ARTICLES linked to a beneficial metabolic outcome. Here we demonstrate Cyp7b1 induction as the main driver for a cold-induced increase in that accelerated metabolism of endogenous and dietary cholesterol bile acid synthesis, which increased energy expenditure. This proc in response to cold exposure leads to increased bile acid synthesis ess of enhanced cholesterol catabolism was triggered by accelerated via the alternative pathway. This results in profoundly elevated bile lipoprotein processing in cold-activated BAT, which stimulated acid excretion and distinct changes in the gut bacterial composition, cholesterol flux toward the liver. Furthermore, we demonstrated that providing a mechanism for how cold-induced increase in energy unlike CYP7Al, the rate-limiting enzyme of the classical pathway l8, expenditure alters the gut microbiome. Moreover, cholesterol intake CYP7Bl was not suppressed by pharmacological FXR agonism. Thus, and its hepatic conversion to bile acids are found to activate brown CYP7B1 is not subject to classical negative feedback control regulated and beige adipocytes, suggesting a role in diet-induced thermogen- by elevated bile acid levels. This is of special note, as it enables the esis. The findings of our study are summarized in a model describ- body to maintain cholesterol homeostasis despite massively increased g how BAT activation stimulates cholesterol flux and conversion to cholesterol conversion to bile acids in the cold. Although the exact bile acids, a process that shapes the gut microbiome and promotes mechanism underlying the induction of Cyp7bl in cold-exposed adaptive thermogenesis (Supplementary Fig 14) mice remains to be elucidated, this is the first report demonstrat- The recent discovery that a substantial number of adult humans ing a physiological regulation of the alternative bile acid synthesis retain functional BAT31-35 has introduced adaptive thermogenesis pathway. Notably, CYP7B1 expression was reduced in subjects with a promising approach to enhance energy expenditurel4, 36. In acti- type 2 diabetes, suggesting a role of the alternative pathway for meta- vated BAT, dietary glucose and triglycerides serve as fuels to meet bolic homeostasis in humans. However, previous studies indicated the high-energy demands during heat production 5, 16.37. This in turn higher levels of CYP8B1-derived bile acids in the plasma of humans 8 causes increased appetite and food intake, which leads to the uptake with type 2 diabetes#. 45. It will be of interest to dissect the bile 4 of potentially harmful food components. Cholesterol, which is an acid synthesis pathways in intervention studies, including BAT abundant ingredient of typical energy-rich diets, in particular can- activation by cold, and compare them in healthy individuals and E not be used for heat production and could, in principle, accumulate those with diabetes in arteries, and hence promote cardiovascular disease. However, we Taken together, our findings imply that increased bile acid o previously showed that sustained activation of BAT in mice resulted levels contribute to beneficial effects of beige and brown adipocytes 3 in reduced atherosclerosis 7. Our present study indicates that excess on obesity-associated comorbidities. Mechanistically, elevated z dietary cholesterol is partially converted to bile acids, which are subse- bile acid levels regulate thermogenic responses, which have been s quently removed by fecal excretion, thereby contributing to systemic described to occur in the BAT of mice and humans, likely via TGR5 a have shown that cold exposure induces alterations in the gut micro- the thermogenic activity of skeletal muscle, which may explain the biota, which are associated with improved metabolic parameters 0, I. increased glucose uptake observed in both the BAT and muscle of sub- However, the trigger for cold-induced microbiota remodeling remains jects with type 2 diabetes after cold acclimation 6. Future studies are unknown. In this context, it is of note that bile acids are not only warranted to address the therapeutically relevant question of wheth involved in energy harvesting by facilitating intestinal lipid uptake beneficial cold-mediated metabolic effects are mediated directly by but they also interact with intestinal bacteria26. Notably, we found bile acids acting on TGR5 and other receptors or indirectly by com- g that cold-triggered elevated fecal bile acid levels and alterations in positional changes in the gut microbior gut microbiota could be attenuated by inhibiting the intestinal trans porter NPCILI using ezetimibe, a cholesterol-lowering drug with METHODS tablished anti-atherosclerotic properties. This demonstrates that Methods, including statements of data availability and any associated the enhanced catabolism of cholesterol, resulting in increased fecal accession codes and references, are available in the online version of bile acids, is important for the observed changes in gut microbiota the paper. in cold-exposed mice. Mechanistically, in addition to direct bacte riostatic effects27, increased bile acid levels may indirectly alter the Note: Any Supplementary Information and Source Data files are available in the online version of the paper. gut microbiome via taurine, which we found to be increased as a nsequence of enhanced bile acid deconjugation. In support of this ACKNOWLEDGMENTS idea, it was recently shown that taurine triggers intestinal secretion We thank S.Ehret,BHenkel,AKuhl and E.-M. Azizi for excellent technical of defensins in an inflammasome-dependent manner. In any event, polyclonal antibody, and J. Nedergard and B Cannon (Wenner-Gren Institute, this mechanism would also depend on increased bile acid synthesis Stockholm University)for the UCPl-spcific polyclonal antibody. This work t in the liver. From a general perspective, we provided evidence for the supported by grants funded by the Deutsche Forschungsgemeinschaft (SFB841 und consequences"(LH and of the gut microbiome ported by Merck Sharp Dohme(MSD)(H ) the I project RESOLVE Recently, an induction of Cyp 7bl and other genes related to bile acid FP7-HEALTH-2012-305707 (.H. ) a University Medical Center Hamburg metabolism was described in WT mice in response to cold exposure. Eppendorf MD/PhD fellowship(CS )and the US National Institutes of Health The induction of Cyp7bl after cold exposure or after B3-adrenergi receptor agonism observed in our study suggest a BAT-liver crosstalk AUTHOR CONTRIBUTIONS that regulates hepatic gene expression and thermogenic capacity 0, 4. Aw,C. L.S. and J.H. designed the study, were involved in all aspects of In addition, because germ-free mice have higher hepatic Cyp7b1 lev- the experiments and wrote the manuscript; M CRE-AH and A.E. were elst24s, factors released by the gut microbiota may either directly, were involved in the metabolic studies: M.E. M.D. A.F.C. K und pw.s or indirectly via BAT activation, regulate Cyp7b1 expression. Here, e involved in study design; and all authors read and commented on using Cyp7b1-- mice, we demonstrated the relevance of hepatic the manuscript.© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Ar t i c l e s 10 advance online publication nature medicine linked to a beneficial metabolic outcome10,11. Here we demonstrate that accelerated metabolism of endogenous and dietary cholesterol in response to cold exposure leads to increased bile acid synthesis via the alternative pathway. This results in profoundly elevated bile acid excretion and distinct changes in the gut bacterial composition, providing a mechanism for how cold-induced increase in energy expenditure alters the gut microbiome. Moreover, cholesterol intake and its hepatic conversion to bile acids are found to activate brown and beige adipocytes, suggesting a role in diet-induced thermogen￾esis. The findings of our study are summarized in a model describ￾ing how BAT activation stimulates cholesterol flux and conversion to bile acids, a process that shapes the gut microbiome and promotes adaptive thermogenesis. (Supplementary Fig. 14). The recent discovery that a substantial number of adult humans retain functional BAT31–35 has introduced adaptive thermogenesis as a promising approach to enhance energy expenditure14,36. In acti￾vated BAT, dietary glucose and triglycerides serve as fuels to meet the high-energy demands during heat production15,16,37. This in turn causes increased appetite and food intake, which leads to the uptake of potentially harmful food components. Cholesterol, which is an abundant ingredient of typical energy-rich diets, in particular can￾not be used for heat production and could, in principle, accumulate in arteries, and hence promote cardiovascular disease. However, we previously showed that sustained activation of BAT in mice resulted in reduced atherosclerosis17. Our present study indicates that excess dietary cholesterol is partially converted to bile acids, which are subse￾quently removed by fecal excretion, thereby contributing to systemic cholesterol homeostasis in cold-exposed mice. Notably, recent studies have shown that cold exposure induces alterations in the gut micro￾biota, which are associated with improved metabolic parameters10,11. However, the trigger for cold-induced microbiota remodeling remains unknown. In this context, it is of note that bile acids are not only involved in energy harvesting by facilitating intestinal lipid uptake but they also interact with intestinal bacteria26. Notably, we found that cold-triggered elevated fecal bile acid levels and alterations in gut microbiota could be attenuated by inhibiting the intestinal trans￾porter NPC1L1 using ezetimibe, a cholesterol-lowering drug with established anti-atherosclerotic properties38. This demonstrates that the enhanced catabolism of cholesterol, resulting in increased fecal bile acids, is important for the observed changes in gut microbiota in cold-exposed mice. Mechanistically, in addition to direct bacte￾riostatic effects27, increased bile acid levels may indirectly alter the gut microbiome via taurine, which we found to be increased as a consequence of enhanced bile acid deconjugation. In support of this idea, it was recently shown that taurine triggers intestinal secretion of defensins in an inflammasome-dependent manner39. In any event, this mechanism would also depend on increased bile acid synthesis in the liver. From a general perspective, we provided evidence for the concept that the processing of dietary ingredients, such as choles￾terol, by the host but not the diet per se determines the composition of the gut microbiome. Recently, an induction of Cyp7b1 and other genes related to bile acid metabolism was described in WT mice in response to cold exposure11. The induction of Cyp7b1 after cold exposure or after β3-adrenergic receptor agonism observed in our study suggest a BAT–liver crosstalk that regulates hepatic gene expression and thermogenic capacity40,41. In addition, because germ-free mice have higher hepatic Cyp7b1 lev￾els42,43, factors released by the gut microbiota may either directly, or indirectly via BAT activation, regulate Cyp7b1 expression. Here, using Cyp7b1−/− mice, we demonstrated the relevance of hepatic Cyp7b1 induction as the main driver for a cold-induced increase in bile acid synthesis, which increased energy expenditure. This proc￾ess of enhanced cholesterol catabolism was triggered by accelerated lipoprotein processing in cold-activated BAT, which stimulated cholesterol flux toward the liver. Furthermore, we demonstrated that unlike CYP7A1, the rate-limiting enzyme of the classical pathway18, CYP7B1 was not suppressed by pharmacological FXR agonism. Thus, CYP7B1 is not subject to classical negative feedback control regulated by elevated bile acid levels. This is of special note, as it enables the body to maintain cholesterol homeostasis despite massively increased cholesterol conversion to bile acids in the cold. Although the exact mechanism underlying the induction of Cyp7b1 in cold-exposed mice remains to be elucidated, this is the first report demonstrat￾ing a physiological regulation of the alternative bile acid synthesis pathway. Notably, CYP7B1 expression was reduced in subjects with type 2 diabetes, suggesting a role of the alternative pathway for meta￾bolic homeostasis in humans. However, previous studies indicated higher levels of CYP8B1-derived bile acids in the plasma of humans with type 2 diabetes44,45. It will be of interest to dissect the bile acid synthesis pathways in intervention studies, including BAT activation by cold, and compare them in healthy individuals and those with diabetes. Taken together, our findings imply that increased bile acid levels contribute to beneficial effects of beige and brown adipocytes on obesity-associated comorbidities. Mechanistically, elevated bile acid levels regulate thermogenic responses, which have been described to occur in the BAT of mice and humans, likely via TGR5 (refs. 11,22,24,25). Furthermore, bile acids can also directly promote the thermogenic activity of skeletal muscle24, which may explain the increased glucose uptake observed in both the BAT and muscle of sub￾jects with type 2 diabetes after cold acclimation46. Future studies are warranted to address the therapeutically relevant question of whether beneficial cold-mediated metabolic effects are mediated directly by bile acids acting on TGR5 and other receptors or indirectly by com￾positional changes in the gut microbiome. Methods Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank S. Ehret, B. Henkel, A. Kuhl and E.-M. Azizi for excellent technical assistance, P. Dawson (Emory University School of Medicine) for the ASBT-specific polyclonal antibody, and J. Nedergaard and B. Cannon (Wenner-Gren Institute, Stockholm University) for the UCP1-spcific polyclonal antibody. This work was supported by grants funded by the Deutsche Forschungsgemeinschaft (SFB841, “Liver inflammation: infection, immune regulation und consequences” (J.H. and M.D.); KFO306, “Primary sclerosing cholangitis (J.H. and to A.F.)), a Heisenberg Professorship (HE3645/7-1 (J.H.) and DA1063/3-2 (M.D.)), an EFSD award supported by Merck Sharp Dohme (MSD) (J.H.), the EU FP7 project RESOLVE FP7-HEALTH-2012-305707 (J.H.), a University Medical Center Hamburg– Eppendorf MD/PhD fellowship (C.S.) and the US National Institutes of Health grant HL087564 (P.W.S.). AUTHOR CONTRIBUTIONS A.W., C.J., L.S. and J.H. designed the study, were involved in all aspects of the experiments and wrote the manuscript; M.C.R., F.-A.H. and A.F. were responsible for the microbiome analysis; M.B., N.S., M.H., I.E., C.S. and C.M. were involved in the metabolic studies; M.F., M.D., A.F., C.K. und P.W.S. were involved in study design; and all authors read and commented on the manuscript