Early mitochondrial adaptations in skeletal muscle to diet-induced obesity are strain dependent and determine oxidative stress and energy expenditure but not insulin sensitivity

Abstract

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake.

Fig. 1.

Resistance to diet-induced obesity and increased energy expenditure in FVB vs. C57B mice. Body weights (A), fat mass/body weight (B), whole-body VO₂ (C), whole-body CO₂ release (D), and heat production (E) in C57B and FVB mice on NC or HF. In C, D, and E, data were collected over 72 h. In B, C, D, and E, animals were fed NC or HFD for 5 wk. Data are mean ± sem *, P < 0.05; **, P < 0.005 vs. NC-fed mice of the same strain; #, P < 0.05; ##, P < 0.005 vs. C57B mice on HF; $$, P < 0.005 vs. C57B mice on NC.

Fig. 2.

Mitochondrial respiration and ATP synthesis in skeletal muscle of C57B and FVB mice fed NC or HF for 2, 5, or 10 wk. State 3 (A and E), state 4 (B and F), ATP synthesis rates (C and G), and ATP/O ratios (D and H) in saponin-permeabilized soleus fibers from C57B and FVB mice, respectively (n = 5–8 per group and per strain). Data are mean ± sem. a, P < 0.05 vs. NC at any time point; b, P < 0.05 comparing NC 2 or 5 wk with NC at 10 wk; c, P < 0.05 comparing HF 2 or 5 wk with HF at 10 wk; d, P < 0.05 comparing NC at 5 or 10 wk with NC at 2 wk; e, P < 0.05 comparing HF at 5 or 10 wk with HF at 2 wk.

Fig. 3.

FVB mice are protected against HFD-induced ROS production in skeletal muscle. DCFDA fluorescence (A), reduced/oxidized glutathione (B), 4-HNE protein adduct blot (C), and quantification of 4-HNE protein normalized to α-tubulin (D) in skeletal muscle of C57B and FVB mice fed NC (n = 4–6 for each strain) or HF (n = 6 for each strain) for 5 wk. Data are mean ± sem. *, P < 0.05; **, P < 0.005 vs. NC-fed mice of the same strain; ##, P < 0.005 vs. C57B mice on HF; $, P < 0.05 vs. C57B mice on NC. GSH/GSSG, Glutathione/Glutathione disulfide; AU, arbitrary units.

Fig. 4.

Increased lipid influx and intracellular lipid metabolites in FVB mice on HFD. Skeletal muscle TG content at 2 wk (A) and 5 wk of HFD (B) and skeletal muscle DAG levels at 5 wk of HFD (C) in C57B and FVB mice (n = 5–6 mice per strain and per diet). Representative Western blotting of LPL expression and Coomassie blue (C.B.) staining (D) and the corresponding densitometry of LPL/C.B. (E) in skeletal muscle homogenates from C57B and FVB fed NC or HFD for 5 wk (n = 4 mice per strain and per diet). Data are mean ± sem **, P < 0.005 vs. NC-fed mice of the same strain; #, P < 0.05; ##, P < 0.005 vs. C57B mice on HF.

Fig. 5.

Development of insulin resistance in both C57B and FVB mice after 5 wk of HFD independently of changes in Akt phosphorylation. A and B, GTT; C and D, Glucose infusion rates (GIR); E and F, 2-Deoxyglucose (2-DoG) uptake; G, Representative Western blottings for insulin-stimulated Akt phosphorylation on Ser473 and Thr308; and H and I, Densitometry analysis of phosphorylated Akt/total Akt for the 473 and 308 sites, respectively, of C57B and FVB mice on NC (n = 4–6 for each strain) and HF (n = 6–7 for each strain). Data are mean ± sem *, P < 0.05; **, P < 0.005 vs. NC-fed mice; a, P < 0.005 vs. noninsulin stimulated; b, P < 0.05 vs. C57B mice under the same feeding condition.

Fig. 6.

Modulation of oxidative stress in C57B mice does not affect diet-induced insulin resistance. A and E, DCFDA fluorescence in hindlimb muscle homogenates; B, ATP synthesis in saponin-permeabilized soleus muscle; C and F, GTT; D, ITT. A–D, Data obtained in C57B mice fed HFD for 5 wk and simultaneously treated with saline or the antioxidant MnTBAP. E and F, Data obtained in wild-type and UCP3KO fed either NC or HFD for 10 wk. Data are mean ± sem. *, P < 0.05; **, P < 0.005 vs. HF-fed saline treated or vs. NC; ##, P < 0.005 vs. wild-type HF. WT, Wild type; AU, arbitrary units.