Insulin gene is a target in activin receptor-like kinase 7 signaling pathway in pancreatic beta-cells

Activins regulate pancreatic development, differentiation and insulin secretion. Activin receptor-like kinase 7 (ALK7) has been identified as a receptor for Nodal and Activin AB and B, and is expressed in pancreatic islets and β-cell lines. In this study, human insulin promoter was activated by Smad2, Smad3 and the pancreatic and duodenal homeobox factor-1 (PDX-1) in the ALK7 pathway. A conserved Smad binding element was related to the promoter activation. Phosphorylated Smad2/Smad3 and PDX-1 were bound to insulin gene with Nodal and Activin AB, and the phosphorylated Smad2/Smad3 interacted with PDX-1. These results indicate that one of the direct target genes of Nodal and Activin AB signals is the insulin gene in pancreatic β-cells and that PDX-1 is directly involved in the ALK7-Smad pathway.     

Resistin induces insulin resistance in pancreatic islets to impair glucose-induced insulin release

An adipokine resistin, a small cysteine-rich protein, is one of the major risk factors of insulin resistance. In the present study, transiently resistin-expressing mice using adenovirus method showed an impaired glucose tolerance due to insulin resistance. We found that resistin-expressing mice exhibited impaired insulin secretory response to glucose. In addition, in vitro treatment with resistin for 1 day induced insulin resistance in pancreatic islets and impaired glucose-stimulated insulin secretion by elevating insulin release at basal glucose (2.8 mM) and suppressing insulin release at stimulatory glucose (8.3 mM). In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets as well as other insulin target organs. Furthermore, resistin induced SOCS-3 expression in beta-cells. In conclusion, resistin induces insulin resistance in islet beta-cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation and impairs glucose-induced insulin secretion.     

MIN6 beta-cell-beta-cell interactions influence insulin secretory responses to nutrients and non-nutrients

Insulin-secreting MIN6 cells show greatly enhanced secretory responsiveness to nutrients when grown as islet-like structures (pseudoislets). Since beta-cells use different mechanisms to respond to nutrient and non-nutrient stimuli, we have now investigated the role of homotypic beta-cell interactions in secretory responses to pharmacological or receptor-operated non-nutrient stimuli in MIN6 pseudoislets. In addition to an enhanced secretory responsiveness to glucose, insulin secretion from MIN6 pseudoislets was also enhanced by non-nutrients, including carbachol, tolbutamide, PMA, and forskolin. The improved secretory responsiveness was dependent on the cells being configured as pseudoislets and was lost on dispersal of the pseudoislets into single cells and regained on the re-formation of pseudoislet structures. These observations emphasise the importance of islet anatomy on secretory responsiveness, and demonstrate that homotypic beta-cell interactions play an important role in generating physiologically appropriate insulin secretory responses to both nutrient and non-nutrient stimuli.     

Autocrine anti-apoptotic and proliferative effects of insulin in pancreatic beta-cells

Insulin and glucose inhibited apoptosis in the MIN6 insulin-secreting cell line. The protective effect of 25 mM glucose was prevented by an anti-insulin antibody and this antibody-induced increase in apoptosis was reversed by the presence of excess insulin. Glucose stimulated MIN6 cell proliferation and this was inhibited by blockade of insulin secretion, by an anti-insulin antibody and by phosphatidylinositol-3 kinase (PI-3K) inhibition. Furthermore, MIN6 cell proliferation was stimulated by depolarising concentrations of KCl and by insulin itself. These data indicate that insulin secreted by β-cells in response to elevated glucose exerts autocrine effects to protect against apoptosis and stimulate proliferation, and suggest that the insulin signalling cascade, through the PI-3K pathway, may be an effective means of maintaining β-cell mass in diabetes.     

PPAR-gamma overexpression selectively suppresses insulin secretory capacity in isolated pancreatic islets through induction of UCP-2 protein

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions, but its physiological role in pancreatic islet cells remains to be investigated. In this study, we confirmed the presence of PPAR-gamma in rat isolated islets and examined its role on insulin and glucagon secretion by using PPAR-gamma-overexpressed islets. PPAR-gamma overexpression significantly suppressed insulin secretion induced by stimulatory concentration of glucose (p<0.05). In addition, insulin secretion evoked by high potassium depolarization also was significantly decreased from PPAR-gamma-overexpressed islets (p<0.05). On the other hand, no significant change in glucagon release was observed after high potassium depolarization between PPAR-gamma-overexpressed and control islets. Insulin and glucagon content in islets was not statistically different between the two groups. In addition, the expression of uncoupling protein-2 (UCP-2) was found to be induced in PPAR-gamma-overexpressed islets. This result clearly indicates that the deteriorative effect of PPAR-gamma overexpression on the secretory machinery is selective for pancreatic beta-cells. And it is possible that its site of action can be located in the energy-consuming exocytotic process of insulin secretory granules, and that the reduction of ATP production through increased UCP-2 reduces insulin exocytosis.     

Regulation of pancreatic duct cell differentiation by phosphatidylinositol-3 kinase

We have previously demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling is essential for pancreatic regeneration after partial pancreatectomy in mice. In the present study, we examined a role of PI3K/Akt signaling for pancreatic duct cell differentiation into insulin-producing cells. Epithelial-like cells were isolated from mouse pancreas and confirmed to be positive for a duct cell marker cytokeratin-20 (CK-20) but negative for insulin. Incubation of these cells with epidermal growth factor, exhibited a gradual increase in Akt phosphorylation and expression of pancreatic duodenal homeobox-1 (PDX-1), a regulator of β-cell differentiation. Three weeks later, these CK-20-positive cells were noted to express insulin as determined by immunofluorescent double-staining. Akt phosphorylation, PDX-1 expression, and insulin production were effectively reduced by blocking the PI3K/Akt pathway using siRNA to the p85α regulatory subunit of PI3K. Our results demonstrate that PI3K/Akt activation has a critical role for pancreatic duct cell differentiation into insulin-producing cells.     

Nutrient–secretion coupling in the pancreatic islet β-cell: recent advances

Insulin secretion from pancreatic islet β-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total β-cell mass is essentially unaltered, but β-cells become progressively “glucose blind” and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K⁺(K_ATP) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca²⁺, then prompt the “first phase” on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K_ATP channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Cyclin D1 activation through ATF-2 in Reg-induced pancreatic beta-cell regeneration

Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.     

Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic beta-cell glucose-sensing, cellular signal transduction and integrity

Homocysteine can be converted to its reactive thioester, homocysteine thiolactone. Cytotoxic properties of these amino thiols have been attributed to protein homocysteinylation, increased oxidative stress, DNA damage and apoptosis. This study used pancreatic BRIN-BD11 beta-cells to examine functional defects caused by acute and long-term exposure to homocysteine thiolactone in comparison with homocysteine. Acute and long-term exposure to both agents caused concentration-dependent inhibitions of glucose-induced insulin secretion while impairing the insulin-secretory responses to alanine, KCl, elevated Ca(2+), forskolin and PMA. Acute exposures also caused significant reduction in the amplitude of KCl-induced membrane depolarisation but no effects on changes of intracellular Ca(2+) induced by alanine or KCl. Cellular insulin content and DNA damage were not altered following culture, however, there were early signs of apoptosis consistent with impaired cellular integrity. In conclusion, exposure to homocysteine thiolactone, like homocysteine, induced beta-cell dysfunction and demise by mechanisms independent of changes in membrane potential and [Ca(2+)](i).     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Improved insulin sensitivity and metabolic flexibility in ghrelin receptor knockout mice

Stimulation of the ghrelin receptor (GhrR) by ghrelin results in a variety of metabolic changes including increased food intake, fat storage and insulin resistance. Loss of ghrelin signaling is protective against diet-induced obesity, suggesting that ghrelin plays a significant homeostatic role in conditions of metabolic stress. We examined glycemic control in GhrR −/− mice fed a high-fat diet, and used indirect calorimetry to assess fuel substrate usage and energy expenditure. GhrR −/− mice fed a high-fat diet had several measures of greater insulin sensitivity, including: lower fasted blood glucose and plasma insulin, lower %Hb_A1c, lower insulin levels during glucose tolerance tests, and improved performance in hyperinsulinemic-euglycemic and hyperglycemic clamp studies. GhrR −/− mice fed a high-fat diet did not develop hepatic steatosis and had lower total cholesterol, relative to controls. Furthermore, GhrR −/− mice demonstrated a lower intestinal triglyceride secretion rate of dietary lipid. GhrR −/− mice have higher respiratory quotients (RQ), indicating a preference for carbohydrate as fuel. The range of RQ values was wider in GhrR −/− mice, indicating greater metabolic flexibility and insulin sensitivity in these animals. We therefore propose that loss of ghrelin signaling promotes insulin sensitivity and metabolic flexibility, and protects against several fatty diet-induced features of metabolic syndrome due to convergent changes in the intake, absorption and utilization of energy.