Bile acid-receptor TGR5 deficiency worsens liver injury in alcohol-fed mice by inducing intestinal microbiota dysbiosis
Madeleine SpatzDragos CiocanGrégory MerlenDominique RainteauLydie HumbertNeuza Félix Gomes-RochetteCindy HugotNicolas TrainelFrançoise Mercier‐NoméSéverine DomenichiniVirginie PuchoisLaura WrzosekGladys FerrereThierry TordjmannGabriel PerlemuterAnne‐Marie Cassard
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Bile-acid metabolism and the intestinal microbiota are impaired in alcohol-related liver disease. Activation of the bile-acid receptor TGR5 (or GPBAR1) controls both biliary homeostasis and inflammatory processes. We examined the role of TGR5 in alcohol-induced liver injury in mice.We used TGR5-deficient (TGR5-KO) and wild-type (WT) female mice, fed alcohol or not, to study the involvement of liver macrophages, the intestinal microbiota (16S sequencing), and bile-acid profiles (high-performance liquid chromatography coupled to tandem mass spectrometry). Hepatic triglyceride accumulation and inflammatory processes were assessed in parallel.TGR5 deficiency worsened liver injury, as shown by greater steatosis and inflammation than in WT mice. Isolation of liver macrophages from WT and TGR5-KO alcohol-fed mice showed that TGR5 deficiency did not increase the pro-inflammatory phenotype of liver macrophages but increased their recruitment to the liver. TGR5 deficiency induced dysbiosis, independently of alcohol intake, and transplantation of the TGR5-KO intestinal microbiota to WT mice was sufficient to worsen alcohol-induced liver inflammation. Secondary bile-acid levels were markedly lower in alcohol-fed TGR5-KO than normally fed WT and TGR5-KO mice. Consistent with these results, predictive analysis showed the abundance of bacterial genes involved in bile-acid transformation to be lower in alcohol-fed TGR5-KO than WT mice. This altered bile-acid profile may explain, in particular, why bile-acid synthesis was not repressed and inflammatory processes were exacerbated.A lack of TGR5 was associated with worsening of alcohol-induced liver injury, a phenotype mainly related to intestinal microbiota dysbiosis and an altered bile-acid profile, following the consumption of alcohol.Excessive chronic alcohol intake can induce liver disease. Bile acids are molecules produced by the liver and can modulate disease severity. We addressed the specific role of TGR5, a bile-acid receptor. We found that TGR5 deficiency worsened alcohol-induced liver injury and induced both intestinal microbiota dysbiosis and bile-acid pool remodelling. Our data suggest that both the intestinal microbiota and TGR5 may be targeted in the context of human alcohol-induced liver injury.Keywords:
Dysbiosis
Deoxycholic acid
The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases. The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases. A mouse is the most commonly used laboratory animal to extrapolate investigations regarding human metabolism. However, numerous differences have been reported between mice and humans. Bile acids (BAs), the end products of cholesterol catabolism, take part in the intestinal digestion and absorption and are recycled via the enterohepatic circulation. The BA composition is a primary indicator of the metabolic difference between mice and humans (1Thakare R. Alamoudi J.A. Gautam N. Rodrigues A.D. Alnouti Y. Species differences in bile acids I. Plasma and urine bile acid composition.J. Appl. Toxicol. 2018; 38: 1323-1335Crossref PubMed Scopus (49) Google Scholar). Certain BAs are ligands of nuclear and transmembrane G protein-coupled receptors and regulate lipid and carbohydrate metabolism, inflammation, fibrosis, and carcinogenesis (2Schaap F.G. Trauner M. Jansen P.L. Bile acid receptors as targets for drug development.Nat. Rev. Gastroenterol. Hepatol. 2014; 11: 55-67Crossref PubMed Scopus (466) Google Scholar). Therefore, the BA composition can be a crucial factor for creating relevant mouse models of human diseases (3Rudling M. Understanding mouse bile acid formation: Is it time to unwind why mice and rats make unique bile acids?.J. Lipid Res. 2016; 57: 2097-2098Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 4Fickert P. Wagner M. Biliary bile acids in hepatobiliary injury - what is the link?.J. 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Tauro α-muricholate is as effective as tauro β-muricholate and tauroursodeoxycholate in preventing taurochenodeoxycholate-induced liver damage in the rat.Hepatology. 1994; 19: 1007-1012Crossref PubMed Scopus (18) Google Scholar) and have antagonistic effects on FXR (10Sayin S.I. Wahlstrom A. Felin J. Jantti S. Marschall H.U. Bamberg K. Angelin B. Hyotylainen T. Oresic M. Backhed F. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-β-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). Second, the primary BAs, cholic acid (CA) and CDCA, are 7α-dehydroxylated by intestinal bacteria and transformed into the secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In mice and rats, these secondary BAs are converted to primary BAs by the hepatic BA 7α-hydroxylase (Fig. 1A). Compared with primary BAs, DCA and LCA are more effective in activating the Takeda G protein-coupled receptor 5 (TGR5) (11Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. et al.A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar). They are also more cytotoxic (6Schölmerich J. Becher M.S. Schmidt K. Schubert R. Kremer B. Feldhaus S. Gerok W. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties–studies on isolated hepatocytes and lipid membrane vesicles.Hepatology. 1984; 4: 661-666Crossref PubMed Scopus (255) Google Scholar) and promote carcinogenesis (12Yoshimoto S. Loo T.M. Atarashi K. Kanda H. Sato S. Oyadomari S. Iwakura Y. Oshima K. Morita H. Hattori M. et al.Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome.Nature. 2013; 499 ([Erratum. 2014. 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The specific rodent genes responsible for the CDCA 6β-hydroxylation and BA 7α-rehydroxylation were not determined for a long time. However, Takahashi et al. (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) recently found that MCAs were not detected in liver samples of Cyp2c-cluster-null mice (17Scheer N. Kapelyukh Y. Chatham L. Rode A. Buechel S. Wolf C.R. Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines.Mol. Pharmacol. 2012; 82: 1022-1029Crossref PubMed Scopus (41) Google Scholar). They, therefore, concluded that Cyp2c70 was necessary for the 6β-hydroxylation of CDCA in mice. However, the specific rodent genes responsible for hepatic BA 7α-rehydroxylation were still not determined until recently. An early study on the purification and characterization of rat liver taurodeoxycholic acid (TDCA) 7α-hydroxylase showed that BA 7α-rehydroxylation was catalyzed by a cytochrome P450 (CYP) enzyme (18Murakami K. Okuda K. Purification and characterization of taurodeoxycholate 7α-monooxygenase in rat liver.J. Biol. Chem. 1981; 256: 8658-8662Abstract Full Text PDF PubMed Google Scholar). Another study using liver-specific CYP oxidoreductase KO mice showed that biliary TDCA levels were markedly elevated after feeding CA (19Kunne C. Acco A. Hohenester S. Duijst S. de Waart D.R. Zamanbin A. Oude Elferink R.P. Defective bile salt biosynthesis and hydroxylation in mice with reduced cytochrome P450 activity.Hepatology. 2013; 57: 1509-1517Crossref PubMed Scopus (20) Google Scholar), suggesting that 7α-rehydroxylation of BAs is catalyzed by CYP enzyme(s). In this study, we first identified the mouse and rat genes encoding BA 7α-hydroxylase and CDCA 6β-hydroxylase through a new approach using orthology, tissue distribution, and sexual dimorphism data of CYP (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar). Then, we generated double KO (DKO) mice to examine BA metabolism. These mice showed BA composition just as we had expected, and the BA pool was markedly reduced. However, much to our surprise, FXR was not activated. αMCA, βMCA, ωMCA, tauro-αMCA, tauro-βMCA, and tauro-ωMCA were purchased from Steraloids (Newport, RI). Taurohyodeoxycholic acid (THDCA), taurolithocholic acid 3-sulfate (TLCA-3S), and LCA 3-sulfate (LCA-3S) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled male mouse liver microsomes (CD1), pooled female mouse liver microsomes (CD1), pooled male rat liver microsomes (Sprague-Dawley), pooled female rat liver microsomes (Sprague-Dawley), and pooled human liver microsomes were purchased from BD Biosciences (Franklin Lakes, NJ), and male CD1 mouse kidney microsomes were purchased from Sekisui XenoTech (Kansas City, KS). Cyp2a12−/−Cyp2c70−/− DKO mice were generated using the CRISPR-Cas9 system by the Laboratory Animal Resource Center, University of Tsukuba (Ibaraki, Japan) and Charles River Laboratories Japan, Inc. (Kanagawa, Japan). Mice were kept under pathogen-free conditions and a regular 12 h light-dark cycle (light period: 0600–1800), with free access to standard chow and water. This project was approved by the Animal Experiment Committees of the University of Tsukuba, Charles River Laboratories Japan, and Tokyo Medical University. The oligos, Cyp2a12 intron 2 CRISPR F (5′-caccATAGTTAGGGGAAGCGACAT-3′) and Cyp2a12 intron 2 CRISPR R (5′-aaacATGTCGCTTCCCCTAACTAT-3′), Cyp2a12 intron 4 CRISPR F (5′-caccGTCTTACAATCCAGGCGAGG-3′) and Cyp2a12 intron 4 CRISPR R (5′-aaacCCTCGCCTGGATTGTAAGAC-3′), Cyp2c70 intron 1 CRISPR F (5′-caccAGATGATTATTAGTGTACAG-3′) and Cyp2c70 intron 1 CRISPR R (5′-aaacCTGTACACTAATAATCATCT-3′), and Cyp2c70 intron 2 CRISPR F (5′-caccTGGAACAGTGACAAGAGCGA-3′) and Cyp2c70 intron 2 CRISPR R (5′-aaacTCGCTCTTGTCACTGTTCCA-3′) were annealed and inserted into the BbsI restriction site of the pX330 vector (Addgene plasmid 42230). Constructed plasmids (circular) were designated pX330-Cyp2a12 intron 2, pX330-Cyp2a12 intron 4, pX330-Cyp2c70 intron 1, and pX330-Cyp2c70 intron 2. Pregnant mare serum gonadotropin and human chorionic gonadotropin were injected into female C57BL/6J mice at 48 h intervals, and the mice were mated with male C57BL/6J mice. Fertilized ova were collected from the oviducts, and 5 ng/μl each of pX330-Cyp2a12 intron 2 and pX330-Cyp2a12 intron 4 or pX330-Cyp2c70 intron 1 and pX330-Cyp2c70 intron 2 were injected into the pronuclei according to standard protocols (22Gordon J.W. Ruddle F.H. Integration and stable germ line transmission of genes injected into mouse pronuclei.Science. 1981; 214: 1244-1246Crossref PubMed Scopus (428) Google Scholar). The injected one-cell embryos were transferred into pseudopregnant CD1 mice. Using genomic DNA obtained from tail clippings, founder (F0) mice were selected by PCR followed by direct sequencing as described by Hoshino et al (23Hoshino Y. Mizuno S. Kato K. Mizuno-Iijima S. Tanimoto Y. Ishida M. Kajiwara N. Sakasai T. Miwa Y. Takahashi S. et al.Simple generation of hairless mice for in vivo imaging.Exp. Anim. 2017; 66: 437-445Crossref PubMed Scopus (8) Google Scholar). The sequences of the oligonucleotide primer pairs used were forward (F): 5′-GAGAGGCAAA-TGGGAACAAA-3′ and reverse (R): 5′-AACAGGCAGAAGCAGG-GATA-3′ for WT Cyp2a12, F: 5′-GAGAGGCAAATGGGAACAAA-3′ and R: 5′-AGGACCTCGGGATGAGAAGT-3′ for mutant Cyp2a12, F: 5′-TCTTCTTGCCTTCAACAGCA-3′ and R: 5′-AACCATTGCACAGAGCACAG-3′ for WT Cyp2c70, F: 5′-TCTTCTTGCCTTCAACAGCA-3′ and R: 5′-GAAAGCCCATGAGAGAGGAA-3′ for mutant Cyp2c70, and F: 5′-AGTTCATCAAGCCCATCCTG-3′ and R: 5′-GAAGTTTCTGTTGGCGAAGC-3′ for Cas9 detection. F0 mice for Cyp2a12-null and Cyp2c70-null were bred with WT C57BL/6J mice to determine their germline competency. A male F1 Cyp2a12+/− mouse was crossed with female C57BL/6J mice by in vitro fertilization using CARD HyperOva (Kyudo Co., Saga, Japan), and the resulting female Cyp2a12+/− mice were crossed with a male F1 Cyp2c70+/− mouse by in vitro fertilization using CARD HyperOva. Then, these double heterozygous Cyp2a12+/−Cyp2c70+/− animals were crossed to obtain Cyp2a12−/−Cyp2c70+/+ (2a12KO), Cyp2a12+/+Cyp2c70−/− (2c70KO), and Cyp2a12−/−Cyp2c70−/− (DKO) mice. All experiments reported here were performed with subsequent generations of these animals. Mice that were 10–12 weeks old [11.1 ± 0.8 (mean ± SD), n = 44] were used. After making them fast for 4 h with free access to water, they were euthanized between 1100 and 1600 under combination anesthesia with medetomidine, midazolam, and butorphanol. Their gallbladder, blood (serum), liver, small intestine, cecal contents, and feces were collected immediately and frozen at −80°C. Serum activities of alanine transaminase (ALT) and alkaline phosphatase (ALP) were determined by colorimetric assays using Transaminase CII-Test Wako and LabAssay ALP (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). BA concentrations were determined as described by Murakami et al. (24Murakami M. Iwamoto J. Honda A. Tsuji T. Tamamushi M. Ueda H. Monma T. Konishi N. Yara S. Hirayama T. et al.Detection of Gut Dysbiosis due to Reduced Clostridium Subcluster XIVa Using the Fecal or Serum Bile Acid Profile.Inflamm. Bowel Dis. 2018; 24: 1035-1044Crossref PubMed Scopus (29) Google Scholar) with minor modifications. Liver, small intestine, cecal contents, and feces were solubilized in 5% KOH/water at 80°C for 20 min; this heating step was omitted for serum and bile samples. After the addition of internal standards and 0.5 M potassium phosphate buffer (pH 7.4), BAs were extracted with Bond Elut C18 cartridges and quantified by LC-MS/MS. Chromatographic separation was performed using a Hypersil GOLD column (200 × 2.1 mm, 1.9 μm; Thermo Fisher Scientific) at 40°C. The mobile phase consisted of (A) 20 mM ammonium acetate buffer (pH 7.5)-acetonitrile-methanol (70:15:15, v/v/v) and (B) 20 mM ammonium acetate buffer (pH 7.5)-acetonitrile-methanol (30:35:35, v/v/v). The following gradient program was used at a flow rate of 150 μl/min: 0–50% B for 20 min, 50–100% B for 10 min, hold 100% B for 15 min, and re-equilibrate to 100% A for 10 min. Detailed LC-MS/MS conditions are presented in supplemental Table S1. Serum and hepatic concentrations of total cholesterol and triglycerides were measured by colorimetric assays using Cholesterol E-Test Wako and Triglyceride E-Test Wako (FUJIFILM Wako Pure Chemical Corporation), respectively. Biliary cholesterol and phospholipid concentrations were determined by Cholesterol E-Test Wako and Phospholipid C-Test Wako, respectively. Sterol and oxysterol concentrations in the liver and serum were quantified using our previously described LC-MS/MS method (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). Briefly, 5 μl of serum or 5 mg of liver tissue were incubated with internal standards in 1 N ethanolic KOH at 37°C for 1 h. Sterols were extracted with n-hexane, derivatized to picolinyl esters, and analyzed by LC-MS/MS. Microsomes and mitochondria were prepared from livers by differential ultracentrifugation (26Honda A. Salen G. Matsuzaki Y. Batta A.K. Xu G. Leitersdorf E. Tint G.S. Erickson S.K. Tanaka N. Shefer S. Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27–/– mice and CTX.J. Lipid Res. 2001; 42: 291-300Abstract Full Text Full Text PDF PubMed Google Scholar). Microsomal activities of BA 6β-hydroxylase and BA 7α-hydroxylase were measured as follows: microsomes (100 μg of protein) were incubated for 20 min at 37°C with 200 μM of each BA (dissolved in 10 μl of 50% acetone in water), NADPH (1.2 mM), glucose-6-phosphate (3.6 mM), 1 unit of glucose-6-phosphate dehydrogenase, and 100 mM of potassium phosphate buffer (pH 7.4) containing 0.1 mM of EDTA in a total volume of 250 μl. The incubation was stopped by the addition of 10 μl of 8.9 M aqueous KOH solution. After the addition of internal standards and 0.5 M potassium phosphate buffer (pH 7.4), BAs were extracted with Bond Elut C18 cartridges and quantified by LC-MS/MS as described above. Instead of using microsomes, recombinant rat CYP2A1 (Supersome) prepared from insect cells (Corning, NY) was used to determine BA 7α-hydroxylation. Microsomal HMG-CoA reductase activity was measured by LC-MS/MS method, as described previously (27Honda A. Mizokami Y. Matsuzaki Y. Ikegami T. Doy M. Miyazaki H. Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS.J. Lipid Res. 2007; 48: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The activities of microsomal cholesterol 7α-hydroxylase (CYP7A1) (28Honda A. Salen G. Shefer S. Batta A.K. Honda M. Xu G. Tint G.S. Matsuzaki Y. Shoda J. Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver.J. Lipid Res. 1999; 40: 1520-1528Abstract Full Text Full Text PDF PubMed Google Scholar), mitochondrial cholesterol 27-hydroxylase (CYP27A1) (28Honda A. Salen G. Shefer S. Batta A.K. Honda M. Xu G. Tint G.S. Matsuzaki Y. Shoda J. Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver.J. Lipid Res. 1999; 40: 1520-1528Abstract Full Text Full Text PDF PubMed Google Scholar), and microsomal oxysterol 7α-hydroxylase (CYP7B1) (29Hirayama T. Honda A. Matsuzaki Y. Miyazaki T. Ikegami T. Doy M. Xu G. Lea M. Salen G. Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol.Hepatology. 2006; 44: 602-611Crossref PubMed Scopus (18) Google Scholar) were measured according to our stable-isotope dilution MS method except that LC-MS/MS was used instead of GC-MS to quantify 7α-hydroxycholesterol, 27-hydroxycholesterol, 7α,27-dihydroxycholesterol, and their isotopic variants (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). Microsomal 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase (CYP8B1) activity was determined as described previously (30Honda A. Salen G. Matsuzaki Y. Batta A.K. Xu G. Leitersdorf E. Tint G.S. Erickson S.K. Tanaka N. Shefer S. Side chain hydroxylations in bile acid biosynthesis catalyzed by CYP3A are markedly up-regulated in Cyp27–/– mice but not in cerebrotendinous xanthomatosis.J. Biol. Chem. 2001; 276: 34579-34585Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) except that [2H7]7α,27-dihydroxycholesterol was used as an internal standard and LC-MS/MS was employed instead of HPLC to quantify 7α,12α-dihydroxy-4-cholesten-3-one (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). An aliquot of the liver and terminal ileum specimen were collected in RNAlater (Thermo Fisher Scientific) and stored at −80°C until RNA isolation. Total RNA was extracted using an RNeasy Plus Mini Kit (QIAGEN). Reverse transcription was performed on 4 μg of total RNA using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany). Real-time quantitative PCR was performed on cDNA aliquots with the FastStart DNA MasterPLUS SYBR Green I and a LightCycler (Roche). The sequences of the oligonucleotide primer pairs used to amplify mRNAs are shown in supplemental Table S2. PCR amplification began with a 10 min preincubation step at 95°C, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 10 s, and elongation at 72°C for 16 s. The relative concentration of the PCR product derived from the target gene was calculated by the comparative Ct method, and results were standardized to the expression of Gapdh. The specificity of each PCR product was assessed by melting curve analysis. Serum concentrations of fibroblast growth factor 15 (FGF15) were measured using mouse FGF15 ELISA kit (catalog #MBS2700661; MyBiosource, Inc., San Diego, CA), according to the manufacturer's instruction. Serum concentrations of lipopolysaccharides (LPSs; endotoxin) and TNFα were quantified using ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (catalog #L00350; GenScript USA Inc., Piscataway, NJ) and LBIS Mouse TNF-α ELISA Kit (catalog #AKMTNFA-011; FUJIFILM Wako Shibayagi, Gunma, Japan), respectively, according to the manufacturers' instructions. An aliquot of the liver was fixed in 10% neutral buffered formalin and embedded in a paraffin block. Each paraffin block was sectioned at 3 μm and the paraffin sections were stained using hematoxylin/eosin. Data are expressed as the mean ± SEM. The statistical significance of differences between the results in the different groups was evaluated using the Tukey-Kramer test or the Dunnett's test. For all analyses, significance was accepted at the level of P < 0.05. Correlations were tested by calculating parametric Pearson's correlation coefficient, r, and nonparametric Spearman's correlation coefficient, rs. All statistical analyses were conducted using JMP (version 10.0) software (SAS Institute, Cary, NC). We first explored the mouse and rat genes encoding BA 7α-hydroxylase. To confirm the usefulness of our new method, genes encoding CDCA 6β-hydroxylase [Cyp2c70 is strongly suggested as a responsible gene (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar)] were also examined along with those encoding BA 7α-hydroxylase. Mouse and rat genes encoding BA 7α-hydroxylase and CDCA 6β-hydroxylase must fall under any of 102 mouse and 87 rat Cyp genes (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar) (Fig. 1B). Species difference, sexual dimorphism, and tissue distribution with respect to both enzymes were determined using commercially available microsomal fractions. Neither of the two enzyme activities was detected in the human liver or mouse kidney. Furthermore, sexual dimorphism of both enzyme activities was not observed in the mouse liver but was apparent in the rat liver (Fig. 1C). From the 102 mouse Cyp genes, we narrowed down to four candidate genes, Cyp2a12, Cyp2a22, Cyp2c70, and Cyp2d40 (Fig. 1D, supplemental Table S3), using previously reported orthology, tissue distribution, and sex difference data (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar) as well as our experimental observations (Fig. 1C). If Cyp2c70 encodes for CDCA 6β-hydroxylase (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), the gene encoding BA 7α-hydroxylase would be either Cyp2a12, Cyp2a22, or Cyp2d40. Because mouse CYP2A12, rat CYP2A1, and hamster CYP2A9 have homologous amino acid sequences and similar testosterone 7α-hydroxylase activity (31Kurose K. Tohkin M. Ushio F. Fukuhara M. Cloning and characterization of Syrian hamster testosterone 7α-hydroxylase, CYP2A9.Arch. Biochem. Biophys. 1998; 351: 60-65Crossref PubMed Scopus (14) Google Scholar), we incubated recombinant rat CYP2A1 with TDCA and TLCA. As a result, CYP2A1 catalyzed the 7α-rehydroxylation of TDCA and TLCA (Fig. 1E), suggesting that Cyp2a12 is responsible for BA 7α-rehydroxylation in mice. It is important to note that Cyp2a22 may also encode for BA 7α-hydroxylase in mice because it is highly homologous to Cyp2a12 (96.2% mRNA identity). However, the hepatic expression level of Cyp2a22 is extremely low (less than 3%) compared with that of Cyp2a12 (21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar). Therefore, we finally concluded that Cyp2a12 was primarily responsible for BA 7α-rehydroxylation in mice. We generated Cyp2a12/Cyp2c70 DKO mice using the CRISPR-Cas9 system (Fig. 2A, B). Founder (F0) mice were selected by PCR genotyping (Fig. 2C, D) followed by the detection of Cas9 (supplemental Fig. S1) and direct sequencing (supplemental Table S4). Finally, #4, #5, #44, and #47 mice for Cyp2a12-null and #17, #19, and #32 mice for Cyp2c70-null were selected as F0 mice. The F0 mice were bred with WT C57BL/6J mice, and PCR genotyping followed by sequencing assay demonstrated that mice from #4, #5, and #47 lines and those from #19 line had DNA sequences at CRISPR target sites, as predicted (supplemental Table S5). Finally, a male F1 Cyp2a12+/− mouse from the #4 line and a male F1 Cyp2c70+/− mouse from the #19 line were used for subsequent
Bile Pigments
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Bile acid receptors regulate the metabolic and immune functions of circulating enterohepatic bile acids. This process is disrupted by administration of parenteral nutrition (PN), which may induce progressive hepatic injury for unclear reasons, especially in the newborn, leading to PN-associated liver disease. To explore the role of bile acid signaling on neonatal hepatic function, we initially observed that Takeda G protein receptor 5 (TGR5)-specific bile acids were negatively correlated with worsening clinical disease markers in the plasma of human newborns with prolonged PN exposure. To test our resulting hypothesis that TGR5 regulates critical liver functions to PN exposure, we used TGR5 receptor deficient mice (TGR5-/-). We observed PN significantly increased liver weight, cholestasis, and serum hepatic stress enzymes in TGR5-/- mice compared with controls. Mechanistically, PN reduced bile acid synthesis genes in TGR5-/-. Serum bile acid composition revealed that PN increased unconjugated primary bile acids and secondary bile acids in TGR5-/- mice, while increasing conjugated primary bile acid levels in TGR5-competent mice. Simultaneously, PN elevated hepatic IL-6 expression and infiltrating macrophages in TGR5-/- mice. However, the gut microbiota of TGR5-/- mice compared with WT mice following PN administration displayed highly elevated levels of Bacteroides and Parabacteroides, and possibly responsible for the elevated levels of secondary bile acids in TGR5-/- animals. Intestinal bile acid transporters expression was unchanged. Collectively, this suggests TGR5 signaling specifically regulates fundamental aspects of liver bile acid homeostasis during exposure to PN. Loss of TGR5 is associated with biochemical evidence of cholestasis in both humans and mice on PN.NEW & NOTEWORTHY Parenteral nutrition is associated with deleterious metabolic outcomes in patients with prolonged exposure. Here, we demonstrate that accelerated cholestasis and parental nutrition-associated liver disease (PNALD) may be associated with deficiency of Takeda G protein receptor 5 (TGR5) signaling. The microbiome is responsible for production of secondary bile acids that signal through TGR5. Therefore, collectively, these data support the hypothesis that a lack of established microbiome in early life or under prolonged parenteral nutrition may underpin disease development and PNALD.
Enterohepatic circulation
Farnesoid X receptor
Deoxycholic acid
Neonatal cholestasis
Liver disease
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Deoxycholic acid
Tauroursodeoxycholic acid
Ursodeoxycholic acid
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Bile acids, amphipathic molecules known for their facilitating role in fat absorption, are also recognized as signalling molecules acting via nuclear and membrane receptors. Of the bile acid-activated receptors, the Farnesoid X Receptor (FXR) and the G protein-coupled bile acid receptor-1 (Gpbar1 or TGR5) have been studied most extensively. Bile acid signaling is critical in the regulation of bile acid metabolism itself, but it also plays a significant role in glucose, lipid and energy metabolism. Activation of FXR and TGR5 leads to reduced hepatic bile salt load, improved insulin sensitivity and glucose regulation, increased energy expenditure, and anti-inflammatory effects. These beneficial effects render bile acid signaling an interesting therapeutic target for the treatment of diseases such as cholestasis, non-alcoholic fatty liver disease, and diabetes. Here, we summarize recent findings on bile acid signaling and discuss potential and current limitations of bile acid receptor agonist and modulators of bile acid transport as future therapeutics for a wide-spectrum of diseases.
Farnesoid X receptor
Ursodeoxycholic acid
Chenodeoxycholic acid
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Bile acids are critical contributors to the regulation of whole body glucose homeostasis; however, the mechanisms remain incompletely defined. While the hydrophilic bile acid subtype, ursodeoxycholic acid, has been shown to attenuate hepatic endoplasmic reticulum (ER) stress and thereby improve glucose regulation in mice, the effect of hydrophobic bile acid subtypes on ER stress and glucose regulation in vivo is unknown. Therefore, we investigated the effect of the hydrophobic bile acid subtype, deoxycholic acid (DCA), on ER stress and glucose regulation. Eight week old C57BL/6J mice were fed a high fat diet supplemented with or without DCA. Glucose regulation was assessed by oral glucose tolerance and insulin tolerance testing. In addition, circulating bile acid profile and hepatic insulin and ER stress signaling were measured. DCA supplementation did not alter body weight or food intake, but did impair glucose regulation. Consistent with the impairment in glucose regulation, DCA increased the hydrophobicity of the circulating bile acid profile, decreased hepatic insulin signaling and increased hepatic ER stress signaling. Together, these data suggest that dietary supplementation of DCA impairs whole body glucose regulation by disrupting hepatic ER homeostasis in mice.
Deoxycholic acid
Homeostasis
Ursodeoxycholic acid
Farnesoid X receptor
Blood sugar regulation
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Bile acids are believed to play a role in the etiology of colorectal cancer, and high fecal excretion of secondary bile acids was correlated with increased incidence of colon cancer. Recently, it was also reported that there is an increase in plasma of the secondary bile acid, deoxycholic acid in men with colorectal adenomas. Since deoxycholic acid is formed in the colon and absorbed into the portal systemic circulation, it was suggested that the blood concentration of this bile acid reflects the level of exposure of colonic cells to deoxycholic acid. The objective of this study was to investigate whether plasma deoxycholic acid level represents the fecal content of this bile acid in several animal species with different bile acid composition and deoxycholic acid contribution to the bile acid pool. Eight rabbits, hamsters, guinea pigs, and rats were used in this study. Blood samples and feces were collected on days 1, 3, 5 and 7. Bile samples were obtained only on day 7. The plasma, fecal and biliary bile acids were analyzed by gas chromatography-mass spectrometry. Bile acid composition and deoxycholic acid content varied greatly between the animal species studied. There was a variation in the concentration of total bile acids in the plasma and feces obtained at different times during the experiments, however, the bile acids profile remained constant throughout the study. The data obtained shows that although plasma bile acid profile was not similar to fecal bile acids profile, however, there was a significant correlation between the level of plasma and fecal deoxycholic acid. Plasma deoxycholic acid concentration might be a reliable biomarker for the degree of exposure of colon cells to this bile acid, and may be useful in further studies on the role of secondary bile acids in colon carcinogenesis.
Deoxycholic acid
Chenodeoxycholic acid
Lithocholic acid
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Retention of bile acids in the blood is a hallmark of liver failure. Recent studies have shown that increased serum bile acid levels correlate with bacterial infection and increased mortality. However, the mechanisms by which circulating bile acids influence patient outcomes still are elusive.Serum bile acid profiles in 33 critically ill patients with liver failure and their effects on Takeda G-protein-coupled receptor 5 (TGR5), an immunomodulatory receptor that is highly expressed in monocytes, were analyzed using tandem mass spectrometry, novel highly sensitive TGR5 bioluminescence resonance energy transfer using nanoluciferase (NanoBRET, Promega Corp, Madison, WI) technology, and in vitro assays with human monocytes.Twenty-two patients (67%) had serum bile acids that led to distinct TGR5 activation. These TGR5-activating serum bile acids severely compromised monocyte function. The release of proinflammatory cytokines (eg, tumor necrosis factor α or interleukin 6) in response to bacterial challenge was reduced significantly if monocytes were incubated with TGR5-activating serum bile acids from patients with liver failure. By contrast, serum bile acids from healthy volunteers did not influence cytokine release. Monocytes that did not express TGR5 were protected from the bile acid effects. TGR5-activating serum bile acids were a risk factor for a fatal outcome in patients with liver failure, independent of disease severity.Depending on their composition and quantity, serum bile acids in liver failure activate TGR5. TGR5 activation leads to monocyte dysfunction and correlates with mortality, independent of disease activity. This indicates an active role of TGR5 in liver failure. Therefore, TGR5 and bile acid metabolism might be promising targets for the treatment of immune dysfunction in liver failure.
Liver disease
Proinflammatory cytokine
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Bariatric surgery alters bile acid metabolism, which contributes to post-operative improvements in metabolic health. However, the mechanisms by which bariatric surgery alters bile acid metabolism are incompletely defined. In particular, the role of the gut microbiome in the effects of bariatric surgery on bile acid metabolism is incompletely understood. Therefore, we sought to define the changes in gut luminal bile acid composition after vertical sleeve gastrectomy (VSG).
Deoxycholic acid
Sleeve gastrectomy
Chenodeoxycholic acid
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Bile acids in the serum of rat portal blood have been examined. Fasting (20 hr) caused a marked increase in the deoxycholic acid concentration, mainly of deoxycholyltaurine. Studies with [24-14C]deoxycholic acid failed to show that this was due to decreased rehydroxylation by the liver. Resection of the terminal ileum caused a three- to fourfold reduction in the total bile acid concentration in male animals, cholyltaurine being most affected although it remained the predominant bile acid. In contrast the concentration of deoxycholic acid increased in half the animals. In female animals the concentrations of chenic acid and lithocholic acid also increased so that the total bile acid concentration only decreased slightly. Ileal resection combined with a right hemicolectomy caused the disappearance of deoxycholic acid.
Deoxycholic acid
Lithocholic acid
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