Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

<div><p>Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in <i>Sod2</i>^<i>+/-</i> mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training <i>Sod2</i>^<i>+/-</i> mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised <i>Sod2</i>^<i>+/-</i> mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.</p> </div

Elevated mitochondrial oxidative stress impairs the improvement in work capacity resulting from exercise training.

<p>Mice were tested for exercise capacity on a treadmill at a 10-degree angle after 4 months of exercise training or remaining sedentary as stated in the Materials and Methods. (A) Total distance run, (B) VO_2max, and (C) total work performed during an exercise test to exhaustion. (For resting data regarding body weight, VO₂, VCO₂, food intake and ambulatory activity see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081879#pone.0081879.s001" target="_blank">Figure S1</a>). Data are mean±SE. <i>n</i> = 7-8. *Indicates a significant difference (<i>p</i> < 0.05) from the indicated group as determined by ANOVA. ^{<b><i>#</i></b>}Indicates a main effect of exercise training. </p

Mitochondrial function is compromised with exercise training in <i>Sod2</i>^<i>+/-</i> mice without influencing organelle biogenesis.

<p>Total protein lysates of tibialis anterior muscle were probed by immunoblotting for individual complex subunits (I: NDUFB8, II: SDHB, III: UQCRC2, IV: COXI, V: ATP5A) or assayed for enzyme activity. (A) The protein expression of mitochondrial proteins determined by densitometry relative to LDHA, (B) representative immunoblots of mitochondrial proteins and (C) enzyme activities of citrate synthase (CS), complex I+III, and complex IV (CIV) per unit of protein (<i>n</i> = 8-10). Data are expressed relative to the Sod2^+/+ SED group. Mitochondrial respiration rates in saponin permeabilized muscle fibers from quadriceps femoris muscle using (D) glutamate and malate without adenylates to indicate a respiratory leak state, (E) glutamate, malate, succinate and ADP to measure maximal coupled oxidative phosphorylation capacity and (F) respiratory coupling ratio of ADP stimulated and non-stimulated respiration using glutamate and malate as substrates (<i>n</i> = 4-5). (G) UCP3 and (H) ANT1 protein expression in isolated mitochondrial lysates from quadriceps femoris muscle relative to VDAC and (I) representative immunoblots for each group (<i>n</i> = 8-10). All data are mean±SE. *Indicates a significant difference (<i>p</i> < 0.05) from the indicated group as determined by 2-way ANOVA. <b><i>#</i></b>Indicates a main effect of exercise training (<i>p</i> < 0.05). <b><i>^†</i></b>Indicates a main effect of genotype (<i>p</i> < 0.05). NS, non-significant.</p

Mitochondrial complex assembly worsens in <i>Sod2</i>^<i>+/-</i> mice subjected to exercise.

<p>Lauryl-maltoside solubilized mitochondria were separated using two-dimensional Blue-Native PAGE in order to visualize native migration of each of four electron transport chain complexes. A lack of intermediate species in assembling the native complex is visualized as a shorter horizontal migration, while unassembled complexes are evident as tails or very broad bands. Note the divergent response to exercise between the two genotypes. Samples were run simultaneously on the same native gel. Image frames are identically sized for each complex and were acquired from the same film exposure. This experiment was repeated twice using different samples from each of the four groups in order to confirm that this was a reproducible observation.</p

Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model

<div><p>Background</p><p>Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.</p><p>Methodology</p><p>C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and <i>ex vivo</i> glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and <i>ex vivo</i> palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.</p><p>Principal Findings/Conclusions</p><p>A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.</p></div

Body Composition.

<p>Body mass and fat mass for normal diet (ND) and high-fat diet (HFD) mice throughout diet intervention up to 8 weeks. Body mass for mice from both 3 and 8 week time-points was included for the 0 week time-point. Data are means ± SEM. Two-way ANOVAs were performed separately for body mass and fat mass, p<0.05,</p>*<p>vs. ND, ^#vs. week 0 for body mass or vs. week 3 for fat mass.</p

Metabolism related changes during HFD transition.

<p>Mice in CLAMS were switched from ND to HFD at the beginning of a light cycle. (A) Light cycle and (B) dark cycle calories consumed per 12 hr cycle (N = 7). (C) Light cycle and (D) dark cycle respiratory exchange ratio (RER; VC0₂:VO₂). (E) Light and (F) dark cycle average movement (x-ambulations) per 12 hour cycle. Data are mean ± SEM. Statistics: Repeated measures one-way ANOVA with Tukey’s multiple comparison test, p<0.05. ND (white bars), HFD (black bars). Average CLAMS measurements per cycle are total (A–B) or means (C–F) of measurements taken every 20 minutes with 1 mouse/CLAMS cage, N = 8 unless otherwise noted. Light cycle = 07∶00–19∶00, dark cycle = 19∶00–07∶00.</p

Pro-inflammatory transcripts in muscle following 8 weeks of HFD.

<p>Relative expression of pro-inflammatory transcripts in (A) TA and (B) soleus muscle after 8 weeks of HFD. Relative expression of <i>Tlr-4</i> in (C) TA and (D) soleus muscle after 8 weeks of HFD. Values were corrected for β-actin expression and ND values were normalized to a value of 1. Statistics: (A and B) Two-way ANOVA with Bonferroni post-tests; (C and D) student’s t-test, *p<0.05. Normal diet (ND, white bars), high-fat diet (HFD; black bars).</p

Indices of adipose, liver, blood and muscle inflammation following 3 weeks of HFD.

<p>Expression of pro-inflammatory indices in (A) adipose tissue (N = 3–6), (B) liver (N = 5–6), (C) TA muscle (N = 6) and (D) soleus muscle (N = 5 ND and 6 HFD). <i>Tlr-4</i> gene expression in (E) TA (N = 6) and (F) soleus muscle (N = 5 ND and 6 HFD). Data are means ± SEM. Statistics: (A–D) All ND data was normalized to 1, Two-way ANOVA with Bonferroni post-tests; (E–F) student’s t-test, * p<0.05. Values were normalized to β-actin (muscle and adipose tissue) or TATA binding protein (TBP; liver tissue). Normal diet (ND, white bars), high-fat diet (HFD; black bars).</p

Measures of oxidative metabolism in muscle.

<p>Following 3 weeks of diet intervention and using palmitate as a substrate EDL and soleus muscle (A) total oxidation [CO2+ oxidative intermediates (OI)], (B) relative esterification (Est) to TAG and DAG vs. total oxidation (Ox) and (C) incomplete (oxidative intermediates, OI) vs. complete (carbon dioxide, CO₂) oxidation (N = 4 in duplicate) were assessed. (D) Fiber type percentage in the oxidative area of the GP muscle following 3 weeks of HFD. Type I fibers were not included in the analysis due to the minimal content in GP muscle. Protein analysis of complexes involved in the electron transport chain (ETC; complexes I–V) in whole GP muscle homogenates after (E) 3 weeks and (F) 8 weeks of HFD. Data are mean ± SEM. Statistics: (A–F) Two-way ANOVA with Bonferonni post-hoc test, *p<0.05. ND, white bars; HFD, black bars. Diacylglycerol (DAG), triacylglycerol (TAG).</p