Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.     

Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes

To understand the mechanisms governing the regulation of nuclear receptor (NR) function, we compared the parameters of activation and repression of two isoforms of the orphan receptor hepatocyte nuclear factor (HNF) 4alpha. HNF4alpha7 and HNF4alpha1 differ only in their N-terminal domains, and their expression in the liver is regulated developmentally. We show that the N-terminal activation function (AF)-1 of HNF4alpha1 possesses significant activity that can be enhanced through interaction with glucocorticoid receptor-interacting protein 1 (GRIP-1) and cAMP response element-binding protein-binding protein (CBP). In striking contrast, HNF4alpha7 possesses no measurable AF-1, implying major functional differences between the isoforms. Indeed, although HNF4alpha1 and HNF4alpha7 are able to interact via AF-2 with GRIP-1, p300, and silencing mediator for retinoid and thyroid receptors (SMRT), only HNF4alpha1 interacts in a synergistic fashion with GRIP-1 and p300. Although both isoforms interact physically and functionally with SMRT, the repression of HNF4alpha7 is less robust than that of HNF4alpha1, which may be caused by an increased ability of the latter to recruit histone deacetylase (HDAC) activity to target promoters. Moreover, association of SMRT with HDACs enhanced recruitment of HNF4alpha1 but not of HNF4alpha7. These observations suggest that NR isoform-specific association with SMRT could affect activity of the SMRT complex, implying that selection of HDAC partners is a novel point of regulation for NR activity. Possible physiological consequences of the multiple interactions with these coregulators are discussed.     

Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha

The role of the constitutive androstane receptor (CAR) in xenobiotic metabolism by inducing expression of cytochromes P450 is well known, but CAR has also been implicated in the down-regulation of key genes involved in bile acid synthesis, gluconeogenesis, and fatty acid beta-oxidation by largely unknown mechanisms. Because a key hepatic factor, hepatic nuclear factor-4 (HNF-4), is crucial for the expression of many of these genes, we examined whether CAR could suppress HNF-4 transactivation. Expression of CAR inhibited HNF-4 transactivation of CYP7A1, a key gene in bile acid synthesis, in HepG2 cells, and mutation of the DNA binding domain of CAR impaired this inhibition. Gel shift assays revealed that CAR competes with HNF-4 for binding to the DR1 motif in the CYP7A1 promoter. TCPOBOP, a CAR agonist that increases the interaction of CAR with coactivators, potentiated CAR inhibition of HNF-4 transactivation. Furthermore, inhibition by CAR was reversed by expression of increasing amounts of GRIP-1 or PGC-1alpha, indicating that CAR competes with HNF-4 for these coactivators. Treatment of mice with phenobarbital or TCPOBOP resulted in decreased hepatic mRNA levels of the reported genes down-regulated by CAR, including Cyp7a1 and Pepck. In vivo recruitment of endogenous CAR to the promoters of Cyp7a1 and Pepck was detected in mouse liver after phenobarbital treatment, whereas association of HNF-4 and coactivators, GRIP-1, p300, and PGC-1alpha, with these promoters was significantly decreased. Our data suggest that CAR inhibits HNF-4 activity by competing with HNF-4 for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha, which may be a general mechanism by which CAR down-regulates key genes in hepatic lipid and glucose metabolism.     

The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction

The orphan nuclear receptor CAR (NR1I3) has been characterized as a central component in the coordinate response to xenobiotic and endobiotic stress. In this study, we demonstrate that CAR plays a pivotal function in energy homeostasis and establish an unanticipated metabolic role for this nuclear receptor. Wild-type mice treated with the synthetic CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) exhibited decreased serum concentration of the thyroid hormone (TH) thyroxine (T(4)). However, treatment of Car(-/-) mice with TCPOBOP failed to elicit these changes. To examine whether CAR played a role in the regulation of TH levels under physiological conditions, wild-type and Car(-/-) mice were fasted for 24 h, a process known to alter TH metabolism in mammals. As expected, the serum triiodothyronine and T(4) concentrations decreased in wild-type mice. However, triiodothyronine and T(4) levels in fasted Car(-/-) mice remained significantly higher than those in fasted wild-type animals. Concomitant with the changes in serum TH levels, both CAR agonist treatment and fasting induced the expression of CAR target genes (notably, Cyp2b10, Ugt1a1, Sultn, Sult1a1, and Sult2a1) in a receptor-dependent manner. Importantly, the Ugt1a1, Sultn, Sult1a1, and Sult2a1 genes encode enzymes that are capable of metabolizing TH. An attenuated reduction in TH levels during fasting, as observed in Car(-/-) mice, would be predicted to increase weight loss during caloric restriction. Indeed, when Car(-/-) animals were placed on a 40% caloric restriction diet for 12 weeks, Car(-/-) animals lost over twice as much weight as their wild-type littermates. Thus, CAR participates in the molecular mechanisms contributing to homeostatic resistance to weight loss. These data imply that CAR represents a novel therapeutic target to uncouple metabolic rate from food intake and has implications in obesity and its associated disorders.     

Apolipoprotein A-IV is regulated by nutritional and metabolic stress: involvement of glucocorticoids, HNF-4 alpha, and PGC-1 alpha

Apolipoprotein A-IV (apoA-IV) is a 46 kDa glycoprotein that associates with triglyceride-rich and high density lipoproteins. Blood levels of apoA-IV generally correlate with triglyceride levels and are increased in diabetic patients. This study investigated the mechanisms regulating the in vivo expression of apoA-IV in the liver and intestine of mice in response to changes in nutritional status. Fasting markedly increased liver and ileal apoA-IV mRNA and plasma protein concentrations. This induction was associated with increased serum glucocorticoid levels and was abolished by adrenalectomy. Treatment with dexamethasone increased apoA-IV expression in adrenalectomized mice. Marked increases of apoA-IV expression were also observed in two murine models of diabetes. Reporter gene analysis of the murine and human apoA-IV/C-III promoters revealed a conserved cooperative activation by the hepatic nuclear factor-4 alpha (HNF-4 alpha) and the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) but no evidence of a direct regulatory role for the glucocorticoid receptor. Consistent with these in vitro data, induction of apoA-IV in response to fasting was accompanied by increases in HNF-4 alpha and PGC-1 alpha expression and was abolished in liver-specific HNF-4 alpha-deficient mice. Together, these results indicate that the induction of apoA-IV expression in fasting and diabetes likely involves PGC-1 alpha-mediated coactivation of HNF-4 alpha in addition to glucocorticoid-dependent actions.     

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).     

Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.