Regulation of acetyl-CoA carboxylase gene expression. Insulin induction of acetyl-CoA carboxylase and differentiation of 30A5 preadipocytes require prior cAMP action on the gene.
30A5 preadipocytes, derived from 10T1/2 mouse fibroblasts, can be induced to differentiate into adipocytes by hormone treatment. In this paper, we introduce a modified procedure to induce differentiation of 30A5 cells by pretreatment with cAMP for a brief period or by a "nutrition deprivation" pretreatment, followed by incubation in medium containing insulin. These procedures accelerate the differentiation of the preadipocytes, so that the cells are fully differentiated within 4 days instead of the 7-8 days normally required. This differentiation is accompanied by the early induction of acetyl-CoA carboxylase (ACC). ACC catalyzes the rate-limiting step in the biogenesis of long chain fatty acids. To analyze the relationship between cAMP and insulin action in the induction of ACC and cell differentiation, we identified the DNA sequences in promoter II of the ACC gene necessary for the action of insulin and cAMP. Chimeric genes between different fragments of the ACC promoter and the promoterless chloramphenicol transacetylase (CAT) gene were constructed, and stable clones containing these chimeric genes were obtained. By analyzing the CAT activities in these stable clones, we established that insulin action in inducing ACC and cell differentiation requires prior treatment of cells with cAMP and the presence of specific DNA regions in the ACC promoter for cAMP action. Stable clones containing a chimeric gene which consists of DNA sequences in promoter II that are required for insulin action, thymidine kinase promoter, and the CAT gene did not respond to insulin. However, when the DNA sequences required for cAMP action were placed in this chimeric gene, it responded to insulin upon prior treatment of 30A5 cells with cAMP. Thus, cAMP and insulin, whose physiological actions generally appear to be antagonistic, are synergistically interacting in the induction of ACC and the differentiation of 30A5 cells.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
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D000103:Acetyl-CoA Carboxylase / Q000096:biosynthesis / Q000235:genetics*; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects*; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000242:Cyclic AMP / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D013937:Thymidine Kinase / Q000235:genetics
D007328:Insulin; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase; D000103:Acetyl-CoA Carboxylase