doi: 1.138/nature7221 Brown fat selective genes 12 1 Control Q-RT-PCR (% of Control) 8 6 4 2 Ntrk3 Cox7a1 Cox8b Cox5b ATPase b2 ATPase f1a1 Sirt3 ERRα Elovl3/Cig3 PPARα Zic1 Supplementary Figure S1. stimulates the expression of brown fat selective genes. Quantitative RT-PCR analysis for brown fat selective genes using total RNA isolated from brown preadipocytes treated with vehicle (control) or various BMPs (3.3 nm) in combination of insulin (2 nm) and T3 (1 nm) for 7 days. Data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between control and experimental groups as determined by ANOVA ( = P <.5, = P <.1, = P <.1). Remarks: The selection of these genes is based on literatures (1-3) and public microarray database (4). The brown fat selective expression of these genes was further validated by quantitative RT-PCR using mrna isolated from interscapular brown fat and epididymal white fat of C57BL/6 mice. 1. Gesta,S., Tseng,Y.H. & Kahn,C.R. Developmental origin of fat: tracking obesity to its source. Cell 131, 242-256 (27). 2. Seale,P. et al. Transcriptional Control of Brown Fat Determination by PRDM16. Cell Metab 6, 38-54 (27). 3. Timmons,J.A. et al. Myogenic gene expression signature establishes that brown and white adipocytes originate from distinct cell lineages. Proc Natl. Acad Sci U S. A. 14, 441-446 (27). 4. http://oribetes.peds.uiowa.edu/resources:sets:bat-wat-sm www.nature.com/nature 1
doi: 1.138/nature7221 Brown Pre-Ad Control BMP-6 UCP-1 β-tubulin White Pre-Ad UCP-1 β-tubulin Supplementary Figure S2., but not BMP-6, induces expression of UCP-1 protein in brown preadipocytes. Western blotting analysis of UCP-1 in brown and white preadipocytes treated with vehicle (control), BMP-6 or (8.3 nm) in combination of insulin (2 nm) and T3 (1 nm) for 13 days. Blots were stripped and re-probed with β-tubulin to normalize for variation in loading and transfer of proteins. www.nature.com/nature 2
doi: 1.138/nature7221 a Control Rosiglitazone SVF in BAT SVF in WAT b 25 25 c -Tx SVF from BAT PPARγ UCP-1 2 2 12 UCP-1 15 15 1 Q-RT-PCR (arbitrary units) 1 5 SVF in BAT SVF in WAT Control Rosiglitazone Q-RT-PCR (arbitrary units) 1 5 SVF in BAT SVF in WAT Q-RT-PCR (arbitrary units) 8 6 4 2 Basal camp Supplementary Figure S3. induces brown adipogenesis in stromo-vascular fraction (SVF) isolated from brown fat. a, SVF isolated from interscapular BAT and subcutaneous WAT of 6-week old C57BL/6 mice were induced to undergo adipocyte differentiation using the protocol described in Methods supplemented with rosiglitazone (1 µg/ml), (3.3 nm) or vehicle (control) for 3 days. Photographs were taken under a bright-field microscope. Original magnification = 4 X. b, Quantitative RT-PCR analysis for PPARγ and UCP-1 using total RNA isolated from SVF treated with vehicle (control), rosiglitazone (rosi), or for 7 days. c, BMP- 7-differentiated adipocytes derived from SVF of BAT were stimulated with camp for 4 hrs. mrnas extracted from these cells were analyzed for UCP-1 expression by Q- RT-PCR. Data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between control and experimental groups as determined by ANOVA ( = P <.5, = P <.1, = P <.1). www.nature.com/nature 3
doi: 1.138/nature7221 Q-RT-PCR (% of Maximum) 14 12 1 8 6 4 Brown Pre-Ad White Pre-Ad 2 BMPR2 BMPR1a (ALK3) BMPR1b (ALK6) ACVR1 (ALK2) Supplementary Figure S4. Relative levels of expression for different BMP receptor isoforms in brown and white preadipocytes. Data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between two groups as determined by ANOVA ( = P <.5). www.nature.com/nature 4
doi: 1.138/nature7221 DMSO Control SB2219 SB2328 PD169316 Control Control Control Supplementary Figure S5. Activation of p38 MAP kinase is not essential for - induced lipid accumulation in brown preadipocytes. Oil Red O staining of brown preadipocytes cultured in growth medium supplemented with vehicle (control) or (3.3 nm) in combination of insulin (2 nm) and T3 (1 nm) for 1 days. Inhibitors p38 MAPK, SB2919, SB2358, PD169316 (1 um), or DMSO were added to the cells 7 hrs prior to and throughout treatment. www.nature.com/nature 5
doi: 1.138/nature7221 a Wild-type PGC-1α/β KO Control BMP-4 b Q-RT-PCR (arbitrary units) 35 3 25 2 15 1 5 PPARγ Wild-type PGC-1α/β KO 8 7 6 5 4 3 2 1 UCP-1 Control BMP-4 Wild-type PGC-1α/β KO Supplementary Figure S6. PGC-1 is essential for -induced UCP-1 expression, but is dispensable for s effect on lipid accumulation. a, Oil Red O staining of wild-type and PGC-1 null brown preadipocytes cultured in growth medium supplemented with vehicle (control), BMP-4 (3.3 nm) or (3.3 nm) in combination of insulin (2 nm) and T3 (1 nm) for 6 days. b, Quantitative RT-PCR analysis for PPARγ and UCP-1 in cells described in (a). Data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between control and experimental groups as determined by ANOVA ( = P <.5, = P <.1, = P <.1). www.nature.com/nature 6
doi: 1.138/nature7221 3 PRDM16 Q-RT-PCR (% of Implant) 25 2 15 1 5 UCP-1 9 Q-RT-PCR (% of Implant) 2 1 ND 45 Leptin Q-RT-PCR (% of Implant) 35 25 15 5 WAT Implant BAT Supplementary Figure S7. Fat pad derived from implanted C3H1T1/2 cells strongly resembles endogenous BAT by gene expression. Quantitative RT-PCR analysis for PRDM16, UCP-1 and leptin in fat pad derived from implanted cells, which were pretreated with ex vivo, relative to the levels of expression for these genes in endogenous BAT and WAT. PRDM16 and UCP-1 are BAT markers and leptin serves as a WAT selective marker. www.nature.com/nature 7
doi: 1.138/nature7221 a Control Induction Induction + Rosi b Induction Induction + Rosi C B7 C B7 FAS UCP-1 β-tubulin Supplementary Figure S8. induces brown adipogenesis in mouse embryonic fibroblasts (MEFs). a, Oil Red O staining of MEFs treated with a combination of induction cocktail, rosiglitazone (rosi) or as indicated for 8 days. Lipid droplets stained by Oil Red O are shown in red. Original magnification is 4 X. b, Western blotting analysis of FAS and UCP-1 in MEFs at day 8 of differentiation in a combination of induction cocktail, rosiglitazone (rosi) or. Blots were stripped and reprobed with β-tubulin to normalize for variation in loading and transfer of proteins. www.nature.com/nature 8
doi: 1.138/nature7221 NIH-3T3 Control BMP-4 Q-RT-PCR Q-RT-PCR 16 14 12 1 8 6 4 2 5 4 3 2 1 PPARγ PRDM16 Q-RT-PCR 3 25 UCP-1 2 15 1 5 Control BMP-4 Supplementary Figure S9. Distinct effects of BMP-4 and in induction of lipid accumulation, and expression of brown fat markers in NIH-3T3 cells. NIH-3T3 cells were grown in growth medium supplemented with rosiglitazone (1 µg/ml) in the presence of BMP-4 (3.3 nm), (3.3 nm), or vehicle for 9 days. Cells were fixed for Oil Red O staining or subjected to RNA extraction. Expression of PPARγ, PRDM16, and UCP-1 was measured by quantitative RT-PCR analysis. All data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between control and BMP groups as determined by ANOVA ( = P <.5, = P <.1, = P <.1). www.nature.com/nature 9
doi: 1.138/nature7221 WT KO Supplementary Figure S1. H&E staining of brown fat from wild-type and BMP- 7 KO embryos at 17.5 dpc. Original magnification is 4 X. www.nature.com/nature 1
doi: 1.138/nature7221 Brown fat selective genes 14 WT 12 KO Q-RT-PCR (% of WT) 1 8 6 4 2 Ntrk3 COX 7a1 COX 8b ERRα ATPase b2 ATPase f1a1 Sirt3 Elovl3/Cig3 PPARα Zic1 Supplementary Figure S11. Expression of brown fat selective genes is decreased in KO embryos. Quantitative RT-PCR analysis for brown fat selective genes using total RNA isolated from interscapular BAT derived from 18.5 dpc wild-type (n= 4) and KO (n= 5) embryos. Data are presented as mean ± SEM. Asterisks depict statistically significant differences between control and experimental groups as determined by Student t test ( = P <.5, = P <.1). www.nature.com/nature 11
doi: 1.138/nature7221 Activity (counts) in serum (pg/ml) a Plasma Concentrations 4 Ad-LacZ 35 Ad- 3 25 2 15 1 5 Day Day 1 Day 3 Days after injection b Activity 9 8 7 6 5 4 3 2 1 Light Cycle Dark Cycle d e Activity (counts) VO2 (ml/kg/hr) 3 25 2 15 1 5 9 8 7 6 5 4 3 Activity Ad-LacZ Ad-BMP-3 Ad- 2 4 6 8 1 12 14 16 18 2 22 24 Time (hours) Oxygen Consumption Ad-LacZ Ad-BMP-3 Ad- p <.1, vs. LacZ Food Intake (g)/mouse/day c 3. 2.5 2. 1.5 1..5 Food Consumption Ad-Lac Z Ad-BMP-3 Ad- f Heat (Kcal/hr) 2.9.8.7.6.5 2 4 6 8 1 12 14 16 18 2 22 24 Energy Expenditure Time (hours)..4.3 p <.1, vs. LacZ.2 2 4 6 8 1 12 14 16 18 2 22 24 Time (hours) Supplementary Figure S12. Adenoviral-mediated expression of in C57BL/6 mice leads to increased oxygen consumption and energy expenditure without alteration in activity and food intake. a, Plasma levels in C57BL/6 mice after tail vein injection with adenoviruses expressing or LacZ. b-f, Adenoviruses expressing, BMP-3 or LacZ control were injected into 4-week old C57BL/6 mice via the tail vein (n = 5 for each group). Locomotor activity, O 2 consumption, and CO 2 production were measured in these mice after 1 day of injection during a 24-h time period using the CLAMS www.nature.com/nature apparatus. Energy Expenditure expressed as Kcal/hr per animal was calculated using equations 12 described in Methods. Food consumption was calculated as average food consumed by each mouse per day.
doi: 1.138/nature7221 a WAT b BAT WAT (% of BW) 2.5 2. 1.5 1..5 Ad-Lac Z Ad- BAT (% of BW).7.6.5.4.3.2.1 c VO2 (ml/kg/hr x 1) 35 33 31 29 Oxygen Consumption. 4-w mice 12-w mice. 4-w mice 12-w mice 27 Light Cycle Dark Cycle Supplementary Figure S13. increases BAT, but not WAT, masses and energy expenditure in C57BL/6 mice. Adenoviruses expressing or LacZ control were injected into 4-week and 12-week old C57BL/6 mice via the tail vein (n = 6 for each group). Mice were sacrificed 15 days after injection. Weights of epididymal WAT (a) and interscapular BAT (b) for each group were determined at the time of sacrifice and expressed as percentage of total body weight. c, Oxygen consumption in 12-w old animals at light and dark cycles was measured in a CLAMS apparatus. Data are presented as mean ± SEM. Asterisks depict statistically significant differences between Adeno-lacZ and Adeno- for each age group as determined by Student s t test ( = P <.5, = P <.1, = P <.1). www.nature.com/nature 13
doi: 1.138/nature7221 WAT PGC-1α UCP-3 Cyto C Tfam Control NRF-1 NRF-2 2 4 6 8 1 12 14 Muscle PGC-1α PGC-1β UCP-3 Cyto C DIO-2 DIO-3 5 1 15 2 Liver PGC-1α PGC-1β Cyto C Tfam FXR SHP 2 4 6 8 1 12 14 Relative mrna Expression Level Supplementary Figure S14. No change in the expression of genes involved in energy metabolism in white fat, muscle and liver of -treated mice. Quantitative RT-PCR analysis for genes involved in energy metabolic in WAT, muscle, and liver from 12-week old C57BL/6 mice received adenoviruses expressing LacZ (control) or. www.nature.com/nature 14
doi: 1.138/nature7221 Supplementary Table S1. Relative expression levels of UCP- 1 in BAT, WAT, undifferentiated and differentiated brown and white adipocytes Cell/Tissue UCP-1 mrna a White PreAd-d 1 White PreAd-d7-Differentiated b 1 Brown PreAd-d 1 Brown PreAd-d7-Differentiated b 289 Brown PreAd-d7-Control c 62 Brown PreAd-d7-BMP4 c 13 Brown PreAd-d7-BMP7 c 356 Tissue-BAT d 1936 Tissue-WAT d 18 a Expression levels of UCP-1 was measured by quantitative RT-PCR analysis using the same amount of total RNA isolated from tissues and cells as indicated. All values were normalized to undifferentiated brown preadipocytes (Brown PreAd-d). b White and brown preadipocytes were differentiated using standard differentiation protocol as described in Methods for 7 days. c Brown preadipocytes were grown in DMEM with 1% FBS supplemented with BMP-4 (3.3 nm), (3.3 nm) or vehicle (control) in combination of insulin (2 nm) and T3 (1 nm) for 7 days d Interscapular BAT and epididymal WAT were isolated from 12-week old C57BL/6 male mice. www.nature.com/nature 15
doi: 1.138/nature7221 Supplementary Table S2: Primer sequences used in quantitative RT-PCR Gene Sequences ap2 Forward GATGCCTTTGTGGGAACCT Reverse CTGTCGTCTGCGGTGATTT Arbp (36B4) Forward TTTGGGCATCACCACGAAAA Reverse GGACACCCTCCAGAAAGCGA ALK-2 Forward TGCTAATGATGATGGCTTTCC Reverse TTCACAGTGGTCCTCGTTCC ALK-3 Forward AATGCAAGGATTCACCGAAAGCCC Reverse ACAGCCATGGAAATGAGCACAACC ALK-6 Forward AGAAGAGCACAGAGGCCCAATTCT Reverse TGCAAGGTACACAGCAGTGCTAGA ATPase b2 Forward ACCTATCCCAGCCTCGTCTC Reverse AGGACTTGCCCACTTCTCTTT BMPR2 Forward AGCAATCGCCCATCGAGACTTGAA Reverse TTCTGGAGGCATATAGCGCTTGGT C/EBPα Forward CAAGAACAGCAACGAGTACCG Reverse GTCACTCGTCAACTCCAGCAC C/EBPβ Forward CAAGTTCCGCAGGGTGCT Reverse CCAAGAAGACGGTGGACAA C/EBPδ Forward GCTTTGTGGTTGCTGTTGAA Reverse ATCGACTTCAGCGCCTACA CIDEA Forward ATCACAACTGGCCTGGTTACG Reverse TACTACCCGGTGTCCATTTCT Cox5b Forward GCTGCATCTGTGAAGAGGACAAC Reverse CAGCTTGTAATGGGTTCCACAGT Cox7a1 Forward CAGCGTCATGGTCAGTCTGT Reverse AGAAAACCGTGTGGCAGAGA Cox8b Forward GAACCATGAAGCCAACGACT Reverse GCGAAGTTCACAGTGGTTCC DIO2 Forward CAGTGTGGTGCACTGCTCCAATC Reverse TGAACCAAAGTTGACCACCAG DIO3 Forward ATGCGTATCAGACGACAACC Reverse AAATTGAGCACCAACGGG Cytochrome c1 Forward GCTACCCATGGTCTCATCGT Reverse CATCATCATTAGGGCCATCC www.nature.com/nature 16
doi: 1.138/nature7221 Elovl3/Cig3 Forward TCCGCGTTCTCATGTAGGTCT Reverse GGACCTGATGCAACCCTATGA ERRα Forward GCAGGGCAGTGGGAAGCTA Reverse CCTCTTGAAGAAGGCTTTGCA FXR Forward CACGGTTGTAAATACAGACTAGATAG Reverse TTGATTTAATTAGGCCAAAAGG Leptin Forward CCTCATCAAGACCATTGTCACC Reverse TCTCCAGGTCATTGGCTATCTG Necdin Forward CACTTCCTCTGCTGGTCTCC Reverse ATCGCTGTCCTGCATCTCAC NRF-1 Forward CAACAGGGAAGAAACGGAAA Reverse GCACCACATTCTCCAAAGGT NRF-2 Forward AGGTTGCCCACATTCCCAAACAAG Reverse TTGCTCCATGTCCTGCTCTATGCT Ntrk3 Forward TGGCTCACACTGATCTCTGG Reverse GCCAGAGCCTTTACTGCATC PGC-1α Forward GTCAACAGCAAAAGCCACAA Reverse TCTGGGGTCAGAGGAAGAGA PGC-1β Forward CTTGCTTTTCCCAGATGAGG Reverse CCCTGTCCGTGAGGAACG PPARα Forward GCGTACGGCAATGGCTTTAT Reverse GAACGGCTTCCTCAGGTTCTT PPARγ Forward TCAGCTCTGTGGACCTCTCC Reverse ACCCTTGCATCCTTCACAAG PRDM 16 Forward GACATTCCAATCCCACCAGA Reverse CACCTCTGTATCCGTCAGCA Pref-1 Forward AGTACGAATGCTCCTGCACAC Reverse CTGGCCCTCATCATCCAC Runx2 Forward AGCCTCTTCAGCGCAGTGAC Reverse CTGGTGCTCGGATCCCAA SHP Forward CTGCCTGGAGTCTTTCTGGA Reverse GGTACCAGGGCTCCAAGACT Tfam Forward GTCCATAGGCACCGTATTGC Reverse CCCATGCTGGAAAAACACTT UCP-1 Purchased from SuperArray (Cat # PPM5164A) UCP3 Forward CCTACGACATCATCAAGGAGAA Reverse GCCACCATCTTCAGCATACA www.nature.com/nature 17
doi: 1.138/nature7221 Wnt1a Forward CACCCGGCCATACTTCCT Reverse CACTTACGCCGCATGTTCT Wnt 1b Forward TCCAAGAAATCCCGAGAGAA Reverse GGATGGAAGGGTAGTGGTGAG Zic1 Forward CTGTTGTGGGAGACACGATG Reverse CCTCTTCTCAGGGCTCACAG www.nature.com/nature 18