Bile Salt Metabolism Research Articles

European Society for Paediatric Gastroenterology, Hepatology, and Nutrition Annual Meeting May 25–28, 2011 Sorrento, Italy

Objectives and Study: Ursodeoxycholate (UDCA) is used as treatment for cystic fibrosis liver disease (CFLD). It is hypothesized that the supposed therapeutic action of UDCA in CF conditions is either mediated via its choleretic activity or via effects on bile salt metabolism. In CF conditions, however, the effects of UDCA on biliary bile salt composition and on the enterohepatic circulation of bile salts have remained unclear. We evaluated, in Cftr knockout mice and wild type controls, the effects of UDCA treatment on the biliary bile salt composition and on the biosynthesis rate and pool size of cholate (CA), which is proportionally the major hydrophobic bile salt in mouse and man. Methods: Cftr-/-and control mice were either fed standard or treated with UDCA enriched chow (0.5% wt/wt) for 3 weeks. We characterized biliary bile salt composition after gallbladder cannulation and determined the CA synthesis rate and bile salt pool size using a microscale stable isotope dilution technique. Results: In non UDCA treated Cftr-/-mice the fractional biliary CA content was significantly higher compared to controls (61% vs. 46%, resp.; P

Bacterial degradation of bile salts

Bile salts are surface-active steroid compounds. Their main physiological function is aiding the digestion of lipophilic nutrients in intestinal tracts of vertebrates. Many bacteria are capable of transforming and degrading bile salts in the digestive tract and in the environment. Bacterial bile salt transformation and degradation is of high ecological relevance and also essential for the biotechnological production of steroid drugs. While biotechnological aspects have been reviewed many times, the physiological, biochemical and genetic aspects of bacterial bile salt transformation have been neglected. This review provides an overview of the reaction sequence of bile salt degradation and on the respective enzymes and genes exemplified with the degradation pathway of the bile salt cholate. The physiological adaptations for coping with the toxic effects of bile salts, recent biotechnological applications and ecological aspects of bacterial bile salt metabolism are also addressed. As the pathway for bile salt degradation merges with metabolic pathways for bacterial transformation of other steroids, such as testosterone and cholesterol, this review provides helpful background information for metabolic engineering of steroid-transforming bacteria in general.

A Role of the Bile Salt Receptor FXR in Atherosclerosis

This study reviews current insights into the role of bile salts and bile salt receptors on the progression and regression of atherosclerosis. Bile salts have emerged as important modifiers of lipid and energy metabolism. At the molecular level, bile salts regulate lipid and energy homeostasis mainly via the bile salt receptors FXR and TGR5. Activation of FXR has been shown to improve plasma lipid profiles, whereas Fxr(-/-) mice have increased plasma triglyceride and very-low-density lipoprotein levels. Nevertheless, high-density lipoprotein cholesterol levels are increased in these mice, suggesting that FXR has both anti- and proatherosclerotic properties. Interestingly, there is increasing evidence for a role of FXR in "nonclassical" bile salt target tissues, eg, vasculature and macrophages. In these tissues, FXR has been shown to influence vascular tension and regulate the unloading of cholesterol from foam cells, respectively. Recent publications have provided insight into the antiinflammatory properties of FXR in atherosclerosis. Bile salt signaling via TGR5 might regulate energy homeostasis, which could serve as an attractive target to increase energy expenditure and weight loss. Interventions aiming to increase cholesterol turnover (eg, by bile salt sequestration) significantly improve plasma lipid profiles and diminish atherosclerosis in animal models. Bile salt metabolism and bile salt signaling pathways represent attractive therapeutic targets for the treatment of atherosclerosis.

Role of small heterodimer partner in lipid homeostasis and its potential as a therapeutic target for obesity

the NRs or inhibition of their interaction with other coactivators or their DNA response ele‐ ments. SHP has been shown to interact with approximately half of all mammalian nuclear receptors, including TR‐β, RAR‐α, PPAR‐α, PPAR‐γ, LXR‐α, LXR‐β, FXR, PXR/SXR, CAR, HNF‐4α, HNF6, RXR‐β, ER‐α, ER‐β, ERR‐α, ERR‐β, ERR‐γ, GR, AR, NGFI‐B, SF‐1 and LRH‐1 [11]. SHP also interacts with other transcriptional coregulators, such as EID1, mSin3A, HDAC1–5, BAF155, Brm, G9a, SMRT, N‐COR, RNAPol II, TFIID, TFIIE, CBP, SRC‐1, SMILE, SMAD3, ARNT, SIRT1 and RNF31 [11–16]. Thus, SHP targets multiple genes in diverse signaling pathways, enabling it to regulate multiple biological processes, including bile salt, lipid and glucose metabolism, obesity and so on.

The hepatic response to FGF19 is impaired in patients with nonalcoholic fatty liver disease and insulin resistance

Intestinal FGF19 has emerged as a novel endocrine regulator of hepatic bile salt and lipid metabolism. In patients with nonalcoholic fatty liver disease (NAFLD) hepatic lipid metabolism is deranged. A possible role of FGF19 in NAFLD has not been reported yet. In this study, we assessed intestinal FGF19 production and the hepatic response to FGF19 in NAFLD patients with and without insulin resistance [homeostasis model of assessment (HOMA) score > or =2.5 (n = 12) and HOMA score <2.5 (n = 8), respectively]. To this end, NAFLD patients received a standardized oral fat challenge. Postprandial excursions of triglycerides, bile salts, and FGF19 were monitored, and plasma levels of a marker for bile salt synthesis (7alpha-hydroxy-4-cholesten-3-one) were determined. Fasted FGF19 levels were comparable in a control group of healthy volunteers (n = 15) and in NAFLD patients (0.26 +/- 0.28 vs. 0.18 +/- 0.09 ng/ml, respectively, P = 0.94). Postprandial FGF19 levels in both controls and NAFLD patients peaked between 3-4 h and were three times higher than baseline levels. The areas under the postprandial FGF19 curve were similar in controls and in the HOMA score-based NAFLD subgroups. In NAFLD patients with HOMA score <2.5, the postprandial increase in plasma FGF19 was accompanied by a lowering of plasma levels of 7alpha-hydroxy-4-cholesten-3-one (-30%, P = 0.015). This anticipated decline was not observed in insulin-resistant NAFLD patients (+10%, P = 0.22). In conclusion, patients with NAFLD show an unimpaired intestinal FGF19 production. However, the hepatic response to FGF19 is impaired in NAFLD patients with insulin resistance (HOMA score > or =2.5). This impaired hepatic response to FGF19 may contribute to the dysregulation of lipid homeostasis in NAFLD.

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.

Bile Salt Sequestration Induces Hepatic De Novo Lipogenesis Through Farnesoid X Receptor– and Liver X Receptorα–Controlled Metabolic Pathways in Mice

Diabetes is characterized by high blood glucose levels and dyslipidemia. Bile salt sequestration has been found to improve both plasma glycemic control and cholesterol profiles in diabetic patients. Yet bile salt sequestration is also known to affect triglyceride (TG) metabolism, possibly through signaling pathways involving farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha). We quantitatively assessed kinetic parameters of bile salt metabolism in lean C57Bl/6J and in obese, diabetic db/db mice upon bile salt sequestration using colesevelam HCl (2% wt/wt in diet) and related these to quantitative changes in hepatic lipid metabolism. As expected, bile salt sequestration reduced intestinal bile salt reabsorption. Importantly, bile salt pool size and biliary bile salt secretion remained unchanged upon sequestrant treatment due to compensation by de novo bile salt synthesis in both models. Nevertheless, lean and db/db mice showed increased, mainly periportally confined, hepatic TG contents, increased expression of lipogenic genes, and increased fractional contributions of newly synthesized fatty acids. Lipogenic gene expression was not induced in sequestrant-treated Fxr(-/-) and Lxralpha(-/-) mice compared with wild-type littermates, in line with reports indicating a regulatory role of FXR and LXRalpha in bile salt-mediated regulation of hepatic lipid metabolism. Bile salt sequestration by colesevelam induces the lipogenic pathway in an FXR- and LXRalpha-dependent manner without affecting the total pool size of bile salts in mice. We speculate that a shift from intestinal reabsorption to de novo synthesis as source of bile salts upon bile salt sequestration affects zonation of metabolic processes within the liver acinus.

Timothy Clive Northfield

Timothy Clive Northfield (“Tim”) was born in London in 1935. He was the son of a distinguished neurosurgeon in the London Hospital. He decided not to follow his father into...

Recent understanding of cholesterol gallstone pathogenesis: implication to non-surgical therapeutic strategy

This article reviews the recent understanding of cholesterol gallstone pathogenesis in light of etiology and molecular mechanisms of the cholesterol gallstone formation process, to provide the future direction of a non-surgical therapeutic strategy. In principle, cholesterol gallstone formation, which is associated with an altered bile salt metabolism, is based primarily upon the impairment of cholesterol metabolism and homeostasis. Cholesterol is eliminated physiologically into bile; thus, the excess cholesterol induces bile metastability due to a relative insufficiency of bile salt, to initiate cholesterol crystal nucleation in the gallbladder with an impaired function. Those crystals grow to become macroscopic stones in the gallbladder mucin gel, accumulated under an arachidonate-prostanoid pathway induced-hypersecretion by the gallbladder wall. These events can be modified by bile salt supplementation, which provides a detergent action. Therefore, oral bile salt administration is a cost-effective, non-surgical therapy under certain circumstances. Understanding the pathogenesis of cholesterol gallstones attributes to the therapeutic guideline based upon scientific and clinical evidence.

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.

Insights into the Roles of Gut Microbes in Obesity

Obesity is a major public health issue as it enhances the risk of suffering several chronic diseases of increasing prevalence. Obesity results from an imbalance between energy intake and expenditure, associated with a chronic low-grade inflammation. Gut microbes are considered to contribute to body weight regulation and related disorders by influencing metabolic and immune host functions. The gut microbiota as a whole improves the host's ability to extract and store energy from the diet leading to body weight gain, while specific commensal microbes seem to exert beneficial effects on bile salt, lipoprotein, and cholesterol metabolism. The gut microbiota and some probiotics also regulate immune functions, protecting the host form infections and chronic inflammation. In contrast, dysbiosis and endotoxaemia may be inflammatory factors responsible for developing insulin resistance and body weight gain. In the light of the link between the gut microbiota, metabolism, and immunity, the use of dietary strategies to modulate microbiota composition is likely to be effective in controlling metabolic disorders. Although so far only a few preclinical and clinical trials have demonstrated the effects of specific gut microbes and prebiotics on biological markers of these disorders, the findings indicate that advances in this field could be of value in the struggle against obesity and its associated-metabolic disorders.

Gene expression profiling in livers of mice after acute inhibition of β-oxidation

Inborn errors of mitochondrial β-oxidation cause ectopic fat accumulation, particularly in the liver. Fatty liver is associated with insulin resistance and predisposes to hepatic fibrosis. The factors underlying the pathophysiological consequences of hepatic fat accumulation have remained poorly defined. Gene expression profiling in a model of acute fatty liver disease induced by blocking long-chain fatty acid β-oxidation was performed to study the early effects of steatosis on the transcriptome. Tetradecylglycidic acid (TDGA) was used to irreversibly inhibit carnitine palmitoyltransferase 1, a key enzyme in the control of mitochondrial β-oxidation. TDGA treatment induced massive microvesicular hepatic steatosis within a 12-h time frame in male C57BL6/J mice. Increased hepatic long-chain acyl-CoA content, particularly of C16:0, C16:1 and C18:1, was associated with profound effects on the transcriptome as revealed by unbiased gene expression profiling and quantitative real-time PCR. The results indicate drastic changes in the expression of genes encoding proteins involved in lipid, carbohydrate, and amino acid metabolism. Pathway analysis identified transcription factors and coregulators such as hepatocyte nuclear factor 4 (HNF4), peroxisome proliferator-activated receptor-α (PPAR-α), and PPAR gamma coactivator 1α (PGC-1α ) as key players in these metabolic adaptations. Apoptotic and profibrotic responses were also affected. Surprisingly, a strong reduction in the expression of genes involved in hepatic bile salt metabolism and transport was observed. Therefore, this transcriptome analysis opens new avenues for research.

Multidrug resistance-associated proteins 3, 4, and 5

We summarize in this paper the recently published results on multidrug resistance-associated proteins 3, 4, and 5 (MRPs 3-5). MRP3 can transport organic compounds conjugated to glutathione, sulfate, or glucuronate, such as estradiol-17beta-glucuronide, bilirubin-glucuronides, and etoposide-glucuronide, and also bile salts and methotrexate. Studies in knockout mice have shown that Mrp3 contributes to the transport of morphine-3-glucuronide and acetaminophen-glucuronide from the liver into blood. There is no evidence for a major role of MRP3 in bile salt metabolism, at least in mice. The function of MRP3 in other tissues, notably the gut and the adrenal cortex, remains to be defined. MRP4 and MRP5 have attracted attention by their ability to transport cyclic nucleotides and many nucleotide analogs. The initial reports that MRP4 and 5 can transport cGMP with microM affinity have not been confirmed in recent work and the physiological importance of cyclic nucleotide transport by MRP4 and 5 remains to be determined. Transfected cells containing high concentrations of MRP4 and 5 are moderately resistant to base, nucleoside, and nucleotide analogs. The affinity of both transporters for nucleotide analogs is low (K (m) around 1 mM) and there is no evidence that the transport of these compounds results in resistance in vivo. The physiological function of MRP4 and 5 remains to be found.

Intestinal Failure–Associated Liver Disease: What Do We Know Today?

Intestinal failure-associated liver disease develops in 40% to 60% of infants who require long-term total parenteral nutrition (TPN) for intestinal failure and 15% to 40% of adults on home parenteral nutrition. The clinical spectrum includes hepatic steatosis, cholestasis, cholelithiasis, and hepatic fibrosis. Progression to biliary cirrhosis and the development of portal hypertension and liver failure occurs in a minority but is more common in infants and neonates than in adults. The pathogenesis is multifactorial. In infants it is related to prematurity, low birth weight, duration of PN, short bowel syndrome requiring multiple laparotomies, and recurrent sepsis. Other important mechanisms include lack of enteral feeding, which leads to reduced gut hormone secretion; reduction of bile flow and biliary stasis, which leads to the development of cholestasis; and biliary sludge and gallstones, which exacerbate hepatic dysfunction. In adults, IFALD is less common and related to age, length of time on PN, total caloric intake, and lipid or glucose overload. In preterm infants, a deficiency of taurine or cysteine may play a role, whereas in both adults and children, choline deficiency may exacerbate IFALD. Lipid emulsions, choline deficiency, and manganese toxicity are associated with both hepatic steatosis and cholestasis in adults and children. Management strategies for the prevention of intestinal failure-induced liver disease include early enteral feeding, a multidisciplinary approach to the management of parenteral nutrition, and aseptic catheter techniques to reduce sepsis. The addition of choline, taurine, and cysteine to PN solutions may also play a role. Oral administration of ursodeoxycholic acid may improve bile flow and reduce gallbladder stasis. Survival after either isolated small bowel or combined liver and small bowel transplantation is approximately 50% at 5 years, making this an acceptable therapeutic option in adults and children with irreversible liver and intestinal failure.

PII-22Structure-activity relationships and in silico modeling of activation of the human pregnane X and vitamin D receptors by bile salts and steroid compounds

BACKGROUND/AIMS The pregnane X receptor (PXR; NR1I2) is a nuclear hormone receptor with broad ligand specificity that regulates the metabolism and elimination of bile salts, steroid hormones, and xenobiotics. PXR is closely related to the vitamin D receptor (VDR; NR1I1), although VDR has much narrower ligand specificity. The purpose of the study was to compare structure-activity relationships for activation of the human PXR and VDR by 47 bile salts and 71 steroid hormones and to use in silico modeling to determine the role of ligand-binding pocket size, ligand orientation, and receptor flexibility in determining ligand activity. METHODS Activation of human PXR and human VDR was studied using an in vitro assay in HepG2 liver cells. In silico modeling of human PXR ligand activation was performed with 4D-QSAR analysis (Chem21 Group, Inc.) and comparative molecular field analysis (SYBYL with CoMFA, Tripos, Inc.). RESULTS Human PXR was activated by 25/47 bile salts and 58/71 steroids tested while human VDR was only activated by 5 bile salts. The concentrations of bile salts and steroids that activate human PXR are at least one, and often several, orders of magnitude higher than concentrations circulating in plasma in healthy humans. CONCLUSIONS Human PXR has very broad specificity but low affinity for bile salts and steroid compounds, consistent with a role in detecting toxic levels of these compounds. This broad specificity present significant challenges for in silico prediction of human PXR ligand activation. Clinical Pharmacology & Therapeutics (2005) 79, P42–P42; doi: 10.1016/j.clpt.2005.12.147

MRP2 and 3 in health and disease

MRPs are membrane proteins transporting organic anions at the expense of ATP hydrolysis. MRP2 is known to be a major transporter of organic anions from the liver into bile. We discuss recent results showing allosteric control of human but not rat MRP2. MRP3 has been considered a major player in bile salt metabolism, but our recent results with Mrp3 KO mice do not support this. Instead, we have found a role for MRP3 in the cellular export of drug–glucuronide conjugates. We discuss problems in extrapolating results obtained for murine MRPs.

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

Evolution of the Pregnane X Receptor: Adaptation to Cross-Species Differences in Biliary Bile Salts

The pregnane X receptor (PXR) regulates the metabolism and elimination of bile salts, steroids, and xenobiotics. The sequence of the PXR ligand-binding domain diverges extensively between different animals, suggesting interspecies differences in ligands. Of the endogenous ligands known to activate PXR, biliary bile salts vary the most across vertebrate species, ranging from 27-carbon (C27) bile alcohol sulfates (early fish, amphibians) to C24 bile acids (birds, mammals). Using a luciferase-based reporter assay, human PXR was activated by a wide variety of bile salts. In contrast, zebrafish PXR was activated efficiently only by cyprinol sulfate, the major zebrafish bile salt, but not by recent bile acids. Chicken, mouse, rat, and rabbit PXRs were all activated by species-specific bile acids and by early fish bile alcohol sulfates. In addition, phylogenetic analysis using maximum likelihood demonstrated evidence for nonneutral evolution of the PXR ligand-binding domain. PXR activation by bile salts has expanded from narrow specificity for C27 bile alcohol sulfates (early fish) to a broader specificity for recent bile acids (birds, mammals). PXR specificity for bile salts has thus paralleled the increasing complexity of the bile salt synthetic pathway during vertebrate evolution, an unusual example of ligand-receptor coevolution in the nuclear hormone receptor superfamily.

Evolution of the pregnane X receptor: Role of cross-species differences in biliary bile salts

Background/Aims The pregnane X receptor (PXR) regulates the metabolism and elimination of xenobiotics, bile salts, and steroids. The PXR ligand-binding domain diverges extensively between different animals, suggesting adaptation to changing endogenous ligands. We hypothesized that variation in biliary bile salts has driven PXR gene divergence during evolution. Biliary bile salts vary significantly among vertebrates and include 27-carbon (C27) bile alcohol sulfates (fish), C27 bile acids (amphibians), and C24 bile acids (birds, mammals). Methods Activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs was studied in an vitro luciferase-based assay in HepG2 liver cells. Bile salts were either obtained commercially or isolated from bile of hagfish, carp, eagle spotted ray, or alligator. Results Mammalian, chicken, and zebrafish PXRs were activated by micromolar concentrations of at least one species-appropriate bile salt, with human PXR showing the broadest specificity. Zebrafish PXR was activated only by 'primitive' bile alcohol sulfates such as cyprinol sulfate (from carp) or scymnol sulfate (from eagle spotted ray) but not by evolutionarily more 'modern' bile acids. Interestingly, all PXRs studied were activated by cyprinol sulfate, suggesting that activation by primitive bile salts is an ancestral property of PXRs. Conclusions We propose that cross-species differences in biliary bile salts have influenced PXR function across 400 million years of evolution. Clinical Pharmacology & Therapeutics (2005) 77, P62–P62; doi: 10.1016/j.clpt.2004.12.129

Mechanisms involved in spironolactone-induced choleresis in the rat: Role of multidrug resistance-associated protein 2

The mechanisms involved in spironolactone (SL, 200 μmol/kg body weight, 3 days i.p.)-induced choleresis were explored in vivo by evaluating bile salt export pump (Bsep)-, multidrug resistance-associated protein 2 (Mrp2)-, and anion exchanger 2 (AE2)-mediated secretory processes in rat liver. Hepatic bile salt metabolism was also analyzed. Total bile flow was significantly increased by SL, primarily due to an increase in bile salt-independent bile flow, whereas bile salt secretion was decreased. SL did not produce any choleresis in TR − rats. SL decreased the de novo bile salt synthesis rate in concordance with impaired microsomal cholesterol 7α-hydroxylase activity, thus leading to a decrease in endogenous bile salt pool size. In contrast, the maximum secretory rate of tauroursodeoxycholate as well as expression of Bsep protein detected by Western blotting were not affected. Thus, decreased bile salt availability for canalicular transport rather than transport capability itself likely explains reduced biliary secretion of bile salts. Biliary secretion of glutathione, an endogenous substrate of Mrp2, and HCO 3 −, the AE2 substrate, were increased by SL, as a main factor explaining enhanced bile salt-independent bile flow. Western blot studies revealed increased expression of Mrp2 in response to SL whereas AE2 content remained unchanged. Enhanced activity and expression of Mrp2 was confirmed by analyzing the excretion rate of dinitrophenyl S-glutathione, an exogenous substrate of Mrp2, in isolated hepatocytes and by immunofluorescence microscopy, respectively. We conclude that SL increased bile flow mainly by increasing the biliary secretion of glutathione species and HCO 3 −; increased expression of Mrp2 is also involved.