Studies of the trafficking of the insulin-responsive glucose transporter, GLUT4, in 3T3-L1 adipocytes
The translocation of GLUT4 from intracellular stores to the plasma membrane in response to insulin accounts for the large insulin-mediated glucose uptake in muscle and fat tissue. A defect of this translocation mechanism is evident in individuals with insulin resistance and type 2 diabetes. Hence, understanding the molecular basis of GLUT4 localisation and recycling is important in order to assist the design of rational therapies for the treatment of this disease. Here, we have used iodixanol gradient analysis to examine the intracellular distribution of GLUT4. By this method intracellular GLUT4 could be separated into two pools one of which is highly insulin sensitive, and corresponds to "GLUT4 storage vesicles" (GSV), while the other is less insulin sensitive and is of endosomal origin. We further show that during differentiation of 3T3-L1 fibroblasts into adipocytes, the formation of the GSV compartment appears to be driven by the expression of GLUT4.SNARE proteins are involved in the fidelity of GLUT4 translocation, but little is known about how these proteins may be altered in insulin resistance. Using insulin-resistant 3T3-L1 adipocytes, we show that SNARE protein levels are increased. The potential importance of this observation is discussed. Using iodixanol gradient analysis we also found that the downregulation of GLUT4 known to occur in insulin-resistant 3T3-L1 adipocytes is predominantly from the GSV compartment. The implications of these data for the aetiology of insulin resistance are discussed.