AMPA receptors regulate exocytosis and insulin release in pancreatic β cells

Ionotropic glutamate receptors (iGluRs) are expressed in islets and insulinoma cells and involved in insulin secretion. However, the exact roles that iGluRs play in β cells remain unclear. Here, we demonstrated that GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were expressed in mouse β cells. Glutamate application increased both cytosolic calcium and the number of docked insulin-containing granules, which resulted in augmentation of depolarization-induced exocytosis and high-glucose-stimulated insulin release. While glutamate application directly depolarized β cells, it also induced an enormous depolarization when K(ATP) channels were available. Glutamate application reduced the conductance of K(ATP) channels and increased voltage oscillations. Moreover, actions of AMPARs were absent in Kir6.2 knock-out mice. The effects of AMPARs on K(ATP) channels were mediated by cytosolic cGMP. Taken together, our experiments uncovered a novel mechanism by which AMPARs participate in insulin release.     

Protective effects of dipeptidyl peptidase-4 (DPP-4) inhibitor against increased β cell apoptosis induced by dietary sucrose and linoleic acid in mice with diabetes

Chronic exposure to high glucose and fatty acid levels caused by dietary sugar and fat intake induces β cell apoptosis, leading to the exacerbation of type 2 diabetes. Oleic acid and linoleic acid are two major dietary fatty acids, but their effects in diabetes are unclear. We challenged β cell-specific glucokinase haploinsufficient (Gck(+/-)) mice with a diet containing sucrose and oleic acid (SO) or sucrose and linoleic acid (SL) and analyzed β cell apoptosis. In Gck(+/-) but not wild-type mice, SL significantly decreased the β cell mass and β cell proportion in islet cells arising from increased apoptosis to a greater degree than did SO. The mRNA expression of SREBP-1c was significantly higher, and that of E-cadherin was significantly lower in the islets of Gck(+/-) mice fed SL compared with mice fed SO. We next evaluated monotherapy with desfluorositagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in these mouse groups. DPP-4 inhibitor protected against β cell apoptosis, restored the β cell mass, and normalized islet morphology in Gck(+/-) mice fed SL. DPP-4 inhibition normalized the changes in the islet expression of SREBP-1c and E-cadherin mRNA induced by the SL diet. Furthermore, linoleic acid induced β cell apoptosis to a greater degree in the presence of high glucose levels than in the presence of low glucose levels in vitro in islets and MIN6 cells, whereas a GLP-1 receptor agonist prevented apoptosis. In conclusion, SL exacerbated β cell apoptosis in diabetic Gck(+/-) mice but not in euglycemic wild-type mice, and DPP-4 inhibition protected against these effects.     

Inhibition of p38 mitogen-activated protein kinase exerts a hypoglycemic effect by improving β cell function via inhibition of β cell apoptosis in db/db mice

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in endoplasmic reticulum stress (ERS) and inflammation, which may play an important role in the pathogenesis of type 2 diabetes (T2DM). This study aimed to investigate whether p38 MAPK contributes to the pathogenesis of T2DM. 6-week-old female db/db mice were randomly assigned to Dmo and Dmi groups, and C57 mice were assigned as controls. The Dmi group was gavaged with the p38 MAPK inhibitor SB203580 for 9?weeks, and the effects on β cell dysfunction and apoptosis were investigated. db/db mice showed higher food intake, body mass, fasting glucose, and plasma insulin levels than C57 mice. After SB203580 administration, blood glucose was significantly lower. HOMA β and HOMA IR were improved. Islet mRNA expression levels of the ERS markers were lower. P38 MAPK inhibition reduced blood glucose and improved β cell function, at least in part by reducing β cell apoptosis.     

Early liraglutide treatment is better in glucose control, β-cell function improvement and mass preservation in db/db mice

Glucagon-like peptide-1 (GLP-1) has been proved to have effects of anti-hyperglycemia and β-cell preservation. However, it is still unclear whether there are differences between early and late GLP-1 intervention in type 2 diabetes mellitus (T2DM). We divided the mice into 5 groups: early treated group (n = 7, 8-week old, fasting glucose > 10 mmol/l), late treated group (n = 7, 10-week old, fasting glucose > 20 mmol/l), early control group (n = 7), late control group (n = 7) and wild type group (n = 7). Treated group was injected with liraglutide (a GLP-1 analog) 300 μg/kg bid for 4 weeks, while control group was given saline at the same time. The results showed that compared with control group, food intake and body weight gain were reduced in both early and late treated group (p < 0.05), and there was no significance between the two treated groups. Early liraglutide intervention showed better improvements in glucose control, acute insulin response to glucose (AIRg) and disposition index (before vs. after treatment, AIRg 1.01 ± 0.53 vs. 2.98 ± 0.63, disposition index 10.81 ± 0.89 vs. 27.4 ± 2.15) than late intervention (AIRg 0.99 ± 0.02 vs. 1.41 ± 0.32, disposition index 3.47 ± 0.38 vs. 6.43 ± 1.62, p = 0.001). The histopathology of the pancreas showed the estimated β-cell mass (BCM) was increased more in early treated group than that in late one (0.03 vs. 0.01 g). Expressions of the proliferation related genes PDX-1, MafA and GLP-1 receptor (GLP-1R) in early treated group were 1.81, 2.57 and 1.59 times as much as that in late treated group. In conclusion, early liraglutide intervention was better in glucose control, β-cell function improvement and β-cell mass preservation.     

Ghrelin inhibits insulin secretion through the AMPK-UCP2 pathway in β cells

Ghrelin inhibits insulin secretion partly via induction of IA-2β. However, the orexigenic effect of ghrelin is mediated by the AMP-activated protein kinase (AMPK)-uncoupling protein 2 (UCP2) pathway. Here, we demonstrate that ghrelin’s inhibitory effect on insulin secretion also occurs through the AMPK-UCP2 pathway. Ghrelin increased AMPK phosphorylation and UCP2 mRNA expression in MIN6 insulinoma cells. Overexpression or downregulation of UCP2 attenuated or enhanced insulin secretion, respectively. Furthermore, AMPK activator had a similar effect to ghrelin on UCP2 and insulin secretion in MIN6 cells. In conclusion, ghrelin’s inhibitory effect on insulin secretion is partly mediated by the AMPK-UCP2 pathway, which is independent of the IA-2β pathway.     

How to make insulin-producing pancreatic β cells for diabetes treatment

Around 400 million people worldwide suffer from diabetes mellitus.The major pathological event for Type 1 diabetes and advanced Type 2 diabetes is loss or impairment of insulin-secreting β cells of the pancreas.For the past 100 years,daily insulin injection has served as a life-saving treatment for these patients.However,insulin injection often cannot achieve full glucose control,and over time poor glucose control leads to severe complications and mortality.As an alternative treatment,islet transplantation has been demonstrated to effectively maintain glucose homeostasis in diabetic patients,but its wide application is limited by the scarcity of donated islets.Therefore,it is important to define new strategies to obtain functional human β cells for transplantation therapies.Here,we summarize recent progress towards the production of β cells in vitro from pluripotent stem cells or somatic cell types including a cells,pancreatic exocrine cells,gastrointestinal stem cells,fibroblasts and hepatocytes.We also discuss novel methods for optimizing β cell transplantation and maintenance in vivo.From our perspective,the future of βcell replacement therapy is very promising although it is still challenging to control differentiation of β cells in vitro and to protect these cells from autoimmune attack in Type 1 diabetic patients.Overall,tremendous progress has been made in understanding βcell differentiation and producing functional β cells with different methods.In the coming years,we believe more clinical trials will be launched to move these technologies towards treatments to benefit diabetic patients.     

Sphk2−/− mice are protected from obesity and insulin resistance

Sphingosine kinases phosphorylate sphingosine to sphingosine 1?phosphate (S1P), which functions as a signaling molecule. We have previously shown that sphingosine kinase 2 (Sphk2) is important for insulin secretion. To obtain a better understanding of the role of Sphk2 in glucose and lipid metabolism, we have characterized 20- and 52-week old Sphk2^?/? mice using glucose and insulin tolerance tests and by analyzing metabolic gene expression in adipose tissue. A detailed metabolic characterization of these mice revealed that aging Sphk2^?/? mice are protected from metabolic decline and obesity compared to WT mice. Specifically, we found that 52-week old male Sphk2^?/? mice had decreased weight and fat mass, and increased glucose tolerance and insulin sensitivity compared to control mice. Indirect calorimetry studies demonstrated an increased energy expenditure and food intake in 52-week old male Sphk2^?/? versus control mice. Furthermore, expression of adiponectin gene in adipose tissue was increased and the plasma levels of adiponectin elevated in aged Sphk2^?/? mice compared to WT. Analysis of lipid metabolic gene expression in adipose tissue showed increased expression of the Atgl gene, which was associated with increased Atgl protein levels. Atgl encodes for the adipocyte triglyceride lipase, which catalyzes the rate-limiting step of lipolysis. In summary, these data suggest that mice lacking the Sphk2 gene are protected from obesity and insulin resistance during aging. The beneficial metabolic effects observed in aged Sphk2^?/? mice may be in part due to enhanced lipolysis by Atgl and increased levels of adiponectin, which has lipid- and glucose-lowering effects.     

Molecular physiology of glucagon-like peptide-1 insulin secretagogue action in pancreatic β cells

Insulin secretion from pancreatic β cells is stimulated by glucagon-like peptide-1 (GLP-1), a blood glucose-lowering hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., Byetta) and GLP-1 analogs (e.g., Victoza) activate the β cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., Januvia, Galvus). These compounds slow metabolic degradation of intestinally released GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating β cell insulin release. In this review, we summarize what is currently known concerning the signal transduction properties of the β cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2-mediated actions of GLP-1 to regulate ATP-sensitive K⁺ channels, voltage-dependent K⁺ channels, TRPM2 cation channels, intracellular Ca²⁺ release channels, and Ca²⁺-dependent exocytosis. We also discuss new evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM.     

Hyperoxia reverses glucotoxicity-induced inhibition of insulin secretion in rat INS-1 β cells

Chronic hyperglycemia has deleterious effects on pancreatic β-cell function, a process known as glucotoxicity. This study examined whether chronic high glucose (CHG) induces cellular hypoxia in rat INS-1 β cells, and whether hyperoxia (35% O₂) can reverse glucotoxicity-induced inhibition of insulin secretion. CHG (33.3?mm, 96?h) reduced insulin secretion, and down-regulated insulin and pancreatic duodenal homeobox factor 1 gene expression. CHG also increased intracellular pimonidazole-protein adducts, a marker for hypoxia. CHG also enhanced hypoxia-inducible factor 1α (HIF-1α) protein expression and its DNA-binding activity, which was accompanied by a decrease in mRNA expression of glucose transporter 2 (GLUT2), glucokinase and uncoupling protein-2 and an increase in mRNA expression of GLUT1 and pyruvate dehydrogenase kinase 1. Hyperoxia restored the decrease in insulin secretion and the gene expression except for GLUT2, and suppressed intracellular hypoxia and HIF-1α activation. These results suggest that glucotoxicity may cause β-cell hypoxia. Hyperoxia might prevent glucotoxicity-induced β-cell dysfunction and improve insulin secretion.     

A kinetic core model of the glucose-stimulated insulin secretion network of pancreatic β cells

The construction and characterization of a core kinetic model of the glucose-stimulated insulin secretion system (GSIS) in pancreatic β cells is described. The model consists of 44 enzymatic reactions, 59 metabolic state variables, and 272 parameters. It integrates five subsystems: glycolysis, the TCA cycle, the respiratory chain, NADH shuttles, and the pyruvate cycle. It also takes into account compartmentalization of the reactions in the cytoplasm and mitochondrial matrix. The model shows expected behavior in its outputs, including the response of ATP production to starting glucose concentration and the induction of oscillations of metabolite concentrations in the glycolytic pathway and in ATP and ADP concentrations. Identification of choke points and parameter sensitivity analysis indicate that the glycolytic pathway, and to a lesser extent the TCA cycle, are critical to the proper behavior of the system, while parameters in other components such as the respiratory chain are less critical. Notably, however, sensitivity analysis identifies the first reactions of nonglycolytic pathways as being important for the behavior of the system. The model is robust to deletion of malic enzyme activity, which is absent in mouse pancreatic β cells. The model represents a step toward the construction of a model with species-specific parameters that can be used to understand mouse models of diabetes and the relationship of these mouse models to the human disease state. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of alogliptin, pioglitazone and glargine on pancreatic β-cells in diabetic db/db mice

Objective: Progressive β-cell dysfunction and loss of β-cell mass are fundamental pathogenic features of type 2 diabetes. To examine if anti-diabetic reagents, such as insulin, pioglitazone (pio), and alogliptin (alo), have protective effects on β-cell mass and function in vivo, we treated obese diabetic db/db mice with these reagents. Methods: Male db/db mice were treated with a chow including pio, alo, or both of them from 8 to 16 weeks of age. Insulin glargine (gla) was daily injected subcutaneously during the same period. Results: At 16 weeks of age, untreated db/db mice revealed marked increase of HbA1c level, whereas those treated with pio, pio + alo, or insulin revealed the almost same HbA1c levels as non-diabetic db/m mice. Islet mass evaluated by direct counting in the whole pancreas and insulin content in isolated islets were preserved in pio, pio + alo and gla groups compared with untreated or alo groups, and there was no difference among pio, pio + alo and gla groups. To precisely evaluate islet β-cell functions, islet perifusion analysis was performed. In pio, pio + alo and gla groups, biphasic insulin secretion was preserved compared with untreated or alo groups. In particular, pio + alo as well as gla therapy preserved almost normal insulin secretion, although pio therapy improved partially. To examine the mechanism how these reagents exerted beneficial effects on β-cells, we evaluated expression levels of various factors which are potentially important for β-cell functions by real-time RT-PCR and immunohistochemistry. The results showed that expression levels of MafA and GLP-1 receptor were markedly decreased in untreated and alo groups, but not in pio, pio + alo and gla groups. Conclusion: Combination therapy with pio and alo almost completely normalized β-cell functions in vivo, which was comparable with gla treatment.     

The mTORC2/PKC pathway sustains compensatory insulin secretion of pancreatic β cells in response to metabolic stress

BackgroundCompensation of the pancreatic β cell functional mass in response to metabolic stress is key to the pathogenesis of Type 2 Diabetes. The mTORC2 pathway governs fuel metabolism and β cell functional mass. It is unknown whether mTORC2 is required for regulating metabolic stress-induced β cell compensation.MethodsWe challenged four-week-old β-cell-specific Rictor (a key component of mTORC2)-knockout mice with a high fat diet (HFD) for 4 weeks and measured metabolic and pancreatic morphological parameters. We performed ex vivo experiments to analyse β cell insulin secretion and electrophysiology characteristics. Adenoviral-mediated overexpression and lentiviral-ShRNA-mediated knocking down proteins were applied in Min6 cells and cultured primary mouse islets.ResultsβRicKO mice showed a significant glucose intolerance and a reduced plasma insulin level and an unchanged level β cell mass versus the control mice under HFD. A HFD or palmitate treatment enhanced both glucose-induced insulin secretion (GIIS) and the PMA (phorbol 12-myristate 13-acetate)-induced insulin secretion in the control islets but not in the βRicKO islets. The KO β cells showed similar glucose-induced Ca^2 + influx but lower membrane capacitance increments versus the control cells. The enhanced mTORC2/PKC proteins levels in the control HFD group were ablated by Rictor deletion. Replenishing PKCα by overexpression of PKCα-T638D restored the defective GIIS in βRicKO islets.ConclusionsThe mTORC2/Rictor pathway modulates β cell compensatory GIIS under nutrient overload mediated by its phosphorylation of PKCα.General significanceThis study suggests that the mTORC2/PKC pathway in β cells is involved in the pathogenesis of T2D.     

Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms

Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in?vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.     

Gaegurin-6 stimulates insulin secretion through calcium influx in pancreatic β Rin5mf cells

Gaegurin-6, an antimicrobial peptide that belongs to the alpha-helix family, was isolated from the skin of Rana rugosa. Gaegurin-6 contains a hydrophobic motif at the N-terminus and a helical region at the C-terminus. Although gaegurin-6 has been implicated in cell signaling, the precise role in insulin secretion is currently unknown. We have attempted to determine whether gaegurin-6 affects insulin secretion and tried to elucidate the relationship between the structural motifs and biological activity. In this study, we have shown that gaegurin-6 stimulates insulin secretion and also increases the intracellular calcium concentration in pancreatic β Rin5mf cells. Moreover, a corollary study revealed that both the hydrophobicity of the N-terminus and the disulfide bridge of the C-terminus of gaegurin-6 are critical for its effects on insulin secretion. Membrane pore-forming ability is also observed in gaegurin-6, but not in the linear form or the N-terminus hydrophobic amino acid-deleted form. We further showed that these regions of gaegurin-6 are responsible for calcium influx in pancreatic β Rin5mf cells. Taken together, these results indicate that gaegurin-6 can affect insulin secretion in pancreatic β cells through the modulation of calcium influx.     

Dietary nucleotides increase the mucosal IgA response and the secretion of transforming growth factor β from intestinal epithelial cells in mice

We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing γδ TCR (TCRγδ⁺ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ⁺ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ⁺ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ⁺ IELs. This revised version was published online in August 2006 with corrections to the Cover Date.