ROLES OF 1-PHOSPHATIDYLINOSITOL 3-KINASE AND RAS IN REGULATING TRANSLOCATION OF GLUT4 IN TRANSFECTED RAT ADIPOSE-CELLS

Insulin stimulates glucose transport in insulin target tissues by recr uiting glucose transporters (primarily GLUT4) from an intracellular co mpartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylat ion of insulin receptor substrate 1 (IRS-1) contribute to mediating th e effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two s ignaling proteins located downstream from tyrosine phosphorylation, Ra t adipose cells were cotransfected with expression vectors that allowe d transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-ki nase lacking the ability to bind and activate the p110 catalytic subun it exerted a dominant negative effect to inhibit insulin-stimulated tr anslocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinas e) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an es sential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, overexpression of a constitutively active mutan t of ras (L61-ras) resulted in high levels of cell surface GLUT4 in th e absence of insulin that were comparable to levels seen in control ce lls treated with a maximally stimulating dose of insulin. However, wor tmannin treatment of cells overexpressing LG1-ras resulted in only a s mall decrease in the amount of cell surface GLUT4 compared with that o f the same cells in the absence of wortmannin. Therefore, while activa ted ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechani sm by which insulin stimulates GLUT4 translocation in physiological ta rget tissues.