Identification and characterization of enhancer sequences in the 5'-flanking region of the fatty acid synthase gene responsible for in vivo rates of gene transcription

Long-term exposure of β-cells and cardiomyocytes to excessive triglycerides enhances the rate of apoptosis with the consequent loss of β-cell mass and the onset of frank non-insulin-dependent diabetes mellitus (NIDDM). Moreover high triglyceride accumulation contributes to the development of fatty liver and insulin resistance in muscle. Fatty acids for triglyceride synthesis are derived from hepatic re-esterification as well as from de novo synthesis. We studied the determinants of de novo fatty acid synthesis using the fatty acid synthase gene (FAS) as a lipogenic gene model. FAS plays a pivotal role in the de novo synthesis of fatty acids and the expression of the FAS gene is finely regulated by nutrients and hormones. Re-feeding carbohydrate to fasted rats greatly induces the transcription rate of the FAS gene. This transcriptional induction is mediated in part by insulin. An insulin response element has been described for the FAS gene region of −600 to +65, but the 2–3 fold increase in FAS promoter activity attributable to this region is small compared to the 20–30 fold induction in FAS gene transcription observed in fasted rats that have been refed carbohydrate. In the present work, we demonstrated that the FAS gene region between −7382 and −6970 is essential for achieving high in vivo rates of gene transcription. Located within the −7382/−6970 region is a carbohydrate response element (ChoRE_FAS) with the sequence (CATGTGn ₅GGCGTG) that is similar to the ChoRE of the L-type pyruvate kinase and S₁₄ genes. The glucose responsiveness imparted by ChoRE _FAS is independent of insulin. Moreover, ChoRE_FAS confers glucose responsiveness to the L-type pyruvate kinase promoter. Electrophoretic mobility shift assays demonstrated that ChoRE_FAS binds a unique hepatic carbohydrate responsive factor (ChoRF), and that ChoRE_FAS competes with the ChoRE of the L-type pyruvate kinase and S14 genes for Cho-RF binding. In vivo footprinting revealed that fasting reduces and re-feeding increases Cho-RF binding to ChoRE_FAS. In summary, carbohydrate/insulin induction of rat liver FAS requires both insulin and glucose signaling pathways which target different regions in the FAS gene promoter.