CoQ imbalance drives reverse electron transport to disrupt liver metabolism

Abstract

Mitochondrial reactive oxygen species (mROS) are central to physiology^1,2. Excess mROS production has been associated with several disease states^2,3; 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 I_Q 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.

Extended Data Fig. 1 |. ROS generation by RET from site I_Q 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 I_F (FET, left) and reverse electron transport (RET, right) from site I_Q. (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.

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.

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 I_Q, II_F, G_Q, D_Q and E_F). 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.

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.

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 ND6^P25L 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 ND6^P25L (n = 10) and wt (n = 11) mice on chow diet for 10 weeks. Inset, area under the curve. (C) Weight gain of wt and ND6^P25L mice over 15 weeks on chow (n = 10 mice per group). (D) Liver section from wt and ND6^P25L on HFD stained with H&E, bars 200 µm. (E) Weight gain of wt (n = 13) and ND6^P25L (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.

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 (ND6^P25L) 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.

Fig. 1 |. RET at complex I drives excess superoxide and H₂O₂ production in livers from obese mice.

a, Left, general overview of mROS generation. Specific sites and mechanistic detail pinpointing mitochondrial sources are not shown. Right, illustration of the 11 sites that form superoxide, which is subsequently reduced to H₂O₂ by SOD2, shown as red circles and a black star. NAD-linked sites are in the dehydrogenases of branched-chain 2-oxoacids (BCOADH, site B_F), 2-oxoadipate (OADH, site A_F), pyruvate (PDH, site P_F) and oxoglutarate (OGDH, site O_F). CoQ-linked (Q) sites are in complex III (site III_Qo), in the dehydrogenases of succinate (site II_F), glycerol phosphate (GPDH, site G_Q), dihydroorotate (DHODH, D_Q) and in the electron transport flavoprotein ubiquinone oxidoreductase (ETF:QOR, site E_F). Complex I generates superoxide during FET and RET and may have one or two sites that prematurely reduce oxygen^,, the flavin site I_F and the Q-binding site I_Q. b, H₂O₂ levels in vivo in mitochondria of livers from lean wild-type (WT) and leptin-deficient obese (ob/ob) mice assessed by MitoB oxidation. n = 8 mice per group (**P = 0.0094, unpaired t-test). c, Superoxide levels in primary hepatocytes from WT (n = 281 cells) and ob/ob (n = 138 cells) mice assessed by MitoSOX oxidation. Two mice per group (****P < 0.0001, unpaired t-test). a.u., arbitrary units. d, Maximum capacity of superoxide and H₂O₂ production from mitochondria isolated from livers of WT and ob/ob mice. n = 13 mitochondrial isolations from n = 13 mice per group (*P < 0.05, ****P < 0.0001, multiple paired t-test not adjusted for multiple comparisons). e, Maximum capacity of superoxide and H₂O₂ production from mitochondrial isolated from livers of WT mice fed a chow diet (CD) or a 60% HFD for 17 weeks. n = 6 mitochondrial isolations from n = 6 mice per group (*P = 0.04, multiple paired t-test not adjusted for multiple comparisons). Sites are described in a. Data are individual values and the mean ± s.e.m. All t-tests were two-tailed.

Fig. 2 |. The CoQH₂/CoQ ratio is increased in livers of obese mice.

a, CoQ₉ content (CoQ₉H₂ + CoQ) in the livers of WT and ob/ob mice. b,c, The CoQ₉H₂/CoQ₉ ratio (b) and per cent of reduced CoQ₉ (CoQ₉H₂/total CoQ₉) (c) in the livers of WT and ob/ob mice. n = 9 livers per group (*P = 0.016, ***P = 0.0005, ****P < 0.0001, two-way analysis of variance (ANOVA)). d, CoQ chemical structure showing the head and tail precursors. e, Relative levels of phenylalanine (Phe), tyrosine (Tyr) and 4-hydroxibenzoate (4HB) in the livers of WT and ob/ob mice. n = 9 for WT vs n = 11 for ob/ob (**P = 0.011, ***P = 0.0001, unpaired t-test). f, Metabolite levels of the mevalonate (blue) and cholesterol (red) pathways and CoQ in the livers of ob/ob mice were normalized to WT livers. n = 9 mice per group except cholesterol (n = 13), lanosterol (n = 4) and CoQ₉ (n = 18) (*P = 0.22, **P = 0.005, one sample t-test). g, Relative expression of CoQ biosynthetic genes in the livers of WT and ob/ob mice. Expression levels were normalized to WT. n = 16 mice per group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one sample t-test). h, Kinetics of ²H-enrichment in the CoQ₉ pool in the livers of WT (n = 14) and ob/ob mice (n = 9) (***P = 0.0005, two-way ANOVA). i, Newly synthesized hepatic CoQ₉ in 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). j, Mass enrichment in the CoQ₉ isoprenoid tail of the different isotopomers (M1–M4) 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). k, Mass enrichment in the CoQ₉ head 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 < 0.003, two-way ANOVA). Data are individual values and the mean ± s.e.m. All t-tests were two-tailed.

Fig. 3 |. mROS generation through RET increases hepatic glucose production and impairs glucose homeostasis.

a, Effect of MitoPQ on mROS production through RET. n = 4 mitochondrial isolations (n = 4 mice) (***P < 0.0003, ****P < 0.0001, one-way ANOVA Dunnett’s post hoc test). b, Effect of MitoPQ on mROS generation through FET and RET. n = 1 mitochondrial isolation. n = 6 (RET) or 3 (FET) replicates. Each dot is an independent measurement. (**P = 0.0015, two-way ANOVA). c, MitoPQ treatment in vivo. d, Glucose tolerance tests (GTTs; 1 g kg⁻¹) in 16-h fasted WT mice treated with 4 nmol MitoPQ or DMSO. Inset, area under the curve (AUC). n = 16 mice per group (*P = 0.045, two-way ANOVA and **P = 0.003, unpaired t-test). e,f, Immunoblot (e) and quantification (f) of proteins in total liver lysates from WT mice 1.5 h after 4 nmol MitoPQ or DMSO treatment. n = 8 mice per group (*P = 0.026, **P < 0.01, unpaired t-test). p, phosphorylated. g, Lactate–pyruvate tolerance tests (1.5 and 0.15 g kg⁻¹, respectively) in 16-h fasted WT mice treated with 4 nmol MitoPQ or DMSO. Inset, AUC. n = 22 mice per group (^##P = 0.0008, two-way ANOVA, **P = 0.006, unpaired t-test). h, Gluconeogenesis assays in primary hepatocytes from WT mice 1.5 h after MitoPQ or DMSO treatment. n = 11 hepatocyte isolations per group (*P = 0.041, paired t-test). i,j, Immunoblot (i) and quantification (j) of hepatic PDSS2 levels in Pdss2^loxP/loxP WT (n = 3), Alb/Cre,Pdss2^WT/loxP heterozygous (Het) (n = 2) and Alb/Cre,Pdss2^loxP/loxP knockout (KO) (n = 4) normalized by tubulin (****P < 0.0001, one-way ANOVA, Dunnett’s post hoc test). k, Hepatic CoQ₉ + CoQ₁₀ content in Pdss2 WT (n = 3), Het (n = 2) and KO (n = 4) mice. (****P < 0.0001, one-way ANOVA, Dunnett’s post hoc test). l, Site I_Q mROS production through RET in Pdss2 WT (n = 3) and Het (n = 2) mice. m, Lactate–pyruvate tolerance tests (1.5 and 0.15 g kg⁻¹, respectively) in 16-h fasted Pdss2 WT (n = 9) and Het (n = 2) mice. Inset, AUC (**P = 0.010, two-way ANOVA and *P = 0.019, unpaired t-test). Data are individual values and the mean ± s.e.m. All t-tests were two-tailed. NS, not significant.

Fig. 4 |. Suppressing RET in vivo improves metabolism in obese mice.

a, Summary of loss-of-function obese mouse models used to suppress RET in vivo and improve glucose homeostasis. b, H₂O₂ release assay in primary hepatocytes expressing GFP or Aox from obese mice. n = 7 hepatocyte isolations per group (*P = 0.028, paired t-test). c, Immunoblot of anti-HA in tissue lysates from ob/ob mice expressing Aox or GFP (n = 2 mice per group). WAT, white adipose tissue. d,e, Immunofluorescence (d) and quantification (e) of colocalization of Aox (anti-HA) and MitoTracker Deep Red in primary hepatocytes from ob/ob mice expressing Aox or GFP. Scale bar, 50 µm. Box plots show median, interquartile range (IQR) and 1.5× the IQR. n = 6 fields per group (*P = 0.015). f, Six-hour fasting blood glucose levels in ob/ob mice expressing Aox or GFP. n = 8 mice per group (*P = 0.030). g, GTTs in ob/ob mice expressing Aox (n = 32) or GFP (n = 31). Inset, AUC (*P = 0.014, two-way ANOVA, **P = 0.0065, one-tailed unpaired t-test). h, Site I_Q mROS production through RET in isolated mitochondria from WT (n = 4) and Nd6^P25L (n = 4) mice. (*P = 0.025, paired t-test). i, Six-hour fasting blood glucose levels in WT (n = 13) and Nd6^P25L (n = 11) mice fed a HFD for 15 weeks. (*P = 0.0289). j, GTTs in WT (n = 13) and Nd6^P25L (n = 11) mice fed a HFD for 10 weeks. Inset, AUC (^#P = 0.0111, two-way ANOVA, *P = 0.0117). k, CoQ₁₀ content in ob/ob mice. n = 6 livers per group (****P < 0.0001). l, CoQ₁₀H₂/CoQ₁₀ ratio in ob/ob mice. n = 6 livers per group (**P = 0.002). m, Site I_Q mROS production through RET in isolated mitochondria from ob/ob mice treated with vehicle (n = 8) or CoQ₁₀ (n = 8). (*P = 0.011, paired t-test). n, GTTs in ob/ob mice treated with vehicle or CoQ₁₀. Inset, AUC. n = 15 mice per group (^##P = 0.0052, two-way ANOVA, **P = 0.0013). For the GTTs, 0.5 g glucose per kg was administered for all mice. Values are individual values and the mean ± s.e.m. All comparisons were unpaired two-tailed t-tests unless otherwise specified.

Fig. 5 |. CoQ–RET axis in hepatic steatosis in humans.

a, Volcano plots showing the transcript levels of enzymes from the CoQ biosythetic pathway in liver biopsy samples from patients with different stages of steatosis (S0–S3) compared with healthy control individuals. Thresholds for the log₂[fold change] and −log₁₀[P] were set to 1.3, dotted vertical and horizontal lines, respectively. Grey quadrants, non-significant; light purple and blue, significantly lower in steatosis and significantly higher in steatosis, respectively. b, Correlation between the CoQ₁₀H₂/CoQ₁₀ ratio and the stages of steatosis in liver biopsies from patients (n = 18). c, Comparison of the CoQ₁₀H₂/CoQ₁₀ ratio between patients with low-grade and high-grade steatosis. n = 16 for low-grade and n = 3 for high-grade steatosis. **P = 0.009, unpaired two-tailed t-test. Values are individual values and the mean ± s.e.m.