ROLES OF INSULIN, GUANOSINE 5'-[GAMMA-THIO]TRIPHOSPHATE AND PHORBOL 12-MYRISTATE 13-ACETATE IN SIGNALING PATHWAYS OF GLUT4 TRANSLOCATION

Insulin, guanosine 5'-[gamma-thio]triphospate (GTP[S]) and phorbol 12- myristate 13-acetate (PMA) trigger the translocation of GLUT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an in tracellular pool to the cell surface. We have developed a highly sensi tive and quantitative method to detect GLUT4 immunologically on the su rface of intact 313-L1 adipocytes and Chinese hamster ovary (CHO) cell s, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translo cation in the CHO cell system. Among small molecular GTP-binding prote ins, ras, rab3D, rad and rho seem to be candidates as signal transmitt ers of insulin-stimulated GLUT4 translocation. Overexpression of wild- type H-ras and the dominant negative mutant H-ras(S17N) in our cell sy stem respectively enhanced and blocked insulin-stimulated activation o f mitogen-activated protein kinase, but did not affect insulin-stimula ted GLUT4 translocation. Overexpression of rab3D or rad in the cells d id not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, h ad no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely t o be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent path ways.