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Fig. 3
Benzoquinone structures and hPPAR activities. (A) Structures of idebenone, CoQ10 and derivatives. Note that the isoprenoid side chain contains ten isoprenoid subunits, whereas idebenone contains only alkyl units and ends with a polar hydroxyl group. (B) 2?dpf PPAR? LT embryos showing limited GFP expression in a small subset of epidermal cells and in the posterior spinal cord in the absence of exogenous ligand. Treatment with idebenone, CoQ2 or CoQ10 induces similar patterns of GFP expression in cells of the epidermis, blood, CNS and posterior spinal cord. Decylubiquinone, which carries a methyl group instead of the hydroxyl group, does not activate the receptor. Benzoquinone, CoQ0, CoQ1, CoQ4, CoQ6, CoQ8 and CoQ9 showed no reporter activation. Pure CoQ10 (Sigma), which has weak water solubility, shows strong receptor activation when mixed together with PEG. Overlay pictures of bright-field and GFP (85% transparent) of 2?dpf embryos are shown. Views are lateral with anterior to the left (mean n=20, replicated three times). (C) Idebenone and CoQ10 are partial dual agonists of PPAR? and PPAR?. Embryos of the PPAR? fish line show no increased GFP expression in the presence of CoQ10 or idebenone. Overlaid bright-field and GFP images (85% transparent) of 2?dpf embryos are shown. Views are lateral with anterior to the left (mean n=10, replicated three times). (D) HEK293 cells were co-transfected with the GAL4-LBD fusion proteins of hPPAR?, hPPAR? and hPPAR? (as indicated) together with a UAS-luciferase reporter. Treatment of the cells with the full agonists GW7647 (hPPAR?), CAY10592 (hPPAR?/?) or rosiglitazone (hPPAR?) resulted in EC50 concentrations of 8?nM, 12.8?nM and 48?nM, respectively. Treatment with idebenone resulted in an EC50 of 2.5?µM for PPAR? and 3.8?µM for PPAR?. Notably, maximal activation levels were far lower than elicited by the full agonists. Luciferase output was normalized to ?-galactosidase to control for transfection efficiency and expressed as normalized luciferase output. Plasmids used were: pCMX, pcDNA3-GAL4-hPPAR?, pcDNA3-GAL4-hPPAR?, pcDNA3-GAL4-hPPAR?, UAS-luc, pGEM, pCMX??-galactosidase. Data represent meanħs.d., n=3 with at least three repeats.