Refeeding carbohydrate to fasted rats induces the transcription of genes encoding enzymes of fatty acid biosynthesis,e.g. fatty-acid synthase (FAS). Part of this transcriptional induction is mediated by insulin. An insulin response element has been described for the fatty-acid synthase gene region of −600 to +65, but the 2–3-fold increase in fatty-acid synthase promoter activity attributable to this region is small compared with the 20–30-fold induction in fatty-acid synthase gene transcription observed in fasted rats refed carbohydrate. We have previously reported that the fatty-acid synthase gene region between −7382 and −6970 was essential for achieving high in vivorates of gene transcription. The studies of the current report demonstrate that the region of −7382 to −6970 of the fatty-acid synthase gene contains a carbohydrate response element (CHO-RE_FAS) with a palindrome sequence (CATGTGn ₅GGCGTG) that is nearly identical to the CHO-RE of the l-type pyruvate kinase and S₁₄ genes. The glucose responsiveness imparted by CHO-RE_FAS was independent of insulin. Moreover, CHO-RE_FAS conferred glucose responsiveness to a heterologous promoter (i.e. l-type pyruvate kinase). Electrophoretic mobility shift assays demonstrated that CHO-RE_FAS readily bound a unique hepatic ChoRF and that CHO-RE_FAS competed with the CHO-RE of thel-type pyruvate kinase and S₁₄ genes for ChoRF binding. In vivo footprinting revealed that fasting reduced and refeeding increased ChoRF binding to CHO-RE_FAS. Thus, carbohydrate responsiveness of rat liver fatty-acid synthase appears to require both insulin and glucose signaling pathways. More importantly, a unique hepatic ChoRF has now been shown to recognize glucose responsive sequences that are common to three different genes: fatty-acid synthase, l-type pyruvate kinase, and S₁₄.

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between −7300 and −7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between −7110 and −7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4α (hepatocyte nuclear factor 4α), and chromatin immunoprecipitation establishes that HNF-4α binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4α transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4α by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4α and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4α in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4α functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4α is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.