Purinergic P2X7 receptors regulate secretion of interleukin-1 receptor antagonist and beta cell function and survival

In obesity, beta cells activate compensatory mechanisms to adapt to the higher insulin demand. Interleukin-1 receptor antagonist (IL-1Ra) prevents obesity-induced hyperglycaemia and is a potent target for the treatment of diabetes, but the mechanisms of its secretion and regulation in obesity are unknown. In the present study, we hypothesise the regulation of IL-1Ra secretion by purinergic P2X7 receptors in islets. Production and regulation of P2X7 were studied in pancreatic sections from lean and obese diabetic patients, non-diabetic controls and in isolated islets. IL-1Ra, IL-1β and insulin secretion, glucose tolerance and beta cell mass were studied in P2x7 (also known as P2Rx7)-knockout mice. P2X7 levels were elevated in beta cells of obese patients, but downregulated in patients with type 2 diabetes mellitus. Elevated glucose and non-esterified fatty acids rapidly activated P2X7 and IL-1Ra secretion in human islets, and this was inhibited by P2X7 blockade. In line with our results in vitro, P2x7-knockout mice had a lower capacity to secrete IL-1Ra. They exhibited severe and rapid hyperglycaemia, glucose intolerance and impaired beta cell function in response to a high-fat/high-sucrose diet, were unable to compensate by increasing their beta cell mass in response to the diet and showed increased beta cell apoptosis. Our study shows a tight correlation of P2X7 activation, IL-1Ra secretion and regulation of beta cell mass and function. The increase in P2X7 production is one mechanism that may explain how beta cells compensate by adapting to the higher insulin demand. Disturbances within that system may result in the progression of diabetes

Hyperglycemia Activates Caspase-1 and TXNIP-Mediated IL-1β Transcription in Human Adipose Tissue

Contains fulltext : 96993.pdf (publisher's version ) (Closed access)OBJECTIVE: Obesity is characterized by elevated levels of proinflammatory cytokines, including interleukin (IL)-1beta, that contribute to the development of insulin resistance. In this study, we set out to investigate whether hyperglycemia drives IL-1beta production and caspase-1 activation in murine and human adipose tissue, thus inducing insulin resistance. RESEARCH DESIGN AND METHODS: ob/ob animals were used as a model to study obesity and hyperglycemia. Human adipose tissue fragments or adipocytes were cultured in medium containing normal or high glucose levels. Additionally, the role of thioredoxin interacting protein (TXNIP) in glucose-induced IL-1beta production was assessed. RESULTS: TXNIP and caspase-1 protein levels were more abundantly expressed in adipose tissue of hyperglycemic ob/ob animals as compared with wild-type mice. In human adipose tissue, high glucose resulted in a 10-fold upregulation of TXNIP gene expression levels (P < 0.01) and a 10% elevation of caspase-1 activity (P < 0.05), together with induction of IL-1beta transcription (twofold, P < 0.01) and a significant increase in IL-1beta secretion. TXNIP suppression in human adipocytes, either by a small interfering RNA approach or a peroxisome proliferator-activated receptor-gamma agonist, counteracted the effects of high glucose on bioactive IL-1 production (P < 0.01) mainly through a decrease in transcription levels paralleled by reduced intracellular pro-IL-1beta levels. CONCLUSIONS: High glucose activates caspase-1 in human and murine adipose tissue. Glucose-induced activation of TXNIP mediates IL-1beta mRNA expression levels and intracellular pro-IL-1beta accumulation in adipose tissue. The concerted actions lead to enhanced secretion of IL-1beta in adipose tissue that may contribute to the development of insulin resistance

Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors

Glucose Induces Pancreatic Islet Cell Apoptosis That Requires the BH3-Only Proteins Bim and Puma and Multi-BH Domain Protein Bax

OBJECTIVE: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress. RESEARCH DESIGN AND METHODS: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c. RESULTS: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis. CONCLUSIONS: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed

In Vivo Conditional Pax4 Overexpression in Mature Islet β-Cells Prevents Stress-Induced Hyperglycemia in Mice

OBJECTIVE To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129 W) in β-cells. RESEARCH DESIGN AND METHODS Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129 W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS Pax4 but not Pax4R129 W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129 W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression

Weight Gain in Early Life Predicts Risk of Islet Autoimmunity in Children With a First-Degree Relative With Type 1 Diabetes

OBJECTIVE—In a prospective birth cohort study, we followed infants who had a first-degree relative with type 1 diabetes to investigate the relationship between early growth and infant feeding and the risk of islet autoimmunity

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB)

Benchmark datasets for 3D MALDI- and DESI-imaging mass spectrometry

BACKGROUND: Three-dimensional (3D) imaging mass spectrometry (MS) is an analytical chemistry technique for the 3D molecular analysis of a tissue specimen, entire organ, or microbial colonies on an agar plate. 3D-imaging MS has unique advantages over existing 3D imaging techniques, offers novel perspectives for understanding the spatial organization of biological processes, and has growing potential to be introduced into routine use in both biology and medicine. Owing to the sheer quantity of data generated, the visualization, analysis, and interpretation of 3D imaging MS data remain a significant challenge. Bioinformatics research in this field is hampered by the lack of publicly available benchmark datasets needed to evaluate and compare algorithms. FINDINGS: High-quality 3D imaging MS datasets from different biological systems at several labs were acquired, supplied with overview images and scripts demonstrating how to read them, and deposited into MetaboLights, an open repository for metabolomics data. 3D imaging MS data were collected from five samples using two types of 3D imaging MS. 3D matrix-assisted laser desorption/ionization imaging (MALDI) MS data were collected from murine pancreas, murine kidney, human oral squamous cell carcinoma, and interacting microbial colonies cultured in Petri dishes. 3D desorption electrospray ionization (DESI) imaging MS data were collected from a human colorectal adenocarcinoma. CONCLUSIONS: With the aim to stimulate computational research in the field of computational 3D imaging MS, selected high-quality 3D imaging MS datasets are provided that could be used by algorithm developers as benchmark datasets

Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

BACKGROUND:High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS:Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE:These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role

Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

BACKGROUND: Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3-4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach. CONCLUSIONS: h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes