Supplementary Data

Home , ADCYAP1R1, CALCRL, Glutamate receptor, GPR1, GPR119, GPR120

SUPPLEMENTARY DATA

Supplementary Table 1. GTDF does not bind to ionotropic glutamate receptors. Wistar rat cerebral cortex (for AMPA receptor, and NMDA receptor agonist, glycine, phencyclidine and polyamine sites), Wistar rat whole brain (minus cerebellum, for Kainate receptor) or whole Wistar rat brain (for non- selective glutamate binding) were incubated with indicated radioligands in absence or presence of indicated doses of GTDF. % inhibition of radioligand binding was calculated using standard formula. Threshold inhibition was set at 50% for consideration of inhibition.

GTDF Receptor, % Radi Specific Kd of Nonspec Incubat Incubati Concentra assay inhibition oliga binding radiolig ific ion on time, tion (µM) of specific nd activity and competit buffer temperat radioligand of or ure binding radiolig and

AMPA 1.0 mM 50 mM L- Tris- 5.0 Glutami HCl, nM 90% KD1= c acid pH 7.4, 10 3 [3H] 200 AMP 0.018 mM A µM KSCN 1 15 KD2 = 0.1 -7 0.99 90min, µM 4°C Kainate 5.0 1.0 mM 50 mM 60 nM 80% KD = L- Tris- minutes, 10 7 [3H] Glutami HCl, 4°C AMP 0.012 c acid pH 7.4 A µM 1 2 0.1 -1 NMDA, 2.0 1.0 mM 50 mM 20 Agonism nM L- Tris- minutes, [3H] 70% KD = Glutami HCl, 4°C 10 -3 CGP c acid pH 7.4 - 3965 0.019 3 µM 1 7 0.1 6 NMDA, 0.33 50 mM 30 Glycine nM HEPES minutes, [3H] 85% KD = 10.0 µM , pH 4°C 10 7 7.7 MDL 105,5 0.006 MDL 19 µM 105,519 1 3

0.1 3

NMDA, 1.0 µM 45 Phencycli Dizocilp minutes, dine 4.0 ine (+)- 25°C nM MK- [3H] 94% KD = 801) 10 2 0.0084 TCP µM 10 mM Tris- 1 -5 HCl, 0.1 -2 pH 7.4 NMDA, 2.0 50 mM 2 hours, Polyamin nM Tris- 4°C e [3H] 80% KD = HCl, 10 4 pH 7.4 Ifenp 0.026 rodil µM 10.0 µM 1 -8 Ifenprod 0.1 -3 il Glutamate 50 mM 30 , Non- 3.75 Tris- minutes, selective nM HCl, 37°C [3H] 90% KD = pH 7.4, 10 17 L- 2.5 mM Gluta CaCl2 mic 0.29 acid µM 50.0 µM L- 1 13 Glutami 0.1 14 c acid

Supplementary Table 2. GTDF does not activate or repress metabotropic glutamate receptors at its active dose but quercetin activates mGlu2, and suppresses mGlu4, -6 and -8 activity. Modulation of metabotropic glutamate receptor activities by GTDF or quercetin at their pharmacologically active concentrations was assessed. mGlu1, -3, -5 activities were assessed by an aequorin assay, mGlu2 and -7 activities were assessed by cAMP assay, mGlu4, -6 and -8 were assessed by GTPγS binding. PAM; positive allosteric modulator. Threshold activation or repression was set at 50%. % activation or repression beyond the threshold is in red.

Assay mode Conc Co- Compound (µM) Agonist Antagonist PAM Addition % % % % activation inhibition activation activation Receptor: mGlu1 GTDF 1 0.15 -8.98 0.951 2.09 0.1 0.35 1.6 0.791 0.87 Quercetin 1000 -0.049 16.41 2.02 -3.38 100 -0.37 -8.911 6.97 23.58 Receptor: mGlu2 GTDF 1 -10.64 19.62 -17.95 0.1 -7.76 9.56 8.59 Quercetin 1000 -2.17 18.32 12.65 100 117.85* -7.6 284.27* Receptor: mGlu3 GTDF 1 2.38 5.06 -3.55 6.39 0.1 0.78 -12.26 -3.52 3.94 Quercetin 1000 0.36 -6.58 -18.96 -0.061 100 4.75 -11.32 -5.068 24.67 Receptor: mGlu4 GTDF 1 5.04 -4.44 4.14 0.1 1.63 -6.16 5.87 Quercetin 1000 -23.15 86.97 -33.79 100 -7.75 31.65 -14.11 Receptor: mGlu5 GTDF 1 0.4 -1.15 -3.35 2.61 0.1 0.04 3.35 -5.14 3.69 Quercetin 1000 0.11 14.66 -0.74 0.64 100 -0.07 -30.92 8.72 3.55 Receptor:

mGlu6 GTDF 1 -2.24 -0.04 -15.39 0.1 -6.65 -4.91 -4.82 Quercetin 1000 -136.684 201.61 -141.74 100 -20.7469 58.63 -39.7 Receptor: mGlu7 GTDF 1 16.46 11.21 8.68 0.1 19.05 0.22 0.836 Quercetin 1000 18.96 13.68 10.19 100 23.22 -4.71 8.94 Receptor: mGlu8 GTDF 1 1.15 15.33 10.26 0.1 -4.24 -1.28 4.6 Quercetin 1000 -72.89 159.53 -64.26 100 -14.3 58.17 -10.91

Supplementary Table 3. GTDF is not a GPCR agonist. GTDF at its pharmacologically active dose (0.1µM) or high dose (10µM) was assessed for its ability to modulate GPCR signalling using pathhunter β-arrestin assay for the indicated GPCRs. Threshold for consideration as an activator was set at 50%.

GTDF GTDF Assay % % GPCR ID Conc Conc Mode Activity Activity (µM) (µM) ADCYAP1R1 Agonist 0.1 -2% 10 -2% ADORA3 Agonist 0.1 -7% 10 -9% ADRA1B Agonist 0.1 -1% 10 -2% ADRA2A Agonist 0.1 -3% 10 -1% ADRA2B Agonist 0.1 -5% 10 -3% ADRA2C Agonist 0.1 -2% 10 -1% ADRB1 Agonist 0.1 -1% 10 -1% ADRB2 Agonist 0.1 0% 10 -1% AGTR1 Agonist 0.1 -3% 10 -2% AGTRL1 Agonist 0.1 -1% 10 -2% AVPR1A Agonist 0.1 -5% 10 -4% AVPR1B Agonist 0.1 -2% 10 -1% AVPR2 Agonist 0.1 -2% 10 -4% BDKRB1 Agonist 0.1 -5% 10 0% BDKRB2 Agonist 0.1 -3% 10 -3% BRS3 Agonist 0.1 -6% 10 -2% C5AR1 Agonist 0.1 -2% 10 -2% C5LR2 Agonist 0.1 -1% 10 -14% CALCR Agonist 0.1 -1% 10 -11% CALCR + RAMP2 Agonist 0.1 -7% 10 -4% CALCR + RAMP3 Agonist 0.1 -6% 10 -25% CALCRL + RAMP1 Agonist 0.1 -4% 10 -1% CALCRL + RAMP2 Agonist 0.1 -4% 10 -1% CALCRL + RAMP3 Agonist 0.1 -3% 10 -1% CCKAR Agonist 0.1 -2% 10 -1% CCKBR Agonist 0.1 -5% 10 -4% CCR10 Agonist 0.1 -3% 10 -3% CCR2 Agonist 0.1 -2% 10 -2% CCR3 Agonist 0.1 -5% 10 -4% CCR4 Agonist 0.1 -5% 10 -1% CCR5 Agonist 0.1 -4% 10 -1% CCR6 Agonist 0.1 -2% 10 -3%

CCR7 Agonist 0.1 8% 10 -5% CCR8 Agonist 0.1 0% 10 -3% CCR9 Agonist 0.1 -5% 10 -5% CHRM1 Agonist 0.1 -4% 10 -5% CHRM2 Agonist 0.1 -3% 10 -1% CHRM3 Agonist 0.1 -5% 10 -2% CHRM4 Agonist 0.1 6% 10 -4% CHRM5 Agonist 0.1 -7% 10 -29% CMKLR1 Agonist 0.1 -2% 10 -4% CMKOR1 Agonist 0.1 -8% 10 -2% CNR1 Agonist 0.1 0% 10 -2% CNR2 Agonist 0.1 0% 10 -4% CRHR1 Agonist 0.1 -2% 10 -1% CRHR2 Agonist 0.1 -3% 10 -1% CRTH2 Agonist 0.1 -3% 10 -5% CX3CR1 Agonist 0.1 -2% 10 0% CXCR1 Agonist 0.1 -10% 10 -1% CXCR2 Agonist 0.1 -2% 10 -2% CXCR3 Agonist 0.1 -3% 10 -3% CXCR4 Agonist 0.1 7% 10 9% CXCR5 Agonist 0.1 -8% 10 -6% CXCR6 Agonist 0.1 -2% 10 -3% DRD1 Agonist 0.1 -6% 10 -3% DRD2L Agonist 0.1 -4% 10 2% DRD2S Agonist 0.1 -5% 10 -3% DRD3 Agonist 0.1 7% 10 -14% DRD4 Agonist 0.1 -2% 10 -2% DRD5 Agonist 0.1 -4% 10 -2% EDG1 Agonist 0.1 -3% 10 -5% EDG2 Agonist 0.1 -1% 10 -2% EDG3 Agonist 0.1 -4% 10 -5% EDG4 Agonist 0.1 2% 10 1% EDG5 Agonist 0.1 -2% 10 7% EDG6 Agonist 0.1 2% 10 -12% EDG7 Agonist 0.1 -4% 10 -2% EDG8 Agonist 0.1 0% 10 1% EDNRA Agonist 0.1 -7% 10 -3% EDNRB Agonist 0.1 -3% 10 -3% F2R Agonist 0.1 9% 10 -2% F2RL1 Agonist 0.1 -1% 10 -1% F2RL3 Agonist 0.1 -3% 10 -1%

FPR1 Agonist 0.1 -10% 10 -4% FPRL1 Agonist 0.1 -2% 10 -1% FSHR Agonist 0.1 6% 10 -11% GALR1 Agonist 0.1 -4% 10 -1% GALR2 Agonist 0.1 -5% 10 -6% GCGR Agonist 0.1 -3% 10 -1% GHSR1A Agonist 0.1 0% 10 -2% GIPR Agonist 0.1 -3% 10 -13% GLP1R Agonist 0.1 -1% 10 -1% GLP2R Agonist 0.1 -1% 10 -2% GPR1 Agonist 0.1 -4% 10 -3% GPR109A Agonist 0.1 -11% 10 -4% GPR119 Agonist 0.1 -4% 10 -4% GPR120 Agonist 0.1 -1% 10 -7% GPR35 Agonist 0.1 -4% 10 -3% GPR92 Agonist 0.1 -7% 10 -6% GRPR Agonist 0.1 -1% 10 -2% HCRTR1 Agonist 0.1 -3% 10 -1% HCRTR2 Agonist 0.1 -3% 10 -1% HRH1 Agonist 0.1 0% 10 -1% HRH2 Agonist 0.1 -2% 10 -1% HRH3 Agonist 0.1 -14% 10 -6% HTR1A Agonist 0.1 -2% 10 -2% HTR1B Agonist 0.1 -4% 10 -4% HTR1E Agonist 0.1 -7% 10 -1% HTR1F Agonist 0.1 -3% 10 -2% HTR2A Agonist 0.1 3% 10 -11% HTR2C Agonist 0.1 -3% 10 -1% HTR5A Agonist 0.1 -1% 10 -13% KISS1R Agonist 0.1 -3% 10 -4% LHCGR Agonist 0.1 1% 10 -8% LTB4R Agonist 0.1 -3% 10 -3% MC1R Agonist 0.1 -1% 10 -1% MC3R Agonist 0.1 7% 10 -10% MC4R Agonist 0.1 -4% 10 -12% MC5R Agonist 0.1 -8% 10 -5% MCHR1 Agonist 0.1 -1% 10 -5% MCHR2 Agonist 0.1 -3% 10 -2% MLNR Agonist 0.1 -3% 10 -4% MRGPRX2 Agonist 0.1 -6% 10 -2% MTNR1A Agonist 0.1 0% 10 -4%

MTNR1B Agonist 0.1 9% 10 -3% NMU1R Agonist 0.1 -5% 10 -12% NPBWR1 Agonist 0.1 -2% 10 -6% NPBWR2 Agonist 0.1 -4% 10 -3% NPFFR1 Agonist 0.1 1% 10 -2% NPSR1 Agonist 0.1 -2% 10 -4% NPY1R Agonist 0.1 -4% 10 -3% NPY2R Agonist 0.1 -2% 10 -1% NTSR1 Agonist 0.1 -7% 10 -9% OPRD1 Agonist 0.1 -1% 10 -3% OPRK1 Agonist 0.1 0% 10 -5% OPRL1 Agonist 0.1 0% 10 -14% OPRM1 Agonist 0.1 -5% 10 -5% OXTR Agonist 0.1 -3% 10 -3% P2RY11 Agonist 0.1 -3% 10 -2% P2RY12 Agonist 0.1 -4% 10 -5% P2RY2 Agonist 0.1 -2% 10 -3% P2RY4 Agonist 0.1 13% 10 -10% P2RY6 Agonist 0.1 1% 10 0% PPYR1 (NPY4) Agonist 0.1 0% 10 -2% PRLHR Agonist 0.1 -7% 10 -3% PROKR1 Agonist 0.1 7% 10 -1% PROKR2 Agonist 0.1 -3% 10 -1% PTAFR Agonist 0.1 -16% 10 -3% PTGER2 Agonist 0.1 0% 10 -4% PTGER3 Agonist 0.1 3% 10 -3% PTGER4 Agonist 0.1 15% 10 -9% PTGIR Agonist 0.1 -6% 10 -7% PTHR1 Agonist 0.1 -3% 10 -1% PTHR2 Agonist 0.1 -3% 10 -1% RXFP3 Agonist 0.1 2% 10 -4% SCTR Agonist 0.1 -3% 10 1% SSTR2 Agonist 0.1 -2% 10 -1% SSTR3 Agonist 0.1 0% 10 -4% SSTR5 Agonist 0.1 0% 10 -6% TACR1 Agonist 0.1 -13% 10 -20% TACR2 Agonist 0.1 -11% 10 -4% TACR3 Agonist 0.1 -2% 10 -1% TBXA2R Agonist 0.1 0% 10 -3% TRHR Agonist 0.1 -2% 10 -2% TSHR Agonist 0.1 -1% 10 2%

UTR2 Agonist 0.1 1% 10 -4% VIPR1 Agonist 0.1 -2% 10 -2% VIPR2 Agonist 0.1 -1% 10 0%

Supplementary Table 4. GTDF is not a GPCR antagonist or inhibitor. GTDF at its pharmacologically active dose (0.1µM) or high dose (10µM) was assessed for its ability to antagonize or inhibit GPCR signaling in presence of EC80 concentrations of cognate agonists of the indicated GPCRs, using pathhunter β-arrestin assay. Threshold inhibition for consideration as an antagonist or inhibitor was set at 50%.

GTDF % GTDF % Assay GPCR ID Conc Inhibiti Conc Inhibiti Mode (µM) on (µM) on ADCYAP1R1 Antagonist 0.1 3% 10 11% ADORA3 Antagonist 0.1 3% 10 6% ADRA1B Antagonist 0.1 -3% 10 12% ADRA2A Antagonist 0.1 -13% 10 12% ADRA2B Antagonist 0.1 1% 10 5% ADRA2C Antagonist 0.1 -2% 10 5% ADRB1 Antagonist 0.1 -13% 10 -4% ADRB2 Antagonist 0.1 7% 10 10% AGTR1 Antagonist 0.1 -14% 10 4% AGTRL1 Antagonist 0.1 -20% 10 5% AVPR1A Antagonist 0.1 -14% 10 13% AVPR1B Antagonist 0.1 -15% 10 14% AVPR2 Antagonist 0.1 4% 10 1% BDKRB1 Antagonist 0.1 0% 10 3% BDKRB2 Antagonist 0.1 -3% 10 -2% BRS3 Antagonist 0.1 11% 10 4% C5AR1 Antagonist 0.1 3% 10 8% C5LR2 Antagonist 0.1 2% 10 12% CALCR Antagonist 0.1 -4% 10 21% CALCR + RAMP2 Antagonist 0.1 -10% 10 3% CALCR + RAMP3 Antagonist 0.1 5% 10 20% CALCRL + RAMP1 Antagonist 0.1 -8% 10 -1% CALCRL + RAMP2 Antagonist 0.1 -6% 10 -1% CALCRL + RAMP3 Antagonist 0.1 -16% 10 0% CCKAR Antagonist 0.1 1% 10 11% CCKBR Antagonist 0.1 -5% 10 13%

CCR10 Antagonist 0.1 27% 10 6% CCR2 Antagonist 0.1 2% 10 4% CCR3 Antagonist 0.1 13% 10 1% CCR4 Antagonist 0.1 11% 10 1% CCR5 Antagonist 0.1 14% 10 1% CCR6 Antagonist 0.1 13% 10 6% CCR7 Antagonist 0.1 18% 10 7% CCR8 Antagonist 0.1 -2% 10 -25% CCR9 Antagonist 0.1 8% 10 17% CHRM1 Antagonist 0.1 -18% 10 10% CHRM2 Antagonist 0.1 0% 10 8% CHRM3 Antagonist 0.1 -1% 10 15% CHRM4 Antagonist 0.1 8% 10 28% CHRM5 Antagonist 0.1 -7% 10 20% CMKLR1 Antagonist 0.1 0% 10 -2% CMKOR1 Antagonist 0.1 1% 10 1% CNR1 Antagonist 0.1 1% 10 0% CNR2 Antagonist 0.1 2% 10 14% CRHR1 Antagonist 0.1 9% 10 0% CRHR2 Antagonist 0.1 7% 10 10% CRTH2 Antagonist 0.1 -14% 10 13% CX3CR1 Antagonist 0.1 -9% 10 4% CXCR1 Antagonist 0.1 9% 10 -8% CXCR2 Antagonist 0.1 8% 10 1% CXCR3 Antagonist 0.1 14% 10 8% CXCR4 Antagonist 0.1 3% 10 -13% CXCR5 Antagonist 0.1 16% 10 17% CXCR6 Antagonist 0.1 -5% 10 6% DRD1 Antagonist 0.1 5% 10 21% DRD2L Antagonist 0.1 6% 10 3% DRD2S Antagonist 0.1 12% 10 -12% DRD3 Antagonist 0.1 14% 10 0% DRD4 Antagonist 0.1 -1% 10 1% DRD5 Antagonist 0.1 12% 10 11% EDG1 Antagonist 0.1 -1% 10 17% EDG2 Antagonist 0.1 -6% 10 -8% EDG3 Antagonist 0.1 0% 10 6% EDG4 Antagonist 0.1 2% 10 -10% EDG5 Antagonist 0.1 -13% 10 4% EDG6 Antagonist 0.1 -8% 10 11% EDG7 Antagonist 0.1 0% 10 -1%

EDG8 Antagonist 0.1 -11% 10 28% EDNRA Antagonist 0.1 1% 10 16% EDNRB Antagonist 0.1 -13% 10 10% F2R Antagonist 0.1 9% 10 3% F2RL1 Antagonist 0.1 -12% 10 0% F2RL3 Antagonist 0.1 3% 10 -2% FPR1 Antagonist 0.1 -5% 10 9% FPRL1 Antagonist 0.1 -5% 10 4% FSHR Antagonist 0.1 7% 10 37% GALR1 Antagonist 0.1 -11% 10 4% GALR2 Antagonist 0.1 -19% 10 10% GCGR Antagonist 0.1 -3% 10 14% GHSR1A Antagonist 0.1 -3% 10 -1% GIPR Antagonist 0.1 9% 10 23% GLP1R Antagonist 0.1 2% 10 1% GLP2R Antagonist 0.1 2% 10 2% GPR1 Antagonist 0.1 -14% 10 8% GPR109A Antagonist 0.1 15% 10 9% GPR119 Antagonist 0.1 -10% 10 25% GPR120 Antagonist 0.1 -27% 10 -3% GPR35 Antagonist 0.1 -6% 10 9% GPR92 Antagonist 0.1 9% 10 10% GRPR Antagonist 0.1 -19% 10 -9% HCRTR1 Antagonist 0.1 9% 10 7% HCRTR2 Antagonist 0.1 -15% 10 9% HRH1 Antagonist 0.1 -7% 10 10% HRH2 Antagonist 0.1 6% 10 0% HRH3 Antagonist 0.1 -1% 10 24% HTR1A Antagonist 0.1 -3% 10 23% HTR1B Antagonist 0.1 -1% 10 11% HTR1E Antagonist 0.1 0% 10 11% HTR1F Antagonist 0.1 -1% 10 12% HTR2A Antagonist 0.1 3% 10 15% HTR2C Antagonist 0.1 -12% 10 15% HTR5A Antagonist 0.1 -19% 10 31% KISS1R Antagonist 0.1 -7% 10 14% LHCGR Antagonist 0.1 -8% 10 8% LTB4R Antagonist 0.1 6% 10 2% MC1R Antagonist 0.1 -16% 10 12% MC3R Antagonist 0.1 3% 10 4% MC4R Antagonist 0.1 -17% 10 20%

MC5R Antagonist 0.1 -9% 10 14% MCHR1 Antagonist 0.1 11% 10 20% MCHR2 Antagonist 0.1 3% 10 21% MLNR Antagonist 0.1 4% 10 6% MRGPRX2 Antagonist 0.1 -1% 10 18% MTNR1A Antagonist 0.1 -3% 10 25% MTNR1B Antagonist 0.1 -25% 10 13% NMU1R Antagonist 0.1 -21% 10 7% NPBWR1 Antagonist 0.1 -9% 10 14% NPBWR2 Antagonist 0.1 -8% 10 -2% NPFFR1 Antagonist 0.1 -5% 10 2% NPSR1 Antagonist 0.1 -4% 10 17% NPY1R Antagonist 0.1 7% 10 16% NPY2R Antagonist 0.1 -8% 10 4% NTSR1 Antagonist 0.1 -5% 10 1% OPRD1 Antagonist 0.1 15% 10 9% OPRK1 Antagonist 0.1 0% 10 26% OPRL1 Antagonist 0.1 -4% 10 34% OPRM1 Antagonist 0.1 -4% 10 16% OXTR Antagonist 0.1 -22% 10 8% P2RY11 Antagonist 0.1 -11% 10 10% P2RY12 Antagonist 0.1 -5% 10 7% P2RY2 Antagonist 0.1 -2% 10 5% P2RY4 Antagonist 0.1 12% 10 10% P2RY6 Antagonist 0.1 0% 10 -3% PPYR1 (NPY4) Antagonist 0.1 -14% 10 15% PRLHR Antagonist 0.1 4% 10 6% PROKR1 Antagonist 0.1 8% 10 14% PROKR2 Antagonist 0.1 -10% 10 4% PTAFR Antagonist 0.1 -10% 10 16% PTGER2 Antagonist 0.1 2% 10 12% PTGER3 Antagonist 0.1 -19% 10 6% PTGER4 Antagonist 0.1 22% 10 -7% PTGIR Antagonist 0.1 3% 10 16% PTHR1 Antagonist 0.1 -15% 10 0% PTHR2 Antagonist 0.1 5% 10 -7% RXFP3 Antagonist 0.1 -15% 10 21% SCTR Antagonist 0.1 13% 10 -4% SSTR2 Antagonist 0.1 10% 10 7% SSTR3 Antagonist 0.1 -16% 10 5% SSTR5 Antagonist 0.1 0% 10 -2%

TACR1 Antagonist 0.1 -3% 10 10% TACR2 Antagonist 0.1 -9% 10 12% TACR3 Antagonist 0.1 -7% 10 3% TBXA2R Antagonist 0.1 -22% 10 12% TRHR Antagonist 0.1 10% 10 11% TSHR Antagonist 0.1 19% 10 2% UTR2 Antagonist 0.1 5% 10 13% VIPR1 Antagonist 0.1 3% 10 -2% VIPR2 Antagonist 0.1 3% 10 1%

Supplementary Table 5. Oligonucleotide sequences. m; mouse. All sequences in 5' to 3' orientation.

Gene name Forward Reverse

mUCP-1 ACAGAAGGATTGCCGAAAC AGCTGATTTGCCTCTGAATG mUCP-2 GTTCCTCTGTCTCGTCTTGC GGCCTTGAAACCAACCA mUCP-3 TGACCTGCGCCCAGC CCCAGGCGTATCATGGCT mPPARα TCTTCACGATGCTGTCCTCCT GGAACTCGCCTGTGATAAAGC mPPARβ TCCAGAAGAAGAACCGCAACA GGATAGCGTTGTGCGACATG mPPARγ CAGGCCGAGAAGGAGAAGCT GGCTCGCAGATCAGCAGACT mPGC-1α AGCCGTGACCACTGACAACGAG CTGCATGGTTCTGAGTGCTAAG mCPT1B CTCCTTTCCTGGCTGAGGT GATCTGGAACTGGGGGATCT mLPL CCAATGGAGGCACTTTCCA TGGTCCACGTCTCCGAGTC mAdiponectin TGTTGGAATGACAGGAGCTGA CACACTGAACGCTGAGCGATAC mLeptin TCTCCGAGACCTCCTCCATCT TTCCAGGACGCCATCCAG mCD36 GGCCAAGCTATTGCGACAT CAGATCCGAACACAGCGTAGA mFABP3 CCCCTCAGCTCAGCACCAT CAGAAAAATCCCAACCCAAGAAT mFAS CGGAAACTTCAGGAAATGTCC TCAGAGACGTGTCACTCCTGG mFACS TCTAGGAGTGAAGGCCAACG GCAATATCTGAGGGCAGTGG mACOX1 GCCCAACTGTGACTTCCATC GCCAGGACTATCGCATGATT mPRDM16 ACAGGCAGGCTAAGAACCAG CGTGGAGAGGAGTGTCTTCAG mCIDEA AAACCATGACCGAAGTAGCC AGGCCAGTTGTGATGACTAAGAC mβ-actin CCTCACCCTCCCAAAAGC GTGGACTCAGGGCATGGA

Mitochondrial DNA MT -cox-II GCCGACTAAATCAAGCAACA CAATGGGCATAAAGCTATGG Cyt-B CATTTATTATCGCGGCCCTA TGTTGGGTTGTTTGATCCTG β-Globin GAAGCAATTCTAGGGAGCAG GGAGCAGCGATTCTGAGTAGA

Supplementary Figure 1. Validation of AdipoR1 and R2 antibodies. 50µg protein extracts from CHO cells transfected with empty plasmid or AdipoR1 and R2 mammalian expression plasmids or C2C12 cells (that express endogenous AdipoRs) were analyzed by immunoblotting with indicated antibodies. AdipoR1 antibody showed some cross-reactivity with over-expressed AdipoR2, which was expected as this antibody was raised against a peptide from the C-terminus of AdipoR1 that shares amino acid similarity with AdipoR2. Image representative of 3 independent experiments.

Supplementary Figure 2. Saturation binding of 125I-gAd to CHO cells transfected with AdipoR1, AdipoR2 or vector only. CHO cells transfected with AdipoR1, R2 or empty expression vector were incubated with increasing concentrations of 125I-gAd in PBS supplemented with 0.1% BSA for 2h, following which cells were washed and lysed. A portion of the lysate was used for protein estimation and rest was quantitated in a gamma-counter. Nonspecific binding for each concentration was determined using 200-fold excess of cold gAd. Specific binding was calculated by subtracting nonspecific binding from the total binding. The count per minute (CPM) was normalized with protein concentration and plotted.

Supplementary Figure 3. GTDF lowers adipose tissue weight in db/db mice. Indicated fat pads from 6 mice in each group were dissected and weighted using an analytical balance (Mettler, Toledo, Osaka, Japan).