INHIBITION OF PI-3-KINASE AND RAS BLOCKS IGF-I AND INSULIN-INDUCED UNCOUPLING PROTEIN-1 GENE-EXPRESSION IN BROWN ADIPOCYTES

Fetal brown adipocytes expressed uncoupling protein 1 (UCP1) mRNA, thi s expression being blunted throughout culture for 24 h in a serum-free medium. At physiological doses, either insulin-like growth factor I ( IGF-I) or insulin turned out to be as potent as dibutyryl cAMP (dbcAMP ) in increasing UCP1 gene transcription rate (1 h) and also UCP1 mRNA accumulation (3 h), their maximal effect (15-fold increase) reached up on treatment for 24 h. Upon treatment with either ICF-I or insulin for 48 h, a 7-fold increase in the UCP1 protein content relative to level s in the control cells was found, this induction being abolished in th e presence of cycloheximide. Moreover, either IGF-I or insulin transac tivates the UCP1-chloramphenicol acetyl transferase (CAT) fusion gene after transient transfection of primary brown adipocytes, these effect s being tissue-specific. Transient transfection of dominant-negative f orm of phosphatidyl inositol (PI) 3-kinase completely blocked the tran sactivation of the fusion gene UCP1-CAT induced by either IGF-I or ins ulin, although inhibition of p70(S6kinase) with rapamycin does not pre clude transactivation of the UCP1 promoter by insulin. Furthermore, tr ansient transfection of dominant-negative form of p21-ras or treatment of cells with a mitogen-activated protein kinase kinase (MEK-1) inhib itor (PD098059) completely abolished insulin-induced UCP1-CAT transact ivation. Cotransfection with dominant-negative p85 or with dominant-ne gative Pas also produced downregulation of the insulin or IGF-I-induce d 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-CAT (fiv e AP-1, activating protein-1, binding sites arranged in tandem) transa ctivation. In addition, insulin induced AP-1 DNA binding activity, thi s effect being totally prevented in the presence of MEK-1 inhibitor. T hese results strongly suggest that either ICF-I or insulin induced the rmogenic-differentiation through AP-1 activity in a PI 3-kinase and Ra s/MAPK dependent manner in brown adipocytes. (C) 1998 Wiley-Liss, Inc.