Induction of farnesoid X receptor signaling in germ-free mice colonized with a human microbiota
2016; Elsevier BV; Volume: 58; Issue: 2 Linguagem: Inglês
10.1194/jlr.m072819
ISSN1539-7262
AutoresAnnika Wahlström, Petia Kovatcheva‐Datchary, Marcus Ståhlman, Muhammad-Tanweer Khan, Fredrik Bäckhed, Hanns‐Ulrich Marschall,
Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoThe gut microbiota influences the development and progression of metabolic diseases partly by metabolism of bile acids (BAs) and modified signaling through the farnesoid X receptor (FXR). In this study, we aimed to determine how the human gut microbiota metabolizes murine BAs and affects FXR signaling in colonized mice. We colonized germ-free mice with cecal content from a mouse donor or feces from a human donor and euthanized the mice after short-term (2 weeks) or long-term (15 weeks) colonization. We analyzed the gut microbiota and BA composition and expression of FXR target genes in ileum and liver. We found that cecal microbiota composition differed between mice colonized with mouse and human microbiota and was stable over time. Human and mouse microbiota reduced total BA levels similarly, but the humanized mice produced less secondary BAs. The human microbiota was able to reduce the levels of tauro-β-muricholic acid and induce expression of FXR target genes Fgf15 and Shp in ileum after long-term colonization. We show that a human microbiota can change BA composition and induce FXR signaling in colonized mice, but the levels of secondary BAs produced are lower than in mice colonized with a mouse microbiota. The gut microbiota influences the development and progression of metabolic diseases partly by metabolism of bile acids (BAs) and modified signaling through the farnesoid X receptor (FXR). In this study, we aimed to determine how the human gut microbiota metabolizes murine BAs and affects FXR signaling in colonized mice. We colonized germ-free mice with cecal content from a mouse donor or feces from a human donor and euthanized the mice after short-term (2 weeks) or long-term (15 weeks) colonization. We analyzed the gut microbiota and BA composition and expression of FXR target genes in ileum and liver. We found that cecal microbiota composition differed between mice colonized with mouse and human microbiota and was stable over time. Human and mouse microbiota reduced total BA levels similarly, but the humanized mice produced less secondary BAs. The human microbiota was able to reduce the levels of tauro-β-muricholic acid and induce expression of FXR target genes Fgf15 and Shp in ileum after long-term colonization. We show that a human microbiota can change BA composition and induce FXR signaling in colonized mice, but the levels of secondary BAs produced are lower than in mice colonized with a mouse microbiota. The gut microbiota influences the development and progression of metabolic diseases such as obesity, diabetes, and atherosclerosis (1.Karlsson F. Tremaroli V. Nielsen J. Backhed F. Assessing the human gut microbiota in metabolic diseases.Diabetes. 2013; 62: 3341-3349Crossref PubMed Scopus (298) Google Scholar). The influence of the gut microbiota on metabolic diseases may in part be mediated by modification of bile acids (BAs), which are now recognized to have a central role as signaling molecules through activation of receptors involved in metabolic pathways such as the nuclear farnesoid X receptor (FXR) and G-protein-coupled receptor TGR5 (2.Lefebvre P. Cariou B. Lien F. Kuipers F. Staels B. Role of bile acids and bile acid receptors in metabolic regulation.Physiol. Rev. 2009; 89: 147-191Crossref PubMed Scopus (1158) Google Scholar, 3.Ma H. Patti M.E. Bile acids, obesity, and the metabolic syndrome.Best Pract. Res. Clin. Gastroenterol. 2014; 28: 573-583Crossref PubMed Scopus (123) Google Scholar, 4.Sato H. Macchiarulo A. Thomas C. Gioiello A. Une M. Hofmann A.F. Saladin R. Schoonjans K. Pellicciari R. Auwerx J. Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies.J. Med. Chem. 2008; 51: 1831-1841Crossref PubMed Scopus (238) Google Scholar). FXR is a powerful regulator of lipid and glucose metabolism and is activated by the primary BAs chenodeoxycholic acid (CDCA) and cholic acid (CA), the secondary BA deoxycholic acid (DCA), and to a lesser extent by the secondary BA lithocholic acid (LCA) (5.Parks 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. et al.Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1829) Google Scholar, 6.Wang 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: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). The conjugated forms of these BAs, amidated with glycine (G) or taurine (T), can also activate FXR (5.Parks 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. et al.Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1829) Google Scholar, 6.Wang 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: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). BA composition differs between humans and mice; in addition to the BAs found in humans, mice have the primary BAs α- and β-muricholic acid (α/βMCA), which in their T-conjugated forms function as FXR antagonists (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 8.Li F. Jiang C.T. Krausz K.W. Li Y.F. Albert I. Hao H.P. Fabre K.M. Mitchell J.B. Patterson A.D. Gonzalez F.J. Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity.Nat. Commun. 2013; 4: 2384Crossref PubMed Scopus (474) Google Scholar). The gut microbiota deconjugates and subsequently metabolizes primary BAs to secondary BAs in the gut and will thereby modify the extent of FXR activation (9.Wahlström A. Sayin S. Marschall H. Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.Cell Metab. 2016; 24: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar). BA synthesis is regulated by FXR mainly via two downstream targets, fibroblast growth factor 19 (FGF19; in mice, FGF15) in ileum and small heterodimer partner (SHP) in the liver, both of which inhibit expression of the rate-limiting enzyme in BA synthesis, cholesterol 7α-hydroxylase (CYP7A1) in liver. We have previously shown that differences in BA metabolism between germ-free (GF) and conventionally raised (CONV-R) mice are dependent on the FXR antagonist tauro-β-muricholic acid (TβMCA) (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). TβMCA is not metabolized in the absence of bacteria. Thus, GF mice show accumulation of TβMCA and reduced FXR signaling (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar) and, consequently, increased BA synthesis. GF mice colonized with a human microbiota (here termed humanized mice) have been used to study the effect of environmental and genetic factors on gut microbiota and host physiology. These studies showed that the human microbiota is able to establish a stable community in the recipient mice with similarities to the community in the donor sample (10.Turnbaugh P.J. Ley R.E. Mahowald M.A. Magrini V. Mardis E.R. Gordon J.I. An obesity-associated gut microbiome with increased capacity for energy harvest.Nature. 2006; 444: 1027-1031Crossref PubMed Scopus (8177) Google Scholar, 11.Turnbaugh P.J. Ridaura V.K. Faith J.J. Rey F.E. Knight R. Gordon J.I. The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice.Sci. Transl. Med. 2009; 1: 6ra14Crossref PubMed Scopus (2061) Google Scholar, 12.Seedorf H. Griffin N.W. Ridaura V.K. Reyes A. Cheng J. Rey F.E. Smith M.I. Simon G.M. Scheffrahn R.H. Woebken D. et al.Bacteria from diverse habitats colonize and compete in the mouse gut.Cell. 2014; 159: 253-266Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). However, it is unclear if a human gut microbiota can metabolize murine BAs and induce FXR signaling. This would be important to elucidate in order to improve translation of results from mouse models into a human setting. Here we aimed to determine whether a human gut microbiota can deconjugate and metabolize primary murine BAs and thus increase FXR activation in the recipient mice. GF female Swiss Webster mice were maintained in flexible plastic gnotobiotic isolators under a strict 12 h light cycle and fed an autoclaved chow diet (Lab diet, St. Louis, MO) ad libitum. GF isolators were routinely tested for sterility by culturing and PCR analysis of feces amplifying the 16S rRNA gene. Colonization was performed by diluting human fecal or mouse cecal samples (∼0.5 g) in 5 ml reduced PBS; 0.2 ml of this suspension was introduced by gavage into each GF mouse. The human fecal sample from the first donor was obtained from a healthy 38-year-old female volunteer, and the sample from the second donor was obtained from a healthy 40-year-old male volunteer. The samples were obtained shortly before colonization and immediately (within 5 min) diluted and introduced into the GF mice by gavage within 2 h after dilution. The mouse cecal sample was obtained from a 9-week-old CONV-R Swiss Webster female mouse. The GF and humanized (CONV-H) mice were maintained in the isolators, and the conventionalized (CONV-M) mice were transferred from the isolators into conventional cages after colonization. Mice were colonized at 8–15 weeks of age and maintained for 2 weeks or 15 weeks after colonization. Blood was collected from the portal vein and inferior caval vein under deep isoflurane-induced anesthesia following 4 h of fasting. The mice were then euthanized, and tissues (liver, gallbladder, ileum, and cecum) were harvested. All tissues were immediately frozen in liquid nitrogen and stored at −80°C until further processed. All experiments were performed in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animal using protocols approved by the University of Gothenburg Animal Studies Committee. The use of human feces from healthy volunteers was approved by the Regional Ethics Committee in Gothenburg. Genomic DNA was isolated, and the V4 region of the bacterial 16S rRNA gene was amplified with PCR (see supplemental data). The amplified DNA was sequenced at Genomic Core Facility at University of Gothenburg, and the sequencing data were analyzed using the software package Quantitative Insights into Microbial Ecology (QIIME), version 1.8.0. Linear discriminant analysis effect size (LEfSe) algorithm (13.Segata N. Izard J. Waldron L. Gevers D. Miropolsky L. Garrett W.S. Huttenhower C. Metagenomic biomarker discovery and explanation.Genome Biol. 2011; 12: R60Crossref PubMed Scopus (7551) Google Scholar) was used to identify taxa that discriminated cecal microbiota profiles according to the colonization origin (see supplemental data). BAs in liver, gallbladder, cecum, and serum from portal and caval veins were analyzed using ultra-performance LC/MS/MS and quantified using a combination of unlabeled standards and deuterium-labeled internal standards [see supplemental data and Ref. (14.Tremaroli V. Karlsson F. Werling M. Stahlman M. Kovatcheva-Datchary P. Olbers T. Fandriks L. le Roux C.W. Nielsen J. Backhed F. Roux-en-Y gastric bypass and vertical banded gastroplasty induce long-term changes on the human gut microbiome contributing to fat mass regulation.Cell Metab. 2015; 22: 228-238Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar)]. The whole gallbladder, ∼50 mg of liver and cecum, and 50 µl serum were used for the analyses. Suspensions with fecal samples from the first human donor were incubated with TCA or CA to test the capacity of a human microbiota to metabolize BAs in vitro (see supplemental data). Approximately 30 mg of liver or distal ileum was homogenized using TissueLyzer (Qiagen), and total RNA was isolated using RNeasy kit (Qiagen). High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize 20 µl cDNA templates from 500 ng purified RNA using random hexamer primers, and the products were diluted 7× before use in subsequent reactions. 1× SYBR Green Master Mix buffer (Thermo Scientific) was used for quantitative RT-PCR at final reaction volumes of 10 µl. Gene-specific primers (900 nM) were used in each reaction, and all results were normalized to the ribosomal protein L32 mRNA [primer sequences were previously described (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar)]. Assays were performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems) or CFX96 Real-Time System (Bio-Rad Laboratories). The reactions were analyzed using the ΔΔCT analysis method. Data are presented as mean ± SEM. Resource equation method was used to determine the adequate sample size (15.Festing M.F. Altman D.G. Guidelines for the design and statistical analysis of experiments using laboratory animals.ILAR J. 2002; 43: 244-258Crossref PubMed Scopus (572) Google Scholar). Significant differences between the groups were analyzed with one-way ANOVA followed by Tukey's honestly significant difference using R for the BA analysis and one-way ANOVA followed by Dunnett's multiple comparison tests using GraphPad Prism 6 software for the gene expression analysis. We analyzed cecal microbiota after short-term (2 weeks) or long-term (15 weeks) colonization of GF mice with cecal content from a mouse donor (recipient mice termed CONV-M) or feces from a human donor (recipient mice termed CONV-H), to investigate if there was a difference in their gut microbiota composition. Principal coordinate analysis of weighted UniFrac distances showed a clear separation between the microbial communities in the mice driven by the origin of the donor microbiota observed at the first principal coordinate (x-axis), which explained more than 66% of the variance (Fig. 1A). The second principal coordinate (y-axis) accounted for 15% of the variance and separated the microbial communities by host (human vs. mouse) (Fig. 1A). In particular, the community of the human donor sample separated from the communities of CONV-H mice, CONV-M mice, and the mouse donor sample. In contrast, the community of the mouse donor sample and the CONV-M recipient mice clustered together (Fig. 1A). Next, we analyzed the microbiota composition of the donors and recipient mice and found that at order level the cecal microbiota of the CONV-M mice had a higher representation of Clostridiales, whereas CONV-H mice were characterized by a more pronounced representation of Verrucomicrobiales, Burkoholderiales, and Erysipelotrichales (Fig. 1B). The lower abundance of Clostridiales in the humanized mice was not caused by low representation of this taxon in the human donor samples. In fact, Clostridiales was highly abundant in the human donor sample, in agreement with a previous report (12.Seedorf H. Griffin N.W. Ridaura V.K. Reyes A. Cheng J. Rey F.E. Smith M.I. Simon G.M. Scheffrahn R.H. Woebken D. et al.Bacteria from diverse habitats colonize and compete in the mouse gut.Cell. 2014; 159: 253-266Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). In contrast, the human donor sample had relatively low abundance of Verrucomicrobiales, Burkoholderiales, Erysipelotrichales, and Bacteroidales. The microbiota composition of CONV-M mice was similar to the mouse donor sample apart from a lower representation of Lactobacillales and higher representation of Campylobacterales. The mice colonized for 15 weeks had similar microbiota composition at the order level as the mice colonized for 2 weeks, which indicates that the microbiota composition remains stable over time (Fig. 1B). These findings were also confirmed in mice colonized with a second human donor (supplemental Fig. S1A). To identify taxonomic differences in the microbiota between mice colonized with mouse and human microbiota, we applied an LEfSe algorithm (13.Segata N. Izard J. Waldron L. Gevers D. Miropolsky L. Garrett W.S. Huttenhower C. Metagenomic biomarker discovery and explanation.Genome Biol. 2011; 12: R60Crossref PubMed Scopus (7551) Google Scholar). After 2 weeks' colonization, the cecal microbiota of CONV-M mice was enriched in members of Bacilli such as Lactobacillus and Staphylococcus and the Epsilonproteobacteria-Helicobacteraceae (supplemental Fig. S2). Several members of the Bacteroidetes phylum such as Prevotella, Parabacteroides, andBacteroides, as well as an unclassified RF39 Mollicutes and a member of the Proteobacteria phylum, Desulfovibrio, were also enriched in the CONV-M mice after 2 weeks of colonization (supplemental Fig. S2). The taxa enriched in the cecal microbiota of CONV-H mice after 2 weeks of colonization were affiliated to Akkermansia from the Verrucomicrobia phylum, several members of class Erysipelotrichi such as Allobaculum, Coprobacillus, Eubacterium dollichum and cc115 and Oxalobacter formigenes, Suturella and Bilophila from the Proteobacteria phylum. Most of the taxonomic clades that were differently enriched after 2 weeks' colonization showed a similar pattern after 15 weeks' colonization demonstrating stability of the bacterial communities in the colonized mice over time. Some of the additional differences we found after 15 weeks' colonization were enrichment of Bifidobacterium, the genus Anaeroplasma from the Tenerecutes phylum and Allobaculum in the CONV-M mice and enrichment of Enterobacterales in the CONV-H mice (supplemental Fig. S3). In summary, we show that the microbiota composition differs between mice colonized with mouse and human microbiota, and the major differences between the groups are preserved over long-term colonization. Next, we evaluated how colonization with mouse or human microbiota affected BA levels and composition in liver, gallbladder, and cecum of the recipient mice. Because of the major differences in relative gallbladder and cecum weights between the GF and colonized mice (supplemental Tables S2 and S3), we calculated the amounts of BAs in the whole organ rather than the amount of BAs per milligram of tissue. Total BA levels (referring to the total amounts of BAs analyzed) in the liver did not change significantly after the short-term colonization, but after 15 weeks' colonization, the total BAs were reduced in both CONV-M and CONV-H mice compared with GF counterparts (Fig. 2A; supplemental Table S1). In gallbladder, total BAs were significantly reduced in all colonized mice at both time points (Fig. 2B; supplemental Table S2), and BAs levels in cecum were reduced in all groups except for CONV-M mice after 15 weeks (Fig. 2C; supplemental Table S3). Analysis of individual BAs showed that TβMCA and TCA were the predominant BAs in all compartments analyzed in GF mice (Fig. 2A–E; supplemental Tables S1–S5). In the liver, short-term colonization with mouse or human microbiota did not change the levels of TβMCA or TCA significantly, but colonization with mouse microbiota resulted in the presence of tauro-ω-muricholic acid (TωMCA), a secondary BA derived from TβMCA (Fig. 2A; supplemental Table S1). After 15 weeks' colonization, the levels of TβMCA decreased in both CONV-M and CONV-H mice, while the levels of TCA remained unchanged. In gallbladder, the levels of TβMCA were significantly reduced after 2 weeks' colonization with either mouse or human microbiota, while the levels of TCA were reduced only in the CONV-H mice (Fig. 2B; supplemental Table S2). In cecum, mouse microbiota had a large effect on BA composition. The major cecal BAs in CONV-M mice were ωMCA and DCA (secondary BAs derived from TβMCA and TCA, respectively) (Fig. 2C; supplemental Table S3). In cecum of the humanized mice, the most prevalent BA was βMCA followed by TβMCA, and there were only small amounts of DCA and almost no ωMCA (Fig. 2C; supplemental Table S3). To rule out the possibility that the low amounts of secondary BAs in the humanized mice were specific to this particular human donor, we colonized GF mice with human feces from a second donor and analyzed the BA composition. Levels of total and secondary BAs in cecum of mice colonized with feces from the second human donor did not differ significantly from the first donor (supplemental Fig. S1B) indicating that low production of secondary BAs in humanized mice might be a general finding. The relative amount of secondary BAs in cecum was low in the humanized mice, while secondary BAs constituted more than 50% of the cecal BAs in mice colonized with a mouse microbiota (supplemental Fig. S4A–F). In addition, the relative amount of unconjugated BAs was higher in the CONV-M mice after 15 weeks' colonization compared with the humanized mice, suggesting a difference in bile salt hydrolase activity between the mouse and human microbiota (supplemental Fig. S5A–F). We also analyzed the BA composition in portal vein serum, which contains reabsorbed BAs from the intestine and thus reflects the BA composition in ileum. Total BA levels and, in particular, TβMCA levels were dramatically reduced in the portal vein serum by colonization with mouse or human microbiota (Fig. 2D; supplemental Table S4). We found a similar pattern of BA changes in caval vein serum, with particularly low BA levels in the CONV-M mice 2 weeks after colonization (Fig. 2E; supplemental Table S5). The total concentration of BAs were >30-fold lower in the peripheral circulation compared with the levels in portal vein demonstrating the capacity of the liver to maintain BAs within the enterohepatic circulation. Furthermore, we found high levels of CA in both portal and caval vein serum after 15 weeks' colonization with mouse microbiota (Fig. 2D, E; supplemental Tables S4 and S5). In addition to the regular BAs we also analyzed 3β-hydroxylated iso-BAs in cecum and found significant amounts of iso-DCA after 2 weeks' colonization both in CONV-M and CONV-H mice and after 15 weeks' colonization the levels were decreased in both groups of colonized mice (Fig. 2F). We also investigated the levels of sulfated BAs in cecum and found that mice colonized with a human microbiota had four times higher levels of CA-7 sulfate than mice colonized with mouse microbiota (Fig. 2G). Mice colonized with the second human donor had similar levels of CA-7 sulfate as mice colonized with the first human donor, but the levels of iso-DCA were lower at 2 weeks' colonization (supplemental Fig. S1C, D). As expected, we did not detect any iso-DCA in the GF mice and only traces of CA-7 sulfate (Fig. 2F, G; supplemental Fig. S1C, D). Thus, CONV-H mice deconjugated primary BAs as CONV-M counterparts but only metabolized CA and not βMCA. In addition, humanized mice generated more CA-7 sulfate than mice colonized with a mouse microbiota. To further investigate the potential of a human microbiota to deconjugate, dehydroxylate, and sulfate TCA, we performed in vitro experiments with fecal samples from the first human donor in culture medium supplemented with TCA or CA. BA analysis showed that the human microbiota sufficiently deconjugated TCA into CA, but there was only minor conversion into DCA and no production of CA-7 sulfate (supplemental Fig. S6A, B). To evaluate if the changes in BAs composition following colonization with a human microbiota would be sufficient to increase FXR signaling, we analyzed gene expression of FXR target genes in ileum and liver. Colonization with mouse microbiota induced expression of both Fgf15 and Shp in ileum already after 2 weeks' colonization (Fig. 3A), and the expression was further increased after 15 weeks' colonization (Fig. 3B), which is in agreement with our previous study (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). Colonization with a human microbiota showed a tendency of induction after 2 weeks' colonization and reached significant levels after 15 weeks' colonization. We also analyzed gene expression of the ileal BA transporter Ibat and found a significant reduction in the mice colonized with a mouse microbiota at both time points and a minor reduction in the humanized mice (Fig. 3A, B). Furthermore, we analyzed gene expression of liver enzymes involved in BA synthesis and found that colonization with mouse microbiota (both short term and long term) promoted decreased gene expression of the rate-limiting enzyme CYP7A1, and the humanized mice showed a similar expression pattern, although the changes were not significant (Fig. 3C, D). The changes in Cyp7a1 expression corresponded inversely with the changes in Fgf15 expression in ileum; this reciprocal pattern has been observed in previous studies (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 8.Li F. Jiang C.T. Krausz K.W. Li Y.F. Albert I. Hao H.P. Fabre K.M. Mitchell J.B. Patterson A.D. Gonzalez F.J. Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity.Nat. Commun. 2013; 4: 2384Crossref PubMed Scopus (474) Google Scholar, 16.Holt J.A. Luo G. Billin A.N. Bisi J. McNeill Y.Y. Kozarsky K.F. Donahee M. Wang D.Y. Mansfield T.A. Kliewer S.A. et al.Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.Genes Dev. 2003; 17: 1581-1591Crossref PubMed Scopus (532) Google Scholar, 17.Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. et al.Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 18.Lundåsen T. Galman C. Angelin B. Rudling M. Circulating intestinal fibroblast growth factor 19 has a pronounced diurnal variation and modulates hepatic bile acid synthesis in man.J. Intern. Med. 2006; 260: 530-536Crossref PubMed Scopus (321) Google Scholar, 19.Kim I. Ahn S.H. Inagaki T. Choi M. Ito S. Guo G.L. Kliewer S.A. Gonzalez F.J. Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine.J. Lipid Res. 2007; 48: 2664-2672Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). The gene expression of sterol 12-α-hydroxylase (CYP8B1), which is required for CA synthesis, was increased after colonization (Fig. 3C, D). The expression of Cyp7a1 and Cyp8b1 is regulated by a negative feedback mechanism involving FXR and SHP in the liver (20.Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar, 21.Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. et al.A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1500) Google Scholar, 22.Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1386) Google Scholar). However, our finding that Cyp7a1 and Cyp8b1 are differently regulated is supported by several previous studies (7.Sayin 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-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 23.Out C. Hageman J. Bloks V.W. Gerrits H. Sollewijn Gelpke M.D. Bos T. Havinga R. Smit M.J. Kuipers F. Groen A.K. Liver receptor homolog-1 is critical for adequate up-regulation of Cyp7a1 gene transcription and bile salt synthesis during bile salt sequestration.Hepatology. 2011; 53: 2075-2085Crossref PubMed Scopus (42) Google Scholar, 24.Inoue Y. Yu A.M. Yim S.H. Ma X. Krausz K.W. Inoue J. Xiang C.C. Brownstein M.J. Eggertsen G. Bjorkhem I. et al.Regulation of bile acid biosynthesis by hepatocyte nucle
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