Bombesin receptor subtype-3 (BRS-3) regulates glucose-stimulated insulin secretion in pancreatic islets across multiple species

Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, and BRS-3 agonism is being explored as a possible therapy for obesity. Here we study the role of BRS-3 in the regulation of glucose-stimulated insulin secretion (GSIS) and glucose homeostasis. We quantified BRS-3 mRNA in pancreatic islets from multiple species and examined the acute effects of Bag-1, a selective BRS-3 agonist, on GSIS in mouse, rat, and human islets, and on oral glucose tolerance in mice. BRS-3 is highly expressed in human, mouse, rhesus, and dog (but not rat) pancreatic islets and in rodent insulinoma cell lines (INS-1 832/3 and MIN6). Silencing BRS-3 with small interfering RNA or pharmacological blockade with a BRS-3 antagonist, Bantag-1, reduced GSIS in 832/3 cells. In contrast, the BRS-3 agonist (Bag-1) increased GSIS in 832/3 and MIN6 cells. The augmentation of GSIS by Bag-1 was completely blocked by U73122, a phospholipase C inhibitor. Bag-1 also enhanced GSIS in islets isolated from wild-type, but not Brs3 knockout mice. In vivo, Bag-1 reduced glucose levels during oral glucose tolerance test in a BRS-3-dependent manner. BRS-3 agonists also increased GSIS in human islets. These results identify a potential role for BRS-3 in islet physiology, with agonism directly promoting GSIS. Thus, in addition to its potential role in the treatment of obesity, BRS-3 may also regulate blood glucose levels and have a role in the treatment of diabetes mellitus.     

Nutrient responsive nesfatin-1 regulates energy balance and induces glucose-stimulated insulin secretion in rats

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic β-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.     

Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets

Aims/hypothesis

Type 2 diabetes is characterised by impaired glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Since erythropoietin-producing hepatoma (Eph)–ephrin bidirectional signalling fine-tunes GSIS from pancreatic beta cells, we investigated Eph receptor tyrosine kinases (RTK) as potential drug targets for selectively increasing GSIS.

Methods

Insulin secretion assays were carried out using mouse and human pancreatic islets as well as mouse insulinoma (MIN6) cells in the presence or absence of two Eph RTK inhibitors. Furthermore, the most potent inhibitor was injected into mice to evaluate its effects on glucose tolerance and plasma insulin levels.

Results

We showed that the Eph RTK inhibitors selectively increased GSIS from MIN6 cells as well as mouse and human islets. Our results also showed that the insulin secretory effects of these compounds required Eph–ephrin signalling. Finally, pharmacological inhibition of Eph receptor signalling improved glucose tolerance in mice.

Conclusions/interpretation

We showed for the first time that Eph RTKs represent targets for small molecules to selectively increase GSIS and improve glucose tolerance.     

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

Aims/hypothesis

Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells.

Methods

We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot.

Results

Insulin secretion was increased following chronic (≥8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases.

Conclusions/interpretation

Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.     

曲格列酮对胰岛β细胞胰岛素分泌的影响及机制研究

研究曲格列酮对胰岛β细胞(MIN6细胞株)胰岛素分泌的影响,并探讨其机制.10μmol/L曲格列酮短期抑制大鼠胰岛和MIN6细胞的葡萄糖刺激的胰岛素分泌(GSIS,P<0.01),增加AMP活化的蛋白激酶(AMPK)、乙酰辅酶A羧化酶(ACC)的磷酸化水平(均P<0.01),而AMPK抑制剂复合物C可使其AMPK、ACC的磷酸化水平以及胰岛素分泌完全恢复.     

α-Lipoic acid regulates AMP-activated protein kinase and inhibits insulin secretion from beta cells

Aims/hypothesis The antioxidant compound α-lipoic acid (α-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, α-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of α-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of α-LA on pancreatic beta cell function.Materials and methods Isolated rat islets and MIN6 beta cells were treated acutely (15–90 min) or chronically (18–24 h) with α-LA or the known AMPK-activating compounds 5′-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed.Results Acute or chronic treatment of islets and MIN6 cells with α-LA led to dose-dependent rises in phosphorylation of the AMPK α-subunit and acetyl CoA carboxylase. Chronic exposure to α-LA, AICAR or metformin caused a reduction in insulin secretion. α-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, α-LA also acutely inhibited insulin secretion. Examination of the effect of α-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca²⁺.Conclusions/interpretation This study is the first to demonstrate that α-LA directly affects beta cell function. The chronic effects of α-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, α-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function.     

MIN6 is not a pure beta cell line but a mixed cell line with other pancreatic endocrine hormones

MIN6 cells retains glucose-stimulated insulin secretion (GSIS) as isolated islets. We comprehensively evaluated the gene expression and production of other islet hormones in MIN6 cells. Islet hormones were demonstrated by immunohistochemical staining and measured by ELISA. The gene expression profiles of MIN6 cells were compared with those in the mouse islets obtained by the laser capture micro-dissection (LCM). MIN6 cells excreted insulin, glucagon, somatostatin and ghrelin. They expressed mRNAs of insulin I and II, proglucagon, somatostatin, pancreatic polypeptide (PP) and ghrelin which were shown in the mouse pancreatic islet core and periphery obtained by LCM. A variety of genes closely related to the islet hormone producing cells were expressed in MIN6. Confocal laser scanning microscopy revealed that MIN6 cells included not only insulin positive cells but also insulin and glucagon or somatostin double positive cells. Glucagon, somatostatin and ghrelin were detectable in the culture medium. The present study clearly demonstrated that MIN6 produce pancreatic endocrine cells. It would be possible to use this cell line as a model to research the development, cell differentiation and function of pancreatic islets.     

Over-expression of AMP-activated protein kinase impairs pancreatic {beta}-cell function in vivo

Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional beta-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 +/- 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 +/- 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 +/- 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits beta-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.     

Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass

Aims/hypothesis

Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition.

Methods

Wistar rats (2 and 6 months old) received a 72 h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry.

Results

A 72 h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets.

Conclusions/interpretation

In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.     

Regulation of insulin secretion by overexpression of Ca2+/calmodulin-dependent protein kinase II in insulinoma MIN6 cells

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia, suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared to controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2, in isolated islets, leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.     

Pim3 negatively regulates glucose-stimulated insulin secretion

Pancreatic β-cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the β cell, and here, we characterize its role in the regulation of β-cell function. Pim3 expression in the β-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the β cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3-/- mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3-/- mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3-/- mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3-/- islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating β-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3-/- islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the β cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.     

Establishment of new clonal pancreatic β‐cell lines (MIN6‐K) useful for study of incretin/cyclic adenosine monophosphate signaling

Incretin/cyclic adenosine monophosphate (cAMP) signaling is critical for potentiation of insulin secretion. Although several cell lines of pancreatic β‐cells are currently available, there are no cell lines suitable for investigation of incretin/cAMP signaling. In the present study, we have newly established pancreatic β‐cell lines (named MIN6‐K) from the IT6 mouse, which develops insulinoma. MIN6‐K8 cells respond to both glucose and incretins, such as glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP), as is the case in pancreatic islets, whereas MIN6‐K20 cells respond to glucose, but not to incretins. Despite the difference in incretin‐potentiated insulin secretion between these two cell lines, the accumulation of cAMP after stimulation of GLP‐1 is comparable in these cells. Interestingly, we also found that incretin responsiveness is drastically induced by the formation of pseudoislets from MIN6‐K20 cells to a level comparable to that of pancreatic islets. Thus, these cell lines are useful for studying incretin/cAMP signaling in β‐cells. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00026.x, 2010)     

Upregulation of β-cell genes and improved function in rodent islets following chronic glucokinase activation

Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to β-cell exhaustion; this study examines the effect of chronic GK activation on β-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key β-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk(del/wt)). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPβ compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk(del/wt) islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key β-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.     

Hyperthyroidism impairs pancreatic beta cell adaptations to late pregnancy and maternal liporegulation in the rat

Aims/hypothesis Hyperthyroidism modifies lipid dynamics (increased oxidation), impairs insulin action and can suppress insulin secretion. We therefore examined the impact of hyperthyroidism on the relationship between glucose-stimulated insulin secretion (GSIS) and insulin action, using late pregnancy as a model of physiological insulin resistance that is associated with compensatory insulin hypersecretion to maintain glucose tolerance. Our aim was to examine whether hyperthyroidism compromises the regulation of insulin secretion and the ability of insulin to modulate circulating lipid concentrations in late pregnancy.Materials and methods Hyperthyroidism was induced by tri-iodothyronine (T₃) administration from day 17 to 19 of pregnancy. GSIS was assessed during an IVGTT and during hyperglycaemic clamps in vivo and in vitro, using step-up and -down islet perifusions.Results Hyperthyroidism in pregnancy elevated the glucose threshold for GSIS and impaired GSIS at low and high glucose concentrations in islet perifusions. In the intact animal, insulin secretion (after bolus glucose) was more rapidly curtailed following removal of the glucose stimulus to secretion. In contrast, GSIS was maintained during protracted hyperglycaemia (hyperglycaemic clamps) in the hyperthyroid pregnant state in vivo.Conclusions/interpretation Hyperthyroidism in vivo during late pregnancy blunts GSIS in subsequently isolated and perifused islets at low and high glucose concentrations. It also adversely affects GSIS under conditions of an acute glucose challenge in vivo. In contrast, GSIS is maintained during sustained hyperglycaemia in vivo, suggesting that in vivo factors can rescue GSIS. The ability of insulin to suppress systemic lipid levels during hyperglycaemic clamps was impaired. We therefore suggest that higher circulating lipids may preserve GSIS under conditions of sustained hyperglycaemia in the hyperthyroid pregnancy.     

Differential synthesis and action of TGFß superfamily ligands in mouse and rat islets

Members of the TGFβ superfamily, including activins and TGFβ, modulate glucose-stimulated insulin secretion (GSIS) in vitro using rat islets while genetic manipulations that reduce TGFβ superfamily signaling in vivo in mice produced hypoplastic islets and/or hyperglycemia. Moreover, deletion of Fstl3, an antagonist of activin and myostatin, resulted in enlarged islets and β-cell hyperplasia. These studies suggest that endogenous TGFβ superfamily ligands regulate β-cell generation and/or function. To test this hypothesis, we examined endogenous TGFβ ligand synthesis and action in isolated rat and mouse islets. We found that activin A, TGFβ1, and myostatin treatment enhanced rat islet GSIS but none of the ligands tested enhanced GSIS in mouse islets. However, follistatin inhibited GSIS, consistent with a role for endogenous TGFβ superfamily ligands in regulating insulin secretion. Endogenous expression of TGF∆ superfamily members was different in rat and mouse islets with myostatin being highly expressed in mouse islets and not detectable in rats. These results indicate that TGFβ superfamily members directly regulate islet function in a species-specific manner while the ligands produced by islets differ between mice and rats. The lack of in vitro actions of ligands on mouse islets may be mechanical or result from species-specific actions of these ligands.     

APPL1 potentiates insulin secretion in pancreatic β cells by enhancing protein kinase Akt-dependent expression of SNARE proteins in mice

Insulin resistance and defective insulin secretion are the two major features of type 2 diabetes. The adapter protein APPL1 is an obligatory molecule in regulating peripheral insulin sensitivity, but its role in insulin secretion remains elusive. Here, we show that APPL1 expression in pancreatic β cells is markedly decreased in several mouse models of obesity and diabetes. APPL1 knockout mice exhibit glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS), whereas transgenic expression of APPL1 prevents high-fat diet (HFD)-induced glucose intolerance partly by enhancing GSIS. In both pancreatic islets and rat β cells, APPL1 deficiency causes a marked reduction in expression of the exocytotic machinery SNARE proteins (syntaxin-1, synaptosomal-associated protein 25, and vesicle-associated membrane protein 2) and an obvious decrease in the number of exocytotic events. Such changes are accompanied by diminished insulin-stimulated Akt activation. Furthermore, the defective GSIS and reduced expression of SNARE proteins in APPL1-deficient β cells can be rescued by adenovirus-mediated expression of APPL1 or constitutively active Akt. These findings demonstrate that APPL1 couples insulin-stimulated Akt activation to GSIS by promoting the expression of the core exocytotic machinery involved in exocytosis and also suggest that reduced APPL1 expression in pancreatic islets may serve as a pathological link that couples insulin resistance to β-cell dysfunction in type 2 diabetes.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1^-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1^-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1^-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1^-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1^-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1^-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.