Fatty acid metabolism and insulin secretion in pancreatic beta cells

Increases in glucose or fatty acids affect metabolism via changes in long-chain acyl-CoA formation and chronically elevated fatty acids increase total cellular CoA. Understanding the response of pancreatic beta cells to increased amounts of fuel and the role that altered insulin secretion plays in the development and maintenance of obesity and Type 2 diabetes is important. Data indicate that the activated form of fatty acids acts as an effector molecule in stimulus-secretion coupling. Glucose increases cytosolic long-chain acyl-CoA because it increases the "switch" compound malonyl-CoA that blocks mitochondrial -oxidation, thus implementing a shift from fatty acid to glucose oxidation. We present arguments in support of the following: (i) A source of fatty acid either exogenous or endogenous (derived by lipolysis of triglyceride) is necessary to support normal insulin secretion; (ii) a rapid increase of fatty acids potentiates glucose-stimulated secretion by increasing fatty acyl-CoA or complex lipid concentrations that act distally by modulating key enzymes such as protein kinase C or the exocytotic machinery; (iii) a chronic increase of fatty acids enhances basal secretion by the same mechanism, but promotes obesity and a diminished response to stimulatory glucose; (iv) agents which raise cAMP act as incretins, at least in part, by stimulating lipolysis via beta-cell hormone-sensitive lipase activation. Furthermore, increased triglyceride stores can give higher rates of lipolysis and thus influence both basal and stimulated insulin secretion. These points highlight the important roles of NEFA, LC-CoA, and their esterified derivatives in affecting insulin secretion in both normal and pathological states.Abbreviations ACS acyl-CoA synthetase - ACC acetyl-CoA carboxylase - BAT brown adipose tissue - CPT carnitine palmitoyl transferase - CL citrate lyase - DAG diacylglycerol - GSIS glucose-stimulated insulin secretion - HSL hormone-sensitive lipase - K_ATP ATP-sensitive K⁺ channel - LC-CoA long chain acyl-CoA - PA phosphatidate - PFK-1 phosphofructokinase-1 - PKC protein kinase C - PMA phorbol myristate acetate - PC pyruvate carboxylase - PS phosphatidylserine - SNAP soluble NSF-associated protein - SNAP-25 synaptosomal-associated protein of 25 kD - t-SNARE target SNAP receptor - v-SNARE vesicle SNAP receptor - VAMP vesicle-associated membrane protein - VDCC voltage-dependent Ca²⁺ channel - WAT white adipose tissue     

Globular adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentrations

Adiponectin plays an important role in improving insulin resistance and preventing atherosclerosis. However it has been rarely reported that adiponectin influences insulin secretion because its receptor was identified in human islet β cells. In order to investigate the direct effect of adiponectin on pancreatic islet β cells, we performed an insulin secretion test in purified rat islets, which were incubated with adiponectin (100 ng/mL) at low (3.3 mM) and high (16.7 mM) glucose concentrations. Furthermore, cell lysates were extracted from the adiponectin-treated islets for p-AMPKα assay. RTPCR and immunohistochemical examination showed both adiponectin receptor 1 (AdipoR1) and receptor 2 (A dipoR2) were expressed in islet cells and AdipoR1 was predominantly expressed. Insulin secretion was significantly increased in the presence of adiponectin for 6 h at high glucose concentration. Meanwhile, the levels of phosphorylated AMPK increased with adiponectin treatment at high glucose concentrations. It is concluded that adiponectin augments insulin secretion from pancreatic islet β cells at high glucose concentration through AMPK activation.     

Impaired insulin secretion in transgenic mice over-expressing calpastatin in pancreatic β-cells

Calpains are a family of calcium-activated proteases involved in a number of cellular functions including cell death, proliferation and exocytosis. The finding that variation in the calpain-10 gene increases type 2 diabetes risk in some populations has increased interest in determining the potential role of calpains in pancreatic β-cell function. In the present study, transgenic mice (Cast^RIP) expressing an endogenous calpain inhibitor, calpastatin, in pancreatic β-cells were used to dissect the role of the calpain system in the regulation insulin secretion in vivo and in vitro. Glucose concentrations after the administration of intraperitoneal glucose were significantly increased in Cast^RIP mice compared with wildtype littermate controls. This was associated with a reduction in glucose-stimulated insulin secretion in vivo. Using pancreas perfusion, static islet incubation and islet perifusion, it was demonstrated that Cast^RIP islets hypersecreted insulin at low glucose, but exhibited significantly impaired insulin responses to high glucose. Examination of insulin release and calcium signals from isolated islets indicated that distal components of the insulin exocytotic pathway were abnormal in Cast^RIP mice. Cast^RIP islets had modestly reduced expression of Rab3a and other critical components in the late steps of insulin exocytosis. These studies provide the first evidence that blocking endogenous calpain activity partially impairs insulin release in vivo and in vitro by targeting distal components of the insulin exocytotic machinery.     

Overexpression of beta 2-microglobulin in transgenic mouse islet beta cells results in defective insulin secretion.

Overexpression of heavy chains of the class I major histocompatibility complex in islet beta cells of transgenic mice is known to induce nonimmune diabetes. We have now overexpressed the secretory protein beta 2-microglobulin in beta cells. Transgenic mice of one lineage had normal islets. Mice of another lineage did not become overtly diabetic but showed significant depletion of beta-cell insulin. When mice were made homozygous for the transgene locus, they developed diabetes. Introduction of the beta 2-microglobulin chain into class I heavy chain transgenic mice resulted in a significant improvement in their islet morphology and insulin content, and the female mice remained normoglycemic. These results suggest that different transgene molecules overexpressed in beta cells can cause islet dysfunction, though not necessarily overt diabetes, and that this effect is mediated by the level of transgene expression. Evidence is provided to show that beta-cell disruption by transgene overexpression occurs at the level of protein and involves a defect in insulin secretion.     

The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

Aims/hypothesis

Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell.

Methods

Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression.

Results

OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca²⁺ influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates.

Conclusions/interpretation

Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.     

Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells

The purpose of this study was to determine the in vitro effect of ovine PRL (oPRL) on the dynamics of insulin secretion and dye coupling among islet B cells. The effect of oPRL (2 micrograms/ml) on insulin secretion was time dependent and reached a maximum on day 4 when there was a 2.4-fold increase in insulin secretion from cultured neonatal rat islets (n = 6, P less than 0.001). When islets cultured in the presence of oPRL for 4 days were perifused, 300 mg/dl glucose stimulation resulted in insulin release of 131 +/- 20 microU/ml.100 micrograms islet tissue as compared to control islets 94 +/- 20 microU/ml.100 micrograms islet tissue (n = 7, P less than 0.02). Stimulation of the islets with a linear 30-250 mg/dl glucose gradient resulted in a threshold for glucose-stimulated insulin secretion of 73 +/- 6 mg/dl glucose for the oPRL treated islets (n = 7) as compared to a threshold of 123 +/- 6 mg/dl glucose for control islets (n = 7, P less than 0.001). Mean islet volume was unchanged after 4 days of oPRL treatment but was 34% greater after 8 days (n = 6, P less than 0.001). Dye coupling among central islet B cells was also increased after in vitro treatment with oPRL for 4 days. The mean projected area of dye spread was 2-fold greater in the oPRL treated islets (n = 33) in comparison to the control islets (n = 33, P less than 0.05). These results indicate that in vitro lactogen treatment, in the form of oPRL, alters insulin secretory behavior and B cell junctional communication and supports our hypothesis that lactogen, insulin secretion, and junctional communication among B cells are related.     

Ribozyme-mediated attenuation of pancreatic beta-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion.

Phosphorylation of glucose to glucose 6-phosphate by glucokinase (GK; EC 2.7.1.2) serves as a glucose-sensing mechanism for regulating insulin secretion in beta cells. Recent findings of heterozygous GK gene mutations in patients with maturity-onset diabetes of the young (MODY), a form of type II (non-insulin-dependent) diabetes characterized by autosomal dominant inheritance, have raised the possibility that a decrease in beta-cell GK activity may impair the insulin secretory response of these cells to glucose. To generate an animal model for MODY we have expressed in transgenic mice a GK antisense RNA with a ribozyme element under control of the insulin promoter. Mice in two independent lineages had about 30% of the normal islet GK activity. Insulin release in response to glucose from in situ-perfused pancreas was impaired; however, the plasma glucose and insulin levels of the mice remained normal. These mice are likely to be predisposed to type II diabetes and may manifest increased susceptibility to genetic and environmental diabetogenic factors. They provide an animal model for studying the interaction of such factors with the reduced islet GK activity.     

Sodium channel beta1 regulatory subunit deficiency reduces pancreatic islet glucose-stimulated insulin and glucagon secretion

Glucose-stimulated insulin and glucagon release regulates glucose homeostasis by an excitation-secretion coupling pathway beginning with ATP-sensitive K(+) channel closure, membrane depolarization, and entry of calcium ions to stimulate exocytosis. The contribution of voltage-gated sodium channels to this release pathway is still being elucidated. We demonstrate that loss of Scn1b, a major regulatory subunit expressed with Na(v)1.7 protein in mouse pancreatic islets, reduces glucose-stimulated insulin and glucagon secretion in vitro and in vivo, resulting in severe fed and fasting hypoglycemia. This genetic mouse model is the first to demonstrate that sodium channelopathy impairs the physiological excitation-release coupling pathway for pancreatic insulin and glucagon release.     

GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion.

GPR40 was originally isolated from human genomic DNA by degenerate PCR. We isolated GPR40 cDNAs from various species, and precisely analyzed its mRNA expression in rat tissues, and found that GPR40 was highly expressed in beta cells in the islets of rat pancreas. When compared to the cell-surface receptors (i.e., choresistokinin receptor, glucagon-like peptide-1 receptor, and sulfonylurea receptor) that are known to predominantly express in the pancreatic beta cells, GPR40mRNA was comparable to these receptors in mRNA expression levels. In addition, all of pancreatic beta cell lines, which we examined, expressed GPR40mRNA at significant levels. Its highest expression was detected in a mouse beta cell line MIN6. To reveal the function of GPR40, we searched for the ligands of GPR40 by screening more than 1500 compounds. As a result we found that CHO cells expressing GPR40 specifically responded to free fatty acids (FFAs), that is, elevation of intracellular Ca(2+) was detected in these cells. Among FFAs tested, apparent stimulatory activities were detected in C12- to 16-length saturated FFAs (e.g., lauric acid, myristic acid, and palmitic acid) and in both C18- and C22-length unsaturated FFAs (e.g., oleic acid, elaidic acid, linoleic acid, a-linolenic acid, g-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid) at EC(50) of micro molar range. We found that FFAs induced Ca(2+) influx and activated MAP kinase in CHO cells expressing GPR40. As it is known that the increase of intracellular Ca(2+) promotes insulin secretion, we expected the stimulation of FFAs through GPR40 would promote insulin secretion from pancreatic beta cells. As we expected, FFAs induced glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Our results indicate that GPR40 is a cell-surface receptor for FFAs and regulates insulin secretion from pancreatic beta cells. FFAs are known not only to provide an important energy source as nutrients for the body but also to act as signaling molecules in various cellular processes including insulin secretion. However, the molecular mechanism behind the relationship between insulin secretion and FFAs is little understood. We believe that the discovery of a cell-surface FFA receptor on pancreatic beta cells will provide a clue to resolve the relation between FFAs and insulin secretion, and thus eventually lead to the development of anti-diabetic drugs.     

Angiotensin receptor blockade increases pancreatic insulin secretion and decreases glucose intolerance during glucose supplementation in a model of metabolic syndrome

Renin-angiotensin system blockade improves glucose intolerance and insulin resistance, which contribute to the development of metabolic syndrome. However, the contribution of impaired insulin secretion to the pathogenesis of metabolic syndrome is not well defined. To assess the contributions of angiotensin receptor type 1 (AT?) activation and high glucose intake on pancreatic function and their effects on insulin signaling in skeletal muscle and adipose tissue, an oral glucose tolerance test (oGTT) was performed in five groups (n = 10/group) of rats: 1) lean strain-control 2) obese Otsuka Long-Evans Tokushima Fatty (OLETF), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg/kg · d olmesartan for 6 wk; OLETF ARB), 4) OLETF + 5% glucose water (HG) for 6 wk (OLETF HG), and 5) OLETF + HG + ARB (OLETF HG/ARB). The glucose response to the oGTT increased 58% in OLETF compared with lean-strain control, whereas glucose supplementation increased it an additional 26%. Blockade of angiotensin receptor reduced the oGTT response 19% in the ARB-treated groups and increased pancreatic insulin secretion 64 and 113% in OLETF ARB and OLETF HG/ARB, respectively. ARB treatment in OLETF ARB and OLETF HG/ARB did not have an effect on insulin signaling proteins in skeletal muscle; however, it reduced pancreatic AT? protein expression 20 and 27%, increased pancreatic glucagon-like peptide-1 (GLP-1) receptor protein expression 41 and 88%, respectively, and increased fasting plasma GLP-1 approximately 2.5-fold in OLETF ARB. The results suggest that improvement of glucose intolerance is independent of an improvement in muscle insulin signaling, but rather by improved glucose-stimulated insulin secretion associated with decreased pancreatic AT? activation and increased GLP-1 signaling.     

Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity

Aims/hypothesis

Insulin secretion from pancreatic beta cells and insulin-stimulated glucose uptake into skeletal muscle are processes regulated by similar isoforms of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) and mammalian homologue of unc-18 (Munc18) protein families. Double C2 domain β (Doc2b), a SNARE- and Munc18-interacting protein, is implicated as a crucial effector of glycaemic control. However, whether Doc2b is naturally limiting for these processes, and whether Doc2b enrichment might exert a beneficial effect upon glycaemia in vivo, remains undetermined.

Methods

Tetracycline-repressible transgenic (Tg) mice engineered to overexpress Doc2b simultaneously in the pancreas, skeletal muscle and adipose tissues were compared with wild-type (Wt) littermate mice regarding glucose and insulin tolerance, islet function in vivo and ex vivo, and skeletal muscle GLUT4 accumulation in transverse tubule/sarcolemmal surface membranes. SNARE complex formation was further assessed using Doc2b overexpressing L6-GLUT4-myc myoblasts to derive mechanisms relatable to physiological in vivo analyses.

Results

Doc2b Tg mice cleared glucose substantially faster than Wt mice, correlated with enhancements in both phases of insulin secretion and peripheral insulin sensitivity. Heightened peripheral insulin sensitivity correlated with elevated insulin-stimulated GLUT4 vesicle accumulation in cell surface membranes of Doc2b Tg mouse skeletal muscle. Mechanistic studies demonstrated Doc2b enrichment to enhance syntaxin-4–SNARE complex formation in skeletal muscle cells.

Conclusions/interpretation

Doc2b is a limiting factor in SNARE exocytosis events pertinent to glycaemic regulation in vivo. Doc2b enrichment may provide a novel means to simultaneously boost islet and skeletal muscle function in vivo in the treatment and/or prevention of diabetes.     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets

The effect of melatonin (0.1 microM) on freshly isolated islets from adult rats was investigated. Melatonin caused a marked decrease of insulin secretion by islets in response to glucose. The mechanism involved was then examined. Melatonin did not interfere with glucose metabolism as indicated by the measurement of glucose oxidation. However, the content of the protein kinase A (PKA) catalytic alpha-subunit was significantly decreased in islets exposed to melatonin for 1 hr in the presence of 8.3 mM glucose, whereas that of the protein kinase C (PKC) alpha-subunit remained unchanged. Melatonin also inhibited forskolin-induced insulin secretion, a well known activator of adenylate cyclase (AC) activity. This may explain the low content of insulin found in islets incubated in the presence of melatonin for 3 hr. In fact, 3',5' -cyclic adenosine monophosphate (cAMP), a product of AC activity, stimulates insulin synthesis. These findings led us to postulate that a down-regulation of the PKA signaling pathway may be the mechanism involved in the melatonin inhibition of the process of glucose-induced insulin secretion.     

Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species

Aims/hypothesis Levels of uncoupling protein-2 (UCP2) are regulated in the pancreatic beta cells and an increase in the protein level has been associated with mitochondrial uncoupling and alteration in glucose-stimulated insulin secretion. However, it is not clear whether an increase in uncoupling protein-2 per se induces mitochondrial uncoupling and affects ATP generation and insulin secretion. Materials and methods Transgenic mice with beta cell-specific overexpression of the human UCP2 gene and INS-1 cells with doxycycline-inducible overproduction of the protein were generated and the consequences of increased levels of UCP2 on glucose-induced insulin secretion and on parameters reflecting mitochondrial uncoupling were determined. Results In transgenic mice, an increase in beta cell UCP2 protein concentration did not significantly modify plasma glucose and insulin levels. Glucose-induced insulin secretion and elevation in the ATP/ADP ratio were unaltered by an increase in UCP2 level. In INS-1 cells, a similar increase in UCP2 level did not modify glucose-induced insulin secretion, cytosolic ATP and ATP/ADP ratio, or glucose oxidation. Increased levels of UCP2 did not modify the mitochondrial membrane potential and oxygen consumption. Increased UCP2 levels decreased cytokine-induced production of reactive oxygen species. Conclusion/interpretation The results obtained in transgenic mice and in the beta cell line do not support the hypothesis that an increase in UCP2 protein per se uncouples the mitochondria and decreases glucose-induced insulin secretion. In contrast, the observation that increased UCP2 levels decrease cytokine-induced production of reactive oxygen species indicates a potential protective effect of the protein on beta cells, as observed in other cell types. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users. N. Produit-Zengaffinen and N. Davis-Lameloise contributed equally to this work.     

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells

Aims/hypothesis  Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure. Methods  To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells. Results  Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP⁺ ratio and activity of the pyruvate–malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion. Conclusions/interpretation  Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. J. Xu and J. Han contributed equally to this study.