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)