Influence of ABCB11 and HNF4α genes on daclatasvir plasma concentration: preliminary pharmacogenetic data from the Kineti-C study.

Background: Daclatasvir is an inhibitor of HCV non-structural 5A protein and is a P-glycoprotein substrate. Pharmacogenetics has had a great impact on previous anti-HCV therapies, particularly considering the association of IL-28B polymorphisms with dual therapy outcome. Objectives: We investigated the association between daclatasvir plasma concentrations at 2 weeks and 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCB11 and HNF4α). Patients and methods: Allelic discrimination was achieved through real-time PCR, whereas plasma concentrations were evaluated through LC-MS/MS. Results: Fifty-two patients were analysed, all enrolled in the Kineti-C study. HNF4α 975 C > G polymorphism was found to be associated with the daclatasvir plasma concentrations at 2 weeks (P = 0.009) and 1 month of therapy (P = 0.006). Linear regression analysis suggested that, at 2 weeks of therapy, age, baseline BMI and haematocrit were significant predictors of daclatasvir concentrations, whereas at 1 month of therapy ABCB111131 CC and HNF4α CG/GG genotypes were significant predictors of daclatasvir concentrations. Conclusions: These are the first and preliminary results from our clinical study focusing on daclatasvir pharmacogenetics, showing that this approach could have a role in the era of new anti-HCV therapies.

The role of hepatocyte nuclear factor 4-alpha in perfluorooctanoic acid- and perfluorooctanesulfonic acid-induced hepatocellular dysfunction.

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.

Impairment of hepatic nuclear factor-4α binding to the Stim1 promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells.

The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca(2+) entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca(2+) entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.

Vanin-1 is a key activator for hepatic gluconeogenesis.

Vanin-1 (VNN1) is a liver-enriched oxidative stress sensor that has been implicated in the regulation of multiple metabolic pathways. Clinical investigations indicated that the levels of VNN1 were increased in the urine and blood of diabetic patients, but the physiological significance of this phenomenon remains unknown. In this study, we demonstrated that the hepatic expression of VNN1 was induced in fasted mice or mice with insulin resistance. Gain- and loss-of-function studies indicated that VNN1 increased the expression of gluconeogenic genes and hepatic glucose output, which led to hyperglycemia. These effects of VNN1 on gluconeogenesis were mediated by the regulation of the Akt signaling pathway. Mechanistically, vnn1 transcription was activated by the synergistic interaction of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and hepatocyte nuclear factor-4α (HNF-4α). A chromatin immunoprecipitation analysis indicated that PGC-1α was present near the HNF-4α binding site on the proximal vnn1 promoter and activated the chromatin structure. Taken together, our results suggest an important role for VNN1 in regulating hepatic gluconeogenesis. Therefore, VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis.

Interleukin-1 controls the constitutive expression of the Cyp7a1 gene by regulating the expression of Cyp7a1 transcriptional regulators in the mouse liver.

Our previous study using interleukin-1α/ß-knockout (IL-1-KO) and wild-type (WT) mice demonstrated that IL-1 acts as a positive factor for constitutive gene expression of hepatic cytochrome P4507a1 (Cyp7a1). In this study, to clarify the role of IL-1 in the expression of the hepatic Cyp7a1 gene, we focused on Cyp7a1 transcriptional regulators such as α-fetoprotein transcription factor (FTF), liver X receptor α (LXRα), hepatocyte nuclear factor 4α (HNF4α) and small heterodimer partner (SHP) and examined the effects of IL-1 on their gene expression by real-time reverse-transcription polymerase chain reaction using IL-1-KO and WT mice. We observed no significant differences between sex-matched IL-1-KO and WT mice with regard to gene expression levels of FTF, LXRα, and HNF4α, all of which are positive transcriptional regulators for the Cyp7a1 gene. However, interindividual differences in hepatic FTF and LXRα expression were closely dependent on the gene expression level(s) of hepatic IL-1 and tumor necrosis factor-α (TNF-α), while interindividual differences in hepatic HNF4α were clearly correlated with the expression of IL-1, but not TNF-α. In contrast, the gene expression level of SHP, which is a negative transcriptional regulator of the Cyp7a1 gene through inhibition of FTF function, was higher in IL-1-KO mice than in sex-matched WT mice. These findings demonstrate that, like TNF-α, IL-1 positively controls the gene expression of Cyp7a1 transcriptional upregulators but, in contrast to the previously reported action of TNF-α, IL-1 also acts to downregulate SHP gene expression.

Hepatic hepatocyte nuclear factor 4α is essential for maintaining triglyceride and cholesterol homeostasis.

OBJECTIVE: Loss-of-function mutations in human hepatocyte nuclear factor 4α (HNF4α) are associated with maturity-onset diabetes of the young and lipid disorders. However, the mechanisms underlying the lipid disorders are poorly understood. In this study, we determined the effect of acute loss or augmentation of hepatic HNF4α function on lipid homeostasis. METHODS AND RESULTS: We generated an adenovirus expressing LacZ (Ad-shLacZ) or short hairpin RNA of Hnf4α (Ad-shHnf4α). Tail vain injection of C57BL/6J mice with Ad-shHnf4α reduced hepatic Hnf4α expression and resulted in striking phenotypes, including the development of fatty liver and a >80% decrease in plasma levels of triglycerides, total cholesterol, and high-density lipoprotein cholesterol. These latter changes were associated with reduced hepatic lipogenesis and impaired very-low-density lipoprotein secretion. Deficiency in hepatic Hnf4α did not affect intestinal cholesterol absorption despite decreased expression of genes involved in bile acid synthesis. Consistent with the loss-of-function data, overexpression of Hnf4α induced numerous genes involved in lipid metabolism in isolated primary hepatocytes. Interestingly, many of these HNF4α-regulated genes were not induced in wild-type mice that overexpressed hepatic Hnf4α. Because of selective gene regulation, mice overexpressing hepatic Hnf4α had unchanged plasma triglyceride levels and decreased plasma cholesterol levels. CONCLUSIONS: Loss of hepatic HNF4α results in severe lipid disorder as a result of dysregulation of multiple genes involved in lipid metabolism. In contrast, augmentation of hepatic HNF4α activity lowers plasma cholesterol levels but has no effect on plasma triglyceride levels because of selective gene regulation. Our data indicate that hepatic HNF4α is essential for controlling the basal expression of numerous genes involved in lipid metabolism and is indispensable for maintaining normal lipid homeostasis.

Nuclear hormone receptors in diabetic nephropathy.

Diabetes is the leading cause of end-stage renal disease in developed countries. In spite of glucose and blood pressure control, for example by use of angiotensin II receptor blockers, diabetic nephropathy still develops and progresses in affected patients and the development of additional protective therapeutic interventions is, therefore, required. Nuclear hormone receptors are transcription factors that regulate carbohydrate metabolism, lipid metabolism, the immune response, and inflammation. These receptors also modulate the development of fibrosis. As a result of their diverse biological effects, nuclear hormone receptors have become major pharmaceutical targets for the treatment of a host of diseases. The increasing prevalence of diabetic nephropathy has led intense investigation into the role that nuclear hormone receptors may have in slowing or preventing the progression of renal disease. This role of nuclear hormone receptors would be associated with improvements in metabolism, the immune response, and inflammation. Eight nuclear receptors have shown a renoprotective effect in the context of diabetic nephropathy. This Review discusses the evidence regarding the beneficial effects of the activation of these receptors in preventing the progression of diabetic nephropathy and describes how the discovery and development of compounds that modulate the activity of nuclear hormone receptors may provide potential additional therapeutic approaches in the management of diabetic nephropathy.

Identification of small molecule regulators of the nuclear receptor HNF4alpha based on naphthofuran scaffolds.

Nuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4alpha (NR2A1) is a recently 'deorphanized' nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4alpha is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4alpha in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4alpha in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4alpha was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4alpha-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4alpha activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans.

Phenobarbital regulates nuclear expression of HNF-4alpha in mouse and rat hepatocytes independent of CAR and PXR.

Phenobarbital is a lipophilic molecule used as a sedative and antiepileptic drug that elicits a multitude of effects in the liver, including gross liver enlargement, hepatocyte hypertrophy, and induced expression of drug-metabolizing enzymes and other liver-specific genes. The constitutive androstane receptor (CAR; NR1I3) and to a lesser extent the pregnane X receptor (PXR; NR1I2) are responsible for mediating induction of many phenobarbital-responsive genes. However, CAR-mediated transcriptional control of some genes is critically dependent on hepatocyte nuclear factor 4 alpha (HNF-4alpha; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4alpha expression in hepatocytes and provide evidence that HNF-4alpha nuclear expression is regulated in response to phenobarbital. Real-time polymerase chain reaction analyses revealed that HNF-4alpha mRNA is modestly up-regulated by phenobarbital. In addition, nuclear expression of HNF-4alpha protein is significantly elevated 3 hours after the administration of phenobarbital in wild-type, CAR-/-, and CAR-/-/PXR-/- mice. In vitro analysis revealed that phenobarbital-induced HNF-4alpha expression is both time- and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4alpha by phenobarbital. Furthermore, HNF-4alpha nuclear expression is enhanced by inhibition of cyclic AMP-dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4alpha and CAR is an integral part of the phenobarbital response, aimed at coordinated regulation of genes involved in drug metabolism and detoxification as well as maintenance of liver function.

Prenatal programming of hepatocyte nuclear factor 4alpha in the rat: A key mechanism in the 'foetal origins of hyperglycaemia'?

AIMS/HYPOTHESIS: Prenatal glucocorticoid exposure causes lifelong hyperglycaemia in rat offspring, associated with permanently increased hepatic phosphoenolpyruvate carboxykinase 2 (PCK2), the rate-controlling enzyme of gluconeogenesis. To elucidate the mechanisms underlying the 'programming' of PCK2, this study examined the effect of prenatal dexamethasone treatment on expression of transcription factors that regulate Pck2. MATERIALS AND METHODS: Real-time RT-PCR and in situ hybridisation were used to measure and localise hepatic mRNA transcribed from the genes for PCK2, hepatocyte nuclear factor 4, alpha (HNF4A), transcription factor 1 (TCF1), CCAAT/enhancer binding protein, alpha (CEBPA), CEBPB, the glucocorticoid receptor (NR3C1) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PPARGC1A) in foetal and adult offspring of dams treated with dexamethasone or vehicle during the last week of gestation. RESULTS: Prenatal dexamethasone exposure significantly elevated Hnf4a mRNA expression in foetal and adult liver. This resulted from increased expression of isoforms derived from the 'adult' (P1) Hnf4a promoter. In contrast, isoforms from the 'foetal' (P2) promoter were markedly suppressed by dexamethasone. Like Pck2, the increase in hepatic Hnf4a mRNA occurred exclusively in the periportal zone. Foetal Tcf1 expression was also increased by dexamethasone treatment, but this did not persist into adulthood. Prenatal dexamethasone did not affect the amounts of foetal and/or adult Cebpa, Cebpb, Nr3c1 or Ppargc1a mRNA. CONCLUSIONS/INTERPRETATION: Prenatal dexamethasone exposure caused a permanent increase in hepatic Hnf4a mRNA. This increase, which was associated with a premature switch from foetal to adult promoter predominance, was congruent with changes in Pck2 expression. These data suggest that HNF4A might mediate Pck2 overexpression and subsequent hyperglycaemia.