《人体的社交网络——共生微生物》课外文献阅读：Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis

ARTICLES natre medicine Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis Anna Worthmann,, Clara John,, Malte C Ruhlemann20, Miriam Baguhl', Femke-Anouska Heinsen2o Nicola Schaltenbergl, Markus Heine, Christian Schlein, Ioannis Evangelakos', Chieko Mineo,, Markus Fischer 3 Maura Dandri, Claus Kremoser6, Ludger Scheja, Andre Franke 2o, Philip W Shaul& Joerg Heerenl( 2 Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it g entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. ere we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue( BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on s hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1(CYP7B1)and results in increased plasma levels,as G well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production 8 bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol 3 metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism The gut microbiota contributes to energy homeostasis, and altera- form or after its conversion into bile acids. The integrative regulation ions in its composition are associated with cardiovascular and meta- of these processes is not fully understood but is of clinical relevance, a bolic diseases such as atherosclerosis, thrombosis, type 2 diabetes as an imbalance leads to increased plasma concentrations of athero- g certain cancers2-7. Microbial colonization of the gut begins directly Bile acids are exclusively synthesized in the liver by a number after birth and develops in response to genetic and environmental of enzymatic reactions using two different routes. The classical bile factors, especially the amount and composition of the diet. 9. Recent acid synthesis pathway starts with the rate-limiting enzyme choles- tudies in mice indicate that decreasing the housing temperature alters terol 7-a-hydroxylase(encoded by CYP7AI), and it prevails under the gut microbiota, which in turn enhances the thermogenic capacity normal conditions. Bile acids can also be formed by the alternative of adipose tissues and, hence, energy expenditure of the host o, The bile acid synthesis pathway, which is initiated by the action of sterol exposure of mammals to temperatures below their thermoneutral 27-hydroxylase(encoded by CYP27AI)followed by 25-hydroxycholes- N threshold (-30oC for mice and C for humans)is regarded as a terol7-a-hydroxylase(encoded by CYP7B1)8.Both synthesis routes cold stimulus, which activates BAT and promotes the appearance of principally generate the same bile acid species, which are subsequently brown-like beige adipocytes in white adipose tissue(WAT), increas- conjugated with glycine or taurine. The physiological relevance of the ing energy expenditure through non-shivering thermogenesis2-14. alternative pathway is not well understood, but it has been postulated Because this process needs ample amounts of energy, BaT activity is to be important for the metabolism of hydroxylated cholesterol that is a major determinant of plasma glucose and triglyceride levels 5, 6. derived from extrahepatic organs, such as the brain 8, 9. In addition, activated BAT protects from atherosclerosis by acceler- One important physiological role of bile acids after their biliary ating the apolipoprotein-E-dependent clearance of cholesterol-rich secretion in response to food ingestion is to mediate the emulsifica remnant particles by the liver. The resulting excess cholesterol in tion and absorption of dietary lipids20. In addition, bile acids function hepatocytes is recycled to the blood circulation as part of lipoproteins. as signaling molecules in various tissues. In enterocytes, they acti Alternatively, cholesterol is secreted into bile either in an unmodified vate the transcription factor farnesoid X receptor(FXR)to generate logy, Department of Pediatrics, University of Texas Southwestern Me nter, Dallas, Texas, USA -Institute of Food Chemistry, Germany. Department of Internal Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5Phenex Pharmaceuticals AG, Heidelberg, Germany. These authors contributed equally to this work. Correspondence should be addressed to J H.(heeren @uke. de) Received 27 June 2016; accepted 17 May 2017: published online 12 June 2017; doi: 10.1038/nm. 4357 DNLINE PUBLICATION

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. a r t i c l e s nature medicine advance online publication  The gut microbiota contributes to energy homeostasis1, and altera￾tions in its composition are associated with cardiovascular and meta￾bolic diseases such as atherosclerosis, thrombosis, type 2 diabetes and non-alcoholic fatty liver disease, as well as the development of certain cancers2–7. Microbial colonization of the gut begins directly after birth and develops in response to genetic and environmental factors, especially the amount and composition of the diet8,9. Recent studies in mice indicate that decreasing the housing temperature alters the gut microbiota, which in turn enhances the thermogenic capacity of adipose tissues and, hence, energy expenditure of the host10,11. The exposure of mammals to temperatures below their thermoneutral threshold (~30 °C for mice and ~24 °C for humans) is regarded as a cold stimulus, which activates BAT and promotes the appearance of brown-like beige adipocytes in white adipose tissue (WAT), increas￾ing energy expenditure through non-shivering thermogenesis12–14. Because this process needs ample amounts of energy, BAT activity is a major determinant of plasma glucose and triglyceride levels15,16. In addition, activated BAT protects from atherosclerosis by acceler￾ating the apolipoprotein-E-dependent clearance of cholesterol-rich remnant particles by the liver17. The resulting excess cholesterol in hepatocytes is recycled to the blood circulation as part of lipoproteins. Alternatively, cholesterol is secreted into bile either in an unmodified form or after its conversion into bile acids. The integrative regulation of these processes is not fully understood but is of clinical relevance, as an imbalance leads to increased plasma concentrations of athero￾genic lipoproteins. Bile acids are exclusively synthesized in the liver by a number of enzymatic reactions using two different routes. The classical bile acid synthesis pathway starts with the rate-limiting enzyme choles￾terol 7-α-hydroxylase (encoded by CYP7A1), and it prevails under normal conditions18. Bile acids can also be formed by the alternative bile acid synthesis pathway, which is initiated by the action of sterol 27-hydroxylase (encoded by CYP27A1) followed by 25-hydroxycholes￾terol 7-α-hydroxylase (encoded by CYP7B1)18. Both synthesis routes principally generate the same bile acid species, which are subsequently conjugated with glycine or taurine. The physiological relevance of the alternative pathway is not well understood, but it has been postulated to be important for the metabolism of hydroxylated cholesterol that is derived from extrahepatic organs, such as the brain18,19. One important physiological role of bile acids after their biliary secretion in response to food ingestion is to mediate the emulsifica￾tion and absorption of dietary lipids20. In addition, bile acids function as signaling molecules in various tissues. In enterocytes, they acti￾vate the transcription factor farnesoid X receptor (FXR) to generate 1Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 2Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany. 3Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 4Institute of Food Chemistry, University of Hamburg, Hamburg, Germany. 5Department of Internal Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 6Phenex Pharmaceuticals AG, Heidelberg, Germany. 7These authors contributed equally to this work. Correspondence should be addressed to J.H. (heeren@uke.de). Received 27 June 2016; accepted 17 May 2017; published online 12 June 2017; doi:10.1038/nm.4357 Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis Anna Worthmann1,7, Clara John1,7, Malte C Rühlemann2 , Miriam Baguhl1, Femke-Anouska Heinsen2 , Nicola Schaltenberg1, Markus Heine1, Christian Schlein1, Ioannis Evangelakos1, Chieko Mineo3, Markus Fischer4, Maura Dandri5, Claus Kremoser6, Ludger Scheja1, Andre Franke2 , Philip W Shaul3 & Joerg Heeren1 Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism

ARTICLES a negative endocrine feedback signal to th ia fibroblast growth warm-housed mice(Fig.). On the genus level, the abundance of factor(fgf)9inh Parabacteroides spp. was higher, whereas the abundance of undlassi cold- ances at the versus differ- cold in an ghe the 16S rR ent with studie exposurel0.. We examine that th Porphyrom uction

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Ar t i c l e s  advance online publication nature medicine a negative endocrine feedback signal to the liver via fibroblast growth factor (FGF) 19 in humans or via the mouse ortholog FGF15 (ref. 21). The mechanism involves the activation of the transcriptional co-repressor small heterodimeric partner (SHP; encoded by Nr0b2) in hepatocytes, which confers suppression of CYP7A1 expression and hence lowers bile acid synthesis. Both in liver and adipose tissue, bile￾acid-activated FXR has additional effects, including such as those on cholesterol, fatty acid and glucose metabolism20,21. Bile acids also stimulate signaling at the plasma membrane via the G-protein-cou￾pled bile acid receptor 1 (GPBAR1; also known as TGR5), thereby regulating diverse metabolic pathways22. In the context of energy metabolism, TGR5 stimulation triggers the secretion of the insulin secretagogue and anorexic hormone glucagon (encoded by Gcg) in L-cells of the intestine23. Moreover, in both mice and humans, bile acids increase energy expenditure via the stimulation of TGR5 on brown adipocytes24,25, which suggests a role of liver-derived bile acids as regulators of adaptive thermogenesis. Bile acids show mutual interactions with gut microbiota. On the one hand, the gut bacteria modify bile acids through both dehydroxy￾lation, to form secondary bile acids such as deoxycholic acid (DCA), and through deconjungation via the enzymatic activity of bile salt hydrolases26. On the other hand, bile acids exert species-dependent bacteriostatic effects, thereby affecting the composition of the intes￾tinal microbiota27. As previously mentioned, cold exposure has been found to induce alterations in the gut microbiota of mice10,11, and transplantation of feces from cold-exposed mice was found to improve the metabolic profile of recipient mice that were housed at ambient temperature10. Thus, these studies provided evidence that cold exposure has distinct effects on the bacterial composition of the intestine and can promote a healthy metabolic phenotype. However, how cold conditions change the composition of the gut microbiota remains elusive. Here we demonstrate that during BAT activation by cold expo￾sure, bile-acid-driven alterations in systemic cholesterol metabolism integrate the organ-specific responses in thermogenic adipose tis￾sues, liver and intestine. Cholesterol homeostasis was achieved by the stimulation of lipoprotein flux, increased bile acid synthesis via the alternative pathway and massive fecal excretion of conjugated bile acids, which ultimately shaped the gut microbiome and promoted adaptive thermogenesis in a cold environment. RESULTS Cold alters the gut microbiome Dietary composition is a determinant of the gut microbiome28,29, and cold exposure requires higher food intake to meet the increased energy demands for thermogenesis12. Here we combined dietary and cold intervention by challenging mice with a cholesterol-enriched high-fat diet (HFD) and keeping the mice under thermoneutral (30 °C; hereafter referred to as warm) versus cold (6 °C) conditions, respectively. First, we profiled the fecal microbiota from warm-housed and cold-housed mice by analyzing the DNA sequence encoding the 16S rRNA. Multidimensional scaling (MDS) based on weighted UniFrac distances revealed distinct clustering between microbiota from warm-housed versus cold-housed mice (Fig. 1a), which is consist￾ent with studies using mice that were subjected to prolonged cold exposure10,11. We examined the changes at the family level and found that the abundance of Lachnospiraceae and Deferribacteraceae fam￾ily members was higher, whereas the abundance of Clostridiales and Porphyromonadaceae family members was lower, in cold- versus warm-housed mice (Fig. 1b). On the genus level, the abundance of Parabacteroides spp. was higher, whereas the abundance of unclassi￾fied Porphyromonadaceae family members was clearly lower in cold￾housed versus warm-housed mice (Fig. 1b). These differences could be traced to operational taxonomic unit (OTU) abundances at the species level, which showed distinct patterns in warm-housed versus cold-housed mice (Fig. 1c). Although we could not observe differ￾ences in α-diversity at the genus level (Fig. 1d), we did note that cold exposure resulted in lower richness and a decreased Shannon index at the species level (Fig. 1e). We detected similar cold-dependent differences in chow-fed wild-type (WT) mice (Supplementary Fig. 1a–e) and in HFD-fed diabetic db/db mice (Supplementary Fig. 2a–e). Thus, these data clearly indicated that cold-housing resulted in an altered gut microbiome in mice that were fed either a chow diet or a cholesterol-enriched HFD. Cold induces the alternative bile acid synthesis pathway In accordance with increased energy demands, we observed higher food intake and fecal mass in cold-exposed mice than in mice that were housed at thermoneutrality (Fig. 1f and Supplementary Table 1a). Despite higher dietary lipid uptake, the cold-exposed mice had lower plasma levels of triglycerides and total cholesterol (Fig. 1g), owing to reduced levels of triglyceride-rich lipoproteins (Fig. 1h) and choles￾terol-transporting low-density lipoprotein (LDL) and high-density lipoprotein (HDL) (Fig. 1i). Next we administered radiolabeled cho￾lesterol by oral gavage and observed higher cholesterol uptake in all BAT depots in cold- versus warm-housed mice, despite the slightly lower levels of radioactivity in plasma (Fig. 1j). Notably, cholesterol uptake was also higher in the liver, which took up more than 10% of the administered dose in cold-exposed mice (Fig. 1k). Consistent with the known inefficient absorption of dietary cholesterol, the main proportion of radioactive tracer was still present in the small intes￾tine, irrespective of cold exposure (Fig. 1k). Taken together, these data demonstrate both accelerated lipoprotein processing in BAT and increased cholesterol transport from thermogenic adipose tissues toward the liver, which is consistent with previous studies17. To determine the fate of cholesterol, we measured bile acid levels in the liver by quantitative liquid chromatography coupled to mass spec￾trometry (LC–MS). We observed that the levels of most of the unconju￾gated bile acid (UBA; Fig. 2a) and conjugated bile acid (CBA; Fig. 2b) species were significantly higher in the cold-housed mice than in the warm-housed control mice and noted that the most prominent induc￾tions were for cholic acid (CA), ursodeoxycholic acid (UDCA) and muricholic acids (MCAs). However, the composition of the bile acid pool was not significantly affected (Supplementary Fig. 3a). In gall bladder, higher levels of CBA were observed in cold- versus warm￾housed mice (Fig. 2c), suggesting that there was efficient biliary CBA excretion via the bile salt export pump (BSEP; encoded by Abcb11). Consistent with normal liver function, cold exposure did not provoke liver inflammation or damage (Supplementary Fig. 3b,c). To determine the basis for cold-induced hepatic bile acid synthesis, we performed mRNA analysis of liver tissue to evaluate the expression of components of both the classical and alternative bile acid syn￾thesis pathways (Fig. 2d,e). Whereas Cyp7a1 expression was unal￾tered, Cyp7b1 expression was fourfold higher in cold-housed mice than in warm-housed mice (Fig. 2e), indicating specific upregulation of the alternative bile acid synthesis pathway. To unravel the poten￾tial role of FXR in cold-induced bile acid synthesis, we treated mice with the FXR agonist Phenex20606 (hereafter referred to as PX)30. As expected, treatment with PX resulted in significant induction

ARTICLES ●cod oTU 32 Palndibacter oTU 897 oTU 3 orphyromonadaceae aludibacter oTU 998 Osciibacter OTU 15 Deferribacteraceae oTu 7 Porphyromonadaceae Parabacteroides OT Ds1(52%) Clostridium xIva OTU 68 小导4杂P98 Genera Mean relative abundance (% Mean relative abundance(%) t Clostridiales ■Unc. Porphyromonas■Uc Anaerotruncus Uncl. Lachnospiraceae UncL. ■Unc. Erysipelotrichac.■uncL zEz9 Cold g8E8型 Food intake Feces Fracto k Figure 1 BAT activation alters the gut microbiome, lipoprotein levels and cholesterol uptake (a-e)MDS plot of weighted UniFrac distance(a), mean relative sundance of gut microbiota on family and genus level( b), hierarchical clustering of significant altered oTUs (c), as well as alpha diversity presented at the (w), n=9 mice)or 6C(cold (c), n=5 mice). (f)Food intake and amount of feces per 24 h period (n= 6 mice per group). (g) Plasma triglyceride (g)and cholesterol(Chol)levels(n= 5 mice per group).(h, i) Profiles of triglyceride-rich lipoproteins(TRLs)and cholesterol-rich LDL and hdl by measurement of triglycerides(h)and cholesterol (i)in FPLC fractions, respectively (pooled plasma samples of n=5 mice per group).( k)Organ uptake of 3H-radiolabeled P<0.05, ""P<0.01.*P<0.001: n.s., not significant; by pairwise Wilcoxon rank-sum test(d, e)or unpaired two-tailed Student's -test (f,gk-mgWAT holesterol 4 h after oral gavage, expressed as fold induction (i or as percentage of injected dose for liver and small intestine ( k)(n=7 mice per group). ing guinal WAT; epiwAT, epididymal WAT; ScBAT, subscapular BAT; iBAT, interscapular BAT: deBAT, deep cervical BAT. Throughout, data are mean +se

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. a r t i c l e s nature medicine advance online publication  MDS2 (20%) MDS1 (52%) Warm Cold Warm Warm *** *** *** *** ** *** *** *** * * Food intake Feces Cold Cold Warm Cold Warm Cold Warm Cold TRL Warm HDL LDL Cold Warm Cold Warm Cold Warm Cold Warm Cold Warm Warm Cold Warm Families Mean relative abundance (%) Porphyromonadaceae Mean relative abundance (%) Parabacteroides Uncl. Porphyromonad. Uncl. Ruminococc. Mucispirillum Anaerotruncus Other Uncl. Marinilabiliac. Uncl. Clostridiales Uncl. Synthrophom. Uncl. Lachnospiraceae Uncl. Erysipelotrichac. Genera Cold Cold log10 scaled abundance Family Lachnospiraceae Lachnospiraceae Porphyromonadaceae Porphyromonadaceae Porphyromonadaceae Ruminococcaceae Ruminococcaceae Erysipelotrichaceae Erysipelotrichaceae Other 30 2.0 160 3 2 1 0 120 80 40 0 1.5 1.0 0.5 0.0 20 10 0 5 200 3 10 8 6 4 2 0 2 1 0 0 10 20 Fraction 30 0 10 20 Fraction 30 150 100 TG Chol 50 0 4 3 2 1 0 5 4 3 2 1 0 n.s. n.s. ** ** Observed genera Amount (g/24 h) Organ uptake (cpm/organ, fold) Organ uptake (% of given dose) Concentration (mg/dl) TG (mg/dl) Chol (mg/dl) Shannon (genera) Observed OTUs Shannon (OTU) Marinilabiliaceae Clostridiales Synthrophomonadaceae Deferribacteraceae Deferribacteraceae Porphyromonadaceae Lachnospiraceae w6 Plasma Liver Spleen Kidney Muscle epiWAT ingWAT scBAT iBAT dcBAT Liver Duodenum Jejunum Ileum Intestine total w1w2 w9 w3 w7 w5w4 w8c2 c3 c1 c4c5 Genus OTU OTU_32 OTU_897 OTU_3 OTU_998 OTU_15 OTU_23 OTU_7 OTU_1 OTU_68 incertae sedis Paludibacter Paludibacter Paludibacter Oscillibacter Allobaculum Mucispirillum Parabacteroides Clostridium XIVa –2 –1 0 1 2 0.10 0.1 0.2 –0.10 –0.2 –0.1 0.05 –0.05 0 0 a b d f j k g h i c Figure 1 BAT activation alters the gut microbiome, lipoprotein levels and cholesterol uptake. (a–e) MDS plot of weighted UniFrac distance (a), mean relative abundance of gut microbiota on family and genus level (b), hierarchical clustering of significant altered OTUs (c), as well as alpha diversity presented at the genus level (d) or at the OTU level (e), on the basis of 16S-rRNA-encoding sequences in feces collected from mice that were housed at thermoneutrality (warm (w), n = 9 mice) or 6 °C (cold (c), n = 5 mice). (f) Food intake and amount of feces per 24 h period (n = 6 mice per group). (g) Plasma triglyceride (TG) and cholesterol (Chol) levels (n = 5 mice per group). (h,i) Profiles of triglyceride-rich lipoproteins (TRLs) and cholesterol-rich LDL and HDL by measurement of triglycerides (h) and cholesterol (i) in FPLC fractions, respectively (pooled plasma samples of n = 5 mice per group). (j,k) Organ uptake of 3H-radiolabeled cholesterol 4 h after oral gavage, expressed as fold induction (j) or as percentage of injected dose for liver and small intestine (k) (n = 7 mice per group). ingWAT, inguinal WAT; epiWAT, epididymal WAT; scBAT, subscapular BAT; iBAT, interscapular BAT; dcBAT, deep cervical BAT. Throughout, data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant; by pairwise Wilcoxon rank-sum test (d,e) or unpaired two-tailed Student’s t-test (f,g,j,k)

ARTICLES O warm●ca owam●cad O Warm 20 001● e 冒 g :: O wT. ● WT cold ● WT cold o cyp7br--cold 0.0 0-a Figure 2 BAT activation induces the alternative bile acid synthesis pathway independently of FXR. (a, b)Relative levels of unconjugated bile acid (UBA) species(a)and conjugated bile acid(CBA)species()in the livers of mice that were housed at thermoneutrality (warm, n=9 mice)or 6C (cold, n=5 nice).(c)Total UBA and CBa levels in gall bladders of warm- housed(n=8)and cold- housed(n= 10)mice. (d) Schematic diagram of the classical and ternative bile acid synthesis pathways. (e, f)Expression of genes involved in cholesterol and bile acid transport and metabolism(e)and hepatic UBA and CBA levels(f)in warm-housed and cold-housed mice that were treated with the fXr agonist PX20606 (PX)or with vehicle for 3 d (warm-housed: vehicle (warm), n=6 mice; PX, n=6 mice; cold-housed: vehicle (cold), n=5 mice; PX, n=6 mice). (g, h)Hepatic mRNA expression of genes involved in bile acid etabolism(g), as well as UBA (left)and CBa (right)levels(h), in mice that were housed at 22C and treated without(mock)or with CL316, 243(CL) for 7 d(n=8 mice per group). (i)Bile acid levels in livers of mice that were housed at thermoneutrality and treated with either AAv-GFP or AAV-Cyp7bl (n=7 mice per group). () Hepatic levels of bile acids in warm- housed and cold-housed WT and Cyp7b1--mice(n= 3 mice per group).(k, l)Expression of Cyp27al in BAT (n= 5 mice per group)(k) from, and plasma levels of 27-hydroxycholesterol (n= 4 mice per group)()in, warm-housed and cold housed mice (m)Levels of 27-hydroxycholesterol in the feces of warm housed and cold-housed WT (n=5 mice per group) and Cyp7b1--(n=4 mice per group)mice. CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; GDCA, glycodeoxycholic acid; HDCA, hyodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; THDCA urohyodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; T-a-MCA, tauro-a-muricholic acid; T-B-MCA, tauro-B-muricholic id: UDCA, ursodeoxycholic acid; a-MCA, a-muricholic acid; B-MCA, B-muricholic acid; o-MCA, o-muricholic acid. Throughout, data are mean ts.e. m. P 0.05, P<0.01, P<0.001; by unpaired two-tailed Students t-test (a-c, g-i, k, I or two-way analysis of variance(ANOvA)(e, f, j, m)

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Ar t i c l e s  advance online publication nature medicine Warm CA β-MCA UDCA α-MCA HDCA T-α/βMCA THDCA TUDCA TCDCA/TDCA TLCA TCA GCDCA GDCA GCA UBA CBA ω-MCA 60 8 200 150 100 50 0 6 4 2 0 40 20 0 Bile acids (ng/mg, fold) Bile acids (ng/mg, fold) Bile acids (mM) *** *** *** ** ** ** ** * * a Cold b Warm Cold c Warm Cold Cyp7a1 Cyp8a1 Cyp7b1 Cyp27a1 Baat Abcb11 Nr0b2 Cholesterol 5-Cholesten-3-β, 7α-diol 5-Cholesten-7-α-ol-one 7-Hydroxycholesterol 27�-Hydroxycholesterol Unconjugated bile acid Conjugated bile acids Baat Cholic acid �-Muricholic acid Classical pathway Alternative pathway Cyp27a1 Cyp7b1 Cyp8b1 Cyp7a1 Gene expression (fold) Bile acids (ng/mg) *** *** *** ** ** ** ** ** ** ** ** ** * * * * * * * * * * Warm 20 15 200 150 100 50 0 10 5 0 15 10 5 0 Warm PX Cold PX UBA CBA Cold d e f Cyp7a1 Cyp8b1 Cyp7b1 Cyp27a1 Baat Abcb11 Nr0b2 Gene expression (fold) Bile acids (ng/mg) Bile acids (ng/mg) * * 200 150 100 50 0 200 150 100 50 0 10 8 6 4 2 0 20 15 10 5 0 4 3 2 1 0 Mock CL Mock CL AAV-GFP AAV-Cyp7b1 UBA CBA UBA CBA g h i Gene expression (fold) Concentration (nmol/mg cholestrol) 27-OH-Chol (ng/mg) Bile acids (ng/mg) ** ** ** * * * * 15 500 WT warm Cyp7b1−/− warm WT cold Cyp7b1−/− cold 400 300 200 100 10 5 0 0 UBA CBA Warm Cold Cyp27a1 27-OH-Chol Feces WT warm WT cold Cyp7b1−/− warm Cyp7b1−/− cold 2.0 0.3 80 60 40 20 0 0.2 0.1 0.0 1.5 1.0 0.5 0.0 Warm Cold j k l m Figure 2 BAT activation induces the alternative bile acid synthesis pathway independently of FXR. (a,b) Relative levels of unconjugated bile acid (UBA) species (a) and conjugated bile acid (CBA) species (b) in the livers of mice that were housed at thermoneutrality (warm, n = 9 mice) or 6 °C (cold, n = 5 mice). (c) Total UBA and CBA levels in gall bladders of warm-housed (n = 8) and cold-housed (n = 10) mice. (d) Schematic diagram of the classical and alternative bile acid synthesis pathways. (e,f) Expression of genes involved in cholesterol and bile acid transport and metabolism (e) and hepatic UBA and CBA levels (f) in warm-housed and cold-housed mice that were treated with the FXR agonist PX20606 (PX) or with vehicle for 3 d (warm-housed: vehicle (warm), n = 6 mice; PX, n = 6 mice; cold-housed: vehicle (cold), n = 5 mice; PX, n = 6 mice). (g,h) Hepatic mRNA expression of genes involved in bile acid metabolism (g), as well as UBA (left) and CBA (right) levels (h), in mice that were housed at 22 °C and treated without (mock) or with CL316,243 (CL) for 7 d (n = 8 mice per group). (i) Bile acid levels in livers of mice that were housed at thermoneutrality and treated with either AAV-GFP or AAV-Cyp7b1 (n = 7 mice per group). (j) Hepatic levels of bile acids in warm-housed and cold-housed WT and Cyp7b1−/− mice (n = 3 mice per group). (k,l) Expression of Cyp27a1 in BAT (n = 5 mice per group) (k) from, and plasma levels of 27-hydroxycholesterol (n = 4 mice per group) (l) in, warm-housed and cold￾housed mice. (m) Levels of 27-hydroxycholesterol in the feces of warm-housed and cold-housed WT (n = 5 mice per group) and Cyp7b1−/− (n = 4 mice per group) mice. CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; GDCA, glycodeoxycholic acid; HDCA, hyodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; THDCA, taurohyodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; T-α-MCA, tauro-α-muricholic acid; T-β-MCA, tauro-β-muricholic acid; UDCA, ursodeoxycholic acid; α-MCA, α-muricholic acid; β-MCA, β-muricholic acid; ω-MCA, ω-muricholic acid. Throughout, data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; by unpaired two-tailed Student’s t-test (a–c,g–i,k,l) or two-way analysis of variance (ANOVA) (e,f,j,m)

ARTICLES of AbcblI expression, as well as that of Nrob2, and downregulation transporter(ASBT), which is required for efficient CBA re-uptake as of the SHP target genes Cyp 7al and Cyp8b1 in warm-housed, as well part of the enterohepatic circulation- Cold exposure led to higher as cold-housed, mice(Fig. 2e), whereas other genes important for ASBT expression at the mRNA (SIc10a2; Fig 3f), as well as protein synthesis( Cyp27a1)and conjugation(Baat)were unaffected by PX (Fig. 3g), level, which was lost in Cyp7b1-/-mice(Supplementary treatment but were slightly higher after cold exposure. Notably, the Fig. 7c, d). Furthermore, cold-housing resulted in a trend toward upregulation of Cyp bl that we observed after cold treatment was higher bile acid levels in portal(Fig. 3h) and systemic(Fig. 3i)blood. not affected by a FXR-mediated negative feedback loop( Fig. 2e). Taken together, these data argue against diminished CBa re-uptake Consistent with sustained Cyp7b1 expression, PX treatment did not capacity as a cause for higher fecal bile acid levels influence the cold-induced increase in hepatic bile acid levels(Fig Another mechanism for increased fecal concentrations of cBa and only species that arose by the action of the PX-sensitive enzyme could be due to reduced bile acid deconjugation by bile salt hydrolases CYP8B1, such as CA, were slightly lower(Supplementary Fig 4a-c).(BSHs), which are expressed by a number of gut bacteria 26. To address In a human cohort, we found lower hepatic expression of CYP7BI this hypothesis we depleted the intestinal microbiota by using an ant in obese subjects with type 2 diabetes than in non-obese controls, biotic cocktail. Relative to that in the untreated controls, this interven whereas expression of CYP7AI and CYP8BI was not different tion resulted in higher fecal CBA content both in warm-housed and (Supplementary Fig. 5a-c), indicating that there was metabolic cold-exposed mice(Fig. 3j, k), which could be explained by the com- 9 regulation of the alternative pathway in humans. plete eradication of gut BSH activity by antibiotic treatment(Fig. 31) To determine the role of BAT in cold-induced bile acid synthesis, we Although antibiotic treatment did not affect the expression of genes g used the B3-adrenergic receptor agonist CL316, 243 to pharmacologi- related to bile acid metabolism(Supplementary Fig. 8a), hepatic e cally activate BAT in mice that were housed at room temperature. As UBA but not CBA levels were lower(Supplementary Fig 8b, c) w compared to the untreated controls, expression of Cyp7bl, but of no Of note, higher BSH activity was observed in cold-exposed mice other genes involved in hepatic bile acid synthesis, was higher in the in the absence of antibiotics than in warm-housed mice(Fig. 31), CL316, 243-treated mice(Fig. 2g)and was associated with increased resulting in higher fecal taurine levels(Fig. 3m). Cumulatively, these levels of hepatic bile acids( Fig. 2h and Supplementary Fig. 6a, b). results suggest that saturation of ASBT rather than diminished decon Genetic intervention by adeno-associated virus(AAV)-mediated jugation by BSH activity is responsible for higher fecal CBAexcretion E overexpression of Cyp 7bl in WT mice resulted in moderately higher after cold exposure z hepatic bile acid levels at thermoneutral conditions, as compared to those in mice that were treated with GFP-expressing control AAv Bile acid excretion depends on a BAT-liver cholesterol axis E(AAV-GFP)(Fig. 2i and Supplementary Fig. 6c). Conversely, the Cold-activated BAT efficiently processes dietary lipids carried by p a cold-dependent induction in hepatic bile acid levels was blunted prandial lipoproteins and promotes receptor-mediated uptake of the in Cyp7b1-- mice, as compared to that in WT mice(Fig. 2j and respective cholesterol-rich remnants by the liver 5, 17. Accordingly, in induced the a CyP27aI in BAT(Fig. 2k)and led to increased amounts of plasma cold(Fig.1g-i), we observed higher amounts of circulating cholesterol 27-hydroxycholesterol(Fig. 21), the substrate for hepatic CYP7B1. rich lipoproteins in cold-housed versus warm-housed mice that were Of note, 27-hydroxycholesterol did not accumulate in the liver and deficient in the LDL receptor (LDLr), which is the main receptor for g plasma of cold-exposed Cyp7b1-/-mice(Supplementary Fig. 6f, g), hepatic remnants(Fig 4a, b). When we combined LdIr-genotype with which could be because much of it ed in the feces( Fig. 21 knockout of the the alternative In conclusion, these data reveal that under conditions of BAT activa- lipoprotein receptor, LDLR-related protein 1 (LrpI), we observed on, the alternative pathway is selectively upregulated and increases even higher lipoprotein levels(Fig. 4c, d), indicating blunted remnant bile acid sy clearance in the absence of both receptors. Notably, the cold-induced increase in fecal bile acid excretion in LdIr-mice was only 50% of that old accelerates fecal bile acid excretion via cyP7b1 in WT mice, and it was nearly abolished in mice that lacked LDLR and To further explore the fate of bile acids, we quantified their levels hepatic LRPI (Fig. 4e-g). Taken together, these findings indicate that N in stool samples from warm-housed and cold-housed mice. The fecal bile acid excretion is dependent on hepatic cholesterol that is deliv- amount of excreted bile acids was much higher in cold-housed ered by postprandial lipoproteins generated by cold-activated BAT than in warm-housed mice(Fig 3a, b). Notably, some CBA sp To assess the contribution of dietary cholesterol to cold-induced cies, especially tauro-a/B-MCA (T-a/B-MCA) and tauro-ca bile acid synthesis, we first blocked dietary cholesterol resorption by TCA), were up to 40-fold higher UBA levels were less affected and inhibiting the intestinal cholesterol transporter Niemann-Pick-1-like were increased up to twofold(Fig. 3a, b ) In Cyp7b1-/- mice, the 1(NPCIL1)with ezetimibe(EZ). Notably, combining cold-exposure cold-induced rise in fecal CBA was abrogated, and concentrations and EZ treatment resulted in lower levels of plasma lipids and choles- of some bile acid species were even lower(Fig. 3c, d). Conversely, terol-rich lipoproteins, even more so than with cold exposure alone AAV-mediated Cyp7b1 overexpression resulted in higher fecal bile (Fig 5a, b). Consistent with diminished dietary cholesterol uptak acid species, as compared to that in the AAv-GFP controls(Fig. 3e and hepatic lipid levels after EZ treatment(Fig. 5c), we observed and Supplementary Fig. 7a, b). Notably, the effect was not present compensatory hepatic upregulation of the gene encoding the rate- at thermoneutral conditions but at 22C, and it was even more pro- limiting enzyme of the cholesterol synthesis pathway(Hmgcr),as bounced at 16C(Fig. 3e and Supplementary Fig. 7a, b), indicat- well as of Ldlr(Fig. 5d), whereas expression of Cyp7b1 was unaf ing that increased dietary intake and BAT-dependent processing of fected( Supplementary Fig 9a). Liver bile acid levels of cold-housed cholesterol at lower temperatures is critical for efficient CYP7B1- EZ-treated mice were comparable to those of cold-housed control mediated bile acid production mice(Fig. 5e and Supplementary Fig 9b, c). In contrast, the fecal mice, wea sate the basis of higher fecal CBA levels in cold-housed bile acid concentrations of EZ-treated mice were not higher after rst quantified the ileal expression of apical sodium bile cold exposure, and bile acid levels were almost identical to those in

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. a r t i c l e s nature medicine advance online publication  of Abcb11 expression, as well as that of Nr0b2, and downregulation of the SHP target genes Cyp7a1 and Cyp8b1 in warm-housed, as well as cold-housed, mice (Fig. 2e), whereas other genes important for synthesis (Cyp27a1) and conjugation (Baat) were unaffected by PX treatment but were slightly higher after cold exposure. Notably, the upregulation of Cyp7b1 that we observed after cold treatment was not affected by a FXR-mediated negative feedback loop (Fig. 2e). Consistent with sustained Cyp7b1 expression, PX treatment did not influence the cold-induced increase in hepatic bile acid levels (Fig. 2f), and only species that arose by the action of the PX-sensitive enzyme CYP8B1, such as CA, were slightly lower (Supplementary Fig. 4a–c). In a human cohort, we found lower hepatic expression of CYP7B1 in obese subjects with type 2 diabetes than in non-obese controls, whereas expression of CYP7A1 and CYP8B1 was not different (Supplementary Fig. 5a–c), indicating that there was metabolic regulation of the alternative pathway in humans. To determine the role of BAT in cold-induced bile acid synthesis, we used the β3-adrenergic receptor agonist CL316,243 to pharmacologi￾cally activate BAT in mice that were housed at room temperature. As compared to the untreated controls, expression of Cyp7b1, but of no other genes involved in hepatic bile acid synthesis, was higher in the CL316,243-treated mice (Fig. 2g) and was associated with increased levels of hepatic bile acids (Fig. 2h and Supplementary Fig. 6a,b). Genetic intervention by adeno-associated virus (AAV)-mediated overexpression of Cyp7b1 in WT mice resulted in moderately higher hepatic bile acid levels at thermoneutral conditions, as compared to those in mice that were treated with GFP-expressing control AAV (AAV-GFP) (Fig. 2i and Supplementary Fig. 6c). Conversely, the cold-dependent induction in hepatic bile acid levels was blunted in Cyp7b1−/− mice, as compared to that in WT mice (Fig. 2j and Supplementary Fig. 6d,e). Cold exposure induced the expression of Cyp27a1 in BAT (Fig. 2k) and led to increased amounts of plasma 27-hydroxycholesterol (Fig. 2l), the substrate for hepatic CYP7B1. Of note, 27-hydroxycholesterol did not accumulate in the liver and plasma of cold-exposed Cyp7b1−/− mice (Supplementary Fig. 6f,g), which could be because much of it was excreted in the feces (Fig. 2m). In conclusion, these data reveal that under conditions of BAT activa￾tion, the alternative pathway is selectively upregulated and increases bile acid synthesis. Cold accelerates fecal bile acid excretion via CYP7B1 To further explore the fate of bile acids, we quantified their levels in stool samples from warm-housed and cold-housed mice. The amount of excreted bile acids was much higher in cold-housed than in warm-housed mice (Fig. 3a,b). Notably, some CBA spe￾cies, especially tauro-α/β-MCA (T-α/β-MCA) and tauro-CA (TCA), were up to 40-fold higher. UBA levels were less affected and were increased up to twofold (Fig. 3a,b). In Cyp7b1−/− mice, the cold-induced rise in fecal CBA was abrogated, and concentrations of some bile acid species were even lower (Fig. 3c,d). Conversely, AAV-mediated Cyp7b1 overexpression resulted in higher fecal bile acid species, as compared to that in the AAV-GFP controls (Fig. 3e and Supplementary Fig. 7a,b). Notably, the effect was not present at thermoneutral conditions but at 22 °C, and it was even more pro￾nounced at 16 °C (Fig. 3e and Supplementary Fig. 7a,b), indicat￾ing that increased dietary intake and BAT-dependent processing of cholesterol at lower temperatures is critical for efficient CYP7B1- mediated bile acid production. To investigate the basis of higher fecal CBA levels in cold-housed mice, we first quantified the ileal expression of apical sodium bile transporter (ASBT), which is required for efficient CBA re-uptake as part of the enterohepatic circulation20. Cold exposure led to higher ASBT expression at the mRNA (Slc10a2; Fig. 3f), as well as protein (Fig. 3g), level, which was lost in Cyp7b1−/− mice (Supplementary Fig. 7c,d). Furthermore, cold-housing resulted in a trend toward higher bile acid levels in portal (Fig. 3h) and systemic (Fig. 3i) blood. Taken together, these data argue against diminished CBA re-uptake capacity as a cause for higher fecal bile acid levels. Another mechanism for increased fecal concentrations of CBA could be due to reduced bile acid deconjungation by bile salt hydrolases (BSHs), which are expressed by a number of gut bacteria26. To address this hypothesis we depleted the intestinal microbiota by using an anti￾biotic cocktail. Relative to that in the untreated controls, this interven￾tion resulted in higher fecal CBA content both in warm-housed and cold-exposed mice (Fig. 3j,k), which could be explained by the com￾plete eradication of gut BSH activity by antibiotic treatment (Fig. 3l). Although antibiotic treatment did not affect the expression of genes related to bile acid metabolism (Supplementary Fig. 8a), hepatic UBA but not CBA levels were lower (Supplementary Fig. 8b,c). Of note, higher BSH activity was observed in cold-exposed mice in the absence of antibiotics than in warm-housed mice (Fig. 3l), resulting in higher fecal taurine levels (Fig. 3m). Cumulatively, these results suggest that saturation of ASBT rather than diminished decon￾jugation by BSH activity is responsible for higher fecal CBA excretion after cold exposure. Bile acid excretion depends on a BAT–liver cholesterol axis Cold-activated BAT efficiently processes dietary lipids carried by post￾prandial lipoproteins and promotes receptor-mediated uptake of the respective cholesterol-rich remnants by the liver15,17. Accordingly, in contrast to the decline in circulating lipids that occurs in WT mice with cold (Fig. 1g–i), we observed higher amounts of circulating cholesterol￾rich lipoproteins in cold-housed versus warm-housed mice that were deficient in the LDL receptor (LDLR), which is the main receptor for hepatic remnants (Fig. 4a,b). When we combined Ldlr−/− genotype with a liver-specific knockout of the gene encoding the alternative hepatic lipoprotein receptor, LDLR-related protein 1 (Lrp1), we observed even higher lipoprotein levels (Fig. 4c,d), indicating blunted remnant clearance in the absence of both receptors. Notably, the cold-induced increase in fecal bile acid excretion in Ldlr−/− mice was only 50% of that in WT mice, and it was nearly abolished in mice that lacked LDLR and hepatic LRP1 (Fig. 4e–g). Taken together, these findings indicate that fecal bile acid excretion is dependent on hepatic cholesterol that is deliv￾ered by postprandial lipoproteins generated by cold-activated BAT. To assess the contribution of dietary cholesterol to cold-induced bile acid synthesis, we first blocked dietary cholesterol resorption by inhibiting the intestinal cholesterol transporter Niemann-Pick-1-like 1 (NPC1L1) with ezetimibe (EZ). Notably, combining cold-exposure and EZ treatment resulted in lower levels of plasma lipids and choles￾terol-rich lipoproteins, even more so than with cold exposure alone (Fig. 5a,b). Consistent with diminished dietary cholesterol uptake and hepatic lipid levels after EZ treatment (Fig. 5c), we observed compensatory hepatic upregulation of the gene encoding the rate￾limiting enzyme of the cholesterol synthesis pathway (Hmgcr), as well as of Ldlr (Fig. 5d), whereas expression of Cyp7b1 was unaf￾fected (Supplementary Fig. 9a). Liver bile acid levels of cold-housed EZ-treated mice were comparable to those of cold-housed control mice (Fig. 5e and Supplementary Fig. 9b,c). In contrast, the fecal bile acid concentrations of EZ-treated mice were not higher after cold exposure, and bile acid levels were almost identical to those in