DIRECT DEMONSTRATION OF INSULIN-INDUCED GLUT4 TRANSLOCATION TO THE SURFACE OF INTACT-CELLS BY INSERTION OF A C-MYC EPITOPE INTO AN EXOFACIAL GLUT4 DOMAIN

Stimulation of glucose transport is the main physiological effect of insulin in target tissues. This effect is linked to translocation of the GLUT4 glucose transporter from an intracellular pool to the cell surface. To elucidate the molecular mechanisms involved in this effect, we developed a simple direct sensitive method to detect GLUT4 immunologically on the cell surface. cDNA containing GLUT4 inserted by a c-myc epitope in the first ectodomain (GLUT4myc) was constructed without disrupting the functions of GLUT4 and was expressed in 3T3-L1 and Chinese hamster ovary fibroblast cells. In response to insulin, the GLUT4myc expressed in 3T3-L1 adipocytes was translocated to the cell surface from the intracellular pool, as shown by assays of exofacial antibody binding against the myc epitope and of the uptake of 2-deoxyglucose. Insulin, guanosine 5'-O-(3-thiotriphosphate), guanylyl imidodiphosphate, NaF, and phorbol 12-myristate 13-acetate also induced the translocation of GLUT4myc in Chinese hamster ovary cells coexpressing the human insulin receptor.