HEPATOLOGY, VOL. 70, NO. 1, 2019 AUTOIMMUNE, CHOLESTATIC AND BILIARY DISEASE Enhanced Microbial Bile Acid Deconjugation and Impaired Ileal Uptake in Pregnancy Repress Intestinal Regulation of Bile Acid Synthesis 1 2 2 3 2 Caroline Ovadia , * Alvaro Perdones-Montero, * Konstantina Spagou, * Ann Smith, Magali H. Sarafian, 2 1 1 4 1 1 1 Maria Gomez-Romero, Elena Bellafante, Louise C.D. Clarke, Fouzia Sadiq, Vanya Nikolova, Alice Mitchell, Peter H. Dixon, 1 5 1 4 5 2 Natalie Santa-Pinter, Annika Wahlström, Shadi Abu-Hayyeh, Julian R.F. Walters, Hanns-Ulrich Marschall, Elaine Holmes, 3,6 1 Julian R. Marchesi, and Catherine Williamson Pregnancy is associated with progressive hypercholanemia, hypercholesterolemia, and hypertriglyceridemia, which can result in metabolic disease in susceptible women. Gut signals modify hepatic homeostatic pathways, linking intesti- nal content to metabolic activity. We sought to identify whether enteric endocrine signals contribute to raised serum bile acids observed in human and murine pregnancies, by measuring fibroblast growth factor (FGF) 19/15 protein and mRNA levels, and 7α-hydroxy-4-cholesten-3-one. Terminal ileal farnesoid X receptor (FXR)-mediated gene ex- pression and apical sodium bile acid transporter (ASBT) protein concentration were measured by qPCR and western blotting. Shotgun whole-genome sequencing and ultra-performance liquid chromatography tandem mass spectrom- etry were used to determine the cecal microbiome and metabonome. Targeted and untargeted pathway analyses were performed to predict the systemic effects of the altered metagenome and metabolite profiles. Dietary CA supplemen- tation was used to determine whether the observed alterations could be overcome by intestinal bile acids functioning as FXR agonists. Human and murine pregnancy were associated with reduced intestinal FXR signaling, with lower FGF19/15 and resultant increased hepatic bile acid synthesis. Terminal ileal ASBT protein was reduced in murine pregnancy. Cecal bile acid conjugation was reduced in pregnancy because of elevated bile salt hydrolase-producing Bacteroidetes. CA supplementation induced intestinal FXR signaling, which was not abrogated by pregnancy, with strikingly similar changes to the microbiota and metabonome as identified in pregnancy. Conclusion: The altered in- testinal microbiota of pregnancy enhance bile acid deconjugation, reducing ileal bile acid uptake and lowering FXR induction in enterocytes. This exacerbates the effects mediated by reduced bile acid uptake transporters in preg- nancy. Thus, in pregnant women and mice, there is reduced FGF19/15-mediated hepatic repression of hepatic bile acid synthesis, resulting in hypercholanemia. (Hepatology 2019;70:276-293). (2) n normal human pregnancy, there is a change in and hypertriglyceridemia in the third trimester. metabolism with advancing gestation that results in These gestational alterations in lipid metabolism are (1) Ielevated serum bile acids, hypercholesterolemia, essential to ensure nutrient provision for the growing Abbreviations: ASBT, apical sodium dependent bile acid transporter; BSH, bile salt hydrolase; C4, 7α-hydroxy-4-cholesten-3-one; CA, cholic acid; CDCA, chenodeoxycholic acid; Cyp7a1, cytochrome P450 family 7 subfamily A member 1; DCA, deoxycholic acid; FGF, fibroblast growth factor; FXR, farnesoid X receptor; LCA, lithocholic acid; LPE, lysophosphatidylethanolamine; MCA, muricholic acid; OST, organic solute transporter; PM5S, epiallopregnanolone sulfate; RXR, retinoid X receptor; SHP, small heterodimer partner; TMCAs, tauromuricholic acids; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass spectrometry. Received June 7, 2018; accepted February 28, 2019. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.30661/suppinfo. *Co-first authors: these authors contributed equally to this work. Supported by Wellcome Trust (Grant P30874), Tommy’s, ICP Support, the National Institute for Health Research Biomedical Research Centres at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and Imperial College Healthcare NHS Trust, and Genesis Research Trust. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, or the Department of Health. According to Wellcome Trust’s Policy on data, software and materials management and sharing, all data supporting this study will be openly available by request of the authors. 276 HEPATOLOGY, Vol. 70, No. 1, 2019 OVADIA, PERDONES-MONTERO, ET AL. fetus and are associated with altered expression of G-protein-coupled receptor 5 (TGR5), expressed by key genes in pathways that control bile acid and lipid enteroendocrine cells within the gut, results in release (12) homeostasis. Serum and hepatocyte bile acid con- of glucagon-like peptide 1 (GLP1). Thus, luminal centrations are elevated on day 18 of murine preg- bile acids have a profound effect on bile acid, lipid, (3,4) nancy, likely secondary to gestational increases and glucose homeostasis through interaction with dif- (5) (13) in 17β-estradiol and sulfated progesterone metab- ferent receptors in a range of tissues. (6) olites. These endocrine changes cause reduced The composition of the gut microbiota influ- function of hepatic farnesoid X receptor (FXR); the ences the luminal concentration and conjugation sulfated progesterone metabolite, epiallopregnanolone of specific bile acids and consequent expression of (7) sulfate (PM5S), is a partial agonist of FXR, whereas enzymes that control intestinal and hepatic bile acid 17β-estradiol–bound estrogen receptor α directly transport and metabolism through FXR-dependent (14) interacts with FXR to repress downstream transcrip- pathways (reviewed in a previous work ). In brief, (5) tion. Bile acids also signal through enterocyte and when compared to germ-free mice, convention- enteroendocrine L-cell receptors to influence bile acid ally reared animals had higher serum and fecal bile homeostasis and the release of hormones that impact acid concentrations, more deconjugated bile acids gestational lipid and bile acid metabolism. Activated in the distal gut, and a lower proportion of taurine- FXR in enterocytes dimerizes with retinoid X recep- conjugated bile acids in the proximal and distal (15) tor (RXR), causing induction of rodent ileal bile acid gut and other tissues, including the kidney and (8) (16) binding protein, organic solute transporters (OSTs; heart. The potency of bile acid species as FXR (9) (17) OSTα and OSTβ), secretion of the hormone fibro- agonists differs, chenodeoxycholic acid (CDCA), blast growth factor (FGF) 15 in mice, FGF19 in cholic acid (CA) and their taurine conjugates are (10) humans, and small heterodimer partner (SHP)- FXR agonists, whereas the taurine-conjugated α and (15) mediated repression of apical sodium-dependent bile β muricholic acids (MCAs) are FXR antagonists. (11) acid transporter (ASBT). Bile-acid–binding and Furthermore, alterations in the luminal concentra- activation of the G-protein-coupled receptor, Takeda tion of specific bile acids will affect the abundance of © 2019 The Authors. HEPATOLOGY published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use and distribution in any medium, provided the original work is properly cited. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.30661 Potential conflict of interest: Nothing to report. ARTICLE INFORMATION: 1 2 From the Division of Women and Children’s Health, King’s College London, London, United Kingdom; Section of Biomolecular Medicine, Division of Computational & Systems Medicine, Department of Surgery & Cancer, Faculty of Medicine, Imperial 3 4 College London, London, United Kingdom; School of Biosciences, Cardiff University, Cardiff, United Kingdom; Division 5 of Digestive Diseases, Hammersmith Hospital, Imperial College London, London, United Kingdom; Institute of Medicine, Department of Molecular and Clinical Medicine and Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden; 6 Centre for Digestive and Gut Health, Department of Surgery and Cancer, Imperial College London, London, United Kingdom. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Catherine Williamson, M.D., F.R.C.P. or Division of Women and Children’s Health Julian R. Marchesi, Ph.D. 2nd Floor, Hodgkin Building, Guy’s Campus Division of Integrative Systems Medicine and Digestive Disease King’s College London QEQM, St Mary’s Hospital, Imperial College London Great Maze Pond Praed Street London, SE1 1UL, United Kingdom London, W2 1NY, United Kingdom E-mail: [email protected] E-mail: [email protected] Tel.: +44 (0) 207 848 6014 Tel.: +44 (0)20 3312 6197 277 OVADIA, PERDONES-MONTERO, ET AL. HEPATOLOGY, July 2019 particular microbes within the gut lumen; for exam- healthy women. Women were restricted to those with ple, Bilophila wadsworthia grows preferentially in the spontaneously conceived third trimester singleton (18) presence of high bile acid concentrations, whereas pregnancies, without pregnancy complications and deoxycholic acid (DCA) and CDCA are toxic to who had not taken antibiotics for the duration of the o the growth of Bifidobacterium breve, Blautia coccoides pregnancy. Fecal samples were frozen at −80 C within T JCM 1395 , and Bacteroides thetaiotaomicron DSM 24 hours of collection. T (19) 2079 . FXR activity in the intestine