Inoue G, Kuzuya H, Hayashi T, Okamoto M, Yoshimasa Y, Kosaki A, Kono S, Okamoto M, Maeda I, Kubota M
Second Division of Internal Medicine, Kyoto University School of Medicine, Japan.
J Biol Chem. 1993 Mar 5;268(7):5272-8.
Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.
用胰岛素处理3T3-L1脂肪细胞可导致2-脱氧葡萄糖转运活性的激活以及葡萄糖转运蛋白（GLUT4和GLUT1）从细胞质空间向质膜的转位。ML-9（一种肌球蛋白轻链激酶抑制剂）在100微摩尔浓度时（IC50 = 27微摩尔）可将胰岛素刺激的2-脱氧葡萄糖转运活性抑制80%，而不影响基础状态下的2-脱氧葡萄糖转运活性。这种抑制作用与细胞外钙离子浓度无关，并且在40微摩尔ML-9时几乎完全可逆。ML-9不抑制麦胚凝集素纯化制剂中胰岛素刺激的95千道尔顿蛋白的酪氨酸磷酸化以及完整细胞中95千道尔顿和160千道尔顿蛋白的酪氨酸磷酸化。然而，ML-9以剂量依赖的方式抑制胰岛素诱导的GLUT4和GLUT1的转位。剂量反应曲线与观察到的抑制胰岛素刺激的2-脱氧葡萄糖转运活性的曲线相似。胰岛素和ML-9均不影响肌球蛋白重链和轻链的磷酸化状态。因此，ML-9似乎是通过一种不同于影响肌球蛋白轻链磷酸化的机制，在胰岛素受体激酶活性之后的步骤抑制胰岛素诱导的葡萄糖转运蛋白转位。胰岛素刺激了微管相关蛋白2和髓鞘碱性蛋白的磷酸化活性，并且这种刺激不受ML-9影响。然而，ML-9以类似于观察到的抑制胰岛素刺激葡萄糖转运的剂量依赖方式在体外抑制磷酸化活性以及完整细胞中胰岛素对核糖体蛋白S6磷酸化活性的刺激。这些结果表明，丝裂原活化蛋白激酶可能是细胞内胰岛素向葡萄糖转运系统信号传导的组成成分之一。
