SUPPLEMENTARY INFORMATION Prdm4 induction by the small molecule butein promotes white adipose tissue browning No-Joon Song 1 , Seri Choi 2,# , Prashant Rajbhandari 3,# , Seo-Hyuk Chang 1 , Suji Kim 1 , Laurent Vergnes 4 So Mi Kwon 1 Jung Hoon Yoon 1 Suk Chan Lee 5 Jin Mo Ku 6 Jeong Laurent Vergnes 4 , So-Mi Kwon 1 , Jung-Hoon Yoon 1 , Suk-Chan Lee 5 , Jin-Mo Ku 6 , Jeong- Soo Lee 7 , Karen Reue 4,8 , Seung-Hoi Koo 2 , Peter Tontonoz 3,9 , Kye Won Park 1,* 1 Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea. 2 Division of Life Sciences, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Korea. 3 Howard Hughes Medical Institute and 9 Departments of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA. 4 Department of Human Genetics, 8 Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA. 5 Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Korea. 6 Natural product research team, Gyeonggi Bio-Center, Gyeonggi Institute of Science and Technology promotion, Suwon 16229, Korea. 7 Korea Research Institute of Bioscience and Biotechnology and Major of Functional Genomics, University of Science and Technology, Daejeon 34141, Korea. # These authors contributed equally These authors contributed equally Corresponding author : [email protected]Nature Chemical Biology: doi:10.1038/nchembio.2081
Transcript
SUPPLEMENTARY INFORMATION
Prdm4 induction by the small molecule butein promotes white adipose tissue browning
No-Joon Song1, Seri Choi2,#, Prashant Rajbhandari3,#, Seo-Hyuk Chang1, Suji Kim1, Laurent Vergnes4 So Mi Kwon1 Jung Hoon Yoon1 Suk Chan Lee5 Jin Mo Ku6 JeongLaurent Vergnes4, So-Mi Kwon1, Jung-Hoon Yoon1, Suk-Chan Lee5, Jin-Mo Ku6, Jeong-Soo Lee7, Karen Reue4,8, Seung-Hoi Koo2, Peter Tontonoz3,9, Kye Won Park1,*
1Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea.2Division of Life Sciences, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Korea.f f , y, , g , ,3 Howard Hughes Medical Institute and 9Departments of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA.4Department of Human Genetics,8Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA. 5Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Korea.6Natural product research team, Gyeonggi Bio-Center, Gyeonggi Institute of Science and Technology promotion, Suwon 16229, Korea.7Korea Research Institute of Bioscience and Biotechnology and Major of Functional Genomics, University of Science f gy j f y fand Technology, Daejeon 34141, Korea.
Nature Chemical Biology: doi:10.1038/nchembio.2081
SUPPLEMENTARY RESULTS
Supplementary Table 1: Ucp1 induction by bioactive compoundsSupplementary Table 1: Ucp1 induction by bioactive compounds.
Supplementary figure1. Identification of butein as an Ucp1 inducer.Supplementary figure 2: Butein inhibits adipogenesis in C3H10T1/2 cells.Supplementary figure 3: Butein induces expression of thermogenic genes in adipocytes.S l fi 4 S l i i d i f U 1 b b iSupplementary figure 4: Selective induction of Ucp1 by butein.Supplementary figure 5. Identification of genes selectively regulated by butein.Supplementary figure 6: Effects on Ucp1 expression by butein-selective genes.Supplementary figure 7: Induction of Prdm4 and Ucp1 by butein in vivo.Supplementary figure 8: Acute induction of Prdm4 by butein.pp y g ySupplementary figure 9: Prdm4 silencing increases lipid accumulation and expression of white adipocyte-selective genes in preadipocytes.Supplementary figure 10: Prdm4 regulates oxygen consumption rates in C3H10T1/2 cells.Supplementary figure 11: Prdm4 induces Ucp1 and thermogenic genes in T37i cells.Supplementary figure 12: Prdm4 overexpressing cells decrease expression of white adipocyte-selective genesSupplementary figure 12: Prdm4 overexpressing cells decrease expression of white adipocyte-selective genes. Supplementary figure 13: Prdm4 is necessary for the effects of butein in thermogenic programming. Supplementary figure 14: Prdm4 expression is reduced in adipose tissues of obese and diabetic mice.Supplementary figure 15: Effects of Prdm4 knockdown on tissue weight gains and insulin sensitivity in HFD fed mice.Supplementary figure 16: Prdm4 knockdown in HFD fed mice does not alter food intake and locomotor activity. S l t fi 17 P d 4 k kd i HFD f d i d ditSupplementary figure 17: Prdm4 knockdown in HFD fed mice decreases energy expenditure.Supplementary figure 18: Effects of Prdm4 knockdown on white and brown adipocyte-selective gene expression in adipose tissues of HFD fed mice. Supplementary figure 19: Effects of Prdm4 knockdown on Ucp1 expression in adipose tissues of HFD fed mice.Supplementary figure 20: Uncropped versions of immunoblots in the figure 1b.Supplementary figure 21: Uncropped versions of immunoblots in the supplementary figures 4b, 7c, and 10d.Supplementary figure 22: Uncropped versions of immunoblots in the supplementary figure 11b and 18b.
Supplementary Notes: Chemical Identity of Butein.Supplementary data set 1: Lists of butein selective genesSupplementary data set 1: Lists of butein selective genes
Nature Chemical Biology: doi:10.1038/nchembio.2081
Number Compound Name Fold change (UCP-1 expression)
Supplemental Table 1. Ucp1 induction by bioactive compounds. C3H10T1/2 adipocytes were treated with bioactive compounds (20 μM) for 6 hours and Ucp1 expression was measured by real time PCR. Fold increase relative to DMSO treatment was shown. r-Oryzanol is a mixture of steryl and other triterpenyl esters of ferulic acids and Conjugated linoleic acids (CLA) is a family of at least 28 isomers of linoleic acid.
42 Tert-butylhydroquinone 0.881284
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Supplementary Figure 1. Identification of butein as an Ucp1 inducer. (a) C3H10T1/2 adipocytes were treated with various polyphenols isolated from herbal products, including the known anti-adipogenic compounds resveratrol, butein, and sulfuretin. Ucp1 mRNA expression was measured by real time PCR. Data represent means ± s.d. (n=3). (b) C3H10T1/2 adipocytes were treated with butein, resveratrol or sulfuretinfor 6 hours and the gene expression profiles were analyzed by microarray. Diagram showing the number of genes (> 1.6 fold) regulated by g p p y y y g g g ( ) g ybutein, sulfuretin (Sul), and resveratrol (Res). (c) Butein but not resveratrol and sulfuretin induces Prdm4 mRNA expression. Data represent means ± s.d. (n=3). Statistical significance was determined relative to a control by the Student’s t-test (* P<0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Supplementary Figure 2. Butein inhibits adipogenesis in C3H10T1/2 cells. (a) Butein suppresses lipid accumulation in C3H10T1/2 cells. Data are representative of two independent experiments. (b) C3H10T1/2 cells were differentiated and treated with butein for 7 days and mRNA expression of Pparγ, aP2, CD36, and adipoq (adiponectin) were measured by realtime PCR. Data represent means ± s.d. (n=3). Statistical significance was determined relative to a control by Student’s t-test (* P<0.05; ** P<0.005; *** P<0.0005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
Supplementary Figure 3. Butein induces expression of thermogenic genes in adipocytes. (a) Mitochondrial staining by mitochondria specific cytopainter (ab112145, Abcam) in C3H10T1/2 adipocytes (left) and quantified by NIH Image J software (right). Data represent means ± s.d.(n=3). (scale bar = 100 μm) (b) C3H10T1/2 adipocytes were treated with butein and stained with bodipy (green). Quantification of changes in ( ) ( μ ) ( ) p y py (g ) Q glipid droplet (LD) size (right). Data represent means ± s.d. (n=3). (scale bar = 50 μm) (c) Primary adipocytes were treated with butein for 24 hours and expression levels of general adipocyte markers (Pparγ and aP2), white adipocyte-selective markers (Nnmt and Retn), and thermogenic-selective markers (Ucp1, Cidea, Ppargc1α, and Dio2) were measured by realtime PCR. Data represent means ± s.d. (n=3). Statistical significance was determined relative to a control by Student’s t-test (* P<0.05; ** P<0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
Supplementary Figure 7. Induction of Prdm4 and Ucp1 by butein in vivo. (a) Treatment with butein at daily doses of 5 and 15mg/kg for 8 weeks in mice (n=7 per each group) increases the expression of Prdm4 in inguinal (iWAT) (top), epididymal white adipose tissues (eWAT) (middle), and brown adipose tissues (BAT) (bottom). (b) Treatment with butein increases the expression of Ucp1 in iWAT (top), eWAT (middle), and BAT (bottom). Dots (open and closed) and bars in scatter plots represent expression levels of individual mice andth ti l St ti ti ll i ifi t diff i i b t th t l d b t i t t d i ( 7the average, respectively. Statistically significant differences in gene expression between the control and butein-treated mice (n=7 per each group) were determined by Student’s t-test (* P < 0.05; ** P<0.005; *** P<0.0005). (c) Expression of Prdm4 and Ucp1 protein in eWAT of control (n=5) and butein-injected mice (15mg/kg, n=5) was determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. 21.
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Supplementary Figure 16. Prdm4 knockdown in HFD fed mice does not alter food intake and locomotor activity. C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. (a) Food intake of HFD-fed control ASO and Prdm4 ASO-treated mice. Data represent means ± s.d. (n=5). (b) Physical activity of HFD-fed control ASO and Prdm4 ASO-treatedASO and Prdm4 ASO treated mice. Data represent means ± s.d. (n 5). (b) Physical activity of HFD fed control ASO and Prdm4 ASO treated mice. Data represent means ± s.d. (n=5). Statistically significant differences in the control ASO and Prdm4 ASO-injected groups were determined by Student’s t-test (NS, not significant).
Nature Chemical Biology: doi:10.1038/nchembio.2081
b
48 Ctrl ASOPrdm4 ASO
min
/kg)
55
50
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a
min
/kg)
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VSupplementary Figure 17. Prdm4 knockdown in HFD fed mice decreases energy expenditure. (a-d) Mice on a high fat diet were treated with Prdm4 ASO or control ASO for 2.5 weeks and energy expenditure was measured. Data represent means ± s.d. (n=5). (a) O2 consumption wasPrdm4 ASO or control ASO for 2.5 weeks and energy expenditure was measured. Data represent means ± s.d. (n 5). (a) O2 consumption was recorded during a 24 hour period. (b) averages of O2 consumption. (c) CO2 production was recorded during a 24 hour period. (d) averages of CO2production. Statistically significant differences in the control and Prdm4 ASO-treated mice were determined by Student’s t-test (* P < 0.05; **P<0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
Supplementary Figure 18. Effects of Prdm4 knockdown on white and brown adipocyte selective gene expression in adipose tissues of HFD fed mice. (a-b) C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. Expression of brown (a) and white adipocyte-selective genes (n=6 per group) (b) in epididymal adipose tissues of control and Prdm4 ASO-treated mice (n=6 per group). Dots (open and closed) and bars in the scatter plots represent individual mice and the average, respectively. Statistically significant differences in the control ASO and Prdm4 ASO-injected groups were determined by Student’s t-test (* P < 0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
P d 4 ASOa b
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S l t Fi 19 Eff t f P d 4 k kd U 1 i i di ti f HFD f d i ( b) C57BL/6 iSupplementary Figure 19. Effects of Prdm4 knockdown on Ucp1 expression in adipose tissues of HFD fed mice. (a-b) C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. (a) Expression of Prdm4 and Ucp1in epididymal adipose tissues (eWAT) of control and Prdm4 ASO-injected mice (n=5 per group). Dots (open and closed) and bars in the scatter plots represent expression levels of individual mice and the average, respectively. (b) Expression of Prdm4 and Ucp1 protein in eWAT of control ASO and Prdm4 ASO-injected mice (n=5 per group) was determined by western blotting. Brown adipose tissues (BAT) were used as controls. Uncropped images of blots are shown in Supplementary Fig. 22.
Nature Chemical Biology: doi:10.1038/nchembio.2081
Original blots
- + - +12h 24h
Butein - + - +12h 24h
Fig. 1b
- + - +12h 24h
(kD )Butein Butein
170100
7055403525
(kDa)170100
70554035
(kDa) 170100
7055403525
(kDa)
WB: Ucp1 WB: ActinWB: Prdm4
25
1025
1010
Supplementary Figure 20 Uncropped versions of immunoblots in the Fig 1bSupplementary Figure 20. Uncropped versions of immunoblots in the Fig 1b.
Nature Chemical Biology: doi:10.1038/nchembio.2081
