(A) Representative trace comparing rates of mitochondrial H₂O₂ emission under state 4 conditions (10 μg/ml oligomycin) in permeabilized skeletal muscle fibers prepared from a red gastrocnemius muscle of rats fed standard chow (Std chow), high-fat diet (HF; lard) for 3 days, or a high-fat (60%) diet for 3 weeks. The experiment was initiated by addition of glutamate/malate (GM, 5 μM/2 μM) to a deenergized fiber bundle (FB), followed by successive additions of succinate (final concentration shown in mM). (B) Quantified rates of experiments shown in A. Data represent mean ± SEM; n = 4, *P < 0.05 vs. Std chow.

(A and B) Dose-response curves for mitochondrial H₂O₂ (mH₂O₂) emission following in vitro or in vivo administration of SS31. (A) Permeabilized fibers were briefly incubated in a range of SS31 concentrations prior to being assayed for maximal H₂O₂ emission under state 4 conditions (5 mM pyruvate/2 mM malate, 10 μg/ml oligomycin) in the presence of the complex III inhibitor antimycin A (10 μM). (B) Permeabilized fibers were assayed for maximal succinate-induced (3 mM) mitochondrial H₂O₂ emission under state 4 conditions (10 μg/ml oligomycin) approximately 2 hours following an acute intraperitoneal injection of SS20 (control peptide, 5 mg/kg) or varied concentrations of SS31. (C and D) SS31 ameliorates the increased mitochondrial H₂O₂ emission caused by high-fat diet. Permeabilized fibers were prepared from rats fed (6 weeks) standard chow, high-fat diet, or high-fat diet with daily SS31 administration, and H₂O₂ emission was measured during state 4 respiration (10 μg/ml oligomycin) supported by (C) succinate (as described in Figure 1) or (D) palmitoylcarnitine (25 μM) and malate (2 mM). (E and F) High-fat diet increases basal respiration with NADH-linked substrates, but SS31 has no effect. Permeabilized fibers were prepared from rats treated as indicated above, and respiration was measured with (E) pyruvate/malate (5 mM/2 mM) or (F) palmitoylcarnitine/malate (25 μM/2 mM) in both basal respiratory state 4 (PM₄, PCM₄) and maximal ADP-stimulated (2 mM) respiratory state 3 (PM₃, PCM₃). Data represent mean ± SEM; n = 4–6, *P < 0.05 vs. Std chow; ^†P < 0.05 vs. Std. chow and SS31-treated.

(A and B) SS31 prevents the increase in GSSG and decrease in GSH/GSSG ratio in response to acute glucose ingestion. GSSG content (A) and GSH/GSSG ratio (B) were measured before (–) and 1 hour after (+) oral glucose ingestion in red gastrocnemius muscle of rats (10-hour-fasted) fed standard chow, high-fat diet (6 weeks), or high-fat diet with daily SS31 treatment. (C) Muscle GSH_t in standard chow–fed, high-fat diet–fed, and high-fat diet–fed plus SS31-treated rats. Data represent mean ± SEM; n = 4–6, *P < 0.05 vs. corresponding fasted state (i.e., before glucose injection); ^†P < 0.05 vs. Std chow before glucose injection; ^‡P < 0.05 vs. all other groups.

(A–D) Whole body insulin sensitivity is preserved in high-fat diet–fed rats treated with SS31. (A) Plasma glucose and (B) plasma insulin concentrations in response to oral glucose challenge. (C) AUC (arbitrary units) for both glucose and insulin and (D) HOMA, a fasting index of insulin action. Data represent mean ± SEM; n = 9–10; *P < 0.05 vs. Std chow; ^†P < 0.05 vs. SS31-treated. (E) Preserved insulin signaling in high-fat diet–fed animals treated with SS31. Phosphorylated Akt relative to total Akt in response to glucose challenge in red gastrocnemius muscle (representative blots shown in Supplemental Figure 2). Data represent mean ± SEM; n = 4–5; ^§P < 0.05 vs. before glucose challenge. (F–H) Targeted expression of MCAT prevents high-fat diet–induced increase in mitochondrial H₂O₂ emission and insulin resistance in transgenic mice. (F) Succinate stimulated H₂O₂ emission (as described in Figure 1) during state 4 respiration (10 μg/ml oligomycin) in permeabilized red gastrocnemius fibers prepared from wild-type and MCAT mice (4- to 5-hour fasted) fed standard chow or high-fat (60%) diet for 12 weeks. (G) Glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamps. (H) Rate of glucose uptake (Rg) in soleus (Sol), gastrocnemius (G), and vastus lateralis (VL) skeletal muscle from mice in G. Data represent mean ± SEM; n = 4 for mH₂O₂ and n = 9–11 for glucose-clamp experiments; ^¶P< 0.05 vs. WT–std chow; ^#P < 0.05 vs. MCAT-HF; ^‡P < 0.05 vs. WT-HF and MCAT–std chow.

(A and B) Mitochondrial H₂O₂ emission in permeabilized fibers prepared from vastus lateralis of obese and lean human males during state 4 respiration (10 μg/ml oligomycin) supported by (A) succinate (as described in Figure 1) or (B) palmitoylcarnitine (25 μM) and malate (2 mM). (C) O₂ consumption rate determined from parallel experiments during glutamate/malate- or palmitoylcarnitine/malate-supported basal state 4 (GM₄, PCM₄) and maximal ADP-stimulated (2 mM) state 3 (GM₃, PCM₃) respiration. (D) Ratio of H₂O₂ emitted to O₂ consumed under state 4 conditions. (E) GSH/GSSG ratio and GSH_t in skeletal muscle from lean verses obese subjects. (F and G) High-fat diet increases mitochondrial H₂O₂ emission in lean males. Mitochondrial H₂O₂ emission measured during (F) succinate- and (G) palmitoylcarnitine-supported state 4 respiration (10 μg/ml oligomycin) in permeabilized fibers prepared from lean males before (10-hour fasted) and 4 hours after a high-fat meal and after 5 days on a high-fat diet (10-hour fasted). (H) GSH/GSSG ratio and muscle GSH_t in lean males in response to both acute and 5-day lipid ingestion. Data represent mean ± SEM; n = 5–9; *P < 0.05 vs. lean or fasted male (control) for that respective experiment.