KISHI Kazuhiro The University of Tokushima, Institute for Enzyme Research, Research Associate, 分子酵素学研究センター, 助手 (70284320)
HAYASHI Hideki The University of Tokushima, Institute for Enzyme Research, Associate Professor, 分子酵素学研究センター, 助教授 (10218589)
|Budget Amount *help
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥5,400,000 (Direct Cost: ¥5,400,000)
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.