Molecular mechanisms of insulin signal transduction and diabetes mellitus

1） Rat 3 Y1细胞，其中有内源性胰岛素样生长因子-1受体（IGF-1-R）和胰岛素受体底物-2（IRS-2），但两者都缺乏胰岛素受体（IR）和IRS-1，没有胰岛素效应。为了研究IR和RIS 1在胰岛素效应中的作用，我们重新考虑了胰岛素信号通路在细胞中的作用。IRS-1在3 Y1-GLUT 4 myc中的表达·IR细胞引导刺激糖原合成，但没有对GLUT 4 myc的转移作出反应，也可以在细胞中处理NaF或PMA触发器GLUT 4 myc转移。这些结果表明，在3 Y1个细胞中对胰岛素的反应中，（i）IRS-1是糖基合成的必要条件，而不是DNA合成、Akt磷酸化和膜起泡的必要条件;（ii）PI-3、4、5-P_3的累积需要Akt磷酸化和膜反应的累积条件;（iii）PI-3、4，5-P_3和Akt的激活不能满足于糖基合成和GLUT 4转换的作用。2）4型葡萄糖输送机（GLUT 4）从细胞表面转移到细胞表面是胰岛素靶细胞中胰岛素刺激的葡萄糖的主要机制。我们开发了一种高度敏感和定量的方法，用于在接触细胞表面上检测GLUT 4，并使用c-myc表位标记GLUT 4（GLUT 4 myc）。由于GLUT 1和GLUT 4有不同的内容分布和不同的胰岛素转运程度，我们研究了GLUT 4的域，使用C-myc表位标记的化学葡萄糖转运器在这两个不同的异构体之间。GLUT 4具有独立的内部目标信号的Intracellular环路和细胞plasmic C终端区域的结果， （2）GLUT 4的内部目标的这些序列对GLUT 4的内部目标是不够的，（3）GLUT 4转换到胰岛素的响应中的N-terminal half of GLUT 4 devoid both of cytoplasmic N-terminal and of midintracellular loop seems to be necessary for insulin-刺激的GLUT 4转换。

1) Rat 3Y1 cells, which have endogenous insulin-like growth factor-1 receptor (IGF-1-R) and insulin receptor substrate-2 (IRS-2), but lack both insulin receptor (IR) and IRS-1, exhibit no insulin effects. To investigate the role of IR and RIS-1 in insulin effects, we reconstituted the insulin signaling pathways in the cells. The expression of IRS-1 in 3Y1-GLUT4myc・IR cells leads to the stimulation of glycogen synthesis but no to the GLUT4myc translocation in response to insulin, although the treatment of NaF or PMA triggers GLUT4myc translocation in the cells. These results indicate that, in 3Y1 cells in response to insulin, i) IRS-1 is necessary for glycogen synthesis, not essential for DNA synthesis, Akt phosphorylation and membrane ruffling, ii) the accumulation of PI-3,4,5-P_3 is required for Akt phosphorylation and membrane reffling, iii) the accumulation of PI-3,4,5-P_3 and activation of Akt are not sufficient for glycogen synthesis and GLUT4 translocation.2) Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The result intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.