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, this 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 upon treatment for 24 h. Upon treatment with either IGF-I or insulin for 48 h, a 7-fold increase in the UCP1 protein content relative to levels in the control cells was found, this induction being abolished in the presence of cycloheximide. Moreover, either IGF-I or insulin transactivates the UCP1-chloramphenicol acetyl transferase (CAT) fusion gene after transient transfection of primary brown adipocytes, these effects being tissue-specific. Transient transfection of dominant-negative form of phosphatidylinositol (PI) 3-kinase completely blocked the transactivation of the fusion gene UCP1-CAT induced by either IGF-I or insulin, although inhibition of p70S6kinase with rapamycin does not preclude transactivation of the UCP1 promoter by insulin. Furthermore, transient transfection of dominant-negative form of p21-ras or treatment of cells with a mitogen-activated protein kinase kinase (MEK-1) inhibitor (PD098059) completely abolished insulin-induced UCP1-CAT transactivation. Cotransfection with dominant-negative p85 or with dominant-negative Ras also produced down-regulation of the insulin or IGF-I-induced 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-CAT (five AP-1, activating protein-1, binding sites arranged in tandem) transactivation. In addition, insulin induced AP-1 DNA binding activity, this effect being totally prevented in the presence of MEK-1 inhibitor. These results strongly suggest that either IGF-I or insulin induced thermogenic-differentiation through AP-1 activity in a PI 3-kinase and Ras/MAPK dependent manner in brown adipocytes.
Teruel
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Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Valverde
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AM
Navarro
P
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Benito
M
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Lorenzo
M
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D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus / Q000378:metabolism; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D011905:Genes, ras / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics*; D024101:Mitochondrial Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007328:Insulin; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; D007334:Insulin-Like Growth Factor I; D017871:Calcium-Calmodulin-Dependent Protein Kinases