Essential roles of bile acids and their nuclear receptors,FXR and PXR,in the cholestatic liver disease

Bile acids (BAs) are steroid acids found predominantly in the bile of mammals and other vertebrates. Though BAs have been known as digestive juice, recent studies have revealed that BAs act as signaling molecules to control metabolism and inflammation. Today, BAs are considered as potential therapeutic molecules for treatment of complex metabolic liver disease. However, the detergent properties of BAs lead to hepatic injury and intrahepatic cholestasis when BAs are accumulated in the liver with impaired bile flow into gall bladder. Cholestasis is a pathological condition of hepatic retention of cytotoxic bile acids. To date, hydrophilic ursodeoxycholic acid has been currently used to treat cholestasis, but the efficacy of UDCA for cholestasis is still limited. Given that BAs are endogenous ligands of several nuclear receptors, including Farnesoid X receptor and Pregnane X receptor, novel synthetic ligands for those nuclear receptors are promising for the treatment of cholestatic liver diseases.     

Farnesoid X receptor: A “homeostat” for hepatic nutrient metabolism

The Farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). BAs are amphipathic molecules that serve as fat solubilizers in the intestine under postprandial conditions. In the post-absorptive state, BAs bind FXR in the hepatocytes, which in turn provides feedback signals on BA synthesis and transport and regulates lipid, glucose and amino acid metabolism. Therefore, FXR acts as a homeostat of all three classes of nutrients, fats, sugars and proteins. Here we re-analyze the function of FXR in the perspective of nutritional metabolism, and discuss the role of FXR in liver energy homeostasis in postprandial, post-absorptive and fasting/starvation states.FXR, by regulating nutritional metabolism, represses autophagy in conditions of nutrient abundance, and controls the metabolic needs of proliferative cells. In addition, FXR regulates inflammation via direct effects and via its impact on nutrient metabolism. These functions indicate that FXR is an attractive therapeutic target for liver diseases.     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

Translating clinical findings into knowledge in drug safety evaluation--drug induced liver injury prediction system (DILIps)

Drug-induced liver injury (DILI) is a significant concern in drug development due to the poor concordance between preclinical and clinical findings of liver toxicity. We hypothesized that the DILI types (hepatotoxic side effects) seen in the clinic can be translated into the development of predictive in silico models for use in the drug discovery phase. We identified 13 hepatotoxic side effects with high accuracy for classifying marketed drugs for their DILI potential. We then developed in silico predictive models for each of these 13 side effects, which were further combined to construct a DILI prediction system (DILIps). The DILIps yielded 60-70% prediction accuracy for three independent validation sets. To enhance the confidence for identification of drugs that cause severe DILI in humans, the "Rule of Three" was developed in DILIps by using a consensus strategy based on 13 models. This gave high positive predictive value (91%) when applied to an external dataset containing 206 drugs from three independent literature datasets. Using the DILIps, we screened all the drugs in DrugBank and investigated their DILI potential in terms of protein targets and therapeutic categories through network modeling. We demonstrated that two therapeutic categories, anti-infectives for systemic use and musculoskeletal system drugs, were enriched for DILI, which is consistent with current knowledge. We also identified protein targets and pathways that are related to drugs that cause DILI by using pathway analysis and co-occurrence text mining. While marketed drugs were the focus of this study, the DILIps has a potential as an evaluation tool to screen and prioritize new drug candidates or chemicals, such as environmental chemicals, to avoid those that might cause liver toxicity. We expect that the methodology can be also applied to other drug safety endpoints, such as renal or cardiovascular toxicity.     

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

A system of primary cultures of postnatal rat hepatocytes has been developed to serve as an experimental model for drug metabolism and toxicity investigations. The purpose of this study was to examine the reported loss of cytochrome P-450 of hepatocytes when placed in culture and to compare activity in culture to intact liver and freshly isolated hepatocytes. A medium enriched with several hormones and a system of floating filters as a substratum for cell attachment were investigated as methods to reduce the expected loss of cytochrome P-450. When compared to initial values of cytochrome P-450 in whole liver and isolated hepatocytes, these methods failed to prevent the reduction of cytochrome P-450 in culture. However, our results compare favorably with other values reported in the literature.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.     

Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS

To evaluate bile acid (BA) metabolism in detail, we established a method for analyzing BA composition in various tissues and intestinal contents using ultra performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Twenty-two individual BAs were determined simultaneously from extracts. We applied this method to define the differences in BA metabolism between two rat strains, WKAH and DA. The amount of total bile acids (TBAs) in the liver was significantly higher in WKAH than in DA rats. In contrast, TBA concentration in jejunal content, cecal content, colorectal content, and feces was higher in DA rats than in WKAH rats. Nearly all BAs in the liver were in the taurine- or glycine-conjugated form in DA rats, and the proportion of conjugated liver BAs was up to 75% in WKAH rats. Similar trends were observed for the conjugation rates in bile. The most abundant secondary BA in cecal content, colorectal content, and feces was hyodeoxycholic acid in WKAH rats and omega-muricholic acid in DA rats. Analyzing detailed BA profiles, including conjugation status, in a single run is possible using UPLC/ESI-MS. This method will be useful for investigating the roles of BA metabolism under physiological and pathological conditions.     

Xanthine oxidoreductase is present in bile ducts of normal and cirrhotic liver

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.     

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.     

Molecular and cellular remodeling of HepG2 cells upon treatment with antitubercular drugs

Drug-induced liver injury (DILI) is an adverse outcome of the currently used tuberculosis treatment regimen, which results in patient noncompliance, poor treatment outcomes, and the emergence of drug-resistant tuberculosis. DILI is primarily caused by the toxicity of the drugs and their metabolites, which affect liver cells, biliary epithelial cells, and liver vasculature. However, the precise mechanism behind the cellular damage attributable to first-line antitubercular drugs (ATDs), as well as the effect of toxicity on the cell survival strategies, is yet to be elucidated. In the current study, HepG2 cells upon treatment with a high concentration of ATDs showed increased perforation within the cell, cuboidal shape, and membrane blebbing as compared with control/untreated cells. It was observed that ATD-induced toxicity in HepG2 cells leads to altered mitochondrial membrane permeability, which was depicted by the decreased fluorescence intensity of the MitoRed tracker dye at higher drug concentrations. In addition, high doses of ATDs caused cell damage through an increase in reactive oxygen species production in HepG2 cells and a simultaneous reduction in glutathione levels. Further, high dose of isoniazid (50–200 mM), pyrazinamide (50–200 mM), and rifampicin (20–100 µM) causes cell apoptosis and affects cell survival during toxic conditions by decreasing the expression of potent autophagy markers Atg5, Atg7, and LC3B. Thus, ATD-mediated toxicity contributes to the reduced ability of hepatocytes to tolerate cellular damage caused by altered mitochondrial membrane permeability, increased apoptosis, and decreased autophagy. These findings further emphasize the need to develop adjuvant therapies that can mitigate ATD-induced toxicity for the effective treatment of tuberculosis.     

Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11)

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin–Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [³H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.     

Turnover of bile acids in liver,serum and caecal content by high-fat diet feeding affects hepatic steatosis in rats

BackgroundBile acids (BAs) participate in lipid absorption and serve as metabolic regulatory factors in gut-liver communication. To date, there are no studies on the systemic patterns of BAs in the serum, liver, and gut in the same non-alcoholic fatty liver disease (NAFLD) model.MethodsA targeted metabolomics approach and 16S rRNA sequencing were used to identify the profile of BAs and connection between BAs and microbiota. The role and mechanism of altered BAs on hepatic steatosis were investigated.FindingsIn the liver, the composition of taurocholic acid (TCA) was increased, but taurohyodeoxycholic acid (THDCA) and ursodeoxycholic acid (UDCA) were decreased. In the gut, the deconjugated form of TCA (cholic acid (CA)) was increased, while the deconjugated forms of THDCA (α-hyodeoxycholic acid (HDCA)) and ω-muricholic acid (ωMCA) were decreased. In the serum, the composition of TCA was increased, while both HDCA and THDCA were decreased. THDCA induced the gene expression of apolipoprotein, bile secretion-related proteins, and cytochrome P450 family but suppressed inflammatory response genes expression in steatotic hepatocytes by RNAseq analysis. THDCA ameliorated neutral lipid accumulation and improved insulin sensitivity in primary rat hepatocytes. The decreased HDCA level correlated with the level of Bacteroidetes, while the level of CA correlated with the levels of Firmicutes and Verrucomicrobia but correlated inversely with Bacteroidetes.ConclusionBAs profiles in the serum, liver and caecal content were altered in a rat NAFLD model, which may affect hepatic lipid accumulation and correlate with gut dysbiosis.     

Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure

Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen-induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety.     

Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.     

Abcb11 deficiency induces cholestasis coupled to impaired β-fatty acid oxidation in mice

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid β-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid β-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.     

Cyp7a1 is continuously increased with disrupted Fxr-mediated feedback inhibition in hypercholesterolemic TALLYHO/Jng mice

TALLYHO/Jng (TH) mice reveal hypercholesterolemia at an early age before their plasma glucose levels have increased. The increased plasma cholesterol should be related to bile acids (BAs) metabolism, because cholesterol is the precursor of BAs and BAs change cholesterol metabolism in a feedback manner. We analyzed the BAs pool size, BAs composition, and expression levels of several proteins that have key roles in BAs synthesis, excretion, and reabsorption and compared them to those of C57BL/6 (B6) mice to study BAs metabolism in TH mice. TH mice exhibited an increased total BAs pool size, increased BAs content in the cecum feces, and an increased ratio of muricholic acid (MCA)/cholic acid (CA). The mRNA and protein levels of cholesterol 7 alpha-hydroxylase (Cyp7a1) and the ATP-binding cassette sub-family G member 5 (Abcg5) were elevated in the liver but not in the apical sodium bile acid transporter (Asbt) and organic solute transporters (Osts) in the ileum. These results indicate that synthesis and the excretion of BAs from the liver to the gallbladder might be elevated, but the reabsorption rate of BAs in the ileum might be reduced. The declined expression of fibroblast growth factor 15 (Fgf15) and fibroblast growth factor receptor 4 (Fgfr4) was respectively identified in the ileum and the liver, indicating the disrupted feedback inhibition of Cyp7a1. Consequently, hypercholesterolemia in TH mice might increase the BAs amounts via the interrupted Fxr/Fgf15/Fgfr4-mediated feedback regulation of Cyp7a1.