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 ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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 ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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: ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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 ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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% ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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% ©2014 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1619/-/DC1 SUPPLEMENTARY DATA 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% ©2014 American Diabetes Association.