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Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2-and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.The binding of insulin to its heterotetrameric integral-membrane receptor activates its intracellular tyrosine kinase domain and thereby triggers a signaling cascade resulting in the translocation and fusion of intracellular GLUT4 1 isoform-containing vesicles to the plasma membrane (1-3). Although most cell types also constitutively express the GLUT1 isoform at the cell surface, the insulin-stimulated increase in plasma membrane-associated GLUT4 protein accounts for the majority of post-prandial glucose disposal in both muscle and adipose tissue (4).The insulin-stimulated translocation of these GLUT4-containing vesicles has several features in common with the regulated exocytosis pathway of synaptic vesicle trafficking in neurotransmitter release (5). The machinery involved in the regulation of synaptic vesicle priming/docking/fusion entails the pairing of protein complexes in the vesicle compartment (v-SNAREs, for vesicle SNAP receptors) with their cognate receptor complexes at the target membrane (t-SNAREs, for target membrane SNAP receptors). Recently, several of the vand t-SNARE proteins have been identified that specifically participate in the insulin-regulated docking and fusion of GLUT4 vesicles with the adipocyte plasma membrane. GLUT4 vesicles co-purify with both the VAMP2 and VAMP3 v-SNARE isoforms and specific proteolytic cleavage of VAMP2, expression of a dominant-interfering VAMP2 mutant or inhibitory peptides impairs insulin-stimulated GLUT4 translocation (6 -11). In addition, transferrin-horseradish peroxidase ablation of recycling endosomes resulted in a selective loss ...

Evidence suggests that chromium supplementation may alleviate symptoms associated with diabetes, such as high blood glucose and lipid abnormalities, yet a molecular mechanism remains unclear. Here, we report that trivalent chromium in the chloride (CrCl3) or picolinate (CrPic) salt forms mobilize the glucose transporter, GLUT4, to the plasma membrane in 3T3-L1 adipocytes. Concomitant with an increase in GLUT4 at the plasma membrane, insulin-stimulated glucose transport was enhanced by chromium treatment. In contrast, the chromium-mobilized pool of transporters was not active in the absence of insulin. Microscopic analysis of an exofacially Myc-tagged enhanced green fluorescent protein-GLUT4 construct revealed that the chromium-induced accumulation of GLUT4-containing vesicles occurred adjacent to the inner cell surface membrane. With insulin these transporters physically incorporated into the plasma membrane. Regulation of GLUT4 translocation by chromium did not involve known insulin signaling proteins such as the insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt. Consistent with a reported effect of chromium on increasing membrane fluidity, we found that chromium treatment decreased plasma membrane cholesterol. Interestingly, cholesterol add-back to the plasma membrane prevented the beneficial effect of chromium on both GLUT4 mobilization and insulin-stimulated glucose transport. Furthermore, chromium action was absent in methyl-beta-cyclodextrin-pretreated cells already displaying reduced plasma membrane cholesterol and increased GLUT4 translocation. Together, these data reveal a novel mechanism by which chromium may enhance GLUT4 trafficking and insulin-stimulated glucose transport. Moreover, these findings at the level of the cell are consistent with in vivo observations of improved glucose tolerance and decreased circulating cholesterol levels after chromium supplementation.

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/ fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin-and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin-and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130 cas , and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125 FAK and p130 cas . These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.The facilitative glucose transporters are a family of related integral membrane proteins that are responsible for the regulation of whole body and cellular glucose homeostasis. Unlike other members of this family, the insulin-responsive glucose transporter isoform (GLUT4) 1 is predominantly expressed in adipose tissue and in skeletal and cardiac muscle (1, 2). In these tissues, insulin increases glucose uptake by regulating the intracellular trafficking of the GLUT4 protein. In the basal state, GLUT4 cycles continuously between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing within the cell interior (3, 4). Activation of the insulin receptor triggers a large increase in the rate of GLUT4 vesicle exocytosis in addition to a smaller decrease in the rate of internalization by endocytosis. This insulin-dependent shift in the cellular dynamics of GLUT4 vesicle trafficking results in a net increase of GLUT4 protein level on the cell surface, thereby increasing the rate of glucose uptake (for recent reviews, see Refs. 5-8).Activation of the insulin receptor by ligand binding initiates a cascade of signaling events, which include activation of the intrinsic receptor tyrosine kinase, autophosphorylation of the receptor, and phosphorylation of cellular substrates such as insulin receptor substrate (IRS)-1/2 and Shc (for recent reviews, see Refs. 9 and 10). Phosphorylation of these substrates provides docking s...

AMP-activated protein kinase (AMPK) enhances glucose transporter GLUT4 regulation. AMPK also suppresses energy-consuming pathways such as cholesterol synthesis. Interestingly, recent in vitro and in vivo data suggest that excess membrane cholesterol impairs GLUT4 regulation. Therefore, this study tested whether a beneficial, GLUT4-regulatory aspect of AMPK stimulation involved cholesterol lowering. Using L6 myotubes stably expressing an exofacial myc-epitope-tagged-GLUT4, AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR; 45 min, 1 mm) or 2,4-dinitrophenol (DNP; 30 min, 200 μm) increased cell surface GLUT4myc labeling by approximately ≈ 25% (P < 0.05). Insulin (20 min, 100 nm) also increased GLUT4myc labeling by about 50% (P < 0.05), which was further enhanced (≈ 25%, P < 0.05) by AICAR or DNP. Consistent with AMPK-mediated suppression of cholesterol synthesis, AICAR and DNP decreased membrane cholesterol by 20-25% (P < 0.05). Whereas AMPK knockdown prevented the enhanced basal and insulin-stimulated GLUT4myc labeling by AICAR and DNP, cholesterol replenishment only blocked the AMPK-associated enhancement in insulin action. Cells cultured in a hyperinsulinemic milieu, resembling conditions in vivo that promote the progression/worsening of insulin resistance, displayed an increase in membrane cholesterol. This occurred concomitantly with a loss of cortical filamentous actin (F-actin) and defects in GLUT4 regulation by insulin. These derangements were prevented by AMPK stimulation. Examination of skeletal muscle from insulin-resistant Zucker rats revealed a similar elevation in membrane cholesterol and loss of F-actin. Lowering cholesterol to control levels restored F-actin structure and insulin sensitivity. In conclusion, these data suggest a novel aspect of GLUT4 regulation by AMPK involves membrane cholesterol lowering. Moreover, this AMPK-mediated process protected against hyperinsulinemia-induced insulin resistance.