| Insulin stimulated glucose uptake is mediated by the GLUT4 glucose transporter. Here we examine three aspects of insulin mediated glucose uptake. This includes SNARE proteins in skeletal muscle, the activation of glucose transport, and the trafficking of GLUT4 to its insulin responsive compartment.; First, we show the development of a transgenic mouse expressing GLUT4-EGFP as a model for examination of insulin stimulated glucose uptake in skeletal muscle. We establish this model showing that the expressed GLUT4-EGFP behaves like endogenous GLUT4. Then, through adenoviral mediated gene transfer, we show that overexpression of Munc18c inhibits GLUT4 translocation to the transverse tubules, and not the sarcolemma. This establishes the physiological difference between the two surface membranes and the stoichiometry between munc18c and syntaxin 4 as an important regulator of SNARE function.; Secondly, we examine the role of temperature on GLUT4 translocation and glucose transport in 3T3L1 adipocytes. Incubation at low temperature decreases the rate at which GLUT4 moves to the plasma membrane, however, the extent of GLUT4 at the PM is eventually equivalent. In contrast, glucose uptake rates at 23°C are decreased throughout the time course, and remain depressed despite an equivalent number of glucose transporters as compared to cells incubated at 37°C. This suggests that temperature can regulate glucose transporter activity. This effect correlates with a temperature specific decrease in Akt phosphorylation and activation, suggesting that Akt may be a mediator of glucose transporter activation.; Finally, we use an electroporation pulse-chase protocol to follow the trafficking of GLUT4 in a 3T3L1 adipocyte. We show that GLUT4 fills the insulin responsive compartment independently of endocytosis, using external and GLUT4 specific inhibitors. Using chimeric proteins of GLUT1 and GLUT4, the amino terminus and the intracellular loop are sufficient to create a chimera that traffics identically to GLUT4. We hypothesize that the amino terminus targets the protein to its storage compartment, while the loop retains the protein in the compartment to prevent the plasma membrane leak. |
