. Reduced ␤-cell mass and altered glucose sensing impair insulin-secretory function in ␤IRKO mice. Am J Physiol Endocrinol Metab 286: E41-E49, 2004. First published September 30, 2003 10.1152/ajpendo.00533.2001.-Pancreatic ␤-cellrestricted 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 ␤-cell insulin receptor knockout (␤IRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, ϳ7-mo-old ␤IRKO 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 ␤-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic ␤IRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic ␤IRKOs and to a lesser degree in the nondiabetic ␤IRKO group. Pancreatic islets of nondiabetic ␤IRKO 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 ␤-cells leads primarily to profound defects in postnatal ␤-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.

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 ␤-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 ␤-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.The pancreatic ␤-cell plays a central role in the pathogenesis of diabetes mellitus. A reduction in ␤-cell mass mediated at least in part by an increase in apoptosis is characteristic of the diabetic state (1-3). It is becoming clear that several pathways can lead to ␤-cell apoptosis, including cytokine signaling, excessive Ca 2ϩ influx during chronic hyperglycemia, high levels of free fatty acids, hypoxia or hypoglycemia, endoplasmic reticulum (ER) 1 stress, and loss of growth factor signaling (1, 3-12). Whether various inducers of apoptosis employ distinct molecular mechanisms has not been systematically studied.Intracellular Ca 2ϩ stores play an important role in the regulation of apoptosis in many cell types (13,14). The present study was undertaken to test the hypothesis that alterations in specific intracellular Ca 2ϩ stores may induce apoptosis in pancreatic ␤-cells. There are at least three classes of intracellular Ca 2ϩ stores in ␤-cells, and these are sensitive, respectively, to inositol trisphosphate (IP 3 )/thapsigargin, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP ribose/ryanodine (15-18). In many cell types, ryanodine receptor Ca 2ϩ channels (RyR) transmit Ca 2ϩ signals directly to closely associated mitochondria (19). In the MIN6 ␤-cell line, RyR were shown to regulate ATP production (20). Because of their role in regulating intracellular Ca 2ϩ and mitochondrial function, we focused specifically on RyR as likely mediators of ␤-cell apoptosis. Of the three RyR subtypes, two have been reported to be present in ␤-cells, RyR1 and RyR2. The latter is more abundant and can be distinguished from the former by its insensitivity to dantrolene (21,22). Ryanodine, a plant alkaloid, is the most specific probe for all RyR subtypes, and its activity is lost in RyR-deficient cells (23,24).In the present study, we examined the role of RyR in the survival of human and mouse pancreatic islets. We uncovered a novel apoptosis pathway that is initiated when Ca 2ϩ flux through RyR2 is blocked. The ...

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Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2؉ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action. Diabetes 50

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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 ϳ60% 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.

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 ϳ80%, GLUT4 mRNA was increased ϳ2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased ϳ2.5-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. Diabetes 54:624 -628, 2005

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an ϳ3-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 ϳ60% 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 contractionsignaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates. adenosine monophosphate-activated protein kinase; calpastatin; calcium/calmodulin-dependent protein kinase-II