Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep −/− mice

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep ^?/? mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep ^?/? mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep ^?/? mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep ^?/? mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep ^?/? mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.     

Effects of feeding bile acids and a bile acid sequestrant on hepatic bile acid composition in mice

An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7α/7β-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7α/β-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Δ⁴-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce α/β-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7β-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Δ⁴-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7α-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Short-term obeticholic acid treatment does not impact cholangiopathy in Cyp2c70-deficient mice with a human-like bile acid composition

Cyp2c70^?/? mice with a human-like bile acid (BA) composition, lacking hydrophilic muricholic acids (MCAs), have been reported to display cholangiopathy and biliary fibrosis with female preponderance that can be reversed by ursodeoxycholic acid (UDCA). Obeticholic acid (OCA), a steroidal BA-like FXR agonist, has been shown to improve liver function in patients with primary biliary cholangitis and is approved as second-line treatment for patients with an inadequate response or intolerance to UDCA. Here, we investigated the impact of OCA on BA hydrophobicity and cholangiopathy in Cyp2c70^?/? mice. Male and female wild-type (WT) and Cyp2c70^?/? mice were fed a chow diet with or without 10 mg/kg/day OCA for 4 weeks. OCA accounted for 1–5% of biliary BAs, with larger enrichments in Cyp2c70^?/? than in WT mice. In WT mice, OCA induced a more hydrophilic, MCA-rich BA pool. In Cyp2c70^?/? mice, however, BA pool became more hydrophobic with a larger proportion of chenodeoxycholic acid, attributable to a reduction of BA 12α-hydroxylation. OCA treatment reduced fecal BA excretion, indicating repression of hepatic BA synthesis in both WT and Cyp2c70^?/? mice. OCA did, however, not impact on markers of liver (dys)function in plasma nor did it ameliorate cholangiopathy and fibrosis in male or female Cyp2c70^?/? mice. OCA treatment also did not affect the expression of genes involved in fibrosis, inflammation and cellular senescence. In conclusion, 4 weeks of OCA treatment oppositely modulates the hydrophobicity of the BA pool in WT and Cyp2c70^?/? mice, but does not improve or worsen the characteristic sex-dependent liver pathology in Cyp2c70^?/? mice.     

Fxr(-/-) mice adapt to biliary obstruction by enhanced phase I detoxification and renal elimination of bile acids

Farnesoid X receptor knockout (Fxr(-/-)) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr(-/-) mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr(-/-) mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr(-/-) mice, in particular hydroxylations of cholic acid in the 1beta, 2beta, 4beta, 6alpha, 6beta, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr(-/-) mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr(-/-) mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.     

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ～80% compared to ～50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.     

Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-alpha/beta in the adaptive response to bile acids

The bile acid receptor farnesoid X receptor (FXR) is a key regulator of hepatic defense mechanisms against bile acids. A comprehensive study addressing the role of FXR in the coordinated regulation of adaptive mechanisms including biosynthesis, metabolism, and alternative export together with their functional significance is lacking. We therefore fed FXR knockout (FXR(-/-)) mice with cholic acid (CA) and ursodeoxycholic acid (UDCA). Bile acid synthesis and hydroxylation were assessed by real-time RT-PCR for cytochrome P-450 (Cyp)7a1, Cyp3a11, and Cyp2b10 and mass spectrometry-gas chromatography for determination of bile acid composition. Expression of the export systems multidrug resistance proteins (Mrp)4-6 in the liver and kidney and the recently identified basoalteral bile acid transporter, organic solute transporter (Ost-alpha/Ost-beta), in the liver, kidney, and intestine was also investigated. CA and UDCA repressed Cyp7a1 in FXR(+/+) mice and to lesser extents in FXR(-/-) mice and induced Cyp3a11 and Cyp2b10 independent of FXR. CA and UDCA were hydroxylated in both genotypes. CA induced Ost-alpha/Ost-beta in the liver, kidney, and ileum in FXR(+/+) but not FXR(-/-) mice, whereas UDCA had only minor effects. Mrp4 induction in the liver and kidney correlated with bile acid levels and was observed in UDCA-fed and CA-fed FXR(-/-) animals but not in CA-fed FXR(+/+) animals. Mrp5/6 remained unaffected by bile acid treatment. In conclusion, we identified Ost-alpha/Ost-beta as a novel FXR target. Absent Ost-alpha/Ost-beta induction in CA-fed FXR(-/-) animals may contribute to increased liver injury in these animals. The induction of bile acid hydroxylation and Mrp4 was independent of FXR but could not counteract liver toxicity sufficiently. Limited effects of UDCA on Ost-alpha/Ost-beta may jeopardize its therapeutic efficacy.     

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.     

Bile acid synthesis in cultured human hepatoblastoma cells.

Biosynthetic pathways to bile acids have been studied in HepG2 cells, a well-differentiated human hepatoblastoma cell line. Cholesterol metabolites, in total 29, were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry. The production rates/concentrations of cholic acid (CA) and chenodeoxycholic acid (CDCA) in media from control cells were 71 and 74 ng/10(7) cells/h, respectively. Major bile acid precursors were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA), 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid, 7 alpha-hydroxy-3-oxo-4-cholenoic acid, and 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, their concentrations being 60, 30, 23, and 10 ng/10(7) cells/h, respectively. These and nine other isolated intermediates formed essentially complete metabolic sequences from cholesterol to CA and CDCA. The remaining steroids were metabolites of the intermediates or autooxidation products of cholesterol. These findings and the observed effect of dexamethasone on production rates suggest that in HepG2 cells the major biosynthetic pathways to primary bile acids start with 7 alpha-hydroxylation of cholesterol and oxidation to 7 alpha-hydroxy-4-cholesten-3-one followed by hydroxylation at either the 26 or 12 alpha position. CDCA is formed by the sequence of 26-hydroxylation, oxidation, and degradation of the side chain and A-ring reduction. CA is formed by the sequence of 12 alpha-hydroxylation, 26-hydroxylation, oxidation, and degradation of the side chain and reduction of the A-ring. An alternative pathway to CA included A-ring reduction of the intermediate 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid to form THCA prior to side chain cleavage. These pathways are not limited to HepG2 cells but may also be important in humans.     

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.     

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.     

Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11β-hydroxysteroid dehydrogenase 1

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.     

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.     

Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis

Background

Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests.

Methods and Findings

In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87).

Conclusions

Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice. Please see later in the article for the Editors'' Summary