Extended Data Fig. 2: The thermodynamic forces driving mROS via RET.
From: CoQ imbalance drives reverse electron transport to disrupt liver metabolism
(A) Representative traces and (B) quantification of mitochondrial membrane potential from lean wildtype (wt) and obese (ob/ob) livers. n = 5 mito isolations from n = 5 mice per group. AU, arbitrary units. (C-E) Complex I, II and II/III activities in wt and ob/ob liver isolated mitochondria. C, n = 3; D, n = 9; E, n = 4 independent mito isolations per group (ns=p > 0.05, unpaired t-test). (F) Oxygen consumption rate (OCR) of wt and ob/ob liver isolated mitochondria oxidizing FAD- and NAD- linked substrates under phosphorylating (state 3) and non-phosphorylating conditions (state 4). n = 4 mito isolations from n = 4 mice per group (*p = 0.01, **p = 0.007, multiple unpaired t-tests not adjusted for multiple comparisons). (G) Immunoblot (top) and quantification analysis (bottom) of complex II-V of the electron transport chain (ETC) in the liver lysates of wt and ob/ob mice normalized by VDAC, run on a separate gel (bottom of panel H). n = 3 liver lysates per group (*p = 0.029, multiple unpaired t-test not adjusted for multiple comparisons). (H) Immunoblot (left) and quantification analysis (right) of complex I subunits in the livers of wt and ob/ob mice normalized by VDAC. n = 3 liver lysates per group, except ND6 which is n = 10 per group (*p < 0.05, **p = 0.004, ****p < 0.0001, multiple unpaired t-tests not adjusted for multiple comparisons). (I) CoQ10 content (CoQ10H2 + CoQ10), (J) Total CoQ content (CoQ9 + CoQ10), (K) Ratio of CoQ10H2/CoQ10, and (L) % of reduced CoQ10 (CoQ10H2/total CoQ10) in the livers of wt and ob/ob mice. n = 9 mice per group (*p < 0.05, ***p = 0.0006; ns, p > 0.05, two-way ANOVA). (M) CoQ9 and CoQ10 content in liver isolated mitochondria from wt (n = 9) and ob/ob mice (n = 10). Each mito isolation represents one mouse (**p = 0.005, ****p < 0.0001, multiple unpaired t-tests not adjusted for multiple comparisons). (N) CoQ10/CoQ9 ratio. n = 9 mice group [liver] and n = 9 for wt vs n = 10 for ob/ob [mitos] (*p < 0.0001, ns=p > 0.05, multiple unpaired t-tests not adjusted for multiple comparisons). (O) Illustration of the enzymes that can generate mROS and feed electrons into the CoQ pool. (P-S) Quantification of the levels of glycerol phosphate, dihydroorotate, acyl-carnitines and succinate in the livers of wt and ob/ob mice. n = 9 for wt vs n = 11 for ob/ob (**p = 0.0084, unpaired t-test). (T) Relative expression levels of the genes in the mevalonate pathway in the livers of ob/ob mice relative to wt. n = 16 mice (*p < 0.05, **p = 0.0085, ****p < 0.0001, one sample t-test). (U-X) Quantitative proteomics of enzymes in the CoQ synthetic pathway, COQ5, COQ7, COQ8a and COQ9. n = 9 mito isolations from n = 9 mice per group (*p = 0.044, **p = 0.003, unpaired t-test). (Y) Kinetics of 2H-enrichement in the CoQ10 tail in the livers of wt (n = 16) and ob/ob (n = 11) (***p = 0.0006, Two-way ANOVA). (Z) Newly synthesized CoQ10 in the livers of wt and ob/ob mice after 24 h of 2H2O administration in the drinking water (4% v/v). n = 6 mice per group (****p < 0.0001, unpaired t-test). (AA) Total 2H-water enrichment in wt and ob/ob livers. n = 6 mice per group (****<0.0001, unpaired t-test) (AB) Mass enrichment in the CoQ10 isoprenoid tail of the different isotopomers (M1-M3) in the livers of wt and ob/ob mice after 24 h of 2H2O administration in the drinking water (4% v/v). n = 6 mice per group ***p = 0.0003, Two-way ANOVA). (AC) 2H-enrichment in cholesterol and (AD) total cholesterol in the liver of wt and ob/ob mice 24 h after 2H2O administration in the drinking water (4% v/v). n = 6 mice (****<0.0001, unpaired t-test). Data are individual values and means ± SEM. All t-tests were two-tailed. ns, not significant.
