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CoQ imbalance drives reverse electron transport to disrupt liver metabolism

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Abstract

Mitochondrial reactive oxygen species (mROS) are central to physiology1,2. Excess mROS production has been associated with several disease states2,3; however, the precise sources, regulation and mechanism of generation in vivo remain unclear, which limits translational efforts. Here we show that in obesity, hepatic coenzyme Q (CoQ) synthesis is impaired, which increases the CoQH2 to CoQ (CoQH2/CoQ) ratio and drives excessive mROS production through reverse electron transport (RET) from site IQ in complex I. Using multiple complementary genetic and pharmacological models in vivo, we demonstrate that RET is crucial for metabolic health. In patients with steatosis, the hepatic CoQ biosynthetic program is also suppressed, and the CoQH2/CoQ ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

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Fig. 1: RET at complex I drives excess superoxide and H2O2 production in livers from obese mice.
Fig. 2: The CoQH2/CoQ ratio is increased in livers of obese mice.
Fig. 3: mROS generation through RET increases hepatic glucose production and impairs glucose homeostasis.
Fig. 4: Suppressing RET in vivo improves metabolism in obese mice.
Fig. 5: CoQ–RET axis in hepatic steatosis in humans.

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Data availability

All data necessary to evaluate the conclusions of this study are present in the article, extended data and/or the Supplementary Information. All the data and raw materials that support the findings of this study have been deposited into FigShare (https://figshare.com/s/0b856e078932282d73d4)68. Metabolomic data are available with the study identifier ST003846 at the NIH Common Fund’s National Metabolomics Data Repository website, the Metabolomics Workbench (https://doi.org/10.21228/M8WJ9W). Source data are provided with this paper.

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Acknowledgements

We thank K. Prentice and A. Orr for critical review of the paper; K. Langston for help with quantitative PCR; N. Snyder, E. Cagampan, N. Min, L. Greene, S. Karzhevsky for their technical assistance; D. Wallace and A. Jurisicova for sharing the Nd6P25L and the Pdss2-floxed mice, respectively; E. Dufour and P. Rustin for providing the initial Aox plasmid and antibody; HSPH animal facility staff for animal husbandry; and M. Rodriguez for laboratory maintenance. This project was supported by the Hotamışlıgil Laboratory. R.L.S.G. was supported by a Barth Syndrome Foundation Idea Grant. G.S.H. was supported by the NIH (DK123458 and HL148137). S.C.B. was supported by the NIH (P30DK127984 and R01DK128168).

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Author notes

  1. Güneş Parlakgül & Ana Paula Arruda

    Present address: Department of Nutritional Sciences and Toxicology, University of California Berkeley, Berkeley, CA, USA

Authors and Affiliations

  1. Department of Molecular Metabolism, Harvard T. H. Chan School of Public Health, Boston, MA, USA

    Renata L. S. Goncalves, Zeqiu Branden Wang, Jillian K. Riveros, Güneş Parlakgül, Karen E. Inouye, Grace Yankun Lee, Jani Saksi, Clement Rosique, Sheng Tony Hui, Ana Paula Arruda, Isabel Graupera & Gökhan S. Hotamışlıgil

  2. Center for Human Nutrition and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiaorong Fu & Shawn C. Burgess

  3. Fundació de Recerca Clínic Barcelona-Institut d’Investigacions Biomèdiques August Pi i Sunyer (FRCB-IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CiberEHD), Barcelona, Spain

    Mar Coll & Isabel Graupera

  4. Liver Unit Hospital Clinic. Faculty of Medicine and Health Services, University of Barcelona, Barcelona, Spain

    Mar Coll & Isabel Graupera

  5. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Gökhan S. Hotamışlıgil

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Contributions

R.L.S.G. and G.S.H. originally conceptualized this project, were responsible for planning its direction and wrote the manuscript. R.L.S.G. formulated the questions, designed the project and performed and analysed all the experimental data unless otherwise stated. Z.B.W. designed, prepared and conducted the experiments with CoQ10 emulsions, assisted in in vivo experiments and revisions of the paper. J.K.R. performed and assisted with in vitro and in vivo experiments and revisions of the paper. G.P. and A.P.A. provided guidance and helped with in vivo and in vitro experiments and revisions of the paper. S.C.B. and X.F. performed the in vivo CoQ synthesis experiments and analyses. C.R. and S.T.H. performed the metabolomic analyses. J.S. provided advice and performed the tail-vein injections. I.G. and M.C. provided human samples. K.E.I. provided advice and helped with all in vivo studies. G.Y.L. provided advice, technical assistance, reagent preparation and validation. G.S.H. provided mentorship, conceived, supervised and supported the project, designed experiments, interpreted results and revised the manuscript. R.L.S.G., G.S.H., G.P., A.P.A., S.C.B., K.E.I. and X.F. contributed to manuscript editing.

Corresponding author

Correspondence to Gökhan S. Hotamışlıgil.

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G.S.H. is a member of the Scientific Advisory Board and holds equity in Crescenta Pharmaceuticals (not related to the contents of this article). All the other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 ROS generation by RET from site IQ is increased in the liver but not the skeletal muscle of obese mice.

(A) Liver sections from wildtype (wt) and ob/ob mice stained with H&E. Obese hepatocytes contain several lipid vacuoles (arrow) and small foci of inflammation (asterisk). Scale bar, 200 µm. (B) Immunoblot analysis and (C) quantification of 4-HNE as an oxidative stress marker in liver homogenates from wt and ob/ob mice. n = 6 livers per group (***p = 0.001, unpaired t-test). Area used for quantification is shown in Supplementary Fig. 1. (D) Immunoblot analysis and (E) quantification of peroxiredoxin 3 (PRDX3) in liver homogenates from wt and ob/ob mice. n = 4 per group (**p = 0.002, unpaired t-test). (F) Quantitative proteomics of PRDX3 in liver isolated mitochondria from wt and ob/ob mice. n = 9 mito isolations from n = 9 mice per group (*p = 0.011, unpaired t-test). (G) Schematic to illustrate the substrates and inhibitors used to assess the two modes of mROS generation from complex I: forward electron transport from site IF (FET, left) and reverse electron transport (RET, right) from site IQ. (H) Representative Amplex UltraRed traces showing that rotenone blocks mROS during succinate oxidation. The rate difference between minus and plus rotenone defines RET, which is higher in mitochondria isolated from ob/ob livers. Representative of n = 13 independent experiments. (I) Representative Amplex UltraRed traces and (J) quantification showing that 5 µM S1QEL 2.2, 2 µM rotenone, 2 µM piericidin A, and 1 µM FCCP decrease the rate of mROS production during RET induced by succinate oxidation in the presence of oligomycin in isolated mitochondria. n = 5 independent experiments, except piericidin A (n = 4) and S1QEL (n = 3) (****p < 0.0001, two-way ANOVA, Dunnett’s post hoc test) (K) Relative mROS production by RET from wt and ob/ob liver mitochondria using the compounds in (J). n = 3 mito isolations from n = 3 mice per group (*p < 0.05, multiple paired t-test not adjusted for multiple comparisons). (L) S1QEL 2.2 (0.15−10 µM) does not inhibit Hepa 1-6 oxygen consumption rates (OCR). n = 5 independent experiments, except DMSO (n = 8) and S1QEL 0.15 and 0.3 µM (n = 4). ns, p = 0.3268 two-way ANOVA, Dunnett’s post hoc test). (M) Effect of S1QEL 2.2 (0.01−10 µM) on mROS production during RET in liver-isolated mitochondria from wildtype and ob/ob mice. n = 3 mito isolations from n = 3 mice per group (*p < 0.0001, two-way ANOVA). (N) Maximum capacity of superoxide/H2O2 production from skeletal muscle-isolated mitochondria from lean wt (n = 12) and ob/ob mice (n = 12). Pooled from four independent experiments (*p = 0.019, multiple unpaired t-test not adjusted for multiple comparisons). Data are individual values and means ± SEM. All t-tests were two-tailed.

Source data

Extended Data Fig. 2 The thermodynamic forces driving mROS via RET.

(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.

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Extended Data Fig. 3 Mitoparaquat promotes mROS via RET and impairs glucose homeostasis in the liver.

(A) Effect of 1 µM MitoPQ on superoxide/H2O2 production from the sites linked to the Q-pool (sites IQ, IIF, GQ, DQ and EF). n = 4 mito isolations from n = 4 mice (**p = 0.004, multiple paired t-tests not adjusted for multiple comparisons). (B) Effect of 10 µM S1QEL 2.2 or 2 µM rotenone on the rate of MitoPQ-induced superoxide/H2O2 production by RET. MitoPQ (n = 6), S1QEL (n = 3) and rotenone (n = 1) mito isolations. Each mito isolation represents one mouse (*p = 0.0125, **p = 0.009, Two-way ANOVA). (C) Effect of 10 µM S1QEL 2.2 on the rate of MitoPQ-induced superoxide/H2O2 production by FET. n = 3 mito isolations from n = 3 mice. (D) MitoPQ-stimulated rate of superoxide/H2O2 production via RET is suppressed by 2.5 µM S1QEL 2.2. n = 3 mito isolations from n = 3 mice (**p = 0.0044, One-way ANOVA, Dunnett’s post hoc test). (E) Effect of MitoPQ on the oxygen consumption rate (OCR) of wt primary hepatocytes. MitoPQ, port A; 1 µM FCCP, port B; and 2 µM rotenone/antimycin A, port C. n = 2 hepatocytes isolations from n = 2 mice (ns, Two-way ANOVA). (F) Immunoblot analysis and quantification of PRDX3 levels in liver homogenates from DMSO or MitoPQ treated mice for 1.5 h and normalized by ponceau from the same samples on a different blot. n = 9 mice per group (**p = 0.006, unpaired t-test). (G) Liver section from wt mice treated with DMSO or MitoPQ stained with H&E, bars 200 µm. (H) Blood glucose levels during i.p. glucose tolerance test (0.5 g • kg−1) in wt mice treated with 2–4 nmol mitoPQ. Inset is area under the curve. n = 28 mice per group, except MitoPQ 2 nmol (n = 4) (*p = 0.0252, Two-way ANOVA. **p = 0.006, One-way ANOVA, Dunnett’s post hoc test). (I) Blood glucose levels during i.p. lactate: pyruvate tolerance test (1.5:0.15 g• kg−1) in wt mice treated with 1–4 nmol mitoPQ. n = 9 mice per group, except mitoPQ 4 nmol n = 8 (*p = 0.0023, Two-way ANOVA, **p = 0.005, One-way ANOVA Dunnett’s post hoc test). (J) Immunoblot analysis and (K) quantification of in vivo insulin signaling in the gastrocnemius muscle (left) and epididymal fat (right) of wt mice 1.5 h after 4 nmol MitoPQ or DMSO treatment. n = 8 mice per group (*p = 0.047, **p = 0.007 and ns, unpaired t-test). (L) Blood glucose levels during insulin tolerance test (0.7 U insulin • kg−1) in 6 h fasted mice treated with 4 nmol MitoPQ or DMSO. Inset: area under the curve. DMSO (n = 15) and mitoPQ (n = 14) mice (ns, Two-way ANOVA, inset: ns, unpaired t-test). (M) Blood glucose levels during i.p. glycerol tolerance test (1 g • kg−1) in 16 h fasted mice treated with 4 nmol mitoPQ or DMSO. Inset is area under the curve. DMSO (n = 10) and MitoPQ (n = 13) mice (ns, Two-way ANOVA, inset: ns, unpaired t-test). (N) Gluconeogenesis assay in primary hepatocytes from wt mice 1.5 h after MitoPQ or DMSO treatment. 20 mM glycerol as substrate. n = 11 hepatocyte isolations from n = 11 mice per group (ns, unpaired t-test). (O) Bodyweights from pdss2 wt, het and kd mice fed regular chow. pdss2 fl/fl-wt (n = 11), het (n = 3) and KO (n = 6) mice. (P) Liver section from pdss2 wt and het mice stained with H&E, bars 200 µm. Data are individual values and means ± SEM. All t-tests were two-tailed. ns, not significant.

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Extended Data Fig. 4 Ectopic expression of Aox in hepatocytes decreases mROS generation via RET and improves systemic glucose homeostasis.

(A) Illustration showing mROS production in ob/ob mice ± Aox expression. Aox oxidizes excess CoQH2 and decreases mROS by RET. (B) Representative traces of cyanide-insensitive oxygen consumption in Hepa 1-6 and (C) AML12 cells expressing Aox. N-propyl gallate (n-PG) inhibits Aox activity. Top, immunoblot confirming Aox expression. Representative data from n = 3 experiments. (D) Representative traces of Amplex UltraRed oxidation to show that Aox-expressing primary hepatocytes generate less mROS by RET. Ad., adenovirus. Representative data from n = 7 experiments. (E) Immunoblot analysis and (F) quantification of 18 nM insulin action in isolated hepatocytes from 9–10-week-old obese mice incubated with ad.Aox-HA (n = 15) or ad.GFP (n = 15) for 24 h. Pooled from five independent experiments (*p = 0.0298, **p = 0.0092, ****p < 0.0001, one-tailed unpaired t-test). (G) Representative immunoblot analysis of tissue homogenates of Aox expressing mice (n = 1 mouse). (H) Immunoblot analysis of different cellular fractions from the liver of GFP or Aox mice. ndufs1 and VDAC, mitochondria; calreticulin, endoplasmic reticulum and tubulin, cytosol (n = 1 mouse per group). (I) Glucose production from primary hepatocytes isolated from obese mice expressing Aox or GFP using 20 mM lactate, 2 mM pyruvate, and 2 mM glutamine as substrates. n = 9 mice per group (*p = 0.0178, one-tailed unpaired t-test). (J) Blood glucose levels during oral glucose tolerance test (OGTT) (0.75 g • kg−1) in ob/ob mice. expressing aav.GFP (n = 7) or aav.Aox (n = 8). Inset, area under the curve. (*p = 0.0496, one-tailed unpaired t-test). (K) Liver sections from ob/ob mice expressing Aox or GFP stained with PAS, bars 200 µm. (L) Quantification of liver areas positive for PAS staining of glycogen in obese mice expressing aav.Aox (n = 7) or aav.GFP (n = 5). (*p = 0.0159, unpaired t-test). (M) Plasma insulin levels during OGTT (GFP, n = 7 vs Aox, n = 8 mice). (N) Blood glucose levels during insulin tolerance test (3.5 U of insulin • kg−1). Inset, area under the curve (GFP, n = 7 vs Aox, n = 8 mice). (O) Liver sections from ob/ob mice expressing Aox or GFP stained with H&E, bars 200 µm. (P-U) Metabolic profile of obese 10 days after Aox expression. (P) Bodyweights following aav.GFP or aav.Aox administration at 6 weeks of age (n = 8 per group). (Q) Body composition of 7-week-old ob/ob mice following AAV administration (n = 6 per group). (R) Energy expenditure (EE) as a function of bodyweight (Aox, n = 9 vs GFP, n = 7). (S) Respiratory exchange ratio measured during metabolic cage housing (Aox, n = 9 vs GFP, n = 7). (T) Analysis of RER during light and dark cycles (Aox, n = 9 vs GFP, n = 7). (U) Metabolite levels in the livers of ob/ob mice expressing Aox vs. GFP (n = 6 mice per group). Panels D and I were created with BioRender.com.Values are individual values and means ± SEM. ns, not significant.

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Extended Data Fig. 5 Suppressing RET in lean mice does not change bodyweight or glucose tolerance.

(A) Six hour fasting blood glucose levels in wildtype (wt) and ND6P25L mice fed chow diet for 15 weeks (n = 10 mice per group). (B) Blood glucose levels during glucose tolerance test (0.5 g • kg−1) in ND6P25L (n = 10) and wt (n = 11) mice on chow diet for 10 weeks. Inset, area under the curve. (C) Weight gain of wt and ND6P25L mice over 15 weeks on chow (n = 10 mice per group). (D) Liver section from wt and ND6P25L on HFD stained with H&E, bars 200 µm. (E) Weight gain of wt (n = 13) and ND6P25L (n = 11) mice over 15 weeks on HFD. (F) CoQ9 and CoQ10 content in isolated mitochondria from ob/ob mice treated with vehicle or CoQ10 emulsion. n = 5 mito isolations from n = 5 mice per group (****p < 0.0001, multi unpaired t-test not adjusted for multiple comparisons). (G) Blood glucose levels during insulin tolerance test (1.5 U of insulin • kg−1) in 6 h fasted ob/ob mice treated with 10 mg • kg−1 CoQ10 (n = 8) or vehicle (n = 7) every other day for 23 days. Inset, area under the curve (****p < 0.0001Two-way ANOVA and *p = 0.019, two-tailed unpaired t-test). (H) Liver sections from ob/ob mice treated for 20 days with CoQ10 or vehicle stained with H&E, bars 200 µm. (I) CoQ9 content and (J) CoQ9H2/CoQ9 ratio in the liver from leptin-deficient ob/ob mice treated with vehicle (n = 6) or CoQ10 emulsion (n = 6). (K) Blood glucose levels during i.p. glucose tolerance test (0.5 g • kg−1) in lean wt mice treated with 10 mg • kg−1 CoQ10 or vehicle every other day for 23 days. Inset, area under the curve (n = 4 mice per group). (L) Bodyweights of lean wildtype mice treated with CoQ10 (n = 3) or vehicle (n = 4). (M) Bodyweights of ob/ob mice treated with CoQ10 or vehicle (n = 12 per group). Values are individual values and means ± SEM. ns, not significant.

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Extended Data Fig. 6 Histological changes in patients with hepatic steatosis.

Liver section from patients with MAFLD and different grades of steatosis stained with H&E. S0, no steatosis grade 0 (less than 5% hepatocyte occupied by fat); S1, grade 1, mild (5–33% hepatocyte occupied by fat); S2, grade 2, moderate (34–66% hepatocyte occupied by fat); S3, grade 3, severe (above 66% hepatocyte occupied by fat). Histological images are representative of the steatosis scores observed in the livers of the 27 patients included in this study. Left panel bars, 500 µm, and right panel bars, 100 µm.

Extended Data Fig. 7 Model Overview of Coenzyme Q (CoQ) Synthesis Imbalance in Obese Livers.

Illustration of the model detailing the impact of CoQ synthesis deficiency on the CoQH2/CoQ ratio, resulting in increased mitochondrial reactive oxygen species (mROS) production via reverse electron transport (RET) and subsequent impairment of glucose homeostasis (black boxes). Different genetic and pharmacological interventions were utilized to modulate specific nodes within the model, ranging from CoQ synthesis and levels to the direct generation of mROS via RET. Approaches highlighted in blue indicate interventions that improved glucose homeostasis, while those in red denote interventions that worsen it. To investigate whether obesity-driven CoQ deficiency contributed to the observed alterations in CoQH2/CoQ ratio, obese mice were supplemented with CoQ10. This treatment restored CoQ10 levels, lowered CoQH2/CoQ ratio, and mitigated mROS production via RET, thereby improving glucose homeostasis. Similarly, manipulating CoQ redox state through ectopic expression of Ciona intestinalis alternative oxidase (Aox) in the livers of obese mice resulted in decreased mROS via RET and improved glucose homeostasis. The point mutation in the ND6 gene (ND6P25L) was found to directly hinder complex I-mediated mROS generation via RET, these mice had enhanced glucose homeostasis in high-fat diet. Conversely, impairing CoQ biosynthesis in lean mice (via pdss2 knockdown) or directly inducing mROS via RET with mitoPQ led to compromised glucose homeostasis. This figure provides a comprehensive overview of the intricate interplay between hepatic CoQ synthesis, redox state, mROS production via RET, and their collective impact on glucose homeostasis in the context of obesity. Created with BioRender.com.

Extended Data Table 1 Characteristics of subjects undergoing liver biopsies for gene expression analysis
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Extended Data Table 2 Characteristics of subjects undergoing liver biopsies for CoQH2/CoQ ratio analysis
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Supplementary information

Supplementary Information

Reporting Summary

Supplementary Fig. 1

Uncropped western blots from main Figs. 3e,i and 4c and Extended Data Figs. 1b,d, 2g,h, 3f,j, and 4b,c,e,g,h.

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Supplementary Table 1

Statistical tests showing the comparisons and exact P values, replicate numbers and sample type from all the panels showing statistical differences.

Supplementary Table 2

Comparison of published mouse liver CoQ levels with findings from this study.

Supplementary Table 3

A guide with the specific substrates and inhibitors to measure the maximum capacity of the different 11 sites of superoxide/H2O2 production.

Supplementary Table 4

Raw data and calculation for CoQ enrichment kinetics.

Supplementary Table 5

Raw data and calculation for newly synthesized CoQ and head and tail enrichment.

Supplementary Table 6

Metabolite reporting check list and metabolite annotation and documentation.

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Goncalves, R.L.S., Wang, Z.B., Riveros, J.K. et al. CoQ imbalance drives reverse electron transport to disrupt liver metabolism. Nature (2025). https://doi.org/10.1038/s41586-025-09072-1

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