Todaka M, Hayashi H, Imanaka T, Mitani Y, Kamohara S, Kishi K, Tamaoka K, Kanai F, Shichiri M, Morii N, Narumiya S, Ebina Y
Department of Enzyme Genetics, University of Tokushima, Kuramoto-cho, Japan.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):875-82. doi: 10.1042/bj3150875.
Insulin, guanosine 5'-[gamma-thio]triphosphate (GTP[S] and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of Gl UT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rass17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.
胰岛素、鸟苷5'-[γ-硫代]三磷酸（GTP[S]）和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）可触发4型葡萄糖转运蛋白（GLUT4；胰岛素敏感型葡萄糖转运蛋白）从细胞内池转运至细胞表面。我们开发了一种高度灵敏且定量的方法，利用c-myc表位标签的GLUT4（GLUT4myc）在完整的3T3-L1脂肪细胞和中国仓鼠卵巢（CHO）细胞表面免疫检测GLUT4。我们研究了胰岛素、GTP[S]和PMA在CHO细胞系统中GLUT4转位信号通路中的作用。在小分子GTP结合蛋白中，ras、rab3D、rad和rho似乎是胰岛素刺激的GLUT4转位信号传递者的候选蛋白。在我们的细胞系统中，野生型H-ras和显性负性突变体H-rass17N的过表达分别增强和阻断了胰岛素刺激的丝裂原活化蛋白激酶的激活，但不影响胰岛素刺激的GLUT4转位。细胞中rab3D或rad的过表达不影响胰岛素、GTP[S]或PMA触发的GLUT4转位。用肉毒杆菌C3外毒素（一种rho的特异性抑制剂）处理对胰岛素、GTP[S]或PMA诱导的GLUT4转位没有影响。因此，这些小分子GTP结合蛋白不太可能参与GLUT4转位。此外，胰岛素、GTP[S]和PMA显然通过独立的途径刺激GLUT4转位。
