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 by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter 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 decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM 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 transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.
Misra
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S
Department of Nutrition, Harvard School of Public Health, Boston, MA 02115.
Sakamoto
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K
Moustaïd
N
N
Sul
H S
HS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000235:genetics*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D007328:Insulin; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D008156:Luciferases; D064429:Fatty Acid Synthases; D015056:1-Methyl-3-isobutylxanthine