Impaired insulin secretion in transgenic mice over-expressing calpastatin in pancreatic ß-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 ß-cell function. In the present study, transgenic mice (Cast (RIP)) expressing an endogenous calpain inhibitor, calpastatin, in pancreatic ß-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.

Inhibition of calpain results in impaired contraction-stimulated GLUT4 translocation in skeletal muscle.

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.

Calcineurin does not mediate exercise-induced increase in muscle GLUT4.

Exercise induces a rapid increase in expression of the GLUT4 isoform of the glucose transporter in skeletal muscle. One of the signals responsible for this adaptation appears to be an increase in cytosolic Ca(2+). Myocyte enhancer factor 2A (MEF2A) is a transcription factor that is involved in the regulation of GLUT4 expression. It has been reported that the Ca(2+)-regulated phosphatase calcineurin mediates the activation of MEF2 by exercise. It has also been shown that the expression of activated calcineurin in mouse skeletal muscle results in an increase in GLUT4. These findings suggest that increases in cytosolic Ca(2+) induce increased GLUT4 expression by activating calcineurin. However, we have obtained evidence that this response is mediated by a Ca(2+)-calmodulin-dependent protein kinase. The purpose of this study was to test the hypothesis that calcineurin is involved in mediating exercise-induced increases in GLUT4. Rats were exercised on 5 successive days using a swimming protocol. One group of swimmers was given 20 mg/kg body weight of cyclosporin, a calcineurin inhibitor, 2 h before exercise. A second group was given vehicle. GLUT4 protein was increased approximately 80%, GLUT4 mRNA was increased approximately 2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased approximately 2.5-fold 18 h after the last exercise bout. The cyclosporin treatment completely inhibited calcineurin activity but did not affect the adaptive increases in GLUT4, MEF2A, or hexokinase expression. We conclude that calcineurin activation does not mediate the adaptive increase in GLUT4 expression induced in skeletal muscle by exercise.

Defective insulin secretion and increased susceptibility to experimental diabetes are induced by reduced Akt activity in pancreatic islet beta cells.

The insulin and IGF signaling pathways are critical for development and maintenance of pancreatic beta cell mass and function. The serine-threonine kinase Akt is one of several mediators regulated by these pathways. We have studied the role of Akt in pancreatic beta cell physiology by generating transgenic mice expressing a kinase-dead mutant of this enzyme in beta cells. Reduction of Akt activity in transgenic animals resulted in impaired glucose tolerance due to defective insulin secretion. The mechanisms involved in dysregulation of secretion in these mice lie at the level of insulin exocytosis and are not the result of abnormalities in glucose signaling or function of voltage-gated Ca2+ channels. Therefore, transgenic mice showed increased susceptibility to developing glucose intolerance and diabetes following fat feeding. These observations suggest that Akt plays a novel and important role in the regulation of distal components of the secretory pathway and that this enzyme represents a therapeutic target for improvement of beta cell function in diabetes.

RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets.

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover.

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.

Reduced beta-cell mass and altered glucose sensing impair insulin-secretory function in betaIRKO mice.

Pancreatic beta-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in beta-cell insulin receptor knockout (betaIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, approximately 7-mo-old betaIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced beta-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic betaIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic betaIRKOs and to a lesser degree in the nondiabetic betaIRKO group. Pancreatic islets of nondiabetic betaIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in beta-cells leads primarily to profound defects in postnatal beta-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.