LOCALIZATION OF SEQUENCES FOR THE BASAL AND INSULIN-LIKE GROWTH-FACTOR-I INDUCIBLE ACTIVITY OF THE FATTY-ACID SYNTHASE PROMOTER IN 3T3-L1 FIBROBLASTS

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS b y transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5 '-flanking region of the FAS gene linked to a luciferase (LUC) reporte r gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise dec rease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulato ry sequences important for the FAS basal promoter activity in 3T3-L1 f ibroblasts are, therefore, located within the -136 to -19 region. Trea tment with 10 nM IGF-I also increased luciferase activity 1.8+/-0.2-, 1.8+/-0.3- and 2.5+/-0.1-fold in 3T3-L1 fibroblasts transiently transf ected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Dele tion of sequences from -67 to -19 resulted in the loss of responsivene ss to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with an y of the above plasmids. Only upon treatment with pharmacological dose s of insulin (1 muM), probably through IGF-I receptor, did luciferase activity increase 4.3+/-0.4-, 3.2+/-0.4- and 3.5+/-0.5-fold when trans fected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; the re was no increase with -19 LUC. The half-maximal effect of IGF-I on F AS promoter activity was observed at 3 nM and a maximal effect was rea ched at 10 nM. These results indicate that the increased promoter acti vities observed are probably mediated through the IGF-I receptor. Furt hermore, sequences responsible for IGF-I regulation of the FAS gene ar e located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.