Abstract
The nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4a in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4a. Initally, the expression of HNF4a and its splice variants was investigated in three human hepatoma cell lines, HuH7, HepG2 and Hep3B, with the latter two cell lines shown to express the same range of HNF4a splice variants as human adult liver. The regulation of specific HNF4a target genes, L-PK, PEPCK and SREBP-1c, was subsequently investigated in HepG2 cells using a reporter gene approach. HNF4a was found to induce expression of reporter genes containing L-PK, PEPCK and SREBP-1c proximal promoter sequences. Insulin (1 ?M), but not high glucose (25 mM), was found to stimulate HNF4a-driven expression of the SREBP-1c reporter gene, while co-expression of HNF4a with the nuclear receptor coactivators, PGC-1a or p300, led to a reduction in SREBP-1c reporter gene expression. The changes in expression of various HNF4a target genes in response to physiological mediators of the fasting-fed cycle were characterised in HepG2 cells using a real-time quantitative PCR approach. The role of HNF4a, p300 and PGC-1a was further investigated by plasmid overexpression. HNF4a and PGC-1a were found to positively regulate PEPCK expression under cell culture conditions simulating fasting (cAMP), whilst overexpression of HNF4a, PGC1a and p300 reduced L-PK mRNA expression under fed conditions. In conclusion, the results indicate that HNF4a is a transcriptional activator of both glucagon- stimulated gluconeogenic gene expression and insulin-stimulated glycolytic and lipogenic gene expression. It is hypothesised HNF4a forms separate multi-protein complexes to differentially regulate metabolic pathways under different metabolic states.