Supporting Information. Supporting Tables. S-Table 1 Primer pairs for RT-PCR. Product size. Gene Primer pairs
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1 Supporting Information Supporting Tables S-Table 1 Primer pairs for RT-PCR. Gene Primer pairs Product size (bp) FAS F: 5 TCTTGGAAGCGATGGGTA R: 5 GGGATGTATCATTCTTGGAC 3 SREBP-1c F: 5 CGCTACCGTTCCTCTATCA R: 5 CACTTCGCAGGGTCAGG 3 ACCα F: 5 AAGGGCTACCTCTAATG R: 5 CACAAACCAGCGTCTCA 3 PPARα F: 5 AGGCTATCCCAGGCTTTGC R: 5 CGTCTGACTCGGTCTTCTTG 3 ACO1 F: 5 TCAAGCAAAGCGAACCAG R: 5 ACCATACCACCCACCAAC 3 MACD F: 5 CTGCGTGACAGAACCCTC R: 5 CAACTTTCATCGCCATTT 3 VLACD F: 5 AGGCTTTGGCGGCTTTCTG R: 5 CTCTGCTGGCACCTTGACT 3 UCP-2 F: 5 TGGGCACCATCCTAACC R: 5 TGGCATTTCGGGCAACA 3 TNFα F: 5 ACATCTCCCTCCGGAAAGGA R: 5 CGCCACGAGCAGGAATGAGA 3 IL-6 F: 5 GGATACCACCCACAACAG R: 5 TGCCGAGTAGACCTCATAG 3 ERα F: 5 GGTCCAATTCTGACAATCGAC R: 5 TTTCGTATCCCGCCTTTCATC 3 ERβ F: 5 GCACCTTGAGTCCAGAG R: 5 TCAGTCCCACCATTAGC 3 AR F: 5 GCTCTGGCAGCAGTGAAGCA R: 5 AGTCCCCATAGCGGCATTGC 3 CPT-1 F: 5 CTCAGCCTCTACGGCAAATC R: 5 CTTCTTGATCAGGCCTTTGC 3 PPARγ F: 5 ATTCTGGCCCACCAACTTCGG R: 5 TGGAAGCCTGATGCTTTATCCCCA 3 LXRα F: 5 TACAACCCTGGGAGTGAGA R: 5 TTAGCATCCGTGGGAACAT 3 DGAT1 F: 5 GGATGGTCCCTACTATCCAG R: 5 CCACCAGTCCCTGTAGAACT 3 DGAT2 F: 5 GGAGGCCACCGAAGTTAGCA R: 5 CACCTCCCACCACGATGACAA 3 1
2 S-Table 2 Antibodies and dilutions Antibody Mol. Wt. Dilution Cat. nr. Source Company ERα 66KD 1:100 sc-542 rabbit polyclonal IgG Santa Cruz TNFα 17KD 1:200 sc-1350 goat polyclonal IgG Santa Cruz AR 87KD 1:200 sc-816 rabbit polyclonal IgG Santa Cruz UCP2 32KD 1:200 sc-6526 goat polyclonal IgG Santa Cruz FAS 207KD 1:200 sc rabbit polyclonal IgG Santa Cruz CPT1 86KD 1:200 sc goat polyclonal IgG Santa Cruz HMGCR 43KD 1:200 sc goat polyclonal IgG Santa Cruz β-actin 42KD 1:500 sc mouse monoclonal IgG Santa Cruz IL-6 26KD 1:1000 ab-6672 rabbit polyclonal IgG Abcam P-ACC 280KD 1:1000 #3661 rabbit polyclonal IgG Cell Signaling ACC 280KD 1:1000 #3676 rabbit monoclonal IgG Cell Signaling P-HMGCR 98KD 1:1000 # rabbit polyclonal IgG Millipore S-Table 3 Total cholesterol and cholesterol ester formation from the rat hepatocytes in vitro with different treatments (concentration expressed in nm/well). Medium with Palmitic acid : Oleic acid (1:2) Vehicle E2 E2+ICI DHT DHT+Flu E2+DHT Total cholesterol 46.67± ±3.32* 40± ±4.22* 42.52± ±7.38* Cholesterol 16.33± ±3.22* 18.46± ± ± ±4.81 ester * p< 0.05 treatments (E2, DHT and/or E2+DHT) vs. vehicle 2
3 Supplemental Figures S-Fig. 1 Accumulation of lipids in hepatocytes by ORO staining (A) Liver from ORX normal diet control group. (B) Liver cells with marked lipid accumulation in all 1, 2, and 3 zones in ORX HFD control group. (C) Liver from E2-treated group; zones 2 and 3 of liver lobules contain normal appearing liver cells rimmed with marked lipid changes in zone 1. (D) Liver from DHT-treated group; zone 2 and 1 of liver lobules contain normal appearing liver cells rimmed with marked lipid changes in zone 3. (E) Liver from E2+DHT-treated group; zone 2 and 3 of liver lobules contain normal appearing liver cells rimmed with also less lipid changes in zone 1. (F) Quantification of ORO staining shown as Mean±SEM, ***, P<0.001 versus HFD-C, high fat diet control. 3
4 S-Fig. 2 Histopathology of the liver tissues by hematoxylin and eosin (H/E) staining from WT gonad intact (non-orx) SD male rats (n=6/group) (A) Liver from the WT normal diet (ND) control group. Zonation is identical as in Fig. 1 (B) Liver cells with lipid accumulation in the HFD control. The inflammation and fatty liver cell presence is significantly less abundant than in HFD ORX liver (Fig. 1); HFD liver from the E2-treated group (C); zones 2 and 3 of liver lobules contain normal appearing liver cells rimmed with fatty changes in zone 3. HFD liver from the DHT-treated group (D) ; zones 1 and 2 of liver lobules contain normal appearing liver cells rimmed with fatty changes in zone 1. (E1-2) HFD liver from the E2+DHT-treated group, normalized liver cells in zones 1, 2 and 3 of liver lobules and scattered with some lipid accumulated liver cells. 4
5 S-Fig. 3 Genes for triglyceride synthesis in SD rat liver tissues No significant changes could be observed in any of the genes involved in TG synthesis among the treatment groups. None of these above shown treated groups versus HFD-C (high fat diet control) were statistically significant. S-Fig. 4 Relative densitometry analysis of the alterations of phosphorylation related to protein levels for P-ACC/total ACC and P-HMGCR/total HMGCR levels after different treatments (calculated from Fig. 3A). The densitometric values were pooled from three animals in each group and repeated three times by Western blot, and are shown as Mean±SEM. *, p<0.05, ***, p<0.001 versus HFD-C, high fat diet control. 5
6 5A) 5B) S-Fig. 5 Expression levels for genes involves in Fatty acid synthesis (A) and β-oxidation (B) in hepatocytes with different treatments. None of these above shown treated groups versus HFD-C (high fat diet control) were statistically significant. 6
7 S-Fig. 6 Human data for serum lipid profile parameters from milder/subnormal (regarded as control) (n=5) and severe (n=5) steatotic liver samples. None of the above values are statistically significant. 7
Supplementary Table 2. Plasma lipid profiles in wild type and mutant female mice submitted to a HFD for 12 weeks wt ERα -/- AF-1 0 AF-2 0
Supplementary Table 1. List of specific primers used for gene expression analysis. Genes Primer forward Primer reverse Hprt GCAGTACAGCCCCAAAATGG AACAAAGTCTGGCCTGTATCCA Srebp-1c GGAAGCTGTCGGGGTAGCGTC CATGTCTTCAAATGTGCAATCCAT
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ZL ZDF ZDF + E2 *** Visceral (g) ZDF
Body Weight (g) 4 3 2 1 ** * ZL ZDF 6 8 1 12 14 16 Age (weeks) B * Sub-cutaneous (g) 16 12 8 4 ZL ZDF Visceral (g) 25 2 15 1 5 ZL ZDF Total fat pad weight (g) 4 3 2 1 ZDF ZL Supplemental Figure 1: Effect
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Males- Western Diet WT KO Age (wks) Females- Western Diet WT KO Age (wks)
Relative Arv1 mrna Adrenal 33.48 +/- 6.2 Skeletal Muscle 22.4 +/- 4.93 Liver 6.41 +/- 1.48 Heart 5.1 +/- 2.3 Brain 4.98 +/- 2.11 Ovary 4.68 +/- 2.21 Kidney 3.98 +/-.39 Lung 2.15 +/-.6 Inguinal Subcutaneous
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Male 30. Female. Body weight (g) Age (weeks) Age (weeks) Atg7 f/f Atg7 ΔCD11c
ody weight (g) ody weight (g) 34 3 Male 3 27 Female 26 24 22 18 7 9 11 13 15 17 19 21 23 21 18 15 7 9 11 13 15 17 19 21 23 Age (weeks) Age (weeks) Supplementary Figure 1. Lean phenotypes in mice regardless
Hmgcoar AGCTTGCCCGAATTGTATGTG TCTGTTGTAACCATGTGACTTC. Cyp7α GGGATTGCTGTGGTAGTGAGC GGTATGGAATCAACCCGTTGTC
Supplement Table I: primers for Real Time RT-PCR Gene Foward Reverse Hmgcoar AGCTTGCCCGAATTGTATGTG TCTGTTGTAACCATGTGACTTC Cyp7α GGGATTGCTGTGGTAGTGAGC GGTATGGAATCAACCCGTTGTC Cyp27a1 GTGGTCTTATTGGGTACTTGC
18s AAACGGCTACCACATCCAAG CCTCCAATGGATCCTCGTTA. 36b4 GTTCTTGCCCATCAGCACC AGATGCAGCAGATCCGCAT. Acc1 AGCAGATCCGCAGCTTG ACCTCTGCTCGCTGAGTGC
Supplementary Table 1. Quantitative PCR primer sequences Gene symbol Sequences (5 to 3 ) Forward Reverse 18s AAACGGCTACCACATCCAAG CCTCCAATGGATCCTCGTTA 36b4 GTTCTTGCCCATCAGCACC AGATGCAGCAGATCCGCAT Acc1
Supplementary Figure 1
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GPR120 *** * * Liver BAT iwat ewat mwat Ileum Colon. UCP1 mrna ***
a GPR120 GPR120 mrna/ppia mrna Arbitrary Units 150 100 50 Liver BAT iwat ewat mwat Ileum Colon b UCP1 mrna Fold induction 20 15 10 5 - camp camp SB202190 - - - H89 - - - - - GW7647 Supplementary Figure
Supplementary Figure 1
VO (ml kg - min - ) VCO (ml kg - min - ) Respiratory exchange ratio Energy expenditure (cal kg - min - ) Locomotor activity (x count) Body temperature ( C) Relative mrna expression TA Sol EDL PT Heart
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Name Animal source Vendor Cat # Dilutions
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control kda ATGL ATGLi HSL 82 GAPDH * ** *** WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi WT/cTg WT/cTg ATGLi AKO/cTg AKO/cTg ATGLi iwat gwat ibat
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