Colesevelam attenuates cholestatic liver and bile duct injury in Mdr2-/- mice by modulating composition, signalling and excretion of faecal bile acids.

BACKGROUND AND AIMS: Interruption of the enterohepatic circulation of bile acids (BAs) may protect against BA-mediated cholestatic liver and bile duct injury. BA sequestrants are established to treat cholestatic pruritus, but their impact on the underlying cholestasis is still unclear. We aimed to explore the therapeutic effects and mechanisms of the BA sequestrant colesevelam in a mouse model of sclerosing cholangitis. METHODS: Mdr2-/- mice received colesevelam for 8 weeks. Gene expression profiles of BA homeostasis, inflammation and fibrosis were explored in liver, intestine and colon. Hepatic and faecal BA profiles and gut microbiome were analysed. Glucagon-like peptide 1 (GLP-1) levels in portal blood were measured by ELISA. Furthermore, Mdr2-/- mice as well as wild-type 3,5-diethoxy-carbonyl-1,4-dihydrocollidine-fed mice were treated with GLP-1-receptor agonist exendin-4 for 2 weeks prior to analysis. RESULTS: Colesevelam reduced serum liver enzymes, BAs and expression of proinflammatory and profibrogenic markers. Faecal BA profiling revealed increased levels of secondary BAs after resin treatment, while hepatic and biliary BA composition showed a shift towards more hydrophilic BAs. Colonic GLP-1 secretion, portal venous GLP-1 levels and intestinal messenger RNA expression of gut hormone Proglucagon were increased, while ileal Fgf15 expression was abolished by colesevelam. Exendin-4 treatment increased bile duct mass without promoting a reactive cholangiocyte phenotype in mouse models of sclerosing cholangitis. Microbiota analysis showed an increase of the phylum δ-Proteobacteria after colesevelam treatment and a shift within the phyla Firmicutes from Clostridiales to Lactobacillus. CONCLUSION: Colesevelam increases faecal BA excretion and enhances BA conversion towards secondary BAs, thereby stimulating secretion of GLP-1 from enteroendocrine L-cells and attenuates liver and bile duct injury in Mdr2-/- mice.

New therapeutic concepts in bile acid transport and signaling for management of cholestasis.

The identification of the key regulators of bile acid (BA) synthesis and transport within the enterohepatic circulation has revealed potential targets for pharmacological therapies of cholestatic liver diseases. Novel drug targets include the bile BA receptors, farnesoid X receptor and TGR5, the BA-induced gut hormones, fibroblast growth factor 19 and glucagon-like peptide 1, and the BA transport systems, apical sodium-dependent bile acid transporter and Na+ -taurocholate cotransporting polypeptide, within the enterohepatic circulation. Moreover, BA derivatives undergoing cholehepatic shunting may allow improved targeting to the bile ducts. This review focuses on the pathophysiological basis, mechanisms of action, and clinical development of novel pharmacological strategies targeting BA transport and signaling in cholestatic liver diseases. (Hepatology 2017;65:1393-1404).

Metabolic preconditioning protects BSEP/ABCB11-/- mice against cholestatic liver injury.

BACKGROUND & AIMS: Cholestasis is characterized by intrahepatic accumulation of potentially cytotoxic bile acids (BAs) subsequently leading to liver injury with disruption of hepatocellular integrity, inflammation, fibrosis and ultimately liver cirrhosis. Bile salt export pump (BSEP/ABCB11) is the main canalicular BA transporter and therefore the rate limiting step for hepatobiliary BA excretion. In this study we aimed to investigate the role of BSEP/ABCB11 in the development of acquired cholestatic liver and bile duct injury. METHODS: Wild-type (WT) and BSEP knockout (BSEP-/-) mice were subjected to common bile duct ligation (CBDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding as models for cholestasis with biliary obstruction and bile duct injury. mRNA expression profile, serum biochemistry, liver histology, immunohistochemistry, hepatic hydroxyproline levels and BA composition as well as biliary pressure were assessed. RESULTS: BSEP-/- mice were protected against acquired cholestatic liver injury induced by 7days of CBDL or 4weeks of DDC feeding, as reflected by unchanged serum levels of liver transaminases, alkaline phosphatase and BAs. Notably, BSEP-/- mice were also protected from cholestasis-induced hepatic inflammation and biliary fibrosis. In line with induced BA detoxification/hydroxylation pathways in BSEP-/- mice, polyhydroxylated BAs were increased 4-fold after CBDL and 6-fold after DDC feeding in comparison with cholestatic WT mice. Finally, following CBDL, biliary pressure in WT mice increased up to 47mmH2O but remained below 11mmH2O in BSEP-/- mice. CONCLUSION: Metabolic preconditioning with subsequent changes in BA metabolism favors detoxification of potentially toxic BAs and thereby protects BSEP-/- mice from cholestatic liver and bile duct injury. LAY SUMMARY: Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP-/- mice from acquired cholestatic liver disease.

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.

BACKGROUND AND AIMS: Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. METHODS: Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. RESULTS: A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-ß-muricholic acid and taurocholic acid. CONCLUSIONS: Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Therapy of Primary Sclerosing Cholangitis--Today and Tomorrow.

Primary sclerosing cholangitis (PSC) represents a fibro-obliterative bile duct disease with unpredictable individual clinical course that may progress to liver cirrhosis and malignancy. Due to our incomplete understanding of the etiology and pathogenesis of this disease, the therapeutic options are still rather limited. Bile acids play a key role in mediating cholangiocellular and hepatocellular injury in cholangiopathies such as PSC. Therefore, strategies targeting bile composition and homeostasis are valid approaches in PSC. Ursodeoxycholic acid (UDCA) is the paradigm therapeutic bile acid and its role in medical therapy of PSC is still under debate. Promising novel bile acid-based therapeutic options include 24-norursodeoxycholic acid (norUDCA), a side chain-shortened C23 homologue of UDCA, and bile acid receptor/farnesoid X receptor agonists (e.g. obeticholic acid). Other nuclear receptors such as fatty acid-activated peroxisome proliferator-activated receptors, vitamin D receptor and vitamin A receptors (retinoic acid receptor, retinoid X receptor) are also of potential interest and can be targeted by already available drugs. Furthermore, drugs targeting the gut-liver axis (e.g. intregrin blockers such as vedolizumab, antibiotics) appear promising, based on the close link of PSC to inflammatory bowel disease and the emerging relevance of the gut microbiome for the development of PSC. Finally, fibrosis represents a valid therapeutic target for anti-fibrotic drugs (e.g. simtuzumab) in PSC as paradigm fibro-obliterative disease. This review summarizes the current status and recent progress in the development of targeted therapeutic approaches based on increasing knowledge about the pathogenesis of this disease.

Potential of nor-Ursodeoxycholic Acid in Cholestatic and Metabolic Disorders.

24-nor-ursodeoxycholic acid (norUDCA) is a side-chain shortened derivate of ursodeoxycholic acid (UDCA). Since norUDCA is only ineffectively conjugated with glycine or taurine, it has specific physicochemical and therapeutic properties distinct from UDCA. Nonamidated norUDCA undergoes cholehepatic shunting enabling 'ductular targeting' and inducing a bicarbonate-rich hypercholeresis, with cholangioprotective effects. At the same time it has direct anti-inflammatory, antilipotoxic, anti fibrotic, and antiproliferative properties targeting various liver cell populations. norUDCA appears to be one of the most promising novel treatment approaches targeting the liver and the bile duct system at multifactorial and multicellular levels. This review article is a summary of a lecture given at the XXIII International Bile Acid Meeting (Falk Symposium 194) on 'Bile Acids as Signal Integrators and Metabolic Modulators' held in Freiburg, October 8-9, 2014, and summarizes the recent progress with norUDCA as a novel therapeutic approach in cholestatic and metabolic (liver) disorders.

Biliary physiology and disease: reflections of a physician-scientist.

A review is presented of Gustav Paumgartner's five decades of research and practice in hepatology focusing on biliary physiology and disease. It begins with studies of the excretory function of the liver including hepatic uptake of indocyanine green, bilirubin, and bile acids. The implications of these studies for diagnosis and understanding of liver diseases are pointed out. From there, the path of scientific research leads to investigations of hepatobiliary bile acid transport and the major mechanisms of bile formation. The therapeutic effects of the hydrophilic bile acid, ursodeoxycholic acid, have greatly stimulated these studies. Although ursodeoxycholic acid therapy for dissolution of cholesterol gallstones and some other nonsurgical treatments of gallstones were largely superseded by surgical techniques, ursodeoxycholic acid is currently considered the mainstay of therapy of some chronic cholestatic liver diseases, such as primary biliary cirrhosis. The major mechanisms of action of ursodeoxycholic acid therapy in cholestatic liver diseases are discussed. An attempt is made to illustrate how scientific research can lead to advances in medical practice that help patients.

Medical treatment of cholestatic liver disease.

In most cholestatic liver diseases the cause of the disease is not known and therapy can only be directed toward suppression of the pathogenetic processes and amelioration of the consequences of cholestasis. The recognition of adaptive-compensatory responses to cholestasis has become of major importance. They tend to minimize retention of bile acids and other potentially toxic solutes in the hepatocyte by limiting hepatocellular uptake, reducing bile acid synthesis, stimulating detoxification, and up-regulating alternative pathways for excretion. Some of the drugs used for the treatment of cholestatic liver diseases in an empiric way turned out to be modulators of nuclear receptors, which regulate these adaptive-compensatory responses. New drugs are being designed and tested along these lines and may be regarded as treatment opportunities of the future.

Medical treatment of cholestatic liver diseases: From pathobiology to pharmacological targets.

Bile secretion is dependent on the coordinated functions of a number of hepatobiliary transport systems. Cholestasis may be caused by an impairment of bile secretion, an obstruction of bile flow or a combination of the two. The common consequence of all forms of cholestasis is retention of bile acids and other potentially toxic compounds in the hepatocytes leading to apoptosis or necrosis of hepatocytes and eventually to chronic cholestatic liver disease. In certain cholestatic disorders there is also leakage of bile acids into the peribiliary space causing portal inflammation and fibrosis. The following pharmacological targets for treatment of intrahepatic cholestasis can be identified: stimulation of orthograde biliary secretion and retrograde secretion of bile acids and other toxic cholephils into the systemic circulation for excretion via the kidneys to reduce their retention in the hepatocytes; stimulation of the metabolism of hydrophobic bile acids and other toxic compounds to more hydrophilic, less toxic metabolites; protection of injured cholangiocytes against toxic effects of bile; inhibition of apoptosis caused by elevated levels of cytotoxic bile acids; inhibition of fibrosis caused by leakage of bile acids into the peribiliary space. The clinical results of ursodeoxcholic acid therapy of primary biliary cirrhosis may be regarded as the first success of this strategy.

Extracorporeal shock wave lithotripsy of gallstones: 20th anniversary of the first treatment.

Twenty years ago, in January 1985, extracorporeal shock wave lithotripsy (ESWL) was first applied successfully in a patient with gallbladder stones. In the following years, the conditions which influence the success rate of ESWL have been extensively investigated. It was shown that the characteristics of the stones, gallbladder emptying and the degree of stone fragmentation are the most important factors which determine the clearance of all fragments from the gallbladder after ESWL. Severe side effects, such as biliary pancreatitis and liver haematoma, were found to be rare and no deaths related to the procedure have been reported. One or more episodes of biliary pain were observed in about one third of patients within the first 3-4 months after ESWL. Follow-up studies after successful treatment, however, have shown that stone recurrence is considerable, limiting the use of ESWL as a non-invasive therapeutic option. Stone recurrence varies between different subgroups of patients indicating that gallbladder motor function and other less well defined factors may be of importance. The recurrence of stones after ESWL is one of the reasons why laparoscopic cholecystectomy has become the standard treatment of symptomatic gallbladder stones today. ESWL has kept its role only in the treatment of bile duct stones resistant to endoscopic extraction. Unless stone recurrence can be decreased by better patient selection and/or other measures to prevent gallstone recurrence, ESWL of gallbladder stones has little chance of surviving.

Mechanisms of action and therapeutic efficacy of ursodeoxycholic acid in cholestatic liver disease.

Ursodeoxycholic acid (UDCA) is widely used for the treatment of cholestatic liver diseases. Multiple mechanisms of action of UDCA have been described aiming at one or more of the pathogenetic processes of cholestatic liver diseases: (1) protection of injured cholangiocytes against toxic effects of bile acids, (2) stimulation of impaired biliary secretion, (3) stimulation of detoxification of hydrophobic bile acids, and (4) inhibition of apoptosis of hepatocytes. Through one or more of these mechanisms, UDCA slows the progression of primary biliary cirrhosis and improves a number of other cholestatic disorders.

Expression of the hepatocyte canalicular multidrug resistance protein (MRP2) in primary biliary cirrhosis.

The canalicular multidrug resistance protein 2 (MRP2; gene symbol: ABCC2) mediates ATP-dependent biliary excretion of organic anions such as bilirubin diglucuronide, glutathione conjugates and sulfated and glucuronidated bile salts. In chronic cholestatic liver diseases, the biliary excretion of cholephilic organic anions is impaired. While the underlying transport defects have been studied in rat models of cholestasis, little is known about the molecular basis of impaired organic anion excretion in human cholestatic liver disease. Our aim, therefore, was to analyze expression of MRP2 in patients with primary biliary cirrhosis (PBC), a chronic cholestatic liver disease characterized by progressive destruction of small intrahepatic bile ducts. Four patients with PBC stages III (n=1) and IV (n=3) were compared with three non-cholestatic patients with alcoholic liver disease, idiopathic liver cirrhosis and cirrhosis from chronic hepatitis C. Immunohistochemistry was performed on paraffin-embedded tissue slides using a monoclonal antibody to MRP2. MRP2 was detected at the canalicular hepatocyte membrane of all patients. In two PBC patients (stages III and IV, respectively), the degree of immunostaining was comparable with controls, whereas in two other PBC patients with stage IV disease immunostaining was decreased. We conclude that MRP2 expression decreases with progressive cholestasis in PBC.

The emerging role of transport systems in liver function tests.

Liver function tests are of critical importance for the management of patients with severe or terminal liver disease. They are also used as prognostic tools for planning liver resections. In recent years many transport systems have been identified that also transport substances employed in liver function tests. Such substances include endogenous bilirubin or exogenously administered indocyanine green, agents for magnetic resonance imaging, agents for single photon emission computed tomography or agents for breath tests. The increasing functional and molecular information on the respective transport systems should improve the management and as a result the outcome of patients scheduled for liver surgery or transplantation. To achieve the latter goal, clinical studies that assess individual patients' liver function over the course of their disease with liver function tests are needed to firmly establish and validate recently introduced and novel liver function markers.

Pharmacotherapy of cholestatic liver diseases.

New insights into the molecular mechanisms of bile formation and cholestasis have provided new concepts for pharmacotherapy of cholestatic liver diseases. The major aim in all forms of cholestasis is the reduction of hepatocellular retention of bile acids and other potentially toxic constituents of bile. Reduction of hepatocellular retention may be achieved by drugs that stimulate hepatocellular secretion via the canalicular route into the bile or via the alternative route across the basolateral membrane into the blood, and by drugs that stimulate the hepatocellular metabolism of hydrophobic bile acids to hydrophilic, less toxic metabolites. In cholestatic liver diseases that start with an injury of the biliary epithelium (e.g., primary biliary cirrhosis; PBC), protection of the cholangiocytes against the toxic effects of hydrophobic bile acids is most important. When hepatocellular retention of bile acids has occurred, the inhibition of bile acid-induced apoptosis becomes another target of therapy. Ursodeoxycholic acid protects the biliary epithelium by reducing the toxicity of bile, stimulates hepatobiliary secretion by upregulating transporters and inhibits apoptosis. It is the mainstay of therapy in PBC but of benefit also in a number of other cholestatic liver diseases. New drugs such as 6-ethyl-chenodeoxycholic acid and 24-nor-ursodeoxycholic acid are being evaluated for the treatment of cholestatic liver diseases.

Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited.

Ursodeoxycholic acid (UCDA) is increasingly used for the treatment of cholestatic liver diseases. Experimental evidence suggests three major mechanisms of action: (1) protection of cholangiocytes against cytotoxicity of hydrophobic bile acids, resulting from modulation of the composition of mixed phospholipid-rich micelles, reduction of bile acid cytotoxicity of bile and, possibly, decrease of the concentration of hydrophobic bile acids in the cholangiocytes; (2) stimulation of hepatobiliary secretion, putatively via Ca(2+)- and protein kinase C-alpha-dependent mechanisms and/or activation of p38(MAPK) and extracellular signal-regulated kinases (Erk) resulting in insertion of transporter molecules (e.g., bile salt export pump, BSEP, and conjugate export pump, MRP2) into the canalicular membrane of the hepatocyte and, possibly, activation of inserted carriers; (3) protection of hepatocytes against bile acid-induced apoptosis, involving inhibition of mitochondrial membrane permeability transition (MMPT), and possibly, stimulation of a survival pathway. In primary biliary cirrhosis, UDCA (13-15 mg/kg/d) improves serum liver chemistries, may delay disease progression to severe fibrosis or cirrhosis, and may prolong transplant-free survival. In primary sclerosing cholangitis, UDCA (13-20 mg/kg/d) improves serum liver chemistries and surrogate markers of prognosis, but effects on disease progression must be further evaluated. Anticholestatic effects of UDCA have also been reported in intrahepatic cholestasis of pregnancy, liver disease of cystic fibrosis, progressive familial intrahepatic cholestasis, and chronic graft-versus-host disease. Future efforts will focus on definition of additional clinical uses of UDCA, on optimized dosage regimens, as well as on further elucidation of mechanisms of action of UDCA at the molecular level.

Tauroursodeoxycholic acid mobilizes alpha-PKC after uptake in human HepG2 hepatoma cells.

BACKGROUND: Tauroursodeoxycholic acid (TUDCA) may exert anticholestatic effects via Ca(++)- and alpha-protein kinase C (alpha-PKC)-dependent apical vesicular insertion of canalicular transporters in cholestatic hepatocytes (Hepatology 2001; 33: 1206-16). Tauroursodeoxycholic acid is mainly taken up into liver cells by Na(+)-taurocholate cotransporting polypeptide (Ntcp). Tauroursodeoxycholic acid selectively translocates alpha-PKC, a key mediator of regulated exocytosis, to hepatocellular membranes. It is unclear whether TUDCA exerts its effects on alpha-PKC after carrier-mediated uptake into liver cells or by interaction with extracellular/membraneous structures. MATERIALS AND METHODS: Human hepatoblastoma HepG2 cells lacking Ntcp were stably transfected with pcDNA3.1/Ntcp or sham-transfected with pcDNA3.1 [+]. Distribution of alpha-PKC was studied using a Western blotting technique. Uptake of [(3)H]taurocholic acid (TCA) was determined radiochemically. RESULTS: [(3)H]taurocholic acid uptake was approximately 180-fold higher in Ntcp-transfected than in sham-transfected cells. Phorbol 12-myristate 13-acetate (1 micromol L(-1); positive control) increased membrane binding of alpha-PKC by 34% in Ntcp-transfected and by 37% in sham-transfected cells. Tauroursodeoxycholic acid (10 micromol L(-1)) increased membrane-associated alpha-PKC by 19% in Ntcp-transfected, but not in sham-transfected cells (-13%). Taurocholic acid (10 micromol L(-1)) did not affect the distribution of alpha-PKC. CONCLUSION: Carrier-mediated uptake is a prerequisite for TUDCA-induced translocation of alpha-PKC to hepatocellular membranes.

Does ursodeoxycholic acid change the proliferation of the colorectal mucosa?. A randomized, placebo-controlled study.

BACKGROUND: In animal models ursodeoxycholic acid (UDCA) showed a chemoprotective effect against colon cancer. To explain this, a reduced proliferation of the colorectal mucosal proliferation was suggested. We, therefore, examined the influence of UDCA on the proliferation of normal colorectal mucosa in humans. METHODS: Following endoscopic polypectomy, 20 patients with colorectal adenomas were randomized to receive either UDCA (750 mg/day, n = 10, group A) or placebo (n = 10, group B) for 6 months in a double-blinded way. Colorectal biopsies were sampled before and at the end of the medication by total colonoscopy. Colorectal mucosal proliferation was measured by FACScan analysis of propidium iodine labeling. Serum was sampled, and serum bile acids were analyzed by gas chromatography. RESULTS: The proliferation rates at the end of the study were similar in both groups (median 15.4%; range 12.0-20.9 in group A; median 16.0%, 14.0-20.2 in group B, p = 0.41). Serum lithocholic acid levels at the end of the study were significantly higher in group A (1.3 micromol/l, 0.9-1.8) than in group B (0.7 micromol/l, 0-1.7, p < 0.02), whereas serum deoxycholic acid levels were similar in both groups. CONCLUSIONS: In this study, UDCA treatment for 6 months does not seem to induce changes in the proliferative behavior of the colorectal mucosa in patients with adenomas. It seems likely that a putative chemopreventive effect of UDCA in humans is not exerted by a reduction of the colorectal proliferation.

Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets.

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.