| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | insulin-induced glucose uptake / phosphatidylinositol / GLUT4 translocation / phosphatidylinositol 3-kinase / PI 3, 4, 5-P_3 / PI 3, 4-P_2 / SHIP(SH2-containing inositol 5'-phosphatase) / PI 3,4,5-P_3 / SHIP (SH2-containing inositol 5'-phosphatase) / GLUT4translocation / PI(3,4,5)P_3 / PI(3,4)P_2 / SHIP(SH2-containing inositol 5-phosphatase) |
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| Research Abstract |
Insulin stimulation leads to rapid activation of phosphatidylinositol 3-kinase(PI3-kinase)and the subsequent formation of phosphatidylinositol(PI)3, 4-P_2 and PI 3, 4, 5-P_3, which are thought to be involved in signaling for insulin-dependent glucose transporter GLUT4 translocation and glucose uptake. However, the specific role of each of these PIs in insulin signaling is still controversial. Therefore, we assessed the effects of wild type SH2-containing inositol 5'-phosphatase(wt-SHIP)expression, which is expected to decrease the cellular levels of PI 3, 4, 5-P_3, on these biological effects. Moreover, we compared the effects of myristoylated SHIP(myr-SHIP), which has a membrane targeting moiety, to those of wt-SHIP, because PI turnover is thought to occur at plasma membrane.
LacZ(as control), wild-SHIP or myr-SHIP is overexpressed into 3T3-L1 adipocytes using recombinant adenovirus system. Overexpression of each protein was confirmed with immunoblotting, and 5'-phosphatase activity of
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the expressed SHIP was verified by dephosphorylation of[3'-^<32>P]PI 3, 4, 5-P_3 to[3'-^<32>P]PI 3, 4-P_2 in vitro. At 10^<-7> M insulin concentration overexpression of wt-SHIP and myr-SHIP did not inhibit but rather stimulated insulin-induced glucose uptake by 1.7-fold and 2.7-fold above the value obtained with overexpression of LacZ, respectively, in 3T3-L1 adipocytes. Overexpression of wt-SHIP and myr-SHIP did not have any significant effect on total GLUT4 expression, but did potentiate insulin-induced GLUT4 translocation.
In summary, our results demonstrated that expression of wt-SHIP and myr-SHIP potentiated insulin-induced GLUT4 translocation and glucose uptake, suggesting that PI 3, 4-P_2, rather than PI 3, 4, 5-P_3, is the major phospholipid product mediating these biological actions.
In addition, we also repored that dominant negative mutant hepatocyte nuclear factor(HNF)-1α influenced insulin secretion, and that troglitazone increased plasma vascular endothelial growth factor levels, which might cause edema and vascular complications. Less
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