Induction of insulin secretion in engineered liver cells by nitric oxide.
ABSTRACT: Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway.Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide.Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.
Project description:Treatment of pancreatic islets with interleukin 1 (IL-1) results in a time-dependent inhibition of glucose-stimulated insulin secretion which has recently been demonstrated to be dependent on the metabolism of L-arginine to nitric oxide. In this report IL-1 beta is shown to induce the accumulation of cyclic GMP (cGMP) in a time-dependent fashion that mimics the time-dependent inhibition of insulin secretion by IL-1 beta. The accumulation of cGMP is dependent on nitric oxide synthase activity, since NG-monomethyl-L-arginine (a competitive inhibitor of nitric oxide synthase) prevents IL-1 beta-induced cGMP accumulation. cGMP formation and nitrite production induced by IL-1 beta pretreatment of islets are also blocked by the protein synthesis inhibitor, cycloheximide. The formation of cGMP does not appear to mediate the inhibitory effects of IL-1 beta on insulin secretion since a concentration of cycloheximide (1 microM) that blocks IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and nitric oxide formation does not prevent cGMP accumulation, thus dissociating the two events. By using e.p.r. spectroscopy, IL-1 beta is shown to induce the formation of a g = 2.04 iron-nitrosyl feature in islets which is prevented by cycloheximide, demonstrating the requirement of protein synthesis for IL-1 beta-induced nitric oxide formation. Iron-nitrosyl complex-formation by islets confirms that IL-1 beta induces the generation of nitric oxide by islets, and provides evidence indicating that nitric oxide mediates destruction of iron-sulphur clusters of iron-containing enzymes. Consistent with the destruction of iron-sulphur centres is the finding that pretreatment of islets with IL-1 beta results in an approx. 60% inhibition of mitochondrial oxidation of D-glucose to CO2. Inhibition of islet glucose oxidation appears to be mediated by nitric oxide since both NMMA and cycloheximide prevent IL-1 beta-induced inhibition of glucose oxidation. These results show that IL-1 beta-induced nitric oxide formation parallels the ability of IL-1 beta to inhibit glucose-stimulated insulin secretion by islets, and that protein synthesis is required for IL-1 beta-induced nitric oxide formation. These results also suggest that nitric oxide mediates IL-1 beta-induced inhibitory effects on the pancreatic beta-cell by functioning as an effector molecule responsible for the destruction of iron-sulphur centres of iron-containing proteins, resulting in an impairment of mitochondrial function.
Project description:Here, we show that commensal bacteria can stimulate intestinal epithelial cells to secrete insulin in response to glucose. Commensal strains were engineered to secrete the insulinotropic proteins GLP-1 and PDX-1. Epithelia stimulated by engineered strains and glucose secreted up to 1 ng ml(-1) of insulin with no significant background secretion.
Project description:Pancreatic islet beta-cells secrete the hormones insulin, amylin and pancreastatin. To search for further beta-cell hormones, we purified peptides from secretory granules isolated from cultured murine beta TC6-F7 beta-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp(69)-Leu(102) of the proinsulin-like growth factor II E-peptide, which we have termed 'preptin'. Preptin, is present in islet beta-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.
Project description:The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic beta-cells are not completely understood. To identify metabolic disturbances in beta-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal beta-cell lines, which are glucose-responsive (832/13 cells) or glucose-unresponsive (832/2 cells). To this end, we analyzed a number of parameters in glycolytic and mitochondrial metabolism, including mRNA expression of genes involved in cellular energy metabolism. We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production, or respiratory chain complex I, III, and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase A, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. This could account for a Warburg-like effect in the 832/2 cell clone, lacking in 832/13 cells as well as primary beta-cells. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with HbA(1c) levels, reflecting perturbed long term glucose homeostasis, whereas that of Slc2a2 (glucose transporter 2) correlated negatively with HbA(1c) and thus better metabolic control. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal beta-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal beta-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human beta-cells. The 832 beta-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 diabetes.
Project description:Pancreatic beta-cells sense the ambient blood-glucose concentration and secrete insulin to signal other tissues to take up glucose. Mitochondria play a key role in this response as they metabolize nutrients to produce ATP and reactive oxygen species (ROS), both of which are involved in insulin secretion signaling. Based on data available in the literature and previously developed mathematical models, we present a model of glucose-stimulated mitochondrial respiration, ATP synthesis, and ROS production and control in beta-cells. The model is consistent with a number of experimental observations reported in the literature. Most notably, it captures the nonlinear rise in the proton leak rate at high membrane potential and the increase in this leak due to uncoupling protein (UCP) activation by ROS. The functional forms used to model ROS production and UCP regulation yield insight into these mechanisms, as many details have not yet been unraveled in the experimental literature. We examine short- and long-term effects of UCP activation inhibition and changes in the mitochondrial density on mitochondrial responses to glucose. Results suggest increasing mitochondrial density while decreasing UCP activity may be an effective way to increase glucose-stimulated insulin secretion while decreasing oxidative stress.
Project description:Intracellular signaling by which pancreatic beta-cells synthesize and secrete insulin in control of glucose homeostasis is not fully understood. Here we show that Shp2, a cytoplasmic tyrosine phosphatase possessing 2 SH2 domains, coordinates signaling events required for insulin biosynthesis in beta-cells. Mice with conditional ablation of the Shp2/Ptpn11 gene in the pancreas exhibited defective glucose-stimulated insulin secretion and impaired glucose tolerance. Consistently, siRNA-mediated Shp2-knockdown in rat insulinoma INS-1 832/13 cells resulted in decreased insulin production and secretion despite an increase in cellular ATP. Shp2 modulates the strength of signals flowing through Akt/FoxO1 and Erk pathways, culminating in control of Pdx1 expression and activity on Ins1 and Ins2 promoters, and forced Pdx1 expression rescued insulin production in Shp2-knockdown beta-cells. Therefore, Shp2 acts as a signal coordinator in beta-cells, orchestrating multiple pathways controlling insulin biosynthesis to maintain glucose homeostasis.
Project description:AIMS/HYPOTHESIS:Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. METHODS:Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (?ACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-?ACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. RESULTS:?ACC1KO and tmx-?ACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in ?ACC1KO mice but not in tmx-?ACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. CONCLUSIONS/INTERPRETATION:Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood.
Project description:Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.
Project description:Advanced glycation end products (AGEs) are implicated in diabetic complications. However, their role in beta-cell dysfunction is less clear. In this study we examined the effects of AGEs on islet function in mice and in isolated islets. AGE-BSA or BSA was administered ip to normal mice twice a day for 2 wk. We showed that AGE-BSA-treated mice exhibited significantly higher glucose levels and lower insulin levels in response to glucose challenge than did BSA-treated mice, although there were no significant differences in insulin sensitivity and islet morphology between two groups. Glucose-stimulated insulin secretion by islets of the AGE-BSA-treated mice or AGE-BSA-treated normal islets was significantly lower than that by islets isolated from the BSA-treated mice or BSA-treated normal islets. Furthermore, AGE treatment of islet beta-cells inhibited ATP production, and glimepiride, a sulfonylurea derivative, restored glucose-stimulated insulin secretion. Further investigation indicated that AGEs inhibited cytochrome c oxidase activity by inducing the expression of inducible nitric oxide synthase (iNOS). Blocking the formation of nitric oxide with an iNOS selective inhibitor aminoguanidine reversed the inhibitory effects of AGEs on ATP production and insulin secretion. We conclude that AGEs inhibit cytochrome c oxidase and ATP production, leading to the impairment of glucose-stimulated insulin secretion through iNOS-dependent nitric oxide production.
Project description:Metabolic engineering of cyanobacteria has the advantage that sunlight and CO(2) are the sole source of energy and carbon for these organisms. However, as photoautotrophs, cyanobacteria generally lack transporters to move hydrophilic primary metabolites across membranes. To address whether cyanobacteria could be engineered to produce and secrete organic primary metabolites, Synechococcus elongatus PCC7942 was engineered to express genes encoding an invertase and a glucose facilitator, which mediated secretion of glucose and fructose. Similarly, expression of lactate dehydrogenase- and lactate transporter-encoding genes allowed lactate accumulation in the extracellular medium. Expression of the relevant transporter was essential for secretion. Production of these molecules was further improved by expression of additional heterologous enzymes. Sugars secreted by the engineered cyanobacteria could be used to support Escherichia coli growth in the absence of additional nutrient sources. These results indicate that cyanobacteria can be engineered to produce and secrete high-value hydrophilic products.