Study on the mechanism of insulin action on glucose transport

利用D^k-(62-85)，一种MHC类衍生肽，在电渗透大鼠脂肪细胞中检测了鸟嘌呤核苷酸对GLUT4胞外或内吞作用的影响。胰岛素刺激的葡萄糖转运活性逆转被完全阻断，在D^k-存在下，活性提高了25%(62-85)，表明肽阻断了GLUT4的内吞作用。与这一观点一致的是，胰蛋白酶切割的GLUT4的35kda片段的内吞作用几乎完全被肽抑制。因此，在肽存在的情况下，可以测量细胞表面上GLUT4的排斥性积聚。在D^k-存在的情况下，胰岛素刺激的葡萄糖转运活性提高了约50%(62-85)，而基础转运活性仅受到轻微刺激。虽然GTP_< γ >S在缺乏D^k-的情况下对葡萄糖转运的增强程度与胰岛素相同（62-85），但gtpgs刺激的葡萄糖转运仅为肽存在时胰岛素效应的60%：胰岛素的作用被肽显著增强，而GTPgammaS诱导的葡萄糖转运不受影响，这表明GTP_< γ >S具有与肽相似的作用。事实上，GLUT4 35kDa片段的内吞作用被GTPgammaS显著抑制。此外，GTP能促进GLUT4的内吞，而gdpbeta则能抑制GLUT4的内吞。这些结果表明GTPgammaS通过刺激胞吐和抑制内吞作用诱导GLUT4易位。不同类型的gtp结合蛋白参与GLUT4的胞吐和内吞作用。

Effects of guanine nucleotides on either exocytosis or endocytosis of GLUT4 were examined in electrically permeabilized rat adipocytes by using D^k- (62-85), a MHC class l-derived peptide. Reversal of glucose transport activity which had been stimulated with insulin was completely blocked and the activity was rather enhanced 25% in the presence of D^k- (62-85), indicating that the peptide blocked endocytosis of GLUT4. In agreement with this notion, endocytosis of trypsin-cleaved 35 kDa fragment of GLUT4 was almost completely inhibited by the peptide. Therefore, in the presence of the peptide, exclusively exocytotic accumulation of GLUT4 on the cell surface could be measured. Insulin-stimulated glucose transport activity was enhanced about 50% in the presence of D^k- (62-85) while the basal transport activity was stimulated only slightly. Although GTP_<gamma>S augmented glucose transport to the same extent as insulin in the absence of D^k- (62-85), GTPgS-stimulated glucose transport was only 60% of the insulin effect in the presence of the peptide : the effects of insulin was markedly enhanced by the peptide whereas glucose transport induced by GTPgammaS was not affected, suggesting that GTP_<gamma>S has a similar effect to the peptide. In fact, endocytosis of 35kDa fragment of GLUT4 was markedly inhibited by GTPgammaS.In addition, GLUT4 endocytosis was accelerated by GTP but was inhibited by GDPbetaS.These results indicate that GTPgammaS induces translocation of GLUT4 by both stimulating exocytosis and inhibiting endocytosis. Distinct types of GTP-binding proteins are involved in exocytosis and endocytosis of GLUT4.