Adenovirus-associated virus(AAv)-mediated overexpression Systems, USA). Pictures were processed with the respective software package to For Cyp7bl overexpression, we intravenously injected 6- to 8-we determine proximal and distal tail surface temperatures. C57BL/6] mice with an AAV vector encoding mouse Cyp7b1( or a control vector encoding the green fluorescent protein(AAV-GFl Histology. We fixed brown adipose tissue in formalin(3.7% in 90% ethanol) intravenous injection of 3.0 x 10 genome copies per mouse was performed. for 24 h and subsequently embedded the samples in paraffin Hematoxylin and eosin(H&E)staining was performed using standard protocols. Western blotting analysis. For protein analysis, we prepared samples for calorimetry. We determined Oz consumption and CO, production SDS-PAGE using a RIPa buffer supplemented with protease inhibitors(Roche) ect calorimetry in a thermally and humidity-controlled environment om snap-frozen ilea. Protein samples(15 ug per lane)were separated on a TSE Phenomaster system (TSE systems, Germany)with a day and 10%Bis-Tris(pH 6.6)polyacrylamide gel using NuPAGE MES SDS Running night cycle of 12 h each. Mice were housed in single cages and had ad libitum Buffer under reducing conditions(Invitrogen). After the transfer to nitro. access to food and water. lulose membranes, blots were blocked for 2 h in PanReac Blocking buffer (AppliChem) and incubated for I h at room temperature with the primary Statistical analysis. We performed all statistical analyses using GraphPad antibody against the apical sodium bile transporter(ASBT, 1: 2,000; rabbit Prism6(Stat Con). When comparing two groups, significance was calculated polyclonal antibody kindly provided by Dr. P. Dawson, Department of using a non-paired two-tailed Student's t-test with no assumption of equal Pediatrics, Emory University School of Medicine), UCPI(rabbit polyclonal variance When comparing more than two groups, two-way ANOVA followed cat.no.A-5441, Sigma).Membranes were incubated for 1.5 h in secondary All values in the figure panels show mean.em Numbers per group inu e body: 1: 1,000; Cell Signaling cat. no. 9272)and mouse B-actin(1: 10,000, group of an experiment was similar. There was no blinding for the experiment horseradish peroxidase(HRP)-conjugated antibodies(goat anti-rabbit; 1: 5,000 figure legends refer to the number of mice per group. Statistical significan cat.no. 111-035-144, Jackson ImmunoResearch). We detected protein bands is indicated by P value ('P<0.05, ""P<0.01, ""P<0.001). We preferentially g using a luminol and para-hydroxycoumarinic acid-based chemiluminescence used group sizes of 5-8 mice, which we determined as optimal in previ substrate and performed densitometric quantification using control software in vivo studies. We used smaller group sizes in studies with genetically modified of Amersham Imager 600. animals. when sufficient numbers were not available. We describe the statis- tics regarding microbiome analysis in the subsection on microbiome analy a Bile salt hydrolase activity. We suspended freshly harvested cecal content in (see above) PBS(40 mg/ml) and filtered it using a cell strainer(100-um mesh size; Falcon) We immediately added 15 ul of taurocholic acid(I mg/ml in PBS: Acros)to Data availability Data are available from the authors upon requ 900 ul of the suspension 0) tonitrile. After centrifugation(15 min, 13,000g, 4C), 95 ul of the supernatant RP in clearance as transferred into a glass vial, 5 ul of D3-methionine (0. 1 mmol/liter)w st101,689-695 added and taurine measurements were taken using HILIC-ESI-QqQ(column: 48. Ruhlemann, M.C. et al. Fec profiles as diagnostic biomar kers in primary HILIC Kinetex(2.6 um, 150 mm x 2.1 mm inner diameter, Phenomenex): QqQ: sclerosing cholangitis. Gut 66, 753-754(201 API2000(ABSCIEX) 9. Wang a sed analysis of multivariate abundance data. Methods Ecol. Evol. 3 g Quantification of fecal cholesterol For cholesterolmeasuremen, we extracted 5o. enirmuin ao o. ch to mrgit i e testionging the. stse sco sey ies e s a ceao and. (0. IM potassium phosphate,PH 7.4, 0.05MNaCl, 5 mM cholic acid, 0. 1%6 Triton 51. John, C. et al. A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids. J. Chromatogr A1371,184-195(2014) e Rectal temperature and tail heat loss. We measured rectal temperature in 52. Eissing. L. et al. De novo lipogenesis in human fat and liver is linked to ChREBP- ice that were housed at different ambient temperatures by using a medical B and metabolic health. Nat. Commun. 4, 1528(2013) 53. Meiss. E. precision thermometer at 20: 00 h(Ellab, Denmark). We determined heat loss etabolite targeting: development of a comprehensive targeted the assessment of diabetes and its complications. indirectly by taking pictures of the tail with an infrared camera(FLIR E60, FLIR Metabolomics 12, 52(2016) doi:10.1038/nm435© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. doi:10.1038/nm.4357 nature medicine Adenovirus-associated virus (AAV)-mediated overexpression of Cyp7b1. For Cyp7b1 overexpression, we intravenously injected 6- to 8-week-old male C57BL/6J mice with an AAV vector encoding mouse Cyp7b1 (AAV-Cyp7b1) or a control vector encoding the green fluorescent protein (AAV-GFP). A single intravenous injection of 3.0 × 1012 genome copies per mouse was performed. Experiments were started 1–2 weeks after injection. Western blotting analysis. For protein analysis, we prepared samples for SDS–PAGE using a RIPA buffer supplemented with protease inhibitors (Roche) from snap-frozen ilea. Protein samples (15 µg per lane) were separated on a 10% Bis-Tris (pH 6.6) polyacrylamide gel using NuPAGE MES SDS Running Buffer under reducing conditions (Invitrogen). After the transfer to nitro￾cellulose membranes, blots were blocked for 2 h in PanReac Blocking buffer (AppliChem) and incubated for 1 h at room temperature with the primary antibody against the apical sodium bile transporter (ASBT, 1:2,000; rabbit polyclonal antibody kindly provided by Dr. P. Dawson, Department of Pediatrics, Emory University School of Medicine), UCP1 (rabbit polyclonal antibody; 1:12,500; kind gift of Dr. Jan Nedergaard and Dr. Barbara Cannon, Wenner-Gren Institute, Stockholm University), AKT (rabbit polyclonal anti￾body; 1:1,000; Cell Signaling cat. no. 9272) and mouse β-actin (1:10,000, cat. no. A-5441, Sigma). Membranes were incubated for 1.5 h in secondary horseradish peroxidase (HRP)-conjugated antibodies (goat anti-rabbit; 1:5,000; cat. no. 111-035-144, Jackson ImmunoResearch). We detected protein bands using a luminol and para-hydroxycoumarinic acid–based chemiluminescence substrate and performed densitometric quantification using control software of Amersham Imager 600. Bile salt hydrolase activity. We suspended freshly harvested cecal content in PBS (40 mg/ml) and filtered it using a cell strainer (100-µm mesh size; Falcon). We immediately added 15 µl of taurocholic acid (1 mg/ml in PBS; Acros) to 900 µl of the suspension and incubated the mix at 37 °C for 10 min, with gen￾tle shaking at 400 r.p.m. We then stopped the reaction by adding 1 ml of ace￾tonitrile. After centrifugation (15 min, 13,000g, 4 °C), 95 µl of the supernatant was transferred into a glass vial, 5 µl of D3-methionine (0.1 mmol/liter) was added and taurine measurements were taken using HILIC-ESI-QqQ (column: HILIC Kinetex (2.6 µm, 150 mm × 2.1 mm inner diameter, Phenomenex); QqQ: API2000 (ABSCIEX))53. Quantification of fecal cholesterol. For cholesterol measurement, we extracted fecal samples using methanol and re-dissolved the dried pellets in a buffer (0.1 M potassium phosphate, pH 7.4, 0.05 M NaCl, 5 mM cholic acid, 0.1% Triton X-100) before cholesterol was quantified by a colorimetric assay (Roche). Rectal temperature and tail heat loss. We measured rectal temperature in mice that were housed at different ambient temperatures by using a medical precision thermometer at 20:00 h (Ellab, Denmark). We determined heat loss indirectly by taking pictures of the tail with an infrared camera (FLIR E60, FLIR Systems, USA). Pictures were processed with the respective software package to determine proximal and distal tail surface temperatures. Histology. We fixed brown adipose tissue in formalin (3.7% in 90% ethanol) for 24 h and subsequently embedded the samples in paraffin. Hematoxylin and eosin (H&E) staining was performed using standard protocols. Indirect calorimetry. We determined O2 consumption and CO2 production via indirect calorimetry in a thermally and humidity-controlled environment using the TSE Phenomaster system (TSE systems, Germany) with a day and night cycle of 12 h each. Mice were housed in single cages and had ad libitum access to food and water. Statistical analysis. We performed all statistical analyses using GraphPad Prism6 (StatCon). When comparing two groups, significance was calculated using a non-paired two-tailed Student’s t-test with no assumption of equal variance. When comparing more than two groups, two-way ANOVA followed by post hoc testing (Tukey correction) was conducted. No statistical method was used to predetermine sample size. The estimated variation within each group of an experiment was similar. There was no blinding for the experiments. All values in the figure panels show mean ± s.e.m. Numbers per group in the figure legends refer to the number of mice per group. Statistical significance is indicated by P value (*P < 0.05, **P < 0.01, ***P < 0.001). We preferentially used group sizes of 5–8 mice, which we determined as optimal in previous in vivo studies. We used smaller group sizes in studies with genetically modified animals, when sufficient numbers were not available. We describe the statis￾tics regarding microbiome analysis in the subsection on microbiome analysis (see above). Data availability. Data are available from the authors upon request. 47. Rohlmann, A., Gotthardt, M., Hammer, R.E. & Herz, J. Inducible inactivation of hepatic LRP gene by Cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants. J. Clin. Invest. 101, 689–695 (1998). 48. Rühlemann, M.C. et al. Fecal microbiota profiles as diagnostic biomarkers in primary sclerosing cholangitis. Gut 66, 753–754 (2017). 49. Wang, Y., Naumann, U., Wright, S.T. & Warton, D.I. mvabund—an R package for model-based analysis of multivariate abundance data. Methods Ecol. Evol. 3, 471–474 (2012). 50. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995). 51. John, C. et al. A liquid chromatography–tandem mass spectrometry–based method for the simultaneous determination of hydroxy sterols and bile acids. J. Chromatogr. A 1371, 184–195 (2014). 52. Eissing, L. et al. De novo lipogenesis in human fat and liver is linked to ChREBP- β and metabolic health. Nat. Commun. 4, 1528 (2013). 53. Meiss, E. et al. Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications. Metabolomics 12, 52 (2016)