Insulin and insulin-like growth factor I up-regulate GLUT4 gene expressionin fetal brown adipocytes, in a phosphoinositide 3-kinase-dependent manner

Fetal brown adipocytes cultured in a serum-free medium, containing 5 mM glu cose, expressed both GLUT4 and GLUT1 glucose transporters at the mRNA and p rotein level. Treatment with either insulin or insulin-like growth factor ( IGF)-I at physiological concentrations up-regulates the expression of the G LUT4 gene, producing a time-dependent mRNA accumulation (7-fold increase at 24 h) and a 2.5-fold increase in the amount of protein in the total membra ne fraction. However, insulin treatment down-regulates GLUT1 mRNA and prote in expression. Moreover, either insulin or IGF-I transactivates a full-prom oter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct transient ly transfected to the cells, without affecting GLUT1-CAT activity. In conse quence, insulin treatment for 24 h increased by 3-fold the basal glucose up take. Inhibition of phosphoinositide (PI) 3-kinase activity with chemical a gents such as wortmannin or LY294002 partially blocked insulin-induced GLUT 4 mRNA accumulation, insulin-induced GLUT4 protein content, GLUT4-CAT trans activation and glucose uptake. Furthermore, co-transfection of brown adipoc ytes with a dominant-negative form of PI 3-kinase precluded the transactiva tion of the GLUT4 promoter by insulin. However, inhibition of p70S6 kinase (p70(s6k)) with rapamycin or of mitogen-activated protein kinase (MAPK) wit h PD098059 does not preclude insulin effects on GLUT4 gene expression or gl ucose uptake. Our results show for the first time a positive effect of insu lin on GLUT4 gene expression in fetal brown adipocytes, suggesting the exis tence of insulin response element(s) in its promoter. Moreover, PI 3-kinase , but not p70(s6k) or MAPK, is an essential requirement for insulin regulat ion of GLUT4 gene expression.