IMMUNOLOGICAL ANALYSIS OF GLUT4-ENRICHED VESICLES - IDENTIFICATION OFNOVEL PROTEINS REGULATED BY INSULIN AND DIABETES

In adipocytes and muscle, insulin stimulates the translocation of gluc ose transporter proteins from an intracellular vesicle pool to the pla sma membrane. To study the molecular basis of this process, we used th e anti-GLUT4 antibody 1F8 to isolate intracellular vesicles from rat a dipocytes that are enriched in the muscle/fat glucose transporter isof orm. These vesicles were then used as immunogens to generate monoclona l antibodies against their protein components. We isolated an antibody , 3F8, that recognizes three polypeptides, designated GTV3, migrating in the 36-40-kDa range as analyzed by SDS-polyacrylamide gel electroph oresis and Western blotting. These proteins are enriched in GLUT4-cont aining vesicles, and the two smallest of the polypeptides recognized b y 3F8 translocate to the cell surface in response to insulin. GTV3 pro teins are also present in plasma membranes of fat cells and liver as w ell as in a wide number of tissues, red blood cells being the only exc eption. In adipocytes from streptozotocin-induced diabetic rats, GTV3 protein levels decrease dramatically and return to normal levels when animals are treated with insulin. The localization of GTV3 in glucose transporter-containing vesicles as well as their wide tissue distribut ion suggests that these proteins may be involved in vesicle mediated t ransport and regulated trafficking between membrane compartments.