P21(RAS) INDUCED DIFFERENTIATION-RELATED GENE-EXPRESSION IN FETAL BROWN ADIPOCYTE PRIMARY-CELLS AND CELL-LINES

Transfection of primary rat fetal brown adipocytes with constructs of SV40 large T antigen, alone and together with lys(12)-mutated H-ras ge ne, gave permanent cell lines showing an immortalized or transformed p henotype, respectively, all of them selected by the expression of the uncoupling protein (UCP), a tissue-specific marker, Primary brown adip ocytes and immortalized cell lines respond to insulin-like growth fact or I (IGF-I) by increasing their lipid content and the mRNA expression of both the adipogenic marker fatty acid synthase (FAS) and the therm ogenic marker UCP. IGF-l-induced differentiation-related gene expressi on at 24 h in both primary and immortalized brown adipocytes was media ted by an increase in p21(ras),GTP active protein content. Transformed cell lines overexpressing exogenous p21(ras) (mainly in its ras,GTP a ctive form) constitutively showed a higher lipid content and a higher FAS and UCP mRNA expression compared to primary and immortalized cells , These transformed cells were IGF-l independent with respect to their studied differentiation-related parameters. Additionally, transient t ransfection of primary brown adipocytes with the transforming ras gene induced UCP and FAS mRNA expression as well gs cotransactivated UCP-c hloramphenicol acetyltransferase fusion gene. Moreover, IGF-I transact ivation of UCP promoter was partially precluded by cotransfection with the dominant-negative ras gene, Our results strongly suggest that IGF -I/p21(ras) induces adipogenic- and thermogenic-related gene expressio n in brown adipocytes.