Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.     

Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist

We report the discovery of strong HNF4α agonists and their use to uncover a previously unknown pathway by which HNF4α controls the level of fat storage in the liver. This involves the induction of lipophagy by dihydroceramides, the synthesis and secretion of which is controlled by genes induced by HNF4α. The HNF4α activators are N-trans caffeoyltyramine (NCT) and N-trans feruloyltyramine (NFT), which are structurally related to the known drugs alverine and benfluorex, which we previously showed to be weak HNF4α activators. In vitro, NCT and NFT induced fat clearance from palmitate-loaded cells. In DIO mice, NCT led to recovery of hepatic HNF4α expression and reduction of steatosis. Mechanistically, increased dihydroceramide production and action downstream of HNF4α occurred through increased expression of HNF4α downstream genes, including SPNS2 and CYP26A1. NCT was completely nontoxic at the highest dose administered and so is a strong candidate for an NAFLD therapeutic.Subject terms: Lipid signalling, Target validation     

Novel Polymorphisms of Nuclear Receptor SHP Associated with Functional and Structural Changes

We identified three heterozygous nonsynonymous single nucleotide polymorphisms in the small heterodimer partner (SHP, NROB2) gene in normal subjects and CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)-like patients, including two novel missense mutations (p.R38H, p.K170N) and one of the previously reported polymorphism (p.G171A). Four novel heterozygous mutations were also identified in the intron (^Intron1265T→A), 3′-untranslated region (^3′-UTR101C→G, ^3′-UTR186T→C), and promoter (^Pro-423C→T) of the SHP gene. The exonic R38H and K170N mutants exhibited impaired nuclear translocation. K170N made SHP more susceptible to ubiquitination mediated degradation and blocked SHP acetylation, which displayed lost repressive activity on its interacting partners ERRγ and HNF4α but not LRH-1. In contrast, G171A increased SHP mRNA and protein expression and maintained normal function. In general, the interaction of SHP mutants with LRH-1 and EID1 was enhanced. K170N also markedly impaired the recruitment of SHP, HNF4α, HDAC1, and HDAC3 to the apoCIII promoter. Molecular dynamics simulations of SHP showed that G171A stabilized the nuclear receptor boxes, whereas K170N promoted the conformational destabilization of all the structural elements of the receptor. This study suggests that genetic variations in SHP are common among human subjects and the Lys-170 residue plays a key role in controlling SHP ubiquitination and acetylation associated with SHP protein stability and repressive function.