Study on the mechanism of insulin action on glucose transport

用L衍生的一种MHC类多肽D^k-(62-85)，观察了鸟嘌呤核苷酸对电通透性大鼠脂肪细胞GLUT4胞吐和内吞的影响。被胰岛素刺激的葡萄糖转运活性的逆转被完全阻断，在D^k-(62-85)的存在下，其活性相当于提高了25%，这表明该肽阻断了GLUT4的内吞作用。与此一致，胰酶切割的35 kDa GLUT4片段的内吞作用几乎完全被该肽抑制。因此，在多肽存在的情况下，可以测量到GLUT4在细胞表面的独占胞吐积累。在D^k-(62-85)存在下，胰岛素刺激的葡萄糖转运活性增加约50%，而基础转运活性仅有轻微的刺激。在没有D^k-(62-85)的情况下，GTP_lt；γ&gt；S对葡萄糖转运的促进作用与胰岛素相同，但GTPgS刺激的葡萄糖转运仅为有该肽时胰岛素作用的60%：GTP_t；γ&gt；S对胰岛素的作用明显增强，而GTP-GammaS诱导的葡萄糖转运不受影响，提示GTP_&t；γ&gt；S具有类似的作用。GTP-Gamma对GLUT4 35 kDa片段的内吞作用有明显的抑制作用，GTP对GLUT4的内吞作用有促进作用，而GDPbetas对GLUT4的内吞作用有抑制作用。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.