Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (-/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (-/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (-/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the beta-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 beta-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the beta-cells and not the alpha-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic beta-cells.

The disruption of Munc18c binding to syntaxin 4 impairs insulin-stimulated GLUT4 vesicle translocation in 3T3L1 adipocytes. To investigate the physiological function and requirement for Munc18c in the regulation of GLUT4 translocation and glucose homeostasis in vivo, we used homologous recombination to generate Munc18c-knockout (KO) mice. Homozygotic disruption of the Munc18c gene resulted in early embryonic lethality, whereas heterozygous KO mice (Munc18c ؊/؉ ) had normal viability. Munc18c ؊/؉ mice displayed significantly decreased insulin sensitivity in an insulin tolerance test and a >50% reduction in skeletal muscle insulinstimulated GLUT4 translocation when compared with wild-type (WT) mice. Furthermore, glucose-stimulated insulin secretion was significantly reduced in islets isolated from Munc18c ؊/؉ mice compared with those from WT mice. Despite the defects in insulin action and secretion, Munc18c ؊/؉ mice demonstrated the ability to clear glucose to the same level as WT mice in a glucose tolerance test when fed a normal diet. However, after consuming a high-fat diet for only 5 weeks, the Munc18c ؊/؉ mice manifested severely impaired glucose tolerance compared with high-fat؊fed WT mice. Taken together, these data suggest that the reduction of Munc18c protein in the Munc18c ؊/؉ mice results in impaired insulin sensitivity with a latent increased susceptibility for developing severe glucose intolerance in response to environmental perturbations such as intake of a high-calorie diet rich in fat and carbohydrate. Diabetes 54:638 -647, 2005 G lucose homeostasis is maintained by a series of regulated vesicle exocytosis events in several cell types and tissues. Vesicle exocytosis entails the pairing of a vesicle-associated membrane protein (VAMP) v-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein [SNAP] receptor) with a binary cognate receptor complex at the target membrane composed of SNAP-25/23 and syntaxin proteins (t-SNAREs) to form the SNARE core complex. Multiple isoforms of each SNARE protein have been identified and it is hypothesized that by specific pairing and compartmentalization of these proteins, specificity of vesicle targeting can be achieved (rev. in 1,2). On the basis of these studies, investigators from diabetes-related fields have discovered that SNARE protein core complexes are also responsible for regulating the secretion of insulin from islet ␤-cells in response to increased blood glucose (3-6), as well as facilitating the downstream action of insulin on peripheral glucose disposal via the translocation to and integration of intracellular glucose transporter (GLUT4) vesicles into the cell surface membranes of adipocytes and skeletal muscle (rev. in 7-9).Concurrent with the discovery of SNARE proteins has come evidence from yeast genetics suggesting that other proteins also participate in exocytosis and play a role in the regulation of the SNARE complex; this has led to the discovery of the Sec1 secretory proteins. Yeast Sec1 interacts directly with the t-SNARE syntaxin,...

Insulin-stimulated translocation of GLUT4 vesicles from an intracellular compartment to the plasma membrane in 3T3L1 adipocytes is mediated through a syntaxin 4 (Syn4)-and Munc18c-dependent mechanism. To investigate the impact of increasing Syn4 protein abundance on glucose homeostasis in vivo, we engineered tetracycline-repressible transgenic mice to overexpress Syn4 by fivefold in skeletal muscle and pancreas and threefold in adipose tissue. Increases in Syn4 caused increases in Munc18c protein, indicating that Syn4 regulates Munc18c expression in vivo. An important finding was that female Syn4 transgenic mice exhibited an increased rate of glucose clearance during glucose tolerance tests that was repressible by the administration of tetracycline. Insulin-stimulated glucose uptake in skeletal muscle was increased by twofold in Syn4 transgenic mice compared with wild-type mice as assessed by hyperinsulinemic-euglycemic clamp analysis, consistent with a twofold increase in insulin-stimulated GLUT4 translocation in skeletal muscle. Hepatic insulin action was unaffected. Moreover, insulin content and glucosestimulated insulin secretion by islets isolated from Syn4 transgenic mice did not differ from that of wild-type mice. In sum, these data suggest that increasing the number of Syn4-Munc18c "fusion sites" at the plasma membrane of skeletal muscle increases the amount of GLUT4 available to increase the overall rate of insulinmediated glucose uptake in vivo. Diabetes 53:2223-2231, 2004 I nsulin resistance is a major factor in the pathogenesis of type 2 diabetes, although the mechanism by which this occurs remains unclear. Insulin resistance affects multiple insulin-sensitive tissue types (skeletal muscle, adipose, and liver); however, it remains uncertain as to whether a defect in one or multiple tissues is required to lead to the progression to diabetes. The majority of insulin-stimulated glucose uptake in skeletal muscle and adipose tissue is attributed to the insulinresponsive glucose transporter GLUT4 (1). In the basal non-insulin-stimulated state, GLUT4 localizes to tubulovesicular elements and small intracellular vesicles throughout the cell cytoplasm (2,3). Upon stimulation with insulin, these GLUT4-containing compartments undergo a series of regulated steps, leading to their eventual fusion with the plasma membrane (4 -9). This ultimately results in a large increase in the number of functional glucose transporters on the cell surface (a process termed "translocation"), which accounts for nearly all of the insulin-stimulated increase in glucose uptake.Insulin-stimulated translocation of GLUT4 vesicles is mediated by the binding of plasma membrane soluble N-ethylmaleimideϪsensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin 4 (Syn4) and SNAP-23 with the GLUT4 vesicle v-SNARE protein vesicleassociated membrane protein 2 (VAMP2). Studies that substantiate this show that GLUT4 vesicles copurify with the VAMP2 v-SNARE, and that specific proteolytic cleavage of VAMP2 and expression of a do...

To investigate the physiological effects of modulating the abundance of Munc18c or syntaxin 4 (Syn4) proteins on the regulation of glucose homeostasis in vivo, we generated tetracycline-repressible transgenic mice that overexpress either Munc18c or Syn4 proteins in skeletal muscle, pancreas and adipose tissue seven-, five-, and threefold over endogenous protein, respectively. Munc18c transgenic mice displayed whole-body insulin resistance during hyperinsulinemic-euglycemic clamp resulting from >41% reductions in skeletal muscle and white adipose tissue glucose uptake, but without alteration of hepatic insulin action. Munc18c transgenic mice exhibited ϳ40% decreases in whole-body glycogen/ lipid synthesis, skeletal muscle glycogen synthesis, and glycolysis. Glucose intolerance in Munc18c transgenic mice was reversed by repression of transgene expression using tetracycline or by simultaneous overexpression of Syn4 protein. In addition, Munc18c transgenic mice had depressed serum insulin levels, reflecting a threefold reduction in insulin secretion from islets isolated therefrom, thus uncovering roles for Munc18c and/or Syn4 in insulin granule exocytosis. Taken together, these results indicate that balance, more than absolute abundance, of Munc18c and Syn4 proteins directly affects whole-body glucose homeostasis through alterations in insulin secretion and insulin action.