Deletion of Protein Kinase D1 in Pancreatic β-Cells Impairs Insulin Secretion in High-Fat Diet-Fed Mice.
ABSTRACT: Ββ-Cell adaptation to insulin resistance is necessary to maintain glucose homeostasis in obesity. Failure of this mechanism is a hallmark of type 2 diabetes (T2D). Hence, factors controlling functional β-cell compensation are potentially important targets for the treatment of T2D. Protein kinase D1 (PKD1) integrates diverse signals in the β-cell and plays a critical role in the control of insulin secretion. However, the role of β-cell PKD1 in glucose homeostasis in vivo is essentially unknown. Using β-cell-specific, inducible PKD1 knockout mice (βPKD1KO), we examined the role of β-cell PKD1 under basal conditions and during high-fat feeding. βPKD1KO mice under a chow diet presented no significant difference in glucose tolerance or insulin secretion compared with mice expressing the Cre transgene alone; however, when compared with wild-type mice, both groups developed glucose intolerance. Under a high-fat diet, deletion of PKD1 in β-cells worsened hyperglycemia, hyperinsulinemia, and glucose intolerance. This was accompanied by impaired glucose-induced insulin secretion both in vivo in hyperglycemic clamps and ex vivo in isolated islets from high-fat diet-fed βPKD1KO mice without changes in islet mass. This study demonstrates an essential role for PKD1 in the β-cell adaptive secretory response to high-fat feeding in mice.
Project description:Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.
Project description:The Znt7 gene encodes a ubiquitously expressed zinc transporter that is involved in transporting cytoplasmic zinc into the Golgi apparatus and a ZnT7-containing vesicular compartment. Overexpression of ZnT7 in the pancreatic ?-cell stimulates insulin synthesis and secretion through regulation of insulin gene transcription. In this study, we demonstrate that ZnT7 is expressed in the mouse skeletal muscle. The activity of the insulin signaling pathway was down-regulated in myocytes isolated from the femoral muscle of Znt7 knock-out (KO) mice. High fat diet consumption (45% kcal) induced weight gain in male Znt7 KO mice but not female Znt7 KO mice. Male Znt7 KO mice fed the high fat diet at 5 weeks of age for 10 weeks exhibited hyperglycemia in the non-fasting state. Oral glucose tolerance tests revealed that male Znt7 KO mice fed the high fat diet had severe glucose intolerance. Insulin tolerance tests showed that male Znt7 KO mice were insulin-resistant. Diet-induced insulin resistance in male Znt7 KO mice was paralleled by a reduction in mRNA expression of Insr, Irs2, and Akt1 in the primary skeletal myotubes isolated from the KO mice. Overexpression of ZnT7 in a rat skeletal muscle cell line (L6) increased Irs2 mRNA expression, Irs2 and Akt phosphorylation, and glucose uptake. We conclude that a combination of decreased insulin secretion and increased insulin resistance accounts for the glucose intolerance observed in Znt7 KO mice.
Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.
Project description:Cholecystokinin (CCK) is released in response to lipid intake and stimulates insulin secretion. We hypothesized that CCK deficiency would alter the regulation of insulin secretion and glucose homeostasis.We used quantitative magnetic resonance imaging to determine body composition and studied plasma glucose and insulin secretion of CCK gene knockout (CCK-KO) mice and their wild-type controls using intraperitoneal glucose and arginine infusions. The area of anti-insulin staining in pancreatic islets was measured by immunohistochemistry. Insulin sensitivity was assessed with euglycemic-hyperinsulemic clamps.CCK-KO mice fed a low-fat diet had a reduced acute insulin response to glucose but a normal response to arginine and normal glucose tolerance, associated with a trend toward greater insulin sensitivity. However, when fed a high-fat diet (HFD) for 10 weeks, CCK-KO mice developed glucose intolerance despite increased insulin sensitivity that was associated with low insulin secretion in response to both glucose and arginine. The deficiency of insulin secretion in CCK-KO mice was not associated with changes in ?-cell or islet size.CCK is involved in regulating insulin secretion and glucose tolerance in mice eating an HFD. The impaired insulin response to intraperitoneal stimuli that do not typically elicit CCK release suggests that this hormone has chronic effects on ?-cell adaptation to diet in addition to acute incretin actions.
Project description:Notch signaling regulates differentiation of the pancreatic endocrine lineage during embryogenesis, but the role of Notch in mature β cells is unclear. We found that islets derived from lean mice show modest β cell Notch activity, which increases in obesity and in response to high glucose. This response appeared maladaptive, as mice with β cell-specific-deficient Notch transcriptional activity showed improved glucose tolerance when subjected to high-fat diet feeding. Conversely, mice with β cell-specific Notch gain of function (β-NICD) had a progressive loss of β cell maturity, due to proteasomal degradation of MafA, leading to impaired glucose-stimulated insulin secretion and glucose intolerance with aging or obesity. Surprisingly, Notch-active β cells had increased proliferative capacity, leading to increased but dysfunctional β cell mass. These studies demonstrate a dynamic role for Notch in developed β cells for simultaneously regulating β cell function and proliferation.
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:Urocortin 3 (Ucn 3), a member of the corticotropin-releasing factor (CRF) family of peptides, is strongly expressed in mammalian pancreatic beta cells and has been shown to stimulate insulin secretion. Here we report the investigation of the hypothesis that endogenous Ucn 3 regulates insulin secretion, particularly in the presence of nutrient excess. Secretion of Ucn 3-like immunoreactivity from cultured beta cells was stimulated by high glucose and insulin secretagogs such as GLP-1; furthermore, 5 pancreatic Ucn 3 mRNA levels in vivo were increased during the positive energy balance caused by high-fat diet and by the absence of leptin. Immunoneutralization of Ucn 3 or pharmacologic blockade of its receptor, the type 2 CRF receptor (CRFR2), attenuated high but not low glucose-induced insulin secretion from isolated islets in vitro. Cultured islets isolated from Ucn 3-null mice also secreted less insulin in response to high glucose concentrations. Consistently, peripheral injection of a selective CRFR2 antagonist before the administration of a glucose challenge significantly attenuated glucose-induced insulin secretion in vivo. Ucn 3-null mice were relatively protected from the hyperinsulinemia, hyperglycemia, glucose intolerance, hepatic steatosis, and hypertriglyceridemia induced by high-fat diet. Additionally, we found that aged Ucn 3-null mice maintained better glucose tolerance than age-matched wild-type littermates. These results suggest that endogenous Ucn 3 in the pancreas is induced under excessive caloric conditions and acts locally to augment insulin production, which in the long-term may contribute to reduced insulin sensitivity and harmful metabolic consequences.
Project description:Apolipoprotein A-IV (apoA-IV) is secreted by the small intestine in response to fat absorption. Here we demonstrate a potential role for apoA-IV in regulating glucose homeostasis. ApoA-IV-treated isolated pancreatic islets had enhanced insulin secretion under conditions of high glucose but not of low glucose, suggesting a direct effect of apoA-IV to enhance glucose-stimulated insulin release. This enhancement involves cAMP at a level distal to Ca(2+) influx into the ? cells. Knockout of apoA-IV results in compromised insulin secretion and impaired glucose tolerance compared with WT mice. Challenging apoA-IV(-/-) mice with a high-fat diet led to fasting hyperglycemia and more severe glucose intolerance associated with defective insulin secretion than occurred in WT mice. Administration of exogenous apoA-IV to apoA-IV(-/-) mice improved glucose tolerance by enhancing insulin secretion in mice fed either chow or a high-fat diet. Finally, we demonstrate that exogenous apoA-IV injection decreases blood glucose levels and stimulates a transient increase in insulin secretion in KKAy diabetic mice. These results suggest that apoA-IV may provide a therapeutic target for the regulation of glucose-stimulated insulin secretion and treatment of diabetes.
Project description:AIMS/HYPOTHESIS:Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. METHODS:Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. RESULTS:From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p < 0.01) or to 17 mmol/l glucose plus 100 nmol/l GLP-1 (44.3 ± 4.9%, p < 0.01) compared with control islets. Glp1r (42 ± 0.08%, p < 0.01) and Ins2 (15.4 ± 4.6%, p < 0.01) expression was significantly lower in pTcf7l2 islets than in controls. Maintained on a high-fat (but not on a normal) diet, pTcf7l2 mice displayed decreased expansion of pancreatic beta cell volume vs control littermates. No differences were observed in plasma insulin, proinsulin, glucagon or GLP-1 concentrations. CONCLUSIONS/INTERPRETATION:Selective deletion of Tcf7l2 in the pancreas replicates key aspects of the altered glucose homeostasis in human carriers of TCF7L2 risk alleles, indicating the direct role of this factor in controlling beta cell function.
Project description:Obesity and consequent insulin resistance are known risk factors for type 2 diabetes. A compensatory increase in beta cell function and mass in response to insulin resistance permits maintenance of normal glucose homeostasis, whereas failure to do so results in beta cell failure and type 2 diabetes. Recent evidence suggests that the circadian system is essential for proper metabolic control and regulation of beta cell function. We set out to address the hypothesis that the beta cell circadian clock is essential for the appropriate functional and morphological beta cell response to insulin resistance.We employed conditional deletion of the Bmal1 (also known as Arntl) gene (encoding a key circadian clock transcription factor) in beta cells using the tamoxifen-inducible CreER(T) recombination system. Upon adulthood, Bmal1 deletion in beta cells was achieved and mice were exposed to either chow or high fat diet (HFD). Changes in diurnal glycaemia, glucose tolerance and insulin secretion were longitudinally monitored in vivo and islet morphology and turnover assessed by immunofluorescence. Isolated islet experiments in vitro were performed to delineate changes in beta cell function and transcriptional regulation of cell proliferation.Adult Bmal1 deletion in beta cells resulted in failed metabolic adaptation to HFD characterised by fasting and diurnal hyperglycaemia, glucose intolerance and loss of glucose-stimulated insulin secretion. Importantly, HFD-induced beta cell expansion was absent following beta cell Bmal1 deletion indicating impaired beta cell proliferative and regenerative potential, which was confirmed by assessment of transcriptional profiles in isolated islets.Results of the study suggest that the beta cell circadian clock is a novel regulator of compensatory beta cell expansion and function in response to increased insulin demand associated with diet-induced obesity.