Glenn D. Wadley

Antioxidant Supplementation Reduces Skeletal Muscle Mitochondrial Biogenesis

PURPOSE Exercise increases the production of reactive oxygen species (ROS) in skeletal muscle, and athletes often consume antioxidant supplements in the belief they will attenuate ROS-related muscle damage and fatigue during exercise. However, exercise-induced ROS may regulate beneficial skeletal muscle adaptations, such as increased mitochondrial biogenesis. We therefore investigated the effects of long-term antioxidant supplementation with vitamin E and α-lipoic acid on changes in markers of mitochondrial biogenesis in the skeletal muscle of exercise-trained and sedentary rats. METHODS Male Wistar rats were divided into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet, and 4) exercise antioxidant diet. Animals ran on a treadmill 4 d · wk at ∼ 70%VO2max for up to 90 min · d for 14 wk. RESULTS Consistent with the augmentation of skeletal muscle mitochondrial biogenesis and antioxidant defenses, after training there were significant increases in peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) messenger RNA (mRNA) and protein, cytochrome C oxidase subunit IV (COX IV) and cytochrome C protein abundance, citrate synthase activity, Nfe2l2, and SOD2 protein (P < 0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P < 0.05) in both sedentary and exercise-trained rats. CONCLUSIONS Vitamin E and α-lipoic acid supplementation suppresses skeletal muscle mitochondrial biogenesis, regardless of training status.

Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short‐term endurance training

•  The discovery of microRNAs (miRNAs) has established new mechanisms that control health, but little is known about the regulation of skeletal muscle miRNAs in response to exercise. •  This study investigated components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin‐5), muscle enriched miRNAs, (miR‐1, ‐133a, ‐133b and 206), and several miRNAs dysregulated in muscle myopathies, and showed that 3 h following an acute exercise bout, Drosha, Dicer and Exportin‐5, as well as miR‐1, ‐133a, ‐133‐b and miR‐181a were all increased, while miR‐9, ‐23a, ‐23b and ‐31 were decreased. •  Short‐term training increased miR‐1 and miR‐29b, while miR‐31 remained decreased. •  Negative correlations were observed between miR‐9 and HDAC4 protein, miR‐31 and HDAC4 protein and between miR‐31 and NRF1 protein, 3 h after exercise. •  miR‐31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. •  Exercise rapidly and transiently regulates several miRNA species potentially involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis.

The relationship between repeated sprint ability and the aerobic and anaerobic energy systems

A large number of team games require participants to repeatedly produce maximal or near maximal sprints of short duration with brief recovery periods. The purpose of the present study was to determine the relationship between a repeated sprint ability (RSA) test that is specific to the energy demands of Australian Rules football (ARF), and the aerobic and anaerobic energy systems. Seventeen ARF players participated in the study. Each participant was assessed for VO2 max, accumulated oxygen deficit (AOD), best 20 m sprint time and RSA. The RSA test involved 12x20 m sprints departing every 20 s. When including the work performed during the time taken to decelerate, the test involved a work to rest ratio of approximately 1:3. Total sprinting time and the percentage decrement of repeated sprinting times were the two derived measures of RSA. The results indicate that the best 20 m sprint time was the only factor to correlate significantly with total sprinting time (r = 0.829, P < 0.001) and percentage decrement (r = -0.722, P < 0.01). VO2 max and AOD were not related to the total sprinting time or the percentage decrement that was produced by the RSA test. This was interpreted to signify that the phosphagen system was the major energy contributor for this test.

Skeletal muscle mitochondria: a major player in exercise, health and disease.

BACKGROUND Maintaining skeletal muscle mitochondrial content and function is important for sustained health throughout the lifespan. Exercise stimulates important key stress signals that control skeletal mitochondrial biogenesis and function. Perturbations in mitochondrial content and function can directly or indirectly impact skeletal muscle function and consequently whole-body health and wellbeing. SCOPE OF REVIEW This review will describe the exercise-stimulated stress signals and molecular mechanisms positively regulating mitochondrial biogenesis and function. It will then discuss the major myopathies, neuromuscular diseases and conditions such as diabetes and ageing that have dysregulated mitochondrial function. Finally, the impact of exercise and potential pharmacological approaches to improve mitochondrial function in diseased populations will be discussed. MAJOR CONCLUSIONS Exercise activates key stress signals that positively impact major transcriptional pathways that transcribe genes involved in skeletal muscle mitochondrial biogenesis, fusion and metabolism. The positive impact of exercise is not limited to younger healthy adults but also benefits skeletal muscle from diseased populations and the elderly. Impaired mitochondrial function can directly influence skeletal muscle atrophy and contribute to the risk or severity of disease conditions. Pharmacological manipulation of exercise-induced pathways that increase skeletal muscle mitochondrial biogenesis and function in critically ill patients, where exercise may not be possible, may assist in the treatment of chronic disease. GENERAL SIGNIFICANCE This review highlights our understanding of how exercise positively impacts skeletal muscle mitochondrial biogenesis and function. Exercise not only improves skeletal muscle mitochondrial health but also enables us to identify molecular mechanisms that may be attractive targets for therapeutic manipulation. This article is part of a Special Issue entitled Frontiers of mitochondrial research.

UCP3 protein expression is lower in type I, IIa and IIx muscle fiber types of endurance trained compared to untrained subjects

Abstract. Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower (P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups (P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects (P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.

Local Nitric Oxide Synthase Inhibition Reduces Skeletal Muscle Glucose Uptake but Not Capillary Blood Flow During In Situ Muscle Contraction in Rats

OBJECTIVE—We have previously shown in humans that local infusion of a nitric oxide synthase (NOS) inhibitor into the femoral artery attenuates the increase in leg glucose uptake during exercise without influencing total leg blood flow. However, rodent studies examining the effect of NOS inhibition on contraction-stimulated skeletal muscle glucose uptake have yielded contradictory results. This study examined the effect of local infusion of an NOS inhibitor on skeletal muscle glucose uptake (2-deoxyglucose) and capillary blood flow (contrast-enhanced ultrasound) during in situ contractions in rats. RESEARCH DESIGN AND METHODS—Male hooded Wistar rats were anesthetized and one hindleg electrically stimulated to contract (2 Hz, 0.1 ms) for 30 min while the other leg rested. After 10 min, the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) (arterial concentration of 5 μmol/l) or saline was infused into the epigastric artery of the contracting leg. RESULTS—Local NOS inhibition had no effect on blood pressure, heart rate, or muscle contraction force. Contractions increased (P < 0.05) skeletal muscle NOS activity, and this was prevented by l-NAME infusion. NOS inhibition caused a modest significant (P < 0.05) attenuation of the increase in femoral blood flow during contractions, but importantly there was no effect on capillary recruitment. NOS inhibition attenuated (P < 0.05) the increase in contraction-stimulated skeletal muscle glucose uptake by ∼35%, without affecting AMP-activated protein kinase (AMPK) activation. CONCLUSIONS—NOS inhibition attenuated increases in skeletal muscle glucose uptake during contraction without influencing capillary recruitment, suggesting that NO is critical for part of the normal increase in skeletal muscle fiber glucose uptake during contraction.

NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle.

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS−/−) or neuronal NOS (nNOS−/−) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS−/−, nNOS−/− and C57Bl/6 (CON) mice (16.3 ± 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min−1, 5% grade) for 60 min (n= 8 per group) and were killed 6 h after exercise. Other eNOS−/−, nNOS−/− and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS−/− mice had significantly higher nNOS protein and nNOS−/− mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2α and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS−/− mice whilst basal citrate synthase activity was higher in the soleus and basal PGC‐1α mRNA higher in the EDL. Also, eNOS−/− mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC‐1α mRNA in soleus and EDL and NRF2α mRNA in the EDL to a similar extent in all genotypes. In addition, short‐term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise‐induced increases in mitochondrial biogenesis in skeletal muscle.

Uteroplacental insufficiency and reducing litter size alters skeletal muscle mitochondrial biogenesis in a sex-specific manner in the adult rat

Uteroplacental insufficiency has been shown to impair insulin action and glucose homeostasis in adult offspring and may act in part via altered mitochondrial biogenesis and lipid balance in skeletal muscle. Bilateral uterine vessel ligation to induce uteroplacental insufficiency in offspring (Restricted) or sham surgery was performed on day 18 of gestation in rats. To match the litter size of Restricted offspring, a separate cohort of sham litters had litter size reduced to five at birth (Reduced Litter), which also restricted postnatal growth. Remaining litters from sham mothers were unaltered (Control). Offspring were studied at 6 mo of age. In males, both Restricted and Reduced Litter offspring had reduced gastrocnemius PPARgamma coactivator-1alpha (PGC-1alpha) mRNA and protein, and mitochondrial transcription factor A (mtTFA) and cytochrome oxidase (COX) III mRNA (P < 0.05), whereas only Restricted had reduced skeletal muscle COX IV mRNA and protein and glycogen (P < 0.05), despite unaltered glucose tolerance, homeostasis model assessment (HOMA) and intramuscular triglycerides. In females, only gastrocnemius mtTFA mRNA was lower in Reduced Litter offspring (P < 0.05). Furthermore, glucose tolerance was not altered in any female offspring, although HOMA and intramuscular triglycerides increased in Restricted offspring (P < 0.05). It is concluded that restriction of growth due to uteroplacental insufficiency alters skeletal muscle mitochondrial biogenesis and metabolic characteristics, such as glycogen and lipid levels, in a sex-specific manner in the adult rat in the absence of impaired glucose tolerance. Furthermore, an adverse postnatal environment induced by reducing litter size also restricts growth and alters skeletal muscle mitochondrial biogenesis and metabolic characteristics in the adult rat.

Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.

Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism

Nitric oxide (NO) is an important vasodilator and regulator in the cardiovascular system, and this link was the subject of a Nobel prize in 1998. However, NO also plays many other regulatory roles, including thrombosis, immune function, neural activity, and gastrointestinal function. Low concentrations of NO are thought to have important signaling effects. In contrast, high concentrations of NO can interact with reactive oxygen species, causing damage to cells and cellular components. A less-recognized site of NO production is within skeletal muscle, where small increases are thought to have beneficial effects such as regulating glucose uptake and possibly blood flow, but higher levels of production are thought to lead to deleterious effects such as an association with insulin resistance. This review will discuss the role of NO in skeletal muscle during and following exercise, including in mitochondrial biogenesis, muscle efficiency, and blood flow with a particular focus on its potential role in regulating skeletal muscle glucose uptake during exercise.