Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2–3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6–9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulinresponsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4-and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.The insulin-responsive glucose transporter GLUT4 is predominantly expressed in both striated muscle and adipose tissue and is responsible for the majority of insulin-stimulated glucose uptake (24). In the basal non-insulin-stimulated state, GLUT4 localizes to tubulovesicular elements and small intracellular vesicles throughout the cell cytoplasm (30, 31). Upon stimulation with insulin these GLUT4-containing compartments undergo a series of regulated steps leading to the trafficking, association, and eventual fusion with the plasma membrane (3,14,25,27). This ultimately results in a large increase in the number of functional glucose transporters on the cell surface, termed translocation, and accounts for the majority, if not all, of the insulin-stimulated increase in glucose uptake. More recently, another cargo protein, insulin-responsive amino peptidase (IRAP), has been demonstrated to colocalize with GLUT4 and undergoes an identical pattern of insulinstimulated translocation (14-16, 18, 20, 28).The insulin-stimulated translocation of GLUT4 and IRAP storage vesicles shares several features with the docking and fusion of synaptic vesicles in neurotransmitter release. For example, the interaction of the GLUT4 vesicle v-SNARE protein, VAMP2, with the plasma membrane t-SNARE proteins, syntaxin 4 and SNAP23, is necessary for insulin-stimulated GLUT4 translocation (2,19,23,33,37). In addition to these tand v-SNAREs, there are several accessory proteins involved in the regulation of the GLUT4 and IRAP vesicle translocation. We and others have observed that increased expression...

It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-␤-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin-and clathrindependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced (ϳ40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by ϳ20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.One of the major acute actions of insulin is enhanced glucose uptake in striated muscle and adipose tissue (1-3). This results from the rapid translocation of the intracellular-sequestered GLUT4 1 glucose transporter isoform to the plasma membrane (4, 5). The increase in plasma membrane GLUT4 occurs due to a large increase in the rate of GLUT4 exocytosis coupled with a smaller decrease in the rate of GLUT4 endocytosis (6, 7). Recent data suggest that two independent signal transduction pathways are necessary for the full extent of insulin-stimulated GLUT4 translocation. In one case, the insulin receptor tyrosine phosphorylates insulin receptor substrate-family proteins, resulting in the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol 3,4,5-triphosphate. Although less well defined, the serine/threonine kinases phosphoinositide-dependent protein kinase 1 and protein kinase B/Akt as well as protein kinase C/ have been implicated in signaling events functioning downstream of phosphatidylinositol 3-kinase (8 -10). This pathway appears to be spatially segregated from a parallel insulin receptor-signaling pathway that results in the tyrosine phosphory...

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin-and exercisestimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.The stimulation of glucose uptake in adipose and muscle tissues primarily occurs through the translocation of the GLUT4 glucose transporter isoform from intracellular storage sites to the cell surface membranes (1-4). Insulin stimulation of this process requires the tyrosine phosphorylation of the insulin receptor substrate family of proteins and subsequent activation of the type 1 phosphatidylinositol (PI) 1 3-kinase (5-12). Although the precise signaling steps that occur downstream of the PI 3-kinase ultimately leading to GLUT4 translocation have remained elusive, recent studies have begun to resolve the specific trafficking, docking, and fusions events. In adipocytes, it is well established that GLUT4 storage compartments contain vesicle-localized proteins (v-SNAREs) that specifically interact with cognate cell surface target proteins (t-SNAREs) at the plasma membrane to promote vesicle docking and fusion (13,14). Insulin-stimulated GLUT4 translocation is dependent upon the interaction of the v-SNARE, VAMP2, with the plasma membrane t-SNAREs, syntaxin 4 and SNAP23 (15-21). Furthermore, the syntaxin 4-binding protein, Munc18c, has been shown to specifically modulate insulinsensitive GLUT4 translocation in 3T3L1 adipocytes (22-24). However, it is important to recognize that the majority of these studies have been performed in cultured 3T3L1 adipocytes and L6 myotubes due to the inherent technical limitations in the study of GLUT4 trafficking in adipocytes and skeletal muscle in vivo. Sk...