Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells

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 β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β 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 β cells to affect insulin secretion.     

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells

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.     

Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BAD_S. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BAD_S, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD_S. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.     

In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic β-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.     

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

While serotonin (5-HT) co-localization with insulin in granules of pancreatic β-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1−/−) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1−/− β-cells, which clearly showed that the secretory defect is downstream of Ca²⁺-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in β-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic β-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.     

Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice

The Sir2 (silent i nformation r egulator 2) family of NAD-dependent deacetylases regulates aging and longevity across a wide variety of organisms, including yeast, worms, and flies. In mammals, the Sir2 ortholog Sirt1 promotes fat mobilization, fatty acid oxidation, glucose production, and insulin secretion in response to nutrient availability. We previously reported that an increased dosage of Sirt1 in pancreatic β cells enhances glucose-stimulated insulin secretion (GSIS) and improves glucose tolerance in be ta cell-specific S ir t 1- o verexpressing (BESTO) transgenic mice at 3 and 8 months of age. Here, we report that as this same cohort of BESTO mice reaches 18–24 months of age, the GSIS regulated by Sirt1 through repression of Ucp2 is blunted. Increased body weight and hyperlipidemia alone, which are observed in aged males and also induced by a Western-style high-fat diet, are not enough to abolish the positive effects of Sirt1 on β cell function. Interestingly, plasma levels of nicotinamide mononucleotide (NMN), an important metabolite for the maintenance of normal NAD biosynthesis and GSIS in β cells, are significantly reduced in aged BESTO mice. Furthermore, NMN administration restores enhanced GSIS and improved glucose tolerance in the aged BESTO females, suggesting that Sirt1 activity decreases with advanced age due to a decline in systemic NAD biosynthesis. These findings provide insight into the age-dependent regulation of Sirt1 activity and suggest that enhancement of systemic NAD biosynthesis and Sirt1 activity in tissues such as β cells may be an effective therapeutic intervention for age-associated metabolic disorders such as type 2 diabetes.     

Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.

beta cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2's role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of beta cell glucose sensing, and as a critical link between obesity, beta cell dysfunction, and type 2 diabetes.     

Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion

Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr^595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion.     

Pharmacological Activation of Sirt1 Ameliorates Polyglutamine-Induced Toxicity through the Regulation of Autophagy

Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington’s disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, β-lapachone (β-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with β-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). β-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, β-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. β-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by β-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of β-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, β-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that β-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, β-lap provides a novel therapeutic opportunity for the treatment of HD.     

Correlation Between Pancreatic Islet Uncoupling Protein-2 (UCP2) mRNA Concentration And Insulin Status in Rats

Hypothesizing that UCP2 may influence insulin secretion by modifying the ATP/ADP ratio within pancreatic islets, we have investigated the expression of intraislet UCP2 gene in rats showing insulin oversecretion (non-diabetic Zucker fa/fa obese rats, glucose-infused Wistar rats) or insulin undersecretion (fasting and mildly diabetic rats). We found that in Zucker fa/fa obese rats, hyperinsulinemia (1222 ± 98 pmol/1 vs. 128 ± 22 pmol/1 in lean Zucker rats) was accompanied by a significant increase in UCP2 mRNA levels. In rat submitted to a 5 day infusion with glucose, hyperinsulinemia (1126 ± 101 pmol/l vs. 215 ± 25 pmol/1 in Wistar control rats), coincided with an enhanced intraislet UCP2 gene expression, whereas a 8h or a 2 day-infusion did not induce significant changes in UCP2 mRNA expression. In rats made hypoinsulinemic and mildly diabetic by the injection of a low dose of streptozotocin, and in 4-day-fasting rats (plasma insulin 28 ± 5 pmol/1) UCP2 gene expression was sharply decreased. A 3-day-fast was ineffective. The data show the existence of a time-dependent correlation between islet mRNA UCP2 and insulin that may be interpreted as an adaptative response to prolonged insulin excess.     

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia

Aims/Hypothesis

Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.

Methods

This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1^Cre; Sirt1^ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.

Results

We found that in the Pdx1^Cre; Sirt1^ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.

Conclusions/Interpretation

This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.     

Activation of AMPKα2 Is Not Crucial for Mitochondrial Uncoupling-Induced Metabolic Effects but Required to Maintain Skeletal Muscle Integrity

Transgenic (UCP1-TG) mice with ectopic expression of UCP1 in skeletal muscle (SM) show a phenotype of increased energy expenditure, improved glucose tolerance and increase substrate metabolism in SM. To investigate the potential role of skeletal muscle AMPKα2 activation in the metabolic phenotype of UCP1-TG mice we generated double transgenic (DTG) mice, by crossing of UCP1-TG mice with DN-AMPKα2 mice overexpressing a dominant negative α2 subunit of AMPK in SM which resulted in an impaired AMPKα2 activity by 90±9% in SM of DTG mice. Biometric analysis of young male mice showed decreased body weight, lean and fat mass for both UCP1-TG and DTG compared to WT and DN-AMPKα2 mice. Energy intake and weight-specific total energy expenditure were increased, both in UCP1-TG and DTG mice. Moreover, glucose tolerance, insulin sensitivity and fatty acid oxidation were not altered in DTG compared to UCP1-TG. Also uncoupling induced induction and secretion of fibroblast growth factor 21 (FGF21) from SM was preserved in DTG mice. However, voluntary physical cage activity as well as ad libitum running wheel access during night uncovered a severe activity intolerance of DTG mice. Histological analysis showed a progressive degenerative morphology in SM of DTG mice which was not observed in SM of UCP1-TG mice. Moreover, ATP-depletion related cellular stress response via heat shock protein 70 was highly induced, whereas capillarization regulator VEGF was suppressed in DTG muscle. In addition, AMPKα2-mediated induction of mitophagy regulator ULK1 was suppressed in DTG mice, as well as mitochondrial respiratory capacity and content. In conclusion, we demonstrate that AMPKα2 is dispensable for SM mitochondrial uncoupling induced metabolic effects on whole body energy balance, glucose homeostasis and insulin sensitivity. But strikingly, activation of AMPKα2 seems crucial for maintaining SM function, integrity and the ability to compensate chronic metabolic stress induced by SM mitochondrial uncoupling.     

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2)

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2^−/−). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in β-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-β-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2^−/− islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2^−/− mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2^−/− mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9–39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca²⁺ entry through L-type voltage-dependent Ca²⁺ channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.     

Uncoupling protein-2 modulates the lipid metabolic response to fasting in mice

Uncoupling protein-2 (UCP2) regulates insulin secretion by controlling ATP levels in beta-cells. Although UCP2 deficiency improves glycemic control in mice, increased expression of UCP2 interferes with glucose-stimulated insulin secretion. These observations link UCP2 to beta-cell dysfunction in type 2 diabetes with a perplexing evolutionary role. We found higher residual serum insulin levels and blunted lipid metabolic responses in fasted ucp2(-/-) mice, supporting the concept that UCP2 evolved to suppress insulin effects and to accommodate the fuel switch to fatty acids during starvation. In the absence of UCP2, fasting initially promotes peripheral lipolysis and hepatic fat accumulation at less than expected rates but culminates in protracted steatosis, indicating diminished hepatic utilization and clearance of fatty acids. We conclude that UCP2-mediated control of insulin secretion is a physiologically relevant mechanism of the metabolic response to fasting.     

Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.