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Bile acid composition regulates the manganese transporter Slc30a10 in intestine

2020; Elsevier BV; Volume: 295; Issue: 35 Linguagem: Inglês

10.1074/jbc.ra120.012792

1083-351X

Tiara R. Ahmad, Sei Higuchi, Enrico Bertaggia, Allison Hung, Niroshan Shanmugarajah, Nicole C. Guilz, Jennifer R. Gamarra, Rebecca A. Haeusler,

Pharmacological Effects and Toxicity Studies

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis. Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis. Bile acids (BAs) are cholesterol catabolites that regulate many biological functions, including multiple aspects of macronutrient metabolism. One of the mechanisms by which they do so is by promoting lipid emulsification and absorption (1de Aguiar Vallim T.Q. Tarling E.J. Edwards P.A. Pleiotropic roles of bile acids in metabolism.Cell Metab. 2013; 17 (23602448): 657-66910.1016/j.cmet.2013.03.013Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 2Chávez-Talavera O. Tailleux A. Lefebvre P. Staels B. Bile acid control of metabolism and inflammation in obesity, type 2 diabetes, dyslipidemia, and nonalcoholic fatty liver disease.Gastroenterology. 2017; 152 (28214524): 1679-1694.e310.1053/j.gastro.2017.01.055Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 3de Boer J.F. Bloks V.W. Verkade E. Heiner-Fokkema M.R. Kuipers F. New insights in the multiple roles of bile acids and their signaling pathways in metabolic control.Curr. Opin. Lipidol. 2018; 29 (29553998): 194-20210.1097/MOL.0000000000000508Crossref PubMed Scopus (40) Google Scholar). A second mechanism is by acting as a ligand for BA receptors, which can regulate lipid and glucose metabolism (1de Aguiar Vallim T.Q. Tarling E.J. Edwards P.A. Pleiotropic roles of bile acids in metabolism.Cell Metab. 2013; 17 (23602448): 657-66910.1016/j.cmet.2013.03.013Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 2Chávez-Talavera O. Tailleux A. Lefebvre P. Staels B. Bile acid control of metabolism and inflammation in obesity, type 2 diabetes, dyslipidemia, and nonalcoholic fatty liver disease.Gastroenterology. 2017; 152 (28214524): 1679-1694.e310.1053/j.gastro.2017.01.055Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 3de Boer J.F. Bloks V.W. Verkade E. Heiner-Fokkema M.R. Kuipers F. New insights in the multiple roles of bile acids and their signaling pathways in metabolic control.Curr. Opin. Lipidol. 2018; 29 (29553998): 194-20210.1097/MOL.0000000000000508Crossref PubMed Scopus (40) Google Scholar, 4Ahmad T.R. Haeusler R.A. Bile acids in glucose metabolism and insulin signalling—mechanisms and research needs.Nat. Rev. Endocrinol. 2019; 15 (31616073): 701-71210.1038/s41574-019-0266-7Crossref PubMed Scopus (115) Google Scholar). It is underappreciated that there is structural diversity among BAs that results in variable capacities to activate BA receptors (5Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284 (10334993): 1365-136810.1126/science.284.5418.1365Crossref PubMed Scopus (1829) Google Scholar, 6Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284 (10334992): 1362-136510.1126/science.284.5418.1362Crossref PubMed Scopus (2145) Google Scholar, 7Maruyama T. Miyamoto Y. Nakamura T. Tamai Y. Okada H. Sugiyama E. Nakamura T. Itadani H. Tanaka K. Identification of membrane-type receptor for bile acids (M-BAR).Biochem. Biophys. Res. Commun. 2002; 298 (12419312): 714-71910.1016/S0006-291X(02)02550-0Crossref PubMed Scopus (726) Google Scholar, 8Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. Hinuma S. Fujisawa Y. Fujino M. A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278 (12524422): 9435-944010.1074/jbc.M209706200Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar, 9Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3 (10360171): 543-55310.1016/S1097-2765(00)80348-2Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar, 10Makishima M. Lu T.T. Xie W. Whitfield G.K. Domoto H. Evans R.M. Haussler M.R. Mangelsdorf D.J. Vitamin D receptor as an intestinal bile acid sensor.Science. 2002; 296 (12016314): 1313-131610.1126/science.1070477Crossref PubMed Scopus (958) Google Scholar). This structural diversity arises from the number and position of hydroxyl groups and the conjugation of the molecule to glycine, taurine, or neither (11Hofmann A.F. Roda A. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem.J. Lipid Res. 1984; 25 (6397555): 1477-1489Abstract Full Text PDF PubMed Google Scholar, 12Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50 (18815433): S120-S12510.1194/jlr.R800026-JLR200Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Thus, the composition of the BA pool may affect the activity of BA receptors. However, the biological consequences of altered BA pool composition are not fully known. One key determinant of BA composition is the hepatic enzyme sterol 12α-hydroxylase (encoded by CYP8B1). By adding a 12α-hydroxylation to an intermediate of the BA synthesis pathway, CYP8B1 determines the hepatic synthesis of cholic acid (CA) instead of chenodeoxycholic acid (CDCA) (Fig. 1A) (12Russell D.W. Fifty years of advances in bile acid synthesis and metabolism.J. Lipid Res. 2009; 50 (18815433): S120-S12510.1194/jlr.R800026-JLR200Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 13Li-Hawkins J. Gåfvels M. Olin M. Lund E.G. Andersson U. Schuster G. Björkhem I. Russell D.W. Eggertsen G. Cholic acid mediates negative feedback regulation of bile acid synthesis in mice.J. Clin. Invest. 2002; 110 (12393855): 1191-120010.1172/JCI0216309Crossref PubMed Scopus (201) Google Scholar). In settings of insulin resistance, there is an increased proportion of CA, its bacterial metabolite deoxycholic acid (DCA), and their conjugates—collectively termed 12α-hydroxylated BAs (12HBAs) in the BA pool (14Haeusler R.A. Astiarraga B. Camastra S. Accili D. Ferrannini E. Human insulin resistance is associated with increased plasma levels of 12α-hydroxylated bile acids.Diabetes. 2013; 62 (23884887): 4184-419110.2337/db13-0639Crossref PubMed Scopus (283) Google Scholar, 15Choucair I. Nemet I. Li L. Cole M.A. Skye S.M. Kirsop J.D. Fischbach M. Gogonea V. Brown J.M. Tang W.H.W. Hazen S.L. Quantification of bile acids: a mass spectrometry platform for studying gut microbe connection to metabolic diseases.J. Lipid Res. 2019; 61 (31818878): 159-17710.1194/jlr.ra119000311Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Rates of 12HBA synthesis and CA conversion to DCA are also higher in insulin resistance and type 2 diabetes (16Brufau G. Stellaard F. Prado K. Bloks V.W. Jonkers E. Boverhof R. Kuipers F. Murphy E.J. Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism.Hepatology. 2010; 52 (20725912): 1455-146410.1002/hep.23831Crossref PubMed Scopus (157) Google Scholar, 17Haeusler R.A. Camastra S. Nannipieri M. Astiarraga B. Castro-Perez J. Xie D. Wang L. Chakravarthy M. Ferrannini E. Increased bile acid synthesis and impaired bile acid transport in human obesity.J. Clin. Endocrinol. Metabol. 2016; 101: 1935-194410.1210/jc.2015-2583Crossref PubMed Scopus (79) Google Scholar). Moreover, even in healthy subjects, increases in 12HBAs are correlated with the characteristic metabolic abnormalities of insulin resistance (14Haeusler R.A. Astiarraga B. Camastra S. Accili D. Ferrannini E. 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Chouinard M.L. Cummings B.P. TGR5 contributes to glucoregulatory improvements after vertical sleeve gastrectomy in mice.Gut. 2017; 66 (26511794): 226-23410.1136/gutjnl-2015-309871Crossref PubMed Scopus (142) Google Scholar). Furthermore, siRNA against Cyp8b1 improved nonalcoholic steatohepatitis in mice (23Chevre R. Trigueros-Motos L. Castaño D. Chua T. Corlianò M. Patankar J.V. Sng L. Sim L. Juin T.L. Carissimo G. Ng L.F.P. Yi C.N.J. Eliathamby C.C. Groen A.K. Hayden M.R. et al.Therapeutic modulation of the bile acid pool by Cyp8b1 knockdown protects against nonalcoholic fatty liver disease in mice.FASEB J. 2018; 32 (29481310): 3792-380210.1096/fj.201701084RRCrossref PubMed Scopus (27) Google Scholar). Thus, CYP8B1 inhibition is a potential therapeutic target for metabolic diseases. However, the biological processes that are regulated by 12HBAs are incompletely understood. A major site of BA signaling is the intestine, which encounters high BA concentrations, ∼2–12 mm after a meal (24Clarysse S. Tack J. Lammert F. Duchateau G. Reppas C. Augustijns P. Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states.J. Pharm. Sci. 2009; 98 (18680176): 1177-119210.1002/jps.21502Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 25Northfield T.C. McColl I. Postprandial concentrations of free and conjugated bile acids down the length of the normal human small intestine.Gut. 1973; 14 (4729918): 513-51810.1136/gut.14.7.513Crossref PubMed Scopus (143) Google Scholar). The intestine epithelium expresses at least three BA receptors. The transcription factor FXR regulates BA transport and feedback suppression of hepatic BA synthesis and also modulates lipid and glucose metabolism (1de Aguiar Vallim T.Q. Tarling E.J. Edwards P.A. Pleiotropic roles of bile acids in metabolism.Cell Metab. 2013; 17 (23602448): 657-66910.1016/j.cmet.2013.03.013Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). The membrane receptor TGR5 regulates glucose homeostasis and colonic motility by promoting the secretion of GLP-1 and serotonin (26Thomas C. Gioiello A. Noriega L. Strehle A. Oury J. Rizzo G. Macchiarulo A. Yamamoto H. Mataki C. Pruzanski M. Pellicciari R. Auwerx J. Schoonjans K. TGR5-mediated bile acid sensing controls glucose homeostasis.Cell Metab. 2009; 10 (19723493): 167-17710.1016/j.cmet.2009.08.001Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 27Alemi F. Poole D.P. Chiu J. Schoonjans K. Cattaruzza F. Grider J.R. Bunnett N.W. Corvera C.U. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice.Gastroenterology. 2013; 144 (23041323): 145-15410.1053/j.gastro.2012.09.055Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 28Bunnett N.W. Neuro-humoral signalling by bile acids and the TGR5 receptor in the gastrointestinal tract.J. Physiol. 2014; 592 (24614746): 2943-295010.1113/jphysiol.2014.271155Crossref PubMed Scopus (64) Google Scholar). Another intestinal receptor responsive to certain BAs is the vitamin D receptor (VDR), whose canonical role is to promote calcium and phosphate absorption (29Bouillon R. Carmeliet G. Verlinden L. van Etten E. Verstuyf A. Luderer H.F. Lieben L. Mathieu C. Demay M. Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr. Rev. 2008; 29 (18694980): 726-77610.1210/er.2008-0004Crossref PubMed Scopus (1308) Google Scholar). For each of these receptors, the best reported endogenous BA agonists are non-12HBAs. For FXR, it is CDCA (5Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284 (10334993): 1365-136810.1126/science.284.5418.1365Crossref PubMed Scopus (1829) Google Scholar, 6Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284 (10334992): 1362-136510.1126/science.284.5418.1362Crossref PubMed Scopus (2145) Google Scholar, 9Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol. Cell. 1999; 3 (10360171): 543-55310.1016/S1097-2765(00)80348-2Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar), and for TGR5 and VDR, it is lithocholic acid (LCA) (7Maruyama T. Miyamoto Y. Nakamura T. Tamai Y. Okada H. Sugiyama E. Nakamura T. Itadani H. Tanaka K. Identification of membrane-type receptor for bile acids (M-BAR).Biochem. Biophys. Res. Commun. 2002; 298 (12419312): 714-71910.1016/S0006-291X(02)02550-0Crossref PubMed Scopus (726) Google Scholar, 8Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. Hinuma S. Fujisawa Y. Fujino M. A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278 (12524422): 9435-944010.1074/jbc.M209706200Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar, 10Makishima M. Lu T.T. Xie W. Whitfield G.K. Domoto H. Evans R.M. Haussler M.R. Mangelsdorf D.J. Vitamin D receptor as an intestinal bile acid sensor.Science. 2002; 296 (12016314): 1313-131610.1126/science.1070477Crossref PubMed Scopus (958) Google Scholar). LCA is formed by 7α-dehydroxylation of CDCA by bacterial enzymes in the gut. Thus, BA composition is predicted to impact signaling through multiple receptors in the intestine. Investigating the effects of BA composition on intestinal BA signaling in vivo is challenging because of the continued presence of endogenous BAs. This is particularly important for experiments in mice, as mice contain a class of BAs—muricholic acids, which are non-12HBAs—that are not found in healthy adult humans (although we note that, conversely, all known BAs in the human BA pool are also present in mice and are transported through the mouse intestinal epithelium to undergo enterohepatic circulation). To fill the gap, we used primary murine intestinal organoids, also called enteroids. These organoids are generated from stem cells of the intestinal crypts and contain all known cell types of the intestinal epithelium (30Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459 (19329995): 262-26510.1038/nature07935Crossref PubMed Scopus (4123) Google Scholar). We investigated the effects of BA pools of different compositions on ileal organoids, with a particular focus on the effects of lowering 12HBAs (to mimic CYP8B1 inhibition). Furthermore, we addressed the interspecies differences in BAs by using BA pools that we designed to mimic the effects of CYP8B1 inhibition in humans and mice. We unexpectedly found that varying 12HBA proportions modulates expression of Slc30a10, a manganese efflux transporter critical for whole-body Mn excretion. Cellular Mn levels are tightly regulated, as Mn is essential for numerous cellular processes, yet its excess is toxic (31Chen P. Bornhorst J. Aschner M. Manganese metabolism in humans.Front. Biosci. 2018; 23 (29293455): 1655-167910.2741/4665Crossref PubMed Scopus (205) Google Scholar). Our data demonstrate a previously unknown role of BAs in intestinal control of metal homeostasis. To test the effects of a low-12HBA pool on intestinal gene expression, we designed four distinct BA pools with which to stimulate primary murine ileal organoids. The differences between the pools were due to two key features: (i) the proportion of 12HBAs, either 10% (low) or 90% (high), and (ii) the BA pool of the species we modeled, either human or mouse (Table 1 and Fig. 1B). In the human pools, there was a larger proportion of DCA, and the BAs were glycine-conjugated, whereas in the mouse pools, BAs were taurine-conjugated, to mimic the natural abundance in those species (4Ahmad T.R. Haeusler R.A. Bile acids in glucose metabolism and insulin signalling—mechanisms and research needs.Nat. Rev. Endocrinol. 2019; 15 (31616073): 701-71210.1038/s41574-019-0266-7Crossref PubMed Scopus (115) Google Scholar, 32Wahlström A. Kovatcheva-Datchary P. Ståhlman M. Khan M.-T. Bäckhed F. Marschall H.-U. Induction of farnesoid X receptor signaling in germ-free mice colonized with a human microbiota.J. Lipid Res. 2017; 58 (27956475): 412-41910.1194/jlr.M072819Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 33Li J. Dawson P.A. Animal models to study bile acid metabolism.Biochim. Biophys. Acta. 2019; 1865 (29782919): 895-91110.1016/j.bbadis.2018.05.011Crossref PubMed Scopus (86) Google Scholar). Mouse BA pools contained muricholic acids, whereas these were not included in the human pools. Thus, the four BA pools are labeled human low 12HBA (H10), human high 12HBA (H90), mouse low 12HBA (M10), and mouse high 12HBA (M90). Importantly, the total BA pool concentration was the same across all treatment groups. We prepared the four BA pools in mixed micelles containing oleic acid, 2-palmitoyl glycerol, phosphatidylcholine, and free cholesterol to mimic conditions of the intestinal lumen. We used the four lipid-emulsified BA pools to treat primary murine ileal organoids. The vehicle control contained all micelle components except BAs. We performed bulk RNA-Seq after 24 h of treatment.Table 1Composition of BA pools. BAs were in their glycine-conjugated forms for human BA pools and taurine-conjugated forms for mouse BA pools, except LCA, which was unconjugatedBA groupBA speciesIn H10In H90In M10In M90mol %mol %mol %mol %12HBACholic acid7639.081.012HBADeoxycholic acid3271.09.0Non-12HBAChenodeoxycholic acid86.859.6513.51.5Non-12HBAUrsodeoxycholic acid2.250.258.10.9Non-12HBALithocholic acid0.900.100.90.1Non-12HBAα-Muricholic acid22.52.5Non-12HBAβ-Muricholic acid45.05.0 Open table in a new tab We focused on the effects of the low-12HBA pools, as this would mimic the effects of CYP8B1 inhibition. We examined all genes that were significantly induced compared with vehicle, setting thresholds of log2FC > 1.0 and padj < 0.05 for differential expression. The low-12HBA pools collectively induced 1361 genes. Of these, 516 reached those thresholds for both H10 and M10 pools, 95 reached those thresholds for H10 only, and 750 reached the thresholds for M10 only (Fig. S1A). Pathway analysis indicates enrichment of genes involved in lipid metabolism, consistent with known effects of BAs (Table 2).Table 2Pathway analysis of 516 genes induced by both human low-12HBA and mouse low-12HBA poolsGO termDescriptionCountPercentagepGenesList totalPopulation hitsPopulation total-Fold enrichmentFalse discovery rateGO:0006629Lipid metabolic process397.881.60E−11HSD17B11, ACOX1, CHKA, PTGS2, ALOXE3, CHKB, EHHADH, ABHD3, ASAH2, APOB, INSIG2, APOBR, ACOT12, ETNK1, MGLL, PLCD1, HSD17B4, PLCB1, ACAA1B, ACSL5, SCD1, SOAT2, MOGAT2, CUBN, NCEH1, LIPA, PLB1, E PHX2, ACACB, LPIN2, LPCAT3, LPCAT4, GDPD2, MTTP, PCX, CLPS, HMGCS2, LIPH, VLDLR42545918,0823.615009612.70E−08GO:0016491Oxidoreductase activity408.081.57E−08HSD17B11, ME1, ACOX1, CYP24A1, ALDH1L1, CYP3A25, PTGS2, ALOXE3, EHHADH, OSGIN1, ALDH1L2, ALDH3A2, MTHFD2, FMO5, ADH1, ALDH1A3, ADH4, GPX3, ALDH1A7, SMOX, HSD17B4, NOS2, QSOX1, CYP3A44, ADH6A, SCD1, SUOX, CYP3A13, CYP3A11, DHRS9, POR, AKR1B8, CYP4A10, DHRS1, AKR1B7, RDH10, HSDL2, TPH1, BCO2, RETSAT41560417,4462.784010212.34E−05GO:0052689Carboxylic ester hydrolase activity132.634.39E−07NCEH1, ACOT2, ABHD3, ACOT1, ACOT5, ACOT4, ACOT3, ACOT12, CES2A, CES1G, MGLL, LIPH, CES2B4158117,4466.746928456.56E−04GO:0055114Oxidation-reduction process387.681.66E−06HSD17B11, ME1, ACOX1, CYP24A1, ALDH1L1, CYP3A25, PTGS2, ALOXE3, EHHADH, ALDH1L2, ALDH3A2, MTHFD2, FMO5, ADH1, ALDH1A3, ADH4, GPX3, ALDH1A7, SMOX, HSD17B4, NOS2, QSOX1, CYP3A44, SCD1, SUOX, CYP3A13, CYP3A11, DHRS9, POR, AKR1B8, DHRS1, CYP4A10, AKR1B7, RDH10, HSDL2, TPH1, BCO2, RETSAT42567618,0822.391632440.00281558GO:0005102Receptor binding275.456.36E−06PVR, ACOX1, FGF15, EHHADH, ACOT2, ACOT4, SCT, ANG, GSTK1, CCDC129, NOS2, HSD17B4, FGF3, MATK, H2-Q2, PLAT, ICOSL, H2-Q1, BTNL2, EPHX2, CD160, PLAUR, LAMA3, GM8909, LAMA5, H2-BL, VEGFA41541217,4462.75495380.00950826GO:0047617Acyl-CoA hydrolase activity61.218.08E−06ACOT12, ACOT2, ACOT1, ACOT5, ACOT4, ACOT34151317,44619.40240960.01207519GO:0006631Fatty acid metabolic process153.031.89E−05SCD1, ACOX1, LIPA, PTGS2, ALOXE3, EHHADH, ACACB, LPIN2, CYP4A10, ACOT12, MGLL, FABP2, HSD17B4, ACAA1B, ACSL542515618,0824.090950230.03207602GO:0000038Very long–chain fatty acid metabolic process61.212.44E−05ACOX1, ACOT2, HSD17B4, ACOT5, ACOT4, ACOT34251618,08215.95470590.04137758 Open table in a new tab Next, we focused on the subset of these genes that are differentially regulated by low- versus high-12HBA pools, with a particular focus on those for which the effects were shared between human and mouse pools. We used a threshold of log2FC > 1.0 and padj < 0.05 for differential expression. The majority of genes did not reach this threshold, indicating that they are similarly regulated by both low- and high-12HBA pools or are differentially regulated by low versus high 12HBAs in human pools only or in mouse pools only (Fig. S1B). These genes included canonical FXR targets such as Fgf15, Fabp6 (encoding the ileal bile acid–binding protein, Ibabp), and Slc51b (encoding the basolateral BA efflux transporter Ostβ), and we validated these by qPCR (Fig. 1C). There were 44 genes that were preferentially induced by H10 and M10 compared with H90 and M90, respectively (Fig. 1D). Among these, we noted that several are known transcriptional targets of VDR. These included Cyp24a1, S100g, and Cyp3a11, and we validated these by qPCR (Fig. 1E). This is consistent with the concepts that (i) certain BAs, especially the non-12HBA LCA and its conjugates, can activate VDR in the micromolar range (10Makishima M. Lu T.T. Xie W. Whitfield G.K. Domoto H. Evans R.M. Haussler M.R. Mangelsdorf D.J. Vitamin D receptor as an intestinal bile acid sensor.Science. 2002; 296 (12016314): 1313-131610.1126/science.1070477Crossref PubMed Scopus (958) Google Scholar), and (ii) the low-12HBA pools (i.e. H10 and M10) contain more LCA (9 μm, as opposed to 1 μm in the high-12HBA pools). Next, we validated the RNA-Seq findings in multiple experimental systems. Using organoids derived from multiple mice, we confirmed that all BA pools induced expression of FXR targets Fgf15 and Fabp6 (Supp. Fig. S1C). We also confirmed that VDR targets Cyp24a1 and S100g were preferentially induced by low-12HBA pools (Fig. S1D). We found that delivery in micelles was not required and that BAs per se were sufficient to induce Fgf15, Fabp6, and S100g (Fig. S1E). 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