Substrate-Specific Respiration of Isolated Skeletal Muscle Mitochondria after 1 h of Moderate Cycling in Sedentary Adults

Abstract

Introduction: Skeletal muscle mitochondria have dynamic shifts in oxidative metabolism to meet energy demands of aerobic exercise. Specific complexes oxidize lipid and nonlipid substrates. It is unclear if aerobic exercise stimulates intrinsic oxidative metabolism of mitochondria or varies between substrates.

Methods: We studied mitochondrial metabolism in sedentary male and female adults (n = 11F/4M) who were free of major medical conditions with mean ± SD age of 28 ± 7 yr, peak aerobic capacity of 2.0 ± 0.4 L·min-1, and body mass index of 22.2 ± 2 kg·m-2. Biopsies were collected from the vastus lateralis muscle on separate study days at rest or 15 min after exercise (1 h cycling at 65% peak aerobic capacity). Isolated mitochondria were analyzed using high-resolution respirometry of separate titration protocols for lipid (palmitoylcarnitine, F-linked) and nonlipid substrates (glutamate-malate, N-linked; succinate S-linked). Titration protocols distinguished between oxidative phosphorylation and leak respiration and included the measurement of reactive oxygen species emission (H2O2). Western blotting determined the protein abundance of electron transfer flavoprotein (ETF) subunits, including inhibitory methylation site on ETF-β.

Results: Aerobic exercise induced modest increases in mitochondrial respiration because of increased coupled respiration across F-linked (+13%, P = 0.08), N(S)-linked (+14%, P = 0.09), and N-linked substrates (+17%, P = 0.08). Prior exercise did not change P:O ratio. Electron leak to H2O2 increased 6% increased after exercise (P = 0.06) for lipid substrates but not for nonlipid. The protein abundance of ETF-α or ETF-β subunit or inhibitory methylation on ETF-β was not different between rest and after exercise.

Conclusion: In sedentary adults, the single bout of moderate-intensity cycling induced modest increases for intrinsic mitochondrial oxidative phosphorylation that was consistent across multiple substrates.

Trial registration: ClinicalTrials.gov NCT02987491.

Copyright © 2021 by the American College of Sports Medicine.

Figures

Figure 1:. F-linked respiration after aerobic exercise

Respiration of isolated mitochondria collected from vastus lateralis biopsies at rest or 15 minutes after aerobic exercise (60 minutes at 65% VO2 peak). There were no major changes in respiration for any respiratory state with tendency for greater oxidative phosphorylation. Labels above graph indicates respiratory states for leak (L), oxidative phosphorylation (P) and electron transfer system (E) for lipid (F-linked) respiration of mitochondria (mitos), palmitoyl carnitine + malate (PC+M), saturated ADP (ADP), Cytochrome C for membrane integrity, oligomycin (LO), uncoupling via FCCP and antimycin a for residual oxygen consumption (Rox). Data displayed as mean and standard deviation with individual data points (n=15 males and females). Data were compared using paired t-test between rest and exercise within each respiratory state.

Figure 2:. N and S-linked respiration after aerobic exercise

Respiration of isolated mitochondria collected from vastus lateralis biopsies at rest or 15 minutes after aerobic exercise (60 minutes at 65% VO2 peak). There were no major changes in respiration for any respiratory state with tendency for greater oxidative phosphorylation. Labels above graph indicates respiratory states for leak (L), oxidative phosphorylation (P) and electron transfer system (E) for glutamate+malate (N-linked) and succinate (S-linked) respiration of mitochondria (mitos), glutamate+malate (GM), ADP, Cytochrome C for membrane integrity, rotenone for complex I inhibition, oligomycin (LO), electron transfer system (E) during uncoupling via FCCP and antimycin a for residual oxygen consumption (Rox ). Data displayed as mean and standard deviation with individual data points (n=15 males and females). Data were compared using paired t-test between rest and exercise within each respiratory state.

Figure 3:. Coupled respiration after aerobic exercise

Coupled respiration determined as oxidative phosphorylation minus leak respiration for each respiratory substrate (A). P:O was calculated as oxygen consumption during a sub-saturating pulse of ADP and was not changed following exercise (B). Data displayed as mean and standard deviation with individual data points (n=15 males and females). Data were compared using paired t-test between rest and exercise within each respiratory state.

Figure 4:. H2O2 emission and electron leak after aerobic exercise

H2O2 emission was measured during respiratory protocols from isolated mitochondria collected from vastus lateralis biopsies at rest or 15 minutes after aerobic exercise (60 minutes at 65% VO2 peak). Prior exercise did not change H2O2 emissions for F-linked (A) or N(S) linked substrates (B). There a small effect of exercise to electron leak to H2O2 during leak respiration of F-linked substrates (C) with no changes in N(S) substrates (D). Note the broken y-axis scales due to the wide range of electron leak during Leak (L) versus oxidative phosphorylation (P). Data displayed as mean and standard deviation with individual data points (n=15 males and females). Data were compared using paired t-test between rest and exercise within each respiratory state.

Figure 5:. Protein abundance after aerobic exercise

Protein abundance via western blotting of biopsy samples collected from vastus lateralis during rest or 15 minutes after aerobic exercise (60 minutes at 65% VO2 peak). There were no changes in protein abundance after exercise for ETF-α or β subunits, nor in the inhibitory modification of tri-methylation of ETF-β (A-C). β-HAD abundance was also not different (D). There were no differences between rest and 15 minutes post-exercise for PDH E1α or phosphorylated PDH Et1α (F &G). Representative images displayed in E and H. Data displayed as mean and standard deviation with individual data points (n=15 males and females). Data were compared using paired t-test between rest and exercise within each blot.