Beta-cell failure in diet-induced obese mice stratified according to body weight gain: secretory dysfunction and altered islet lipid metabolism without steatosis or reduced beta-cell mass.
ABSTRACT: C57Bl/6 mice develop obesity and mild hyperglycemia when fed a high-fat diet (HFD). Although diet-induced obesity (DIO) is a widely studied model of type 2 diabetes, little is known about beta-cell failure in these mice.DIO mice were separated in two groups according to body weight gain: low- and high-HFD responders (LDR and HDR). We examined whether mild hyperglycemia in HDR mice is due to reduced beta-cell mass or function and studied islet metabolism and signaling.HDR mice were more obese, hyperinsulinemic, insulin resistant, and hyperglycemic and showed a more altered plasma lipid profile than LDR. LDR mice largely compensated insulin resistance, whereas HDR showed perturbed glucose homeostasis. Neither LDR nor HDR mice showed reduced beta-cell mass, altered islet glucose metabolism, and triglyceride deposition. Insulin secretion in response to glucose, KCl, and arginine was impaired in LDR and almost abolished in HDR islets. Palmitate partially restored glucose- and KCl-stimulated secretion. The glucose-induced rise in ATP was reduced in both DIO groups, and the glucose-induced rise in Ca(2+) was reduced in HDR islets relatively to LDR. Glucose-stimulated lipolysis was decreased in LDR and HDR islets, whereas fat oxidation was increased in HDR islets only. Fatty acid esterification processes were markedly diminished, and free cholesterol accumulated in HDR islets.beta-Cell failure in HDR mice is not due to reduced beta-cell mass and glucose metabolism or steatosis but to a secretory dysfunction that is possibly due to altered ATP/Ca(2+) and lipid signaling, as well as free cholesterol deposition.
Project description:Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of ?-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase C?, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany ?-cell failure in HDR islets. The ?-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.
Project description:Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.
Project description:AIMS/HYPOTHESIS:It has been suggested that the transcription factor ARNT/HIF1? is critical for maintaining in vivo glucose homeostasis and pancreatic beta cell glucose-stimulated insulin secretion (GSIS). Our goal was to gain more insights into the metabolic defects seen after the loss of ARNT/HIF1? in beta cells. METHODS:The in vivo and in vitro consequences of the loss of ARNT/HIF1? were investigated in beta cell specific Arnt/Hif1? knockout mice (?-Arnt (fl/fl/Cre) mice). RESULTS:The only in vivo defects found in ?-Arnt (fl/fl/Cre) mice were significant increases in the respiratory exchange ratio and in vivo carbohydrate oxidation, and a decrease in lipid oxidation. The mitochondrial oxygen consumption rate was unaltered in mouse ?-Arnt (fl/fl/Cre) islets upon glucose stimulation. ?-Arnt (fl/fl/Cre) islets had an impairment in the glucose-stimulated increase in Ca(2+) signalling and a reduced insulin secretory response to glucose in the presence of KCl and diazoxide. The glucose-stimulated increase in the NADPH/NADP(+) ratio was reduced in ?-Arnt (fl/fl/Cre) islets. The reduced GSIS and NADPH/NADP(+) levels in ?-Arnt (fl/fl/Cre) islets could be rescued by treatment with membrane-permeable tricarboxylic acid intermediates. Small interfering (si)RNA mediated knockdown of ARNT/HIF1? in human islets also inhibited GSIS. These results suggest that the regulation of GSIS by the KATP channel-dependent and -independent pathways is affected by the loss of ARNT/HIF1? in islets. CONCLUSIONS/INTERPRETATION:This study provides three new insights into the role of ARNT/HIF1? in beta cells: (1) ARNT/HIF1? deletion in mice impairs GSIS ex vivo; (2) ?-Arnt (fl/fl/Cre) mice have an increased respiratory exchange ratio; and (3) ARNT/HIF1? is required for GSIS in human islets.
Project description:AIMS/HYPOTHESIS:Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. METHODS:We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. RESULTS:In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. CONCLUSIONS/INTERPRETATION:The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.
Project description:Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight.We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice.TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls.We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.
Project description:The NF-κB pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic beta cell dysfunction in the metabolic syndrome. While canonical NF-κB signaling is well studied, there is little information on the divergent non-canonical NF-κB pathway in the context of pancreatic islet dysfunction in diabetes. Here, we demonstrate that pharmacological activation of the non-canonical NF-κB inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. Further, we identify NIK as a critical negative regulator of beta cell function as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of non-canonical NF-κB components p100 to p52, and accumulation of RelB. Tumor necrosis factor α (TNFα) and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive beta cell intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the non-canonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to beta cell failure. These studies reveal that NIK contributes a central mechanism for beta cell failure in diet-induced obesity. We identify a role for Nuclear Factor inducing κB (NIK) in pancreatic beta cell failure. NIK activation disrupts glucose homeostasis in zebrafish in vivo and impairs glucose-stimulated insulin secretion in mouse and human islets in vitro. NIK activation also perturbs beta cell insulin secretion in a diet-induced obesity mouse model. These studies reveal that NIK contributes a central mechanism for beta cell failure in obesity. To uncover the role of NIK in pancreatic beta cells, we performed a gene expression microarray analysis comparing pancreatic islets with constitutive beta cell intrinsicNIK activation from the 16 week old mice (beta cell specific TRAF2 and TRAF2 knockout mice) to their controls (n=3 per group).
Project description:The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.
Project description:Pancreatic beta cell (?) dysfunction is an outcome of malnutrition. We assessed the role of the amplifying pathway (AMP PATH) in ? cells in malnourished obese mice. C57Bl-6 mice were fed a control (C) or a low-protein diet (R). The groups were then fed a high-fat diet (CH and RH). AMP PATH contribution to insulin secretion was assessed upon incubating islets with diazoxide and KCl. CH and RH displayed increased glucose intolerance, insulin resistance and glucose-stimulated insulin secretion. Only RH showed a higher contribution of the AMP PATH. The mitochondrial membrane potential of RH was decreased, and ATP flux was unaltered. In RH islets, glutamate dehydrogenase (GDH) protein content and activity increased, and the AMP PATH contribution was reestablished when GDH was blunted. Thus, protein malnutrition induces mitochondrial dysfunction in ? cells, leading to an increased contribution of the AMP PATH to insulin secretion through the enhancement of GDH content and activity.
Project description:Most people with type 2 diabetes (T2D) have reduced beta-cell mass, and apoptosis is a key factor for this reduction. Previously, we showed that ATF3, an adaptive-response gene, is induced by various stress signals relevant to T2D, such as high glucose and high fatty acid. Because ATF3 is proapoptotic in beta-cells, we tested the hypothesis that ATF3 plays a detrimental role and contributes to the development of T2D. We compared wild-type (WT) and ATF3 knockout (KO) mice in an animal model for T2D, high-fat diet-induced diabetes. We also used INS-1 beta-cells and primary islets to analyze the roles of ATF3 in beta-cell function, including insulin gene expression and glucose-induced insulin secretion. Surprisingly, WT mice performed better in glucose tolerance test than KO mice, suggesting a protective, rather than detrimental, role of ATF3. At 12 wk on high-fat diet, no beta-cell apoptosis was observed, and the WT and KO mice had comparable beta-cell areas. However, ATF3 deficiency significantly reduced serum insulin levels in the KO mice without affecting insulin sensitivity, suggesting reduced beta-cell function in the KO mice. Analyses using INS-1 cells and primary islets support the notion that this defect is due, at least partly, to reduced insulin gene transcription in the KO islets without detectable reduction in glucose-induced calcium influx, a critical step for insulin secretion. In conclusion, our results support a model in which, before apoptosis becomes obvious, expression of ATF3 can be beneficial by helping beta-cells to cope with higher metabolic demand.
Project description:AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic beta-cell function remains unclear. In the present study, we therefore investigated whether AMPK plays a critical function in beta-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPK alpha2 in beta-cells and a population of hypothalamic neurons (RIPCre alpha2KO mice) and RIPCre alpha2KO mice lacking AMPK alpha1 (alpha1KORIPCre alpha2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured beta-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCre alpha2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in alpha1KORIPCre alpha2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCre alpha2KO and alpha1KORIPCre alpha2KO mice, and conversely GSIS was impaired. Beta-cells lacking AMPK alpha2 or expressing a kinase-dead AMPK alpha2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCre alpha2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse beta-cell hyperpolarization, mimicking the effect of AMPK alpha2 loss. These results show that AMPK alpha2 activity is necessary to maintain normal pancreatic beta-cell glucose sensing, possibly by maintaining high beta-cell levels of UCP2.