Misra S, Sakamoto K, Moustaïd N, Sul H S
Department of Nutrition, Harvard School of Public Health, Boston, MA 02115.
Biochem J. 1994 Mar 15;298 Pt 3(Pt 3):575-8. doi: 10.1042/bj2980575.
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.
脂肪酸合酶（FAS）在脂肪酸合成中起核心作用，其表达受营养和激素调控。我们通过将包含与荧光素酶（LUC）报告基因相连的FAS基因5'侧翼区的嵌合基因转染到3T3-L1成纤维细胞中，研究了胰岛素样生长因子-I（IGF-I）对FAS的调控。首先，比较了FAS基因从核苷酸-318至-19的5'系列缺失的基础启动子活性。启动子序列从-136至-19的缺失导致启动子活性逐步降低，-67 LUC和-19 LUC质粒分别保留了-136 LUC荧光素酶活性的40%和16%。因此，3T3-L1成纤维细胞中对FAS基础启动子活性重要的调控序列位于-136至-19区域内。用10 mM IGF-I处理也分别使瞬时转染-136 LUC、-110 LUC和-67 LUC质粒的3T3-L1成纤维细胞中的荧光素酶活性增加1.8±0.2倍、1.8±0.3倍和2.5±0.1倍。从-67至-19的序列缺失导致对IGF-I的反应性丧失。然而，生理剂量的胰岛素（10 nM）并未增加转染上述任何质粒的3T3-L1成纤维细胞中的荧光素酶活性。仅在用药理剂量的胰岛素（1 μM）处理时，可能通过IGF-I受体，当分别转染-136 LUC、-110 LUC和-67 LUC质粒时，荧光素酶活性才分别增加4.3±0.4倍、3.2±0.4倍和3.5±0.5倍；-19 LUC则无增加。IGF-I对FAS启动子活性的半最大效应在3 nM时观察到，最大效应在10 nM时达到。这些结果表明，观察到的启动子活性增加可能是通过IGF-I受体介导的。此外，负责IGF-I对FAS基因调控的序列位于FAS基因核苷酸-67和-19之间的近端启动子内。
