Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation

Abstract

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1⁺ with free fatty acid anions resulting in uncoupling; ii) SkQ1H₂ ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Q_i), involving the reduction of SkQ1 to SkQ1H₂ by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (J_m) and cytosolic H₂O₂ (J_c) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of J_m and J_c but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished J_m established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H₂ at Complex I I_Q or Complex III Q_i site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H₂ oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ₁₀. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H₂ oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.

Keywords: HepG2 cells; Mitochondria-targeted antioxidant SkQ1; Mitochondrial Complex I superoxide formation; Mitochondrial Complex III superoxide formation; NAD(P)H fluorescence lifetime imaging microscopy.