Interaction of gut microbiota with bile acid metabolism and its influence on disease states.

Is CYP2C70 the key to new mouse models to understand bile acids in humans?

Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5alpha-reductase, 5beta-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and Cyp3a11), transporters [bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter alpha/beta (Ostalpha/Ostbeta)], and nuclear receptors [fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4alpha (HNF-4alpha)], determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5alpha-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and alpha-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span.

Bile acids as signaling molecules in inflammatory bowel disease: Implications for treatment strategies

Bile Acids as Modulators of Gut Microbiota Linking Dietary Habits and Inflammatory Bowel Disease: A Potentially Dangerous Liaison

Interactions between gut bacteria and bile in health and disease

What Can We Learn From Mouse Models About Bile Acid-Mediated Changes After Bariatric Surgery?

BackgroundThe human gut microbiome performs important functions in human health and disease. A classic example for host-gut microbial co-metabolism is host biosynthesis of primary bile acids and their subsequent deconjugation and transformation by the gut microbiome. To understand these system-level host-microbe interactions, a mechanistic, multi-scale computational systems biology approach that integrates the different types of omic data is needed. Here, we use a systematic workflow to computationally model bile acid metabolism in gut microbes and microbial communities.ResultsTherefore, we first performed a comparative genomic analysis of bile acid deconjugation and biotransformation pathways in 693 human gut microbial genomes and expanded 232 curated genome-scale microbial metabolic reconstructions with the corresponding reactions (available at https://vmh.life). We then predicted the bile acid biotransformation potential of each microbe and in combination with other microbes. We found that each microbe could produce maximally six of the 13 secondary bile acids in silico, while microbial pairs could produce up to 12 bile acids, suggesting bile acid biotransformation being a microbial community task. To investigate the metabolic potential of a given microbiome, publicly available metagenomics data from healthy Western individuals, as well as inflammatory bowel disease patients and healthy controls, were mapped onto the genomes of the reconstructed strains. We constructed for each individual a large-scale personalized microbial community model that takes into account strain-level abundances. Using flux balance analysis, we found considerable variation in the potential to deconjugate and transform primary bile acids between the gut microbiomes of healthy individuals. Moreover, the microbiomes of pediatric inflammatory bowel disease patients were significantly depleted in their bile acid production potential compared with that of controls. The contributions of each strain to overall bile acid production potential across individuals were found to be distinct between inflammatory bowel disease patients and controls. Finally, bottlenecks limiting secondary bile acid production potential were identified in each microbiome model.ConclusionsThis large-scale modeling approach provides a novel way of analyzing metagenomics data to accelerate our understanding of the metabolic interactions between the host and gut microbiomes in health and diseases states. Our models and tools are freely available to the scientific community.

The functions of gut microbiota-mediated bile acid metabolism in intestinal immunity

Analysis of metabolome changes in the bile acid pool in feces and plasma of antibiotic-treated rats

*Abbreviations: ACA, "apparent choleretic activity"; CsA, cyclosporine; EHC, enterohepatic circulation; STBA, serum total bile acids. The first successful human liver transplantation was reported by Starzl(1) in 1967, but the procedure did not become the accepted treatment for end-stage liver disease until the introduction of cyclosporine (CsA*) over a decade later. Survival has continued to improve, with up to 90% and 80% patient survival for chronic liver disease at 1 and 5 years after transplantation, respectively. The success of liver transplantation has led to a widening of the indications for treatment and thus to a donor organ shortage. Over the past few years, the use of more marginal grafts has been increasing to try to compensate for this rising demand. Clinicians have been interested in developing indices that reliably predict graft nonfunction and allow for early recognition of reversible causes of graft dysfunction to enable the maximum numbers of organs to be utilized. Bile secretion is generally accepted as an early sign of hepatic recovery following transplantation (2,3), and attempts have been made use it to assess graft function and recognize early complications after transplantation. Normally bile is secreted continuously by the liver, and during fasting, more than 75% enters the gallbladder, where it is concentrated and stored. Following meal-induced gall-bladder contraction, bile is expelled into the duodenum, where it plays a major role in the digestion and absorption of fat, protein, and fat-soluble vitamins. It is also important for the bioavailability of many drugs with enteral absorption. Bile acids are quantitatively the major component of human bile and their hepatic flux markedly influences bile flow and the concentrations of other biliary lipids, particularly phospholipids and cholesterol. Hepatic bile formation occurs at two levels, canalicular and ductular. Canalicular bile flow is mainly dependent on bile acid secretion (bile acid-dependent bile flow) and accounts for 70% to 85% of the total canalicular flow. Bile acid-independent bile flow is probably driven by inorganic ion transport (4) and is both canalicular and ductular. "Apparent choleretic activity" (ACA) is a concept that expresses the relationship between the bile acid secretion rates and the bile flow and is calculated from the equation of a positive slope of a linear regression, indicating that more bile acid secretion produces more bile flow (bile acid-dependent bile flow). Bile acid-independent bile flow is calculated from the estimated intercept of the slope (5,6). More than 95% of the bile acids entering the duodenum are absorbed from the intestinal lumen and returned to the liver via the portal vein to be re-secreted at the canalicular level (enterohepatic circulation[EHC]). The bile acid uptake by the hepatocytes is very efficient, with a high first-pass extraction rate, but bile acids can still be found in systemic blood bound mainly to albumin (5). Serum bile acids had been considered a simple but reliable method for assessing hepatic function in liver disease, and it can help to understand the dynamics of bile acid metabolism (7). "In vivo" analysis of bile secretory function in humans is far from easy, and many measurement techniques have been described, including those of bile flow and bile acid concentrations of hepatic bile per se, bile secretion using duodenal aspirates or markers of water fluxes, and more indirectly, the measurement of bile acid concentrations in serum. The evolution of these investigations has paralleled the developments of surgical techniques and clinical management in liver transplantation and is the subject of this Overview. BILE STUDIES Javitt et al. (8), in 1971, were among the first to recognize the potential of bile measurements to monitor liver function after transplantation. A T tube placed in the common bile duct allowed the collection of bile from one patient during the first 20 days after transplantation. They found a rapid increase in bile salt "synthesis" within the first 24 hr which was attributed to the loss of bile salt through the T tube, such as occurs when the EHC is interrupted by an external biliary drainage, ileal bypass, or administration of cholestyramine. Deoxycholate was still present for several days after transplant, indicating that not all the bile was diverted by the T tube, despite the authors' suggestion that this represented residual bile salt left within the donor biliary tree at the time of organ retrieval. As the number of liver transplants increased, biliary tract obstruction caused by bile cast formation became recognized as a major postoperative complication. Waldram et al. (9,10), studying T tube bile, suggested that cast formation was caused by an abnormal bile composition (bile supersaturated with cholesterol), possibly potentiated by infection and mucosal damage. Starzl et al. (11) considered it to be a surgical problem related to bile duct reconstruction rather than supersaturated bile. New surgical techniques were developed to try to reduce the incidence of biliary complications, including Roux-en-Y anastomosis to the gallbladder and the gallbladder conduit (Calne et al.[12]), but these modifications did not resolve the problem. McMaster et al. (13,14) studied the biochemical and histological features of biliary sludge, which occurred in up to 60% of patients, and found that during the first days after transplantation, bile supersaturated with cholesterol was primarily attributable to low levels of bile acids following interruption of the EHC and depletion of the bile acid pool. However, there was poor correlation between the development of supersaturated bile and biliary obstruction. McMaster et al. showed (15) that the primary mechanism of bile sludge formation was necrosis of donor bile duct mucosa caused by ischemic injury and the presence of bile within the biliary tree during the period of cold preservation. Flushing of the biliary tree during retrieval was introduced, with resolution of the problem (15). Bile Flow and Bile Composition Studies In 1987, Haagsma et al. (16) reported a study of 20 patients during the first 3 weeks after transplantation and attempted to establish a relationship between T tube bile composition and patient outcome, studying the possible differences between surviving and nonsurviving patients. Although there was no significant correlation at 1-year follow-up, they recognized that the composition of bile produced by the transplanted liver was determined not only by the metabolic state of the recipient, but also by that of the donor. They detected low levels of both cholesterol and bile acids in T tube bile which were though to be due to "cholestasis" following early severe acute rejection. These findings contradicted earlier reports of supersaturation of bile with cholesterol (9,13,14) and may be explained by the introduction of intrabiliary flushing during organ retrieval. Nevertheless, attention was directed toward the ability of the transplanted liver to secrete bile; in a preliminary report, Shiffman et al. (17) introduced the idea of measurement of bile flow, bile acid concentration, and bile acid output as potentially useful parameters for assessing graft function and, in particular, acute rejection. One year later, Bowers et al.(18) measured bile flow and composition after transplantation using a Soloway T tube. The Soloway T tube has two additional lumens when compared with the conventional T tube, one for balloon occlusion of the distal duct to allow complete bile collection and the other for reinfusion of bile distal to the balloon to maintain the EHC. Bowers et al. found that bile flow was depressed initially after orthotopic liver transplantation but then increased significantly over the next few days. Furthermore, one patient was noted to have a fall in bile flow, which signaled the development of hepatic artery thrombosis. Using the same Soloway T tube, Farouk et al. (19) studied biliary stone formation and showed persistent supersaturation of bile with cholesterol in the first week after transplantation, when bile acids were depleted. Of their six patients, three developed choledocholithiasis 15 to 42 months later; however, there was no correlation between the degree of cholesterol saturation after surgery and later development of biliary stones. Tisone et al. (20) used daily monitoring of biliary bile acids and lipid composition to try to differentiate patients with primary nonfunction from those with moderate graft dysfunction or normal function. They showed that patients with primary nonfunction had lower bile acid concentrations and higher biliary lecithin concentrations compared with patients with normal graft function in whom there was a rapid increase in the bile secretion of primary bile acids. They concluded that it is possible to recognize primary nonfunction; however, the use of T tube bile and the small number of patients (n=16) studied limit the value of this study. "Apparent Choleretic Activity" Studies Based on earlier studies, Ericzon et al. (2,21), using T tube bile samples taken from 10 patients with liver grafts retrieved from nonheartbeating donors, demonstrated a significant correlation between bile flow and bile acid output (ACA) and also applied, for the first time in liver transplantation, the concept of bile acid-independent bile flow. Total bile flow, concentration of bile acids, phospholipids, and cholesterol were low during the first week after transplant, reflecting graft recovery from the preservation, ischemia, and reperfusion. Analysis of bile acid composition was not reliable because of EHC interruption, and although changes in biliary lipid composition occurred rapidly with graft dysfunction, it did not provide more information than standard liver function tests. They did, however, detect improved bile lipid concentrations following successful antirejection treatment. Shiffman et al. (22) carefully selected patients considered to have "healthy hepatic grafts" with a "normal" pattern of recovery who were thought to provide a "baseline" of "adequate bile flow" to study the recovery of hepatic function following transplantation. However, bile was collected from T tubes with different degrees of interruption of the EHC, and unpredictable variation of interpatient bile composition should be expected. Despite this, they found a linear relationship between bile flow and bile salt output (ACA), which represents the bile salt-dependent flow, and they described two phases of graft recovery over a 10- to 12-day period: an early phase with rapid increase of bile salt concentration and a later phase with more prolonged recovery of bile salt output, suggesting that bile salt-independent flow recovered more slowly than bile salt-dependent flow. They suggested that variations in the ACA may be related to alterations in the hydrophobicity and structure of the bile salt pool and that the presence of increased bile flow was associated with hypoxia following donor organ retrieval and vagal deinnervation of the transplanted graft. McCashland et al. (6), in 1994, characterized bile acid metabolism and biliary secretion in the first 2 weeks after liver transplantation using T tube bile and compared the results with those from patients who had undergone cholecystectomy. They measured the ACA in patients with interruption of the EHC for whom three or more bile samples were available and showed a linear correlation between bile flow and bile acid output with a markedly elevated ACA value and a low concentration of bile acids after transplantation. They suggested that this anomalous choleresis may indicate the presence of an increased proportion of other osmotically active solutes in bile probably due to impaired biliary epithelial cell function, and they drew attention to causes of disturbances in biliary secretion, such as the removal of the circulating bile acid pool, graft dysfunction and denervation of the graft, and the effect on bile flow. Other Procedures for the Assessment of Bile Flow Interpretation and comparison of the results of the above studies of bile secretion are difficult, particularly if the EHC has been interrupted by use of a T tube for varying periods and at different times after transplant. Measurement of biliary bile acid secretion without interruption of EHC was performed by Theilman et al. (23), in 1991, using duodenal perfusion with a nonabsorbable marker in patients at least 6 weeks after transplant. Biliary secretion was assessed at hourly intervals, and the mean biliary output of bile acids was higher in comparison with their controls. In the bile of the transplant recipients, the majority of bile acids were cholate and chenodeoxycholate with only low percentages of deoxycholate and lithocholate, presumably because of changes in intestinal bacterial flora secondary to perioperative antibiotics. Friman et al. (24) indirectly measured canalicular bile flow using 3H-labeled polyethylene glycol 900, a marker of water fluxes, in patients 3-6 weeks after transplantation. A linear relationship was found between bile flow and the clearance of the polyethylene glycol 900. A reduced canalicular flow was noted (including a reduced bile acid-independent bile flow), despite the patients receiving ursodeoxycholic acid, which has a well-defined choleretic effect. They suggested that these findings were related to immunological, preservation, or reperfusion injury. Bile Studies and Immunosuppressive Drugs Immunosuppressive drugs represent a cornerstone of liver transplantation, but their secondary effects have a marked influence on graft function. CsA was noted to have an apparent cholestatic effect and inhibition of bile acid secretion in experimental studies with rats (25) and in vitro (26), and tacrolimus has been related with hepatotoxicity when administered at higher doses in animals(27). In humans, McCashland et al.(6) found decreased bile acid secretion and bile flow in patients receiving either CsA or tacrolimus and described a reduction in chenodeoxycholic acid biosynthesis related only to patients receiving CsA. However, Ericzon et al. (28,29) concluded that patients with tacrolimus showed a more rapid recovery of biliary secretion, and this observation was supported by Sauer et al. (3), using a duodenal perfusion method, 3-6 weeks after transplantation. They showed that in patients receiving tacrolimus, there was complete recovery of biliary secretion, despite the presence of elevated serum alkaline phosphatase and γ-glutamyl transferase levels. The effect of CsA on the kinetics of primary bile acids at 6-20 months after transplantation using isotope dilution to measure pool size, synthesis, and turnover rates of cholic and chenodeoxycholic acids (30) showed no change. Recently, Ericzon et al. (31), using T tube bile samples, showed that bile secretion and composition were not different in patients receiving either CsA or tacrolimus after transplantation. The ACA assessment showed that bile acid-dependent bile flow was lower in the tacrolimus group than in the CsA group. This was interpreted as evidence of a more rapid recovery of bile secretion capacity in the first group and may due to a lower incidence of acute rejection or a greater hepatotrophic effect of tacrolimus as compared with CsA. SERUM BILE ACIDS In 1987, Mora et al. (32), along with Codoceo et al.(33) and Herrera et al. (34) in 1989, investigated the potential value of monitoring serum total bile acids(STBA) as a guide to early graft function. They found that STBA levels increased during the anhepatic phase of liver transplantation and were rapidly corrected after revascularization. Kohlhaw et al.(35) noted a prompt drop of STBA levels after reperfusion in 24 of 30 posttransplant patients, which correlated with good early graft function. The other six patients either showed no fall or a rise in STBA, and all subsequently lost their graft. Although STBA levels failed to correlate with other parameters of liver function, such as serum transaminases, bilirubin, and coagulation factors, they concluded that STBA monitoring could be helpful in assessing early graft function. Muraca et al.(36) extended this work and suggested that STBA measurement could be used as a sensitive and specific indicator of hepatic graft dysfunction, particularly during graft rejection when levels increased 3.6-fold and as a marker of successful antirejection therapy when levels fell rapidly. Azer et al. (37) also suggested that individual serum bile acids are highly sensitive and specific in assessing graft function, and changes in the added serum concentration of glycocholic and glycodeoxycholic acids and the taurocholic/taurodeoxycholic ratio usually antedated more traditional biochemical indicators of graft rejection. These studies demonstrated the value of STBA levels in liver transplantation; however, their routine use has not been adopted in clinical practice probably because of the lack of reliable and automated measuring techniques. COMBINED SERUM AND BILIARY BILE ACID STUDIES Baumgartner et al. (38) and Baiocchi et al.(39) measured STBA and biliary bile acids in liver transplant recipients and found progressive falls in STBA levels and concomitant increases in biliary bile acids after transplantation. The effect of marked depletion of the bile acid pool and the use of T tube diversion was only reversed by day 15 after transplantation, when normal concentrations of bile acids were achieved. They concluded that determination of STBA and biliary bile acid levels could be used simultaneously to detect graft dysfunction 1-3 days earlier than would be indicated by other "standard" liver function tests. DISCUSSION The evolution of liver transplantation has been closely related to the study of bile production by the graft (Table 1). The early problems of bile cast formation and biliary obstruction led to the introduction of bile duct flushing at the time of organ retrieval. Improved patient survival following the introduction of CsA made the study of bile feasible. The idea that bile analysis can predict graft function was attractive, and studies of T tube bile composition started to appear. The analysis of biliary bile composition was not sufficient to understand the complex pattern of bile acid metabolism, and measurements of bile flow and bile acid concentrations offered the opportunity of studying bile secretion. It is now possible to apply physiological concepts of canalicular secretion as bile acid-dependent bile flow, bile acid-independent bile flow, and apparent choleretic activity. However, the limitations of using T tube bile samples are evident, and although the results of the duodenal perfusion method are more reliable, the test is inappropriate for use in the early posttransplant period. The ability to assess early graft outcome by analysis of bile composition and more recently the effect of immunosuppressive drugs, such as CsA and tacrolimus, on bile flow may have practical implications in the future. The value of STBA levels in the early diagnosis of acute cellular rejection, particularly when combined with analysis of biliary bile acids, is evident, but they have not been introduced into clinical practice because of a lack of automated measuring techniques.Table 1: Bile analysis in human liver transplantationaIt has been recognized that the characteristics of bile production by the liver graft are determined by many donor and recipient factors(16). There are no studies of bile acid secretion in the donor liver prior to transplantation, and all posttransplant studies are based on the consideration that donor livers have a similar functional quality at the outset. Furthermore, the placement of a T tube after biliary reconstruction, although providing a direct source of hepatic bile, interrupts the EHC, causing chronic bile pool depletion. The study of hepatic bile flow and bile acid composition in donors livers prior to retrieval is a potential way forward to assess the graft's future function or dysfunction and may help explain some of the contradictory findings that have been published.

Ursodeoxycholic acid (commercially available as ursodiol) is a naturally occurring bile acid that is used to treat a variety of hepatic and gastrointestinal diseases. Ursodiol can modulate bile acid pools, which have the potential to alter the gut microbiota community structure. In turn, the gut microbial community can modulate bile acid pools, thus highlighting the interconnectedness of the gut microbiota-bile acid-host axis. Despite these interactions, it remains unclear if and how exogenously administered ursodiol shapes the gut microbial community structure and bile acid pool in conventional mice. This study aims to characterize how ursodiol alters the gastrointestinal ecosystem in conventional mice. C57BL/6J wildtype mice were given one of three doses of ursodiol (50, 150, or 450 mg/kg/day) by oral gavage for 21 days. Alterations in the gut microbiota and bile acids were examined including stool, ileal, and cecal content. Bile acids were also measured in serum. Significant weight loss was seen in mice treated with the low and high dose of ursodiol. Alterations in the microbial community structure and bile acid pool were seen in ileal and cecal content compared to pretreatment, and longitudinally in feces following the 21-day ursodiol treatment. In both ileal and cecal content, members of the Lachnospiraceae Family significantly contributed to the changes observed. This study is the first to provide a comprehensive view of how exogenously administered ursodiol shapes the healthy gastrointestinal ecosystem in conventional mice. Further studies to investigate how these changes in turn modify the host physiologic response are important.

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

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.

Impairment of peroxisomal BA synthesis is a characteristic feature of Scp2 (-/-) mice. This study investigated how this may impact BA metabolism. In separate groups of male mice (n=6–9) lipid analysis of hepatic and gallbladder bile was performed. Livers and sequential intestinal fractions were harvested to isolate total RNA, homogenate and plasma membranes. Steady-state gene expression of basolateral (Slc10a1, Slc21a1) and canalicular (Abcb4, Abcb11, Abcc2) transporters was studied and correlated with serum and biliary lipids. Size and composition of the BA pool was measured by GCMS and complemented with measurement of fecal BAs. Finally, gene expression of intestinal BA transporters (Fabp6, Slc10a2) were measured. Serum BAs were higher in -/- mice and composed of nor-BAs and bile alcohols which were absent in controls. Compared with controls, the BA pool of -/- mice was reduced 3-fold and enriched more than 30-fold (p<0.01) with nor-cholate whereas chenodeoxycholic acid was reduced 7-fold (p<0.01). Despite unchanged Abcc2 gene expression, bile flow was 1.5-fold higher (p<0.05) in -/- mice. BA output was reduced 68% (p<0.01) which correlated with a significant suppression of Abcb11 gene expression. Biliary excretion of cholesterol and phospholipids were unchanged in -/- mice and no significant change of Abcb4 gene expression could be detected. In contrast to controls, Slc10a1 gene expression was induced by 40% (p<0.05) whereas no significant changed was observed for Slc21a1. Fecal BA excretion was reduced to 50% (p<0.001) in -/- mice and bile alcohols were present -/- mice but not in controls. In all mice intestinal Slc10a2 and Fabp6 expression increased from proximal to distal. However, Slc10a2 and Fabp6 expression was significantly (p<0.01) lower in -/-mice. Our findings suggest that impaired peroxisomal BA synthesis is associated with adaptive changes of BA metabolism. More hydrophilic BAs of a smaller BA pool size are poor substrates for the BA export pump and explain the lower Abcb11 expression with a reduced BA output. This may further result in increased serum BA concentrations although we were unable to document inhibition of Slc10a1 or Slc21a1 expression. Decreased Slc10a2 and Fabp6 expression implies that conservation of the more hydrophilic BA pool was achieved by inducing BA absorption in more proximal parts of the intestine.

The orphan nuclear hormone receptor SHP (gene designation NROB2) is an important component of a negative regulatory cascade by which high levels of bile acids repress bile acid biosynthesis. Short term studies in SHP null animals confirm this function and also reveal the existence of additional pathways for bile acid negative feedback regulation. We have used long term dietary treatments to test the role of SHP in response to chronic elevation of bile acids, cholesterol, or both. In contrast to the increased sensitivity predicted from the loss of negative feedback regulation, the SHP null mice were relatively resistant to the hepatotoxicity associated with a diet containing 0.5% cholic acid and the much more severe effects of a diet containing both 0.5% cholic acid and 2% cholesterol. This was associated with decreased hepatic accumulation of cholesterol and triglycerides in the SHP null mice. There were also alterations in the expression of a number of genes involved in cholesterol and bile acid homeostasis, notably cholesterol 12alpha-hydroxylase (CYP8B1), which was strongly reexpressed in the SHP null mice, but not the wild type mice fed either bile acid containing diet. This contrasts with the strong repression of CYP8B1 observed with short term bile acid feeding, as well as the effects of long term feeding on other bile acid biosynthetic enzymes such as cholesterol 7alpha-hydroxylase (CYP7A1). CYP8B1 expression could contribute to the decreased toxicity of the chronic bile acid treatment by increasing the hydrophilicity of the bile acid pool. These results identify an unexpected role for SHP in hepatotoxicity and suggest new approaches to modulating effects of chronically elevated bile acids in cholestasis.