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Molecular mechanisms of insulin signal transduction and diabetes mellitus

胰岛素信号转导与糖尿病的分子机制

基本信息

批准号：
08457050
负责人：
EBINA Yousuke
金额：
$ 4.99万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

1)大鼠3Y1细胞具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)，但缺乏胰岛素受体(IR)和IRS-1，不显示胰岛素效应。为了研究IR和RIS-1在胰岛素效应中的作用，我们重构了细胞内的胰岛素信号通路。IRS-1在3Y1-GLUT4myc·IR细胞中的表达可刺激糖原合成，但不能刺激胰岛素引起的GLUT4myc转位，而NaF或PMA处理后可引起细胞内GLUT4myc转位。这些结果表明，在胰岛素应答的3Y1细胞中，IRS-1是糖原合成所必需的，而不是DNA合成、Akt磷酸化和膜褶皱所必需的，ii)Akt磷酸化和膜重排需要PI-3，4，5-P3的积聚，III)PI-3，4，5-P3的积聚和Akt的激活不足以进行糖原合成和GLUT4转位。2)4型葡萄糖转运体(GLUT4)从细胞内池转移到细胞表面是胰岛素刺激胰岛素靶细胞摄取葡萄糖的主要机制。我们利用c-myc表位标记的GLUT4(GLUT4myc)建立了一种高灵敏的定量检测完整细胞表面GLUT4的方法。由于GLUT1和GLUT4具有不同的细胞内分布和不同程度的胰岛素易位，我们使用c-myc表位标记的嵌合葡萄糖转运体来检测GLUT4的结构域。GLUT4的胞内环和胞浆C末端区域具有独立的胞内靶向信号，(2)GLUT4的胞内靶向序列不足以确定GLUT4在胰岛素刺激下的易位，(3)GLUT4的N端一半没有胞浆N端和胞内中环，似乎是胰岛素刺激的GLUT4易位所必需的。