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Dysregulation of lipid metabolism is a major factor contributing to atherosclerotic cardiovascular disease (ACVD), which is the number one cause of death in western countries. The liver plays a central role in maintaining whole body cholesterol homeostasis via catabolism of cholesterol to bile acids, as well as biliary cholesterol excretion. The liver synthesizes lipoproteins that transport dietary cholesterol and fats to muscle and adipose tissue, directs reverse cholesterol transport of excess cholesterol from extrahepatic tissues and macrophages to the liver to convert to bile acids, and thus, protects against metabolism-related nonalcoholic fatty liver disease (NAFLD) and ACVD. Liver fibrosis/nonalcoholic steatohepatitis increases the risk and prevalence of cardiovascular disease morbidity and mortality. Bile acids are signaling molecules and metabolic regulators that activate farnesoid X receptor and G protein-coupled bile acid receptor-1 to regulate lipid, glucose, and energy metabolism. The bidirectional regulation of bile acids and the gut microbiota determine the rate of bile acid synthesis, the bile acid pool size, and the composition of the circulating bile acid pool. The liver-intestine-heart axis regulates lipid metabolism, inflammation, and the pathogenesis of metabolic diseases such as ACVD, NAFLD, diabetes, and obesity. This review focuses on the roles of liver-to-intestine, liver-to-heart and intestine-to-heart axes in cholesterol, lipoprotein, and bile acid metabolism; signaling in heart health and ACVD; and drug therapies for atherosclerosis.

Purpose: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5β-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). Methods: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Results: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. Conclusion: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.

Bile acid synthesis is the most significant pathway for catabolism of cholesterol and for maintenance of whole body cholesterol homeostasis. Bile acids are physiological detergents that absorb, distribute, metabolize, and excrete nutrients, drugs, and xenobiotics. Bile acids also are signal molecules and metabolic integrators that activate nuclear farnesoid X receptor (FXR) and membrane Takeda G protein-coupled receptor 5 (TGR5; i.e., G protein-coupled bile acid receptor 1) to regulate glucose, lipid, and energy metabolism. The gut-to-liver axis plays a critical role in the transformation of primary bile acids to secondary bile acids, in the regulation of bile acid synthesis to maintain composition within the bile acid pool, and in the regulation of metabolic homeostasis to prevent hyperglycemia, dyslipidemia, obesity, and diabetes. High-fat and high-calorie diets, dysbiosis, alcohol, drugs, and disruption of sleep and circadian rhythms cause metabolic diseases, including alcoholic and nonalcoholic fatty liver diseases, obesity, diabetes, and cardiovascular disease. Bile acid-based drugs that target bile acid receptors are being developed for the treatment of metabolic diseases of the liver.

Activation of the nuclear bile acid receptor farnesoid X receptor (FXR) protects against hepatic inflammation and injury, while Takeda G protein-coupled receptor 5 (TGR5) promotes adipose tissue browning and energy metabolism. Here, we examined the physiological and metabolic effects of the deficiency of these two bile acid receptors on hepatic metabolism and injury in mice. Fxr/Tgr5 double knockout mice (DKO) were generated for metabolic phenotyping. Male DKO mice fed a chow diet had reduced liver lipid levels but increased serum cholesterol levels. Liver cholesterol 7α-hydroxylase (Cyp7a1) activity and sterol 12α-hydroxylase mRNA levels were induced, while ileum FXR target genes were suppressed in DKO mice compared to wild-type (WT) mice. Bile acid pool size was increased in DKO mice, with increased taurocholic acid and decreased tauromuricholic acids. RNA sequencing analysis of the liver transcriptome revealed that bile acid synthesis and fibrosis gene expression levels are increased in chow-fed DKO mice compared to WT mice and that the top regulated pathways are involved in steroid/cholesterol biosynthesis, liver cirrhosis, and connective tissue disease. Cholestyramine treatment further induced Cyp7a1 mRNA and protein in DKO mice and increased bile acid pool size, while cholic acid also induced Cyp7a1 in DKO mice, suggesting impaired bile acid feedback regulation. A Western diet containing 0.2% cholesterol increased oxidative stress and markers of liver fibrosis but not hepatic steatosis in DKO mice. Conclusion: FXR and TGR5 play critical roles in protecting the liver from inflammation and fibrosis, and deficiency of both of these bile acid receptors in mice increased cholic acid synthesis and the bile acid pool, liver fibrosis, and inflammation; FXR and TGR5 DKO mice may be a model for liver fibrosis.

ObjectivesTRPV1 channels have been linked to the development and progression of diabetes at multiple levels, including control of appetite and weight, regulation of pancreatic function, thermogenesis, metabolism and energy homeostasis. Despite this, little information is known regarding its role in liver homeostasis and nonalcoholic fatty liver disease (NAFLD).Methods and ResultsTo better understand the role of TRPV1 in liver metabolism, we explored the effects of a high fat/sugar diet (Western, 24-week regimen) in male and female wild type (WT) and TRPV1-null (V1KO) mice. Our data reveal that loss of the TRPV1 gene makes mice susceptible to diet-induced obesity and induces NAFLD. V1KO mice displayed gross phenotypic and gross morphological changes including insulin resistance, glucose intolerance, increased body mass and central adiposity on a western diet compared to WT counterparts. Western fed V1KO mice exhibited gross changes in liver morphology and size compared to western fed WT mice, which were supported with histological H&E and Oil Red O staining. Accompanying the liver changes, Western fed V1KO mice exhibited altered lipid profiles as demonstrated by elevated hepatic triglyceride, cholesterol and free fatty acid levels compared to western fed WT mice. Interestingly, female V1KO mice fed a western diet displayed significant protection against diet-induced obesity and the progression of NAFLD compared to their male counterparts. Taken together, these data suggest that loss of TRPV1 promotes fat accumulation, NAFLD development and changes in liver lipid profiles in male mice, the extent to which is less severe in female V1KO mice.ConclusionIn conclusion, TRPV1 may be a protective therapeutic target for the prevention of NAFLD development in diet-induced obesity.

Mounting research evidence demonstrates a significant negative impact of circadian disruption on human health. Shift work, chronic jet lag and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, type 2 diabetes and dyslipidemia. Here, these associations are reviewed with respect to liver metabolism and disease., Graphical abstract Shift work, chronic jet lag, and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, Type 2 diabetes and dyslipidemia. Here, these associations are reviewed with respect to liver metabolism and disease.

Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr(ΔIEpC)) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr(ΔIEpC) mice to produce the Vdr(ΔIEpC)/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr(ΔIEpC), and Vdr(ΔIEpC)/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr(ΔIEpC) mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr(ΔIEpC)/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7α-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species. —Chen, W., E. Owsley, Y. Yang, D. Stroup, and J. Y. L. Chiang. Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids.

Bile acids concentration in liver is tightly regulated to prevent cell damage. Previous studies have demonstrated that deregulation of bile acid homeostasis can lead to cholestatic liver disease. Recently, we have shown that ER-bound transcription factor Crebh is a downstream effector of hepatic Cb1r signaling pathway. In this study, we have investigated the effect of alcohol exposure on hepatic bile acid homeostasis and elucidated the mediatory roles of Cb1r and Crebh in this process. We found that alcohol exposure or Cb1r-agonist 2-AG treatment increases hepatic bile acid synthesis and serum ALT, AST levels in vivo alongwith significant increase in Crebh gene expression and activation. Alcohol exposure activated Cb1r, Crebh, and perturbed bile acid homeostasis. Overexpression of Crebh increased the expression of key bile acid synthesis enzyme genes via direct binding of Crebh to their promoters, whereas Cb1r knockout and Crebh-knockdown mice were protected against alcohol-induced perturbation of bile acid homeostasis. Interestingly, insulin treatment protected against Cb1r-mediated Crebh-induced disruption of bile acid homeostasis. Furthermore, Crebh expression and activation was found to be markedly increased in insulin resistance conditions and Crebh knockdown in diabetic mice model (db/db) significantly reversed alcohol-induced disruption of bile acid homeostasis. Overall, our study demonstrates a novel regulatory mechanism of hepatic bile acid metabolism by alcohol via Cb1r-mediated activation of Crebh, and suggests that targeting Crebh can be of therapeutic potential in ameliorating alcohol-induced perturbation of bile acid homeostasis.

The transcriptional regulation of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) by hormones and signal transduction pathways was studied by transient transfection assay of the promoter activity. HepG2 cells were transfected with deletion mutants of the CYP7 upstream region linked to the luciferase reporter gene. The transcription of CYP7/luciferase chimeric genes was higher in confluent than in subconfluent cultures of HepG2 cells. Glucocorticoid receptors, in the presence of dexamethasone, up-regulated the CYP7 gene through two regions located between -3262 and -2803, and between -344 and -222, respectively. Thyroid hormones did not have any effect on the promoter activity. Insulin inhibited the promoter activity through sequences located between -344 and -222, and abolished the stimulation by dexamethasone. Hence, the insulin effect was dominant over that of glucocorticoids. Treatment of transfected HepG2 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C (PKC), resulted in a time-dependent inhibition of the CYP7 promoter activity. The negative phorbol ester-response sequences were mapped between -344 and -222, and between -200 and -161, respectively. The CYP7 promoter activity was induced nearly 5-fold by all-trans-retinoic acid through sequences in the region from -200 to -129. Finally, cyclic AMP and protein kinase A (PKA) stimulated the expression of the CYP7/luciferase gene through multiple sequences in the distal and proximal regions, and both positive and negative response regions were mapped. Our results revealed that the -416 fragment of the rat CYP7 gene confers the activation by glucocorticoids and retinoic acid, and inhibition by insulin, phorbol esters and cAMP. It appears that this proximal promoter may contain a pleiotropic domain that regulates the effects of multiple signals.

Activation of farnesoid X receptor (Fxr, Nr1h4) is a major mechanism in suppressing bile-acid synthesis by reducing the expression levels of genes encoding key bile-acid synthetic enzymes (e.g., cytochrome P450 [CYP]7A1/Cyp7a1 and CYP8B1/Cyp8b1). FXR-mediated induction of hepatic small heterodimer partner (SHP/Shp, Nr0b2) and intestinal fibroblast growth factor 15 (Fgf15; FGF19 in humans) has been shown to be responsible for this suppression. However, the exact contribution of Shp/Fgf15 to this suppression, and the associated cell-signaling pathway, is unclear. By using novel genetically modified mice, the current study showed that the intestinal Fxr/Fgf15 pathway was critical for suppressing both Cyp7a1 and Cyp8b1 gene expression, but the liver Fxr/Shp pathway was important for suppressing Cyp8b1 gene expression and had a minor role in suppressing Cyp7a1 gene expression. Furthermore, in vivo administration of Fgf15 protein to mice led to a strong activation of extracellular signal-related kinase (ERK) and, to a smaller degree, Jun N-terminal kinase (JNK) in the liver. In addition, deficiency of either the ERK or JNK pathway in mouse livers reduced the basal, but not the Fgf15-mediated, suppression of Cyp7a1 and Cyp8b1 gene expression. However, deficiency of both ERK and JNK pathways prevented Fgf15-mediated suppression of Cyp7a1 and Cyp8b1 gene expression. Conclusion: The current study clearly elucidates the underlying molecular mechanism of hepatic versus intestinal Fxr in regulating the expression of genes critical for bile-acid synthesis and hydrophobicity in the liver. (HEPATOLOGY 2012;56:1034–1043)

Obesity, diabetes, and metabolic syndromes are increasingly recognized as health concerns worldwide. Overnutrition and insulin resistance are the major causes of diabetic hyperglycemia and hyperlipidemia in humans. Studies in the past decade provide evidence that bile acids are not just biological detergents facilitating gut nutrient absorption, but also important metabolic regulators of glucose and lipid homeostasis. Pharmacological alteration of bile acid metabolism or bile acid signaling pathways such as using bile acid receptor agonists or bile acid binding resins may be a promising therapeutic strategy for the treatment of obesity and diabetes. On the other hand, bile acid signaling is complex, and the molecular mechanisms mediating the bile acid effects are still not completely understood. This paper will summarize recent advances in our understanding of bile acid signaling in regulation of glucose and lipid metabolism, and the potentials of developing novel therapeutic strategies that target bile acid metabolism for the treatment of metabolic disorders.

Cholesterol 7 alpha-hydroxylase (P450c7) is the first and rate-limiting enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7. We have previously reported the cloning of a full-length human cholesterol 7 alpha-hydroxylase cDNA (Karam, W. G., and J. Y. L. Chiang. 1992. Biochem. Biophys. Res. Commun. 185: 588-595). Using this clone in a polymerase chain reaction, we have generated a cDNA (H7 alpha 1.5) in which the codons for the N-terminal 24 amino acid residues were deleted. The translational product of this cDNA would be a truncated protein, P450c7(delta 2-24) with a hydrophilic NH2-terminal sequence, Met-Ala-Arg-Arg-Arg-Gln... This cDNA was cloned into the expression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat cholesterol 7 alpha-hydroxylase cDNA obtained previously (Li, Y. C., and J. Y. L. Chiang. 1991. J. Biol. Chem. 266: 19186-19191) into the pJL vector and have transformed this construct (pJL/R7 alpha 1.5) into E. coli strain MV1304. Both of these systems expressed functional cholesterol 7 alpha-hydroxylase in E. coli. A fivefold improvement in the expression of rat enzyme over the previous expression system was obtained. About 70-80% of the truncated human P450 in the clear lysate was localized in the cytosol. The truncated human and rat P450c7(delta 2-24) were purified to homogeneity. Reconstitution of cholesterol 7 alpha-hydroxylase activity using purified rat or human P450c7(delta 2-24) showed a similar Km of 6 and 7 microM for cholesterol, a Vmax of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectively. Immunoblotting experiment revealed that a polyclonal antibody raised against rat microsomal cholesterol 7 alpha-hydroxylase recognized both rat and human P450c7(delta 2-24). This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.

Cholesterol 7 alpha-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7 alpha-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7 alpha-hydroxylase specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)

This special issue of PPAR Research is dedicated to “PPARs, RXRs and Drug Metabolizing Enzymes”. Knowledge of PPAR biology, over the past five years, has dramatically increased our understanding of the potential therapeutic usefulness of these receptors in metabolic alterations associated with disease process of alcoholic and non-alcoholic fatty liver disease and metabolic syndrome. In addition, the utility of PPAR agonist in the treatment of liver disease by normalizing hypertriglyceridemia, dyslipidemia, and the toxic effects of bile acids has a sound scientific basis in the ability of PPAR receptors to control lipid oxidation and disposal as well as regulators of hepatic inflammation. Further insight into both the indirect and direct effects of dual and pan PPAR agonist may potentiate the development of new therapeutic modalities to treat fatty acid oxidation disorders, dyslipidemia, inflammation, and bile acid accumulation associated with several liver diseases and metabolic syndrome. Articles included in this special issue highlight the importance of PPARs and RXR in drug metabolism and hepatic diseases associated with metabolic disorders. Alterations in drug metabolizing gene expression in different disease states due to the differential expression of PPAR isoforms highlights the importance of PPAR in disease progression and as therapeutic target in the amelioration of disease progression. The first review summarizes past and new developments in alcoholic fatty liver disease (ALD) and how PPAR/RXR regulates phase I enzymes in alcohol metabolism and the redox balance of cells. The direct impairment of PPAR by acetaldehyde results in reduction of NAD+ pool leading to alterations in lipid metabolism, increased oxidative stress, and increased pro-inflammatory cytokines, chemokines and acute phase proteins, which may be central in the onset and perpetuation of mechanism in the clinical progression of alcoholic liver disease (ALD). A second review focuses on the role of PPAR isoforms in the regulation of bile acid and cholesterol metabolism, with specific insight into how PPARα regulates bile acid synthesis, conjugation, and transport by phase II and III enzymes. This review also provides an updated report on how PPARs regulate cholesterol synthesis, absorption, and reverse cholesterol transport, and how PPAR agonist may be used to treat cholestatic liver disease. This review also raises important questions concerning the use of PPAR agonist in treating bile acid accumulation in several liver diseases, which leads to hepatocyte injury, impaired liver metabolic function, progressing to liver fibrosis and cirrhosis. Further research needs to focus on how selective PPAR agonist and isoforms regulated bile acid conjugation and thereby prevent bile acid toxicity observed in cholestastic disease. The conjugation of bile acids by UDP-glucuronosyltransferase, a PPAR target gene further emphasizes the importance of PPAR in bile acid toxicity. In addition, induction of the sulfotransferase genes by PPAR and sulfonation of cholesterol in keratinocyte differentiation suggests that these phase II genes have an important role in epidermal wound healing. Finally, although numerous studies in animal models of disease have dramatically increased our understanding of how PPARs not only regulate drug metabolism and the elimination of drugs and toxic endogenous metabolities in disease progression, the application of these results to human in the design of effective PPAR agonist has all too often led to adverse drug interactions and unacceptable drug toxicity. A fourth review in this series details the exciting possibility of using a human hepatocyte chimeric mouse model to predict metabolism and possible effectiveness of new PPAR agonist in humans. The investigators clearly show the utility of this model system by the increased phosphatidylcholine transport into bile canaliculi through induction of human ABCB4 transporter by benzafibrate activation of PPAR. There are marked species differences in genes and proteins associated with the absorption, distribution, metabolism, and excretion (ADME) of xenobiotics and drugs, thus the human hepatocyte chimeric mice may not only be used for increasing the safety and effectiveness of lead drugs, but may also serve as a model system to study human liver disease and assess the potency and efficacy of dual and pan PPAR agonist in preventing or delaying disease progression. The final review in these series details how fatty acids are partitioned by fatty acid transport proteins to either anabolic or catabolic pathways regulated by PPARs, and explores how medium chain fatty acid (MCFA) CYP4A and long chain fatty acid (LCFA) CYP4Fω-hydroxylase genes are regulated in fatty liver. The authors propose a hypothesis that increased CYP4A expression with a decrease in CYP4F genes may promote the progression of steatosis to steatohepatitis. We thank the editors for the opportunity to share with other investigators these interesting reviews on drug metabolizing genes regulated by PPAR/RXR. It is apparent that drug metabolizing genes not only play a pivotal role in drug efficacy, drug toxicity, and adverse drug reactions, but also a critical and crucial role in the development and progression liver disease. Therefore, understanding how the PPAR genes and other hormone nuclear receptors are regulated during disease processes will provide us the opportunity to design effective therapeutic modalities to treat disease by the inactivation, conjugation, and transport of toxic endogenous metabolities. James P. Hardwick John Y. L. Chiang

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7α-hydroxylase ( CYP7A1 ) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a ∼ 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

The hepatocyte nuclear factor 4alpha (HNF4alpha) is a liver-enriched nuclear receptor that plays a critical role in early morphogenesis, fetal liver development, liver differentiation and metabolism. Human HNF4alpha gene mutations cause maturity on-set diabetes of the young type 1, an autosomal dominant non-insulin-dependent diabetes mellitus. HNF4alpha is an orphan nuclear receptor because of which the endogenous ligand has not been firmly identified. The trans-activating activity of HNF4alpha is enhanced by interacting with co-activators and inhibited by corepressors. Recent studies have revealed that HNF4alpha plays a central role in regulation of bile acid metabolism in the liver. Bile acids are required for biliary excretion of cholesterol and metabolites, and intestinal absorption of fat, nutrients, drug and xenobiotics for transport and distribution to liver and other tissues. Bile acids are signaling molecules that activate nuclear receptors to control lipids and drug metabolism in the liver and intestine. Therefore, HNF4alpha plays a central role in coordinated regulation of bile acid and xenobiotics metabolism. Drugs that specifically activate HNF4alpha could be developed for treating metabolic diseases such as diabetes, dyslipidemia and cholestasis, as well as drug metabolism and detoxification.

Background & Aims Bile acids are physiologic detergents that also activate nuclear receptors to regulate glucose and lipid homeostasis. Cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme that converts cholesterol to bile acids, is transcriptionally regulated by bile acids and circadian rhythms. Fasting, nutrients, and the circadian clock critically control hepatic bile acid and lipid homeostasis, and circadian misalignment is associated with the metabolic syndrome in humans. To delineate these interactions, we employed a sleep disruption model to induce circadian disruption and examined hepatic metabolism with respect to bile acids, lipids, and clock gene expression. Methods B6xC57 mice were maintained on chow or Western diet and were sleep disrupted for 6 hours/day for 5 days. Mice were sacrificed at 4-hour intervals over 24 hours. Hepatic metabolic genes were examined, and bile acid pool and lipid profiles were measured over 24 hours. Results Sleep disruption significantly suppressed circadian expression of core clock genes, genes involved in lipid metabolism, and key regulators of Cyp7a1 as well as Cyp7a1 expression itself. Sleep disruption abolished the peak in serum cholesterol and increased liver and serum free fatty acids. Bile acid pool size was increased while liver bile acids were decreased. Chromatin immunoprecipitation assay revealed that hepatocyte nuclear factor 4α (HNF4α) and D-site binding protein (Dbp) occupancies were suppressed at the Cyp7a1 promoter in sleep-disrupted mice. When coupled with Western diet, sleep disruption abolished liver clock rhythms and elevated free fatty acids. Conclusions Even short-term circadian disruption dramatically alters hepatic clock gene expression, bile acid metabolism, and lipid homeostasis to contribute to dyslipidemia.