Extended Data Fig. 3: Mitoparaquat promotes mROS via RET and impairs glucose homeostasis in the liver.
From: CoQ imbalance drives reverse electron transport to disrupt liver metabolism
(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.
