Supplementary Figure 1 b a Families Genera d c e Supplementary Figure 1 BAT activation alters the gut microbiome in chow-fed wild type mice. (a-e) MDS plot of weighted UniFrac distance (a), hierarchical clustering of significant altered OTUs (b), mean relative abundance of gut microbiota on family (c) and genus (d) level, as well as alpha diversity presented on genus level and on OTU level (e) based on 16S rDNA sequencing of feces collected from chow fed mice housed at thermoneutrality (warm: n = 8) or 6°C (cold: n = 7). Pairwise Wilcoxon Rank Sum Tests were used to determine significant differences between treatment groups. **P < 0.01, n.s. = not significant. Nature Medicine: doi:10.1038/nm.4357
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Supplementary Figure 1
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Supplementary Figure 1 BAT activation alters the gut microbiome in chow-fed wild type mice. (a-e)MDS plot of weighted UniFrac distance (a), hierarchical clustering of significant altered OTUs (b),mean relative abundance of gut microbiota on family (c) and genus (d) level, as well as alphadiversity presented on genus level and on OTU level (e) based on 16S rDNA sequencing of fecescollected from chow fed mice housed at thermoneutrality (warm: n = 8) or 6°C (cold: n = 7). PairwiseWilcoxon Rank Sum Tests were used to determine significant differences between treatment groups.**P < 0.01, n.s. = not significant.
Nature Medicine: doi:10.1038/nm.4357
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Supplementary Figure 2
Supplementary Figure 2 BAT activation alters the gut microbiome in db/db mice. (a-e) MDS plot ofweighted UniFrac distance (a), hierarchical clustering of significant altered OTUs (b), mean relativeabundance of gut microbiota on family (c) and genus (d) level, as well as alpha diversity presented ongenus level and on OTU level (e) based on 16S rDNA sequencing of feces collected from db/db micehoused at thermoneutrality (warm: n = 7) or 16°C (n = 8). In this experiment, 16°C were used insteadof 6°C because db/db mice are intolerant to harsh cold exposure. Pairwise Wilcoxon Rank Sum Testswere used to determine significant differences between treatment groups. *P < 0.05, n.s. = notsignificant.
Supplementary Figure 3 Hepatic bile acid composition, liver inflammation and damage did notchange upon cold exposure. (a) Bile acid composition in livers of warm- and cold-housed micedepicted as % of pool (n = 10). (b) Hepatic expression of inflammatory genes (n = 6). (c) Plasmaactivities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (warm n = 7, coldn = 9). Data represent means ± s.e.m. *P < 0.05 on the basis of unpaired two-tailed Student’s t-test(b,c). Abbreviations: Chenodeoxycholic acid (CDCA); Cholic acid (CA); Deoxycholic acid (DCA);Glycochenodeoxycholic acid (GCDCA); Glycocholic acid (GCA); Glycodeoxycholic acid (GDCA);Glycolithocholic acid (GLCA); Glycoursodeoxycholic acid (GUDCA); Hyodeoxycholic acid (HDCA);Taurochenodeoxycholic acid (TCDCA); Taurocholic acid (TCA); Taurodeoxycholic acid (TDCA);Taurohyodeoxycholic acid (THDCA); Taurolithocholic acid (TLCA); Tauroursodeoxycholic acid(TUDCA); Tauro-α-muricholic acid (T-α-MCA); Tauro-β-muricholic acid (T-β-MCA); Ursodeoxycholicacid (UDCA); α-Muricholic acid (α-MCA); β-Muricholic acid (β-MCA); ω-Muricholic acid (ω-MCA).
Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 4
Supplementary Figure 4 Cold-induced bile acid levels are largely unaffected by FXR activation. (a-c)Bile acid species determined in liver (a), plasma (b) and feces (c) of mice housed at thermoneutrality(warm) or 6°C (cold) and treated with or without the FXR agonist PX20606 (PX) for three days (warm,warm PX, cold PX: n = 6; cold: n = 5). Data represent means ± s.e.m. *P < 0.05, **P < 0.01, ***P <0.001 on the basis of two-way ANOVA.
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Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 5
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nSupplementary Figure 5 CYP7B1 expression is reduced in livers of obese subjects with type 2diabetes. (a-c) Expression of CYP7B1 (a), CYP7A1 (b) and CYP8B1 (c) normalized to thehousekeeper TAF1 determined in livers of metabolically healthy subjects (non-obese, n = 19), obesesubjects without diabetes (obese, n = 21) and obese subjects with type 2 diabetes (obese+T2D, n =21). Data represent means ± s.e.m. *P < 0.05 on the basis of unpaired two-tailed Student’s t-test.
Supplementary Figure 6 Pharmacological beta-3-adrenergic activation and Cyp7b1 expressioninfluence hepatic bile acids. (a-e) Hepatic levels of unconjugated (a) and conjugated (b) bile acidspecies in mice housed at 22°C and treated with or without the beta-3-adrenergic receptor agonistCL316,243 (CL) for seven days (n = 8). (c) Hepatic bile acid levels determined in livers of AAV-GFPor AAV-Cyp7b1 treated mice housed at thermoneutrality (n = 7). Hepatic levels of unconjugated (d)and conjugated (e) bile acid species of warm- and cold-housed wild-type (wt) and Cyp7b1-/- mice (n =3). (f) Liver (n = 9) and (g) plasma (n = 4) levels of 27-OH-cholesterol of cold-housed wt and Cyp7b1-/-
mice. Data represent means ± s.e.m. *P < 0.05, **P < 0.01 on the basis of unpaired two-tailedStudent’s t-test (a,b,c,f,g) or two-way ANOVA (d,e).
Supplementary Figure 7 Cyp7b1 influences fecal bile acid loss but not ileal reabsorption in responseto cold. (a,b) Fecal bile acid species determined in AAV-GFP or AAV-Cyp7b1 treated mice housed atindicated ambient temperatures (n = 7). (c) Expression of Slc10a2 encoding ASBT determined in ileaof warm- and cold-housed wt and Cyp7b1-/- mice (n = 5 for wt warm, wt cold and Cyp7b1-/- cold, n = 4mice for Cyp7b1-/- warm). (d) ASBT protein determined in ileal homogenates from warm- and cold-housed wt and Cyp7b1-/- mice (n = 3). One representative out of two technical replicates of 3biological replicates is shown. Uncropped Western blot images are shown in Supplementary Figure15. Data represent means ± s.e.m. *P < 0.05, **P < 0.01 on the basis of two-way ANOVA.
Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 8
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Supplementary Figure 8 Increased hepatic bile acid synthesis with cold exposure is independent ofthe gut microbiota. (a-c) Relative expression of cholesterol and bile acid transport and metabolismgenes in the liver (a, n = 6), as well as fecal unconjugated (b) and conjugated (c) bile acid levels fromwarm- and cold-housed mice treated with antibiotics (AB) (warm, warm AB, cold: n = 10; cold AB: n =9). Data represent means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 on the basis of two-wayANOVA.
Nature Medicine: doi:10.1038/nm.4357
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Supplementary Figure 9
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Supplementary Figure 9 Ezetimibe (EZ) effects on hepatic bile acids, fecal cholesterol and the gutmicrobiome. (a-g) Relative expression of bile acid transport and metabolism genes in the liver (n = 8)(a), hepatic unconjugated bile acid species (b), hepatic conjugated bile acid species (warm n = 6, coldn = 8, warm EZ n = 7, cold EZ n = 8) (c), daily fecal cholesterol excretion (warm n = 6, cold n = 5,warm EZ n = 6, cold EZ n = 6) (d), food intake (n = 6) (e), daily feces production (warm n = 6, cold n =5, warm EZ n = 6, cold EZ n = 6) (f), and hierarchical clustering of significant altered operationaltaxonomic units (OTUs) determined in feces (warm n = 8, cold n = 7, warm EZ n = 10, cold EZ n = 9)(g) in warm- and cold-housed mice treated without and with EZ. Data represent means ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001 on the basis of two-way ANOVA (a-f).
Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 10
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Supplementary Figure 10. Influence of dietary cholesterol and beta-3-adrenergic activation on gutmicrobiota and fecal bile acids. (a-g) Hierarchical clustering of significant altered OTUs (a), alphadiversity presented on genus level and on OTU level (warm n = 8, cold n = 7, warm Chol n = 8, coldChol n = 8) (b), fecal unconjugated bile acid species (c), fecal conjugated bile acid species (warm n =7, cold n = 6, warm Chol n = 8, cold Chol n = 8) (d), fecal levels of unconjugated (e) and conjugated(f) bile acid species as well as microbiome analysis shown as MDS plot of weighted UniFrac distancebased on fecal 16S rDNA sequencing (g) determined in mice housed at 22°C and treated with orwithout the beta-3-adrenergic receptor agonist CL316,243 (CL) for seven days (n = 8 mice). Datarepresent means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 on the basis of pairwise Wilcoxon RankSum tests (b), two-way ANOVA (c,d) or unpaired two-tailed Student’s t-test (e,f).
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Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 11
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Supplementary Figure 11 Effects of Cyp7b1 overexpression and Mdr2-deficiency on fecal bile acidsand gut microbiota. (a-c) Mean relative abundance of gut microbiota on family and genus level (a),alpha diversity presented on genus and OTU level (b) and abundance of significantly regulated OTU(c) in mice housed at 22°C infected with AAV-GFP or AAV-Cyp7b1 (n = 7). (d,e) Fecal bile acid levels(warm n = 6, cold n = 8) (d) and alpha diversity presented on genus and OTU level (e) determined inwarm- and cold-housed Mdr2-/- mice (warm n = 7, cold n = 8). Data represent means ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001 on the basis of pairwise Wilcoxon Rank Sum tests (b,c,e) or unpairedtwo-tailed Student’s t-test (d).
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Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 12
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Supplementary Figure 12. Influence of dietary cholesterol on plasma bile acids in response to cold.(a,b) Plasma unconjugated bile acid species (a) and conjugated bile acid species (b) in cold-housed(6°C) mice treated without and with EZ (n = 4). (c,d) Plasma unconjugated bile acid species (c) andplasma conjugated bile acid species (d) (cold n = 7, cold Chol n = 8) determined in mice housed at6°C (cold) and fed a chow diet supplemented with or without cholesterol. Data represent means ±
Supplementary Figure 13 AAV-mediated overexpression of Cyp7b1 enhances thermogenicresponses. (a-c) Relative expression of bile acid genes in liver (a), thermogenic genes in BAT (b) andinguinal WAT (c) determined in mice treated with AAV-GFP or AAV-Cyp7b1 housed at indicatedtemperatures (AAV-GFP 22°C: n = 7, AAV-Cyp7b1 22°C: n = 7, AAV-GFP 16°C: n = 6, AAV-Cyp7b116°C: n = 6). (d) Temperature determined at proximal and distal tails in mice treated with AAV-GFPand AAV-Cyp7b1 housed at 30°C and 16°C (AAV-GFP n = 7, AAV-Cyp7b1 n = 4) as well as at 22°C(n = 7). Data represent means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 on the basis of two-wayANOVA (a-c) or unpaired two-tailed Student’s t-test (d).
Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 14
Supplementary Figure 14. Model describing the effects of BAT activation on hepatic cholesterol andbile acid metabolism, gut microbiome and adaptive thermogenesis. Cold exposure increases dietarycholesterol intake and triggers postprandial lipoprotein processing in BAT, which accelerates cholesteroluptake into the liver via lipoprotein-receptor mediated endocytosis. BAT activation causes increasedsynthesis of bile acids via CYP7B1 and the alternative pathway, thus promoting their biliary excretion.Subsequently, higher bile acid levels in the gut lead to an altered microbiome. This may modulate therelease gut-derived endocrine factors or metabolites that together with higher plasma bile acids sustainefficient energy expenditure for heat production in the cold. Thus, cholesterol processing via a BAT-liveraxis determines the composition of the gut microbiome and promotes adaptive thermogenesis.
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Nature Medicine: doi:10.1038/nm.4357
Supplementary Figure 15
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Supplementary Figure 15. Full-scans of Western blots. (a-c) Full-sized images of Western blots fromFigure 3g (a), Figure 6g (b) and Supplementary Figure 7d (c). Red boxes highlight areas that werecropped and are displayed in the indicated figures. Molecular weights are given kDa. Western blotmembranes of Figure 6g were cut out between 40 kDa and 70 kDa to detect AKT and cut under 40kDa to detect UCP1 on separated parts of the membranes.
Supplementary Table 1 (a) Food intake, feces production and plasma lipids in wild type andtransgenic mice under various experimental conditions. (b) Cholesterol balance in warm and cold-housed mice fed a cholesterol-enriched high fat diet.
