Mechanism of 4-Bromocrotonic Acid Modulation of Insulin-Stimulated Glucose Transport

• Main Authors: Shin-Pei Chai, 柴欣沛
• Other Authors: Jim-C Fong
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/56900855580716378853

博士 === 國立陽明大學 === 生物化學研究所 === 91 === Inhibitors of fatty acid oxidation, 4-bromocrotonic acid (Br-C4) was examined for its effect on glucose transport in 3T3-L1 adipocytes. Br-C4 augmented basal but inhibited insulin-stimulated 2-deoxyglucose uptake in a dose- and time-dependent manner. Immunoblot analysis indicated that following Br-C4 pretreatment, the content of GLUT1 in plasma membranes was increased whereas insulin-induced translocation of GLUT4 was greatly eliminated. The total cellular amount of GLUT1 or GLUT4, on the other hand, was not altered. In the present study, we further examined the underlying mechanism involved. Since insulin-induced insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity was not altered by Br-C4, we determined and found insulin activation of protein kinase B (PKB) and protein kinase Cl (PKCl) were both inhibited. However, time-course studies showed that only the inhibition of PKB activation correlated with the inhibition of insulin-stimulated glucose transport. In concert, insulin-stimulated Ser(473/474) phosphorylation on PKB(a/b) were similarly decreased by Br-C4. Since okadaic acid-stimulated glucose transport and PKCl activity were both inhibited by Br-C4, suggesting that the effect of Br-C4 on Ser(473/474) phosphorylation is not mediated by protein phosphatase 2A. Furthermore, whereas Br-C4 nearly abolished insulin-stimulated integrin-linked kinase (ILK) activity, it only inhibited insulin-stimulated PKB activity by 20%, implying that ILK was not the major kinase for Ser(473/474) phosphorylation. We further investigate whether Br-C4 exert its inhibitory influence by exchange the composition of membrane phospholipid, when added to the medium and incubated for 1 hr, phospholipid obtained from Br-C4-pretreated cells could inhibit insulin-stimulated activities of PKB and PKCl and also insulin-stimulated PKB Ser473 phosphorylation whereas phospholipid obtain from control cells was without effect. Phospholipid obtained from Br-C4-pretreated cells was further separated using stepwise-TLC system; we found that Br-C4 may exchange the composition of phosphoinostide leading to inhibit the activation of PKB. Phosphoinostide and phosphotidylserine were exchanged by Br-C4 could inhibit activation of PKCl. Taken together, these results support the notion that PKB is involved in insulin-stimulated glucose transport. In addition, Br-C4 seems to inhibit insulin-stimulated glucose transport via inhibiting insulin activation of PKB, probably by interfering with insulin activation of an upstream kinase responsible for the phosphorylation of Ser(473/474) residue. Evidence strong suggest that Br-C4 may exert its influence to PI(3,4,5)P3 leading to inhibit activation of PKB(a/b) and PKCl in response to insulin.