Mutations in the coding region of hepatocyte nuclear factor 4α (HNF4α)

Mutations in the coding region of hepatocyte nuclear factor 4α (HNF4α) and its upstream promoter (P2) that drives expression in the pancreas are known to lead to maturity-onset diabetes of the small 1 (MODY1). mice. Because insulin increases the activity SB-207499 of sterol regulatory element-binding proteins (SREBP)-1c and -2 we also examined the effect of SREBPs on hepatic HNF4α gene expression and found that like insulin ectopic expression of SREBPs decreases the level of hepatic HNF4α protein and mRNA both in primary hepatocytes and in the liver of C57BL/6 mice. Finally we use gel shift chromatin immunoprecipitation small interfering RNA and reporter gene analysis to show that SREBP2 binds the human HNF4α P1 promoter and negatively regulates its expression. These data indicate that hyperinsulinemia down-regulates HNF4α in the liver through the up-regulation of SREBPs thereby establishing a link between these two crucial transcription factor pathways that regulate lipid and glucose metabolism in the liver. These findings also provide new insights into diabetes-associated complications such as fatty liver disease. Hepatocyte nuclear factor 4α (HNF4α) (NR2A1) is usually a highly conserved member of the nuclear PDGFB receptor superfamily of ligand-dependent transcription factors (1 2 Expression of the gene is usually driven by two distinct promoters the P1 promoter that drives expression of splice variants HNF4α1-6 in the liver kidney and intestine/colon and the P2 promoter that drives expression of splice variations HNF4α7-9 in the intestine/digestive tract tummy and β-cells from the pancreas but notably not really the adult liver organ or kidney. A huge selection of HNF4α focus on genes have already been SB-207499 discovered in liver organ pancreas and digestive tract (3 4 5 also find In the liver organ a few of these goals are genes involved with blood sugar [PEPCK (gene result in maturity-onset diabetes from the youthful 1 (MODY1) an illness seen as a a lack of insulin secretion in the pancreatic β-cells in response to blood sugar (7). The result from the MODY1 mutations are presumably in the pancreatic type of HNF4α because single-nucleotide polymorphisms in the P2 promoter that drives β-cell appearance are also associated with MODY1 (find Ref. 8 and sources therein); simply no such mutations have been recognized in the P1 promoter that drives expression in the liver. Consequently several studies have investigated the role of pancreatic HNF4α in diabetes (5 9 10 but much less is known about the role of hepatic HNF4α in diabetes. Sterol regulatory element-binding proteins (SREBPs) are also well characterized transcription factors that regulate genes involved in cholesterol and fatty acid synthesis and metabolism (11 12 13 You will find two SREBP genes SREBP1 (and effects of insulin under physiological conditions we fasted C57BL/6 mice for 24 h and then refed them for an additional 12 h. We observed an appreciable increase in HNF4α mRNA levels in the livers of fasted animals when insulin levels are presumably low and a significant decrease back to basal levels upon refeeding when insulin levels are expected to be elevated (Fig. 3A?3A).). We also observed a concomitant decrease in HNF4α protein levels upon refeeding (Fig. 3B?3B)) and a SB-207499 corresponding decrease in the HNF4α target gene PEPCK (Fig. 3C?3C).). Finally upon refeeding we noted elevated levels of the mature forms of SREBP1c and SREBP2 proteins (Fig. 3B?3B)) as well as mRNA levels of SREBPs (Fig. 3A?3A)) and their target genes LDLR HMGCR FAS and SCD-1 (Fig. 3C?3C).). Although comparable results regarding insulin effects on HNF4α mRNA and SREBP protein levels have been observed previously (23 29 30 31 32 our results show for the first time in the same liver samples the inverse relationship between the levels of HNF4α and SREBP upon fasting and refeeding. [Comparable results were also observed when the experiments were repeated with 6 h of refeeding after a 24-h fast (supplemental Fig. S1 SB-207499 published as supplemental data around the Endocrine Society’s Journals Online web site at] Together with the decrease in HNF4α levels upon treatment of the hepatocytes with insulin (Fig. SB-207499 2?2)) and the disparate levels of HNF4α in the db/db and STZ mice (Fig. 1?1) ) these results suggest that insulin HNF4α mRNA and SREBP protein levels might be linked in a hierarchical fashion. Physique 3 Inverse relationship between HNF4α gene expression and SREBPs in the livers of fasted and refed mice. A Quantitative RT-PCR detecting HNF4α SREBP1c and SREBP2 mRNA levels in the livers of 8-wk-old male C57BL/6J mice fed normal chow fasted … Ectopic expression of SREBPs decrease HNF4α.