Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

Abstract

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

Figures

Figure 1. High-fat diet increases the mitochondrial H2O2 emission potential in skeletal muscle.

(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.

Figure 2. The mitochondrial-targeted antioxidant SS31 prevents the increase in mitochondrial H2O2-emitting potential caused by high-fat diet in red gastrocnemius skeletal muscle of rats.

(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.

Figure 3. High-fat diet shifts the intracellular redox environment to a more oxidized state in skeletal muscle.

(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 GSHt 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.

Figure 4. Mitochondrial-targeted scavenging of H2O2 prevents high-fat diet–induced insulin resistance in skeletal muscle.

(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.

Figure 5. Mitochondrial H2O2 emission is higher in human obese males and acutely increases in lean males following a high-fat meal.

(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 GSHt 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 GSHt 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.