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