR]

Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60–70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway. [ABSTRACT FROM AUTHOR]

Leptin plays an important role in the control of food intake, energy expenditure, metabolism, and body weight. This hormone also has a key function in the regulation of glucose homeostasis. Although leptin acts through central and peripheral mechanisms to modulate glucose metabolism, the pancreatic &bgr;-cell of the endocrine pancreas is a critical target of leptin actions. Leptin receptors are present in the &bgr;-cell, and their activation directly inhibits insulin secretion from these endocrine cells. The effects of leptin on insulin occur also in the long term, since this hormone inhibits insulin gene expression as well. Additionally, &bgr;-cell mass can be affected by leptin through changes in proliferation, apoptosis, or cell size. All these different functions in the &bgr;-cell are triggered by leptin as a result of the large diversity of signaling pathways that this hormone is able to activate in the endocrine pancreas. Therefore, leptin can participate in glucose homeostasis owing to different levels of modulation of the pancreatic &bgr;-cell population. Furthermore, it has been proposed that alterations in this level of regulation could contribute to the impairment of &bgr;-cell function in obesity states. In the present review, we will discuss all these issues with special emphasis on the effects and pathways of leptin signaling in the pancreatic &bgr;-cell. [ABSTRACT FROM AUTHOR]

Objective: Leptin released from adipocytes plays a key role in the control of food intake, energy balance, and glucose homeostasis. In addition to its central action, leptin directly affects pancreatic beta-cells, inhibiting insulin secretion, and, thus, modulating glucose homeostasis. However, despite the importance of glucagon secretion in glucose homeostasis, the role of leptin in alpha-cell function has not been studied in detail. In the present study, we have investigated this functional interaction.Research Design and Methods: The presence of leptin receptors (ObR) was demonstrated by RT-PCR analysis, Western blot, and immunocytochemistry. Electrical activity was analyzed by patch-clamp and Ca(2+) signals by confocal microscopy. Exocytosis and glucagon secretion were assessed using fluorescence methods and radioimmunoassay, respectively.Results: The expression of several ObR isoforms (a-e) was detected in glucagon-secreting alphaTC1-9 cells. ObRb, the main isoform involved in leptin signaling, was identified at the protein level in alphaTC1-9 cells as well as in mouse and human alpha-cells. The application of leptin (6.25 nmol/l) hyperpolarized the alpha-cell membrane potential, suppressing the electrical activity induced by 0.5 mmol/l glucose. Additionally, leptin inhibited Ca(2+) signaling in alphaTC1-9 cells and in mouse and human alpha-cells within intact islets. A similar result occurred with 0.625 nmol/l leptin. These effects were accompanied by a decrease in glucagon secretion from mouse islets and were counteracted by the phosphatidylinositol 3-kinase inhibitor, wortmannin, suggesting the involvement of this pathway in leptin action.Conclusions: These results demonstrate that leptin inhibits alpha-cell function, and, thus, these cells are involved in the adipoinsular communication. [ABSTRACT FROM AUTHOR]

Abstract: Viral hemorrhagic septicemia virus (VHSV) is one of the most ubiquitous viruses in salmonid aquaculture in Europe. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Thus, accurate methods to test new antiviral compounds need to be developed. Our goal was to establish a model system for testing novel antivirals with potential applications to aquaculture. In a previous study, a TaqMan® real-time RT-PCR assay was designed to detect and quantitate VHSV in rainbow trout tissues [Chico, V., Gomez, N., Estepa, A., Perez, L., 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132, 154–159]. In this report, we applied the real-time RT-PCR assay to the evaluation of the inhibitory effect of ribavirin, a well-known broad spectrum antiviral drug, in a cell culture system. When added from the beginning of the infection, ribavirin caused a dose-dependent reduction of VHSV RNA accumulation. Real-time RT-PCR measurements showed 99.8% inhibition at 25μg/ml ribavirin, with an IC50 of 0.43μg/ml. Ribavirin maintained its inhibitory activity against VHSV when added at 6h post-infection. Quantitation of N protein messenger RNA and plus-stranded RNA showed a substantial decrease of viral transcription in ribavirin-treated cells. Partial reversion of the effect of ribavirin by addition of GTP was observed, confirming that ribavirin targets the synthesis of guanidine nucleotides in the cells. This is the first report of a real-time PCR-based assay for addressing the efficacy and mechanism of action of an antiviral agent for rainbow trout. [Copyright &y& Elsevier]

Pancreatic β cell dysfunction is a key feature of type 2 diabetes, and novel regulators of insulin secretion are desirable. Here, we report that succinate receptor 1 (SUCNR1) is expressed in β cells and is upregulated in hyperglycemic states in mice and humans. We found that succinate acted as a hormone-like metabolite and stimulated insulin secretion via a SUCNR1-GqPKC–dependent mechanism in human β cells. Mice with β cell–specific Sucnr1 deficiency exhibited impaired glucose tolerance and insulin secretion on a high-fat diet, indicating that SUCNR1 is essential for preserving insulin secretion in diet-induced insulin resistance. Patients with impaired glucose tolerance showed an enhanced nutrition-related succinate response, which correlates with the potentiation of insulin secretion during intravenous glucose administration. These data demonstrate that the succinate/SUCNR1 axis is activated by high glucose and identify a GPCR-mediated amplifying pathway for insulin secretion relevant to the hyperinsulinemia of prediabetic states. [ABSTRACT FROM AUTHOR]

The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome. [ABSTRACT FROM AUTHOR]

Obesity is frequently associated with insulin resistance. To compensate for this situation and maintain normoglycaemia, pancreatic beta-cells undergo several morphofunctional adaptations, which result in insulin hypersecretion and hyperinsulinaemia. However, no information exists about pancreatic alpha-cells during this compensatory stage of obesity. Here, we studied alpha-cells in mice fed a high-fat diet (HFD) for 12 weeks. These animals exhibited hyperinsulinaemia and normoglycaemia compared with control animals in addition to hypoglucagonaemia. While the in vivo response of glucagon to hypoglycaemia was preserved in the obese mice, the suppression of glucagon secretion during hyperglycaemia was impaired. Additionally, in vitro glucagon release at low glucose levels and glucagon content in isolated islets were decreased, while alpha-cell exocytosis remained unchanged. Assessment of morphological parameters revealed that alpha-cell area was reduced in the pancreas of the obese mice in association with alpha-cell hypotrophy, increased apoptosis and decreased proliferation. HFD feeding for 24 weeks led to significant deterioration in beta-cell function and glucose homeostasis. Under these conditions, the majority of alpha-cell changes were reversed and became comparable to controls. These findings indicate that pancreatic compensatory adaptations during obesity may also involve pancreatic alpha-cells. Additionally, defects in alpha-cell function during obesity may be implicated in progression to diabetes. [ABSTRACT FROM AUTHOR]

Diabetes mellitus can be subdivided into several categories based on origin and clinical characteristics. The most common forms of diabetes are type 1 (T1D), type 2 diabetes (T2D) and gestational diabetes mellitus (GDM). T1D and T2D are chronic diseases affecting around 537 million adults worldwide and it is projected that these numbers will increase by 12% over the next two decades, while GDM affects up to 30% of women during pregnancy, depending on diagnosis methods. These forms of diabetes have varied origins: T1D is an autoimmune disease, while T2D is commonly associated with, but not limited to, certain lifestyle patterns and GDM can result of a combination of genetic predisposition and pregnancy factors. Despite some pathogenic differences among these forms of diabetes, there are some common markers associated with their development. For instance, gut barrier impairment and inflammation associated with an unbalanced gut microbiota and their metabolites may be common factors in diabetes development and progression. Here, we summarize the microbial signatures that have been linked to diabetes, how they are connected to diet and, ultimately, the impact on metabolite profiles resulting from host-gut microbiota-diet interactions. Additionally, we summarize recent advances relating to promising preventive and therapeutic interventions focusing on the targeted modulation of the gut microbiota to alleviate T1D, T2D and GDM.

Background: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated., Methods: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-βH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib., Findings: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1β, induced PDL1 expression in vitro in human islet cells and EndoC-βH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction., Interpretation: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

We describe a new syndrome characterized by early-onset diabetes associated with bone marrow failure, affecting mostly the erythrocytic lineage. Using whole-exome sequencing in a remotely consanguineous patient from a family with two affected siblings, we identified a single homozygous missense mutation (chr15.hg19:g.48,626,619A>G) located in the dUTPase ( DUT ) gene (National Center for Biotechnology Information Gene ID 1854), affecting both the mitochondrial (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) isoforms. We found the same homozygous mutation in an unrelated consanguineous patient with diabetes and bone marrow aplasia from a family with two affected siblings, whereas none of the >60,000 subjects from the Exome Aggregation Consortium (ExAC) was homozygous for this mutation. This replicated observation probability was highly significant, thus confirming the role of this DUT mutation in this syndrome. DUT is a key enzyme for maintaining DNA integrity by preventing misincorporation of uracil into DNA, which results in DNA toxicity and cell death. We showed that DUT silencing in human and rat pancreatic β-cells results in apoptosis via the intrinsic cell death pathway. Our findings support the importance of tight control of DNA metabolism for β-cell integrity and warrant close metabolic monitoring of patients treated by drugs affecting dUTP balance., (© 2017 by the American Diabetes Association.)

The mechanism by which protein malnutrition impairs glucose-stimulated insulin secretion in the pancreatic beta-cell is not completely known but may be related to alterations in the signaling events involved in insulin release. Here, we aimed to study the stimulus-secretion coupling of beta-cells from mice fed with low-protein (LP) diet or normal-protein (NP) diet for 8 wk after weaning. Patch-clamp measurements in isolated cells showed that beta-cells from LP mice had a resting membrane potential that was more hyperpolarized than controls. Additionally, depolarization and generation of action potentials in response to stimulatory glucose concentrations were also impaired in beta-cells of LP mice. All these alterations in the LP group were most likely attributed to higher ATP-dependent K(+) (K(ATP)) channel activity in resting conditions and lower efficiency of glucose to induce the closure of these channels. Moreover, a Western blot analysis revealed higher protein levels of the sulphonylurea receptor of the K(ATP) channel in islets of LP mice. Because beta-cell Ca(2+) signals depend on electrical activity, intracellular Ca(2+) oscillations were measured by fluorescence microscopy in intact islets, indicating a lower response to glucose in the LP group. Finally, cell-to-cell synchrony of Ca(2+) signals was analyzed by confocal microscopy. Islets from LP mice exhibited a decreased level of coupling among beta-cells, which was probably due to the low expression levels of connexin 36. Therefore, low-protein diet leads to several alterations in the stimulus-secretion coupling of pancreatic beta-cells that might explain the diminished insulin secretion in response to glucose in this malnutrition state.

Atrial natriuretic peptide (ANP) and its guanylyl cyclase-A (GC-A) receptor are being involved in metabolism, although their role in the endocrine pancreas is still greatly unknown. The aim of this work is to study a possible role for the ANP/GC-A system in modulating pancreatic beta-cell function. The results presented here show a direct effect of the GC-A receptor in regulating glucose-stimulated insulin secretion (GSIS) and beta-cell mass. GC-A activation by its natural ligand, ANP, rapidly blocked ATP-dependent potassium (K(ATP)) channel activity, increased glucose-elicited Ca(2+) signals, and enhanced GSIS in islets of Langerhans. The effect in GSIS was inhibited in islets from GC-A knockout (KO) mice. Pancreatic islets from GC-A KO mice responded to increasing glucose concentrations with enhanced insulin secretion compared with wild type (WT). Remarkably, islets from GC-A KO mice were smaller, presented lower beta-cell mass and decreased insulin content. However, glucose-induced Ca(2+) response was more vigorous in GC-A KO islets, and basal K(ATP) channel activity in GC-A KO beta-cells was greatly diminished compared with WT. When protein levels of the two K(ATP) channel constitutive subunits sulfonylurea receptor 1 and Inward rectifier potassium channel 6.2 were measured, both were diminished in GC-A KO islets. These alterations on beta-cell function were not associated with disruption of glucose tolerance or insulin sensitivity in vivo. Glucose and insulin tolerance tests were similar in WT and GC-A KO mice. Our data suggest that the ANP/GC-A system may have a modulating effect on beta-cell function.