Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle.

The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.

Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.

Breast Cancer gene 1 (BRCA1) is a large, multifunctional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

PFKFB3-mediated glycolysis rescues myopathic outcomes in the ischemic limb.

Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hind limb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared with the C57BL/6 (BL6) parental strain. AAV-mediated overexpression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the glucose metabolism Reactome. Muscles from these patients express lower PFKFB3 protein, and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here, we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow-related mitochondrial function is compromised preclinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of the failing CLI patient limb skeletal muscle.

Estrogen receptor-α in female skeletal muscle is not required for regulation of muscle insulin sensitivity and mitochondrial regulation.

OBJECTIVE: Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. METHODS: A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women. RESULTS: Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. CONCLUSIONS: Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.

Serum endotoxin, gut permeability and skeletal muscle metabolic adaptations following a short term high fat diet in humans.

BACKGROUND: Our previous work demonstrated that a short-term high fat diet (HFD) increased fasting serum endotoxin, altered postprandial excursions of serum endotoxin, and led to metabolic and transcriptional responses in skeletal muscle in young, healthy male humans. PURPOSE: The purpose of the present study was to determine if a short-term high fat diet: 1) increases intestinal permeability and, in turn, fasting endotoxin concentrations and 2) decreases postprandial skeletal muscle fat oxidation. METHODS: Thirteen normal weight young adult males (BMI 23.1 ±â€¯0.8 kg/m2, age 22.2 ±â€¯0.4 years) were fed a control diet (55% carbohydrate, 30% fat, 9% of which was saturated, 15% protein) for two weeks, followed by 5 days of an isocaloric HFD (30% carbohydrate, 55% fat, 25% of which was saturated, 15% protein, isocaloric to the control diet). Intestinal permeability (via four sugar probe test) was assessed in the fasting state. Both before and after the HFD, a high fat meal challenge (HFM, 820 kcal, 25% carbohydrate, 63% fat, 26% of which was saturated, and 12% protein) was administered. After an overnight fast, blood samples were collected before and every hour for 4 h after the HFM to assess endotoxin, and other serum blood measures. Muscle biopsies were obtained from the vastus lateralis before and 4 h after the HFM in order to assess substrate oxidation (glucose, fatty acid and pyruvate) using radiolabeled techniques. Insulin sensitivity was assessed via intravenous glucose tolerance test. Intestinal permeability, blood samples and muscle biopsies were assessed in the same manner before and following the HFD. MAIN FINDINGS: Intestinal permeability was not affected by HFD (p > 0.05), but fasting endotoxin increased two fold following the HFD (p = 0.04). Glucose oxidation and fatty acid oxidation in skeletal muscle homogenates significantly increased after the HFM before the HFD (+97%, and +106% respectively) but declined after the HFM following 5 days of the HFD (-24% and +16% respectively). Fatty acid suppressibility of pyruvate oxidation increased significantly after the HFM (+32%) but this physiological effect was abolished following 5 days of the HFD (+7%). Insulin sensitivity did not change following the HFD. CONCLUSION: These findings demonstrate that in healthy young men, consuming an isocaloric HFD for 5 days increases fasting endotoxin, independent of changes in gut permeability. These changes in endotoxin are accompanied by a broad effect on skeletal muscle substrate metabolism including increases in postprandial fat oxidation. Importantly, the latter occurs independent of changes in body weight and whole-body insulin sensitivity.

Doxorubicin causes lesions in the electron transport system of skeletal muscle mitochondria that are associated with a loss of contractile function.

Doxorubicin is an anthracycline-based chemotherapeutic that causes myotoxicity with symptoms persisting beyond treatment. Patients experience muscle pain, weakness, fatigue, and atrophy, but the underlying mechanisms are poorly understood. Studies investigating doxorubicin-induced myotoxicity have reported disrupted mitochondrial function. Mitochondria are responsible for regulating both cellular energy status and Ca2+ handling, both of which impact contractile function. Moreover, loss of mitochondrial integrity may initiate muscle atrophy. Skeletal muscle mitochondrial dysregulation may therefore contribute to an overall loss of skeletal muscle quality and performance that may be mitigated by appropriately targeted mitochondrial therapies. We therefore assessed the impact of doxorubicin on muscle performance and applied a multiplexed assay platform to diagnose alterations in mitochondrial respiratory control. Mice received a clinically relevant dose of doxorubicin delivered systemically and were euthanized 72 h later. We measured extensor digitorum longus and soleus muscle forces, fatigue, and contractile kinetics in vitro, along with Ca2+ uptake in isolated sarcoplasmic reticulum. Isolated skeletal muscle mitochondria were used for real-time respirometry or frozen for protein content and activity assays. Doxorubicin impaired muscle performance, which was indicated by reduced force production, fatigue resistance, and sarcoplasmic reticulum-Ca2+ uptake, which were associated with a substrate-independent reduction in respiration and membrane potential but no changes in the NAD(P)H/NAD(P)+ redox state. Protein content and dehydrogenase activity results corroborated these findings, indicating that doxorubicin-induced mitochondrial impairments are located upstream of ATP synthase within the electron transport system. Collectively, doxorubicin-induced lesions likely span mitochondrial complexes I-IV, providing potential targets for alleviating doxorubicin myotoxicity.

International Society of Sports Nutrition Position Stand: nutritional considerations for single-stage ultra-marathon training and racing.

Background In this Position Statement, the International Society of Sports Nutrition (ISSN) provides an objective and critical review of the literature pertinent to nutritional considerations for training and racing in single-stage ultra-marathon. Recommendations for Training. i) Ultra-marathon runners should aim to meet the caloric demands of training by following an individualized and periodized strategy, comprising a varied, food-first approach; ii) Athletes should plan and implement their nutrition strategy with sufficient time to permit adaptations that enhance fat oxidative capacity; iii) The evidence overwhelmingly supports the inclusion of a moderate-to-high carbohydrate diet (i.e., ~ 60% of energy intake, 5-8 g·kg- 1·d- 1) to mitigate the negative effects of chronic, training-induced glycogen depletion; iv) Limiting carbohydrate intake before selected low-intensity sessions, and/or moderating daily carbohydrate intake, may enhance mitochondrial function and fat oxidative capacity. Nevertheless, this approach may compromise performance during high-intensity efforts; v) Protein intakes of ~ 1.6 g·kg- 1·d- 1 are necessary to maintain lean mass and support recovery from training, but amounts up to 2.5 g.kg- 1·d- 1 may be warranted during demanding training when calorie requirements are greater; Recommendations for Racing. vi) To attenuate caloric deficits, runners should aim to consume 150-400 Kcal·h- 1 (carbohydrate, 30-50 g·h- 1; protein, 5-10 g·h- 1) from a variety of calorie-dense foods. Consideration must be given to food palatability, individual tolerance, and the increased preference for savory foods in longer races; vii) Fluid volumes of 450-750 mL·h- 1 (~ 150-250 mL every 20 min) are recommended during racing. To minimize the likelihood of hyponatraemia, electrolytes (mainly sodium) may be needed in concentrations greater than that provided by most commercial products (i.e., > 575 mg·L- 1 sodium). Fluid and electrolyte requirements will be elevated when running in hot and/or humid conditions; viii) Evidence supports progressive gut-training and/or low-FODMAP diets (fermentable oligosaccharide, disaccharide, monosaccharide and polyol) to alleviate symptoms of gastrointestinal distress during racing; ix) The evidence in support of ketogenic diets and/or ketone esters to improve ultra-marathon performance is lacking, with further research warranted; x) Evidence supports the strategic use of caffeine to sustain performance in the latter stages of racing, particularly when sleep deprivation may compromise athlete safety.

Induced in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness.

KEY POINTS: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. ABSTRACT: Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced-loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle-specific Brca1 homozygote knockout (Brca1KOsmi ) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age-matched wild-type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction-induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle-specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health.

Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants.

The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51-84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.

Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle function.

BACKGROUND: The ability to assess skeletal muscle function and delineate regulatory mechanisms is essential to uncovering therapeutic approaches that preserve functional independence in a disease state. Skeletal muscle provides distinct experimental challenges due to inherent differences across muscle groups, including fiber type and size that may limit experimental approaches. The flexor digitorum brevis (FDB) possesses numerous properties that offer the investigator a high degree of experimental flexibility to address specific hypotheses. To date, surprisingly few studies have taken advantage of the FDB to investigate mechanisms regulating skeletal muscle function. The purpose of this study was to characterize and experimentally demonstrate the value of the FDB muscle for scientific investigations. METHODS: First, we characterized the FDB phenotype and provide reference comparisons to skeletal muscles commonly used in the field. We developed approaches allowing for experimental assessment of force production, in vitro and in vivo microscopy, and mitochondrial respiration to demonstrate the versatility of the FDB. As proof-of principle, we performed experiments to alter force production or mitochondrial respiration to validate the flexibility the FDB affords the investigator. RESULTS: The FDB is made up of small predominantly type IIa and IIx fibers that collectively produce less peak isometric force than the extensor digitorum longus (EDL) or soleus muscles, but demonstrates a greater fatigue resistance than the EDL. Unlike the other muscles, inherent properties of the FDB muscle make it amenable to multiple in vitro- and in vivo-based microscopy methods. Due to its anatomical location, the FDB can be used in cardiotoxin-induced muscle injury protocols and is amenable to electroporation of cDNA with a high degree of efficiency allowing for an effective means of genetic manipulation. Using a novel approach, we also demonstrate methods for assessing mitochondrial respiration in the FDB, which are comparable to the commonly used gastrocnemius muscle. As proof of principle, short-term overexpression of Pgc1α in the FDB increased mitochondrial respiration rates. CONCLUSION: The results highlight the experimental flexibility afforded the investigator by using the FDB muscle to assess mechanisms that regulate skeletal muscle function.

Strain-Dependent Variation in Acute Ischemic Muscle Injury.

Limited efficacy of clinical interventions for peripheral arterial disease necessitates a better understanding of the environmental and genetic determinants of tissue pathology. Existing research has largely ignored the early skeletal muscle injury response during hind limb ischemia (HLI). We compared the hind limb muscle response, after 6 hours of ischemia, in two mouse strains that differ dramatically in their postischemic extended recovery: C57BL/6J and BALB/cJ. Perfusion, measured by laser Doppler and normalized to the control limb, differed only slightly between strains after HLI (<12% across all measures). Similar (<10%) effect sizes in lectin-perfused vessel area and no differences in tissue oxygen saturation measured by reflectance spectroscopy were also found. Muscles from both strains were functionally impaired after HLI, but greater muscle necrosis and loss of dystrophin-positive immunostaining were observed in BALB/cJ muscle compared with C57BL/6J. Muscle cell-specific dystrophin loss and reduced viability were also detected in additional models of ischemia that were independent of residual perfusion differences. Our results indicate that factors other than the completeness of ischemia alone (ie, background genetics) influence the magnitude of acute ischemic muscle injury. These findings may have implications for future development of therapeutic interventions for limb ischemia and for understanding the phasic etiology of chronic and acute ischemic muscle pathophysiology.

Induced Cre-mediated knockdown of Brca1 in skeletal muscle reduces mitochondrial respiration and prevents glucose intolerance in adult mice on a high-fat diet.

The breast cancer type 1 susceptibility protein (Brca1) is a regulator of DNA repair in mammary gland cells; however, recent cell culture evidence suggests that Brca1 influences other processes, including those in nonmammary cells. In this study, we sought to determine whether Brca1 is necessary for metabolic regulation of skeletal muscle using a novel in vivo mouse model. We developed an inducible skeletal muscle-specific Brca1knockout (BRCA1KOsmi) model to test whether Brca1 expression is necessary for maintenance of metabolic function of skeletal muscle when exposed to a high-fat diet (HFD). Our data demonstrated that deletion of Brca1 prevented HFD-induced alterations in glucose and insulin tolerance. Irrespective of diet, BRCA1KOsmi mice exhibited significantly lower ADP-stimulated complex I mitochondrial respiration rates compared to age-matched wild-type (WT) mice. The data show that Brca1 has the ability to localize to the mitochondria in skeletal muscle and that BRCA1KOsmi mice exhibit higher whole-body CO2 production, respiratory exchange ratio, and energy expenditure, compared with the WT mice. Our results demonstrate that loss of Brca1 in skeletal muscle leads to dysregulated metabolic function, characterized by decreased mitochondrial respiration. Thus, any condition that results in loss of Brca1 function could induce metabolic imbalance in skeletal muscle.-Jackson, K. C., Tarpey, M. D., Valencia, A. P., Iñigo, M. R., Pratt, S. J., Patteson, D. J., McClung, J. M., Lovering, R. M., Thomson, D. M., Spangenburg, E. E. Induced Cre-mediated knockdown of Brca1 in skeletal muscle reduces mitochondrial respiration and prevents glucose intolerance in adult mice on a high-fat diet.

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms.

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic ßcyto - or γcyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific ßcyto - and γcyto -actin KO mice. We found ßcyto - and γcyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking ßcyto - and/or γcyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific ßcyto - and γcyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old ßcyto - and γcyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.

Skeletal muscle autophagy and mitophagy in endurance-trained runners before and after a high-fat meal.

OBJECTIVE: We tested the hypothesis that skeletal muscle of endurance-trained male runners would exhibit elevated autophagy and mitophagy markers, which would be associated with greater metabolic flexibility following a high-fat meal (HFM). METHODS: Muscle biopsies were collected to determine differences in autophagy and mitophagy protein markers and metabolic flexibility under fasting conditions and 4 h following a HFM between endurance-trained male runners (n = 10) and sedentary, non-obese controls (n = 9). RESULTS: Maximal oxygen consumption (ml·kg·min-1) was approximately 50% higher (p < 0.05) in endurance-trained runners compared with sedentary controls (65.8 ± 2.3 and 43.1 ± 3.4, respectively). Autophagy markers were similar between groups. Mitophagy and mitochondrial dynamics protein markers were significantly higher in skeletal muscle of endurance-trained runners compared with sedentary controls in the fasted state, although unaffected by the HFM. Skeletal muscle metabolic flexibility was similar between groups when fasted (p > 0.05), but increased in response to the HFM in endurance-trained athletes only (p < 0.005). Key mitophagy markers, phospho-Pink1Thr257 and phospho-ParkinS65 (r = 0.64, p < 0.005), and phospo-ParkinSer65 and phospho-Drp1Ser616 (r = 0.70, p < 0.05) were correlated only within the endurance-trained group. Autophagy and mitophagy markers were not correlated with metabolic flexibility. CONCLUSION: In summary, mitophagy may be enhanced in endurance-trained runners based on elevated markers of mitophagy and mitochondrial dynamics. The HFM did not alter autophagy or mitophagy in either group. The absence of a relationship between mitophagy markers and metabolic flexibility suggests that mitophagy is not a key determinant of metabolic flexibility in a healthy population, but further investigation is warranted.

The effect of a decaffeinated green tea extract formula on fat oxidation, body composition and exercise performance.

BACKGROUND: The cardio-metabolic and antioxidant health benefits of caffeinated green tea (GT) relate to its catechin polyphenol content. Less is known about decaffeinated extracts, particularly in combination with exercise. The aim of this study was therefore to determine whether a decaffeinated green tea extract (dGTE) positively influenced fat oxidation, body composition and exercise performance in recreationally active participants. METHODS: Fourteen, recreationally active males participated in a double-blind, placebo-controlled, parallel design intervention (mean ± SE; age = 21.4 ± 0.3 yrs; weight = 76.37 ± 1.73 kg; body fat = 16.84 ± 0.97%, peak oxygen consumption [[Formula: see text]] = 3.00 ± 0.10 L·min(-1)). Participants were randomly assigned capsulated dGTE (571 mg·d(-1); n = 7) or placebo (PL; n = 7) for 4 weeks. Following body composition and resting cardiovascular measures, participants cycled for 1 hour at 50% [Formula: see text], followed by a 40 minute performance trial at week 0, 2 and 4. Fat and carbohydrate oxidation was assessed via indirect calorimetry. Pre-post exercise blood samples were collected for determination of total fatty acids (TFA). Distance covered (km) and average power output (W) were assessed as exercise performance criteria. RESULTS: Total fat oxidation rates increased by 24.9% from 0.241 ± 0.025 to 0.301 ± 0.009 g·min(-1) with dGTE (P = 0.05; ηp(2) = 0.45) by week 4, whereas substrate utilisation was unaltered with PL. Body fat significantly decreased with dGTE by 1.63 ± 0.16% in contrast to PL over the intervention period (P < 0.001; ηp(2) = 0.84). No significant changes for FFA or blood pressure between groups were observed. dGTE resulted in a 10.9% improvement in performance distance covered from 20.23 ± 0.54 km to 22.43 ± 0.40 km by week 4 (P < 0.001; ηp(2) = 0.85). CONCLUSIONS: A 4 week dGTE intervention favourably enhanced substrate utilisation and subsequent performance indices, but did not alter TFA concentrations in comparison to PL. The results support the use of catechin polyphenols from dGTE in combination with exercise training in recreationally active volunteers.

Assessing a commercially available sports drink on exogenous carbohydrate oxidation, fluid delivery and sustained exercise performance.

BACKGROUND: Whilst exogenous carbohydrate oxidation (CHOEXO) is influenced by mono- and disaccharide combinations, debate exists whether such beverages enhance fluid delivery and exercise performance. Therefore, this study aimed to ascertain CHOEXO, fluid delivery and performance times of a commercially available maltodextrin/ fructose beverage in comparison to an isocaloric maltodextrin beverage and placebo. METHODS: Fourteen club level cyclists (age: 31.79 ± 10.02 years; height: 1.79 ± 0.06 m; weight: 73.69 ± 9.24 kg; VO2max: 60.38 ± 9.36 mL · kg·-1 min-1) performed three trials involving 2.5 hours continuous exercise at 50% maximum power output (Wmax: 176.71 ± 25.92 W) followed by a 60 km cycling performance test. Throughout each trial, athletes were randomly assigned, in a double-blind manner, either: (1) 1.1 g · min-1 maltodextrin + 0.6 g · min-1 fructose (MD + F), (2) 1.7 g · min-1 of maltodextrin (MD) or (3) flavoured water (P). In addition, the test beverage at 60 minutes contained 5.0 g of deuterium oxide (2H2O) to assess quantification of fluid delivery. Expired air samples were analysed for CHOEXO according to the 13C/12C ratio method using gas chromatography continuous flow isotope ratio mass spectrometry. RESULTS: Peak CHOEXO was significantly greater in the final 30 minutes of submaximal exercise with MD + F and MD compared to P (1.45 ± 0.09 g · min-1, 1.07 ± 0.03 g · min-1and 0.00 ± 0.01 g · min-1 respectively, P < 0.0001), and significantly greater for MD + F compared to MD (P = 0.005). The overall appearance of 2H2O in plasma was significantly greater in both P and MD + F compared to MD (100.27 ± 3.57 ppm, 92.57 ± 2.94 ppm and 78.18 ± 4.07 ppm respectively, P < 0.003). There was no significant difference in fluid delivery between P and MD + F (P = 0.078). Performance times significantly improved with MD + F compared with both MD (by 7 min 22 s ± 1 min 56 s, or 7.2%) and P (by 6 min 35 s ± 2 min 33 s, or 6.5%, P < 0.05) over 60 km. CONCLUSIONS: A commercially available maltodextrin-fructose beverage improves CHOEXO and fluid delivery, which may benefit individuals during sustained moderate intensity exercise. The greater CHOEXO observed when consuming a maltodextrin-fructose beverage may support improved performance times.

The ingestion of protein with a maltodextrin and fructose beverage on substrate utilisation and exercise performance.

The study investigated the ingestion of maltodextrin, fructose, and protein on exogenous carbohydrate oxidation (CHOEXO) and exercise performance. Seven trained cyclists and (or) triathletes (maximal oxygen consumption, 59.20 ± 9.00 mL · kg(-1) · min(-1)) performed 3 exercise trials that consisted of 150 min of cycling at 50% maximal power output (160 ± 11 W), followed by a 60-km time trial. One of 3 beverages were randomly assigned during each trial and consumed at 15-min intervals: (i) 0.84 g · min(-1) maltodextrin + 0.52 g · min(-1) fructose + 0.34 g · min(-1) protein (MD+F+P); (ii) 1.10 g · min(-1) maltodextrin + 0.60 g · min(-1) fructose (MD+F); or (iii) 1.70 g · min(-1) maltodextrin (MD). CHO(EXO) and fuel utilisation were assessed via measurement of expired air (13)C content and indirect calorimetry, respectively. Mean total CHO oxidation (CHOTOT) rates were 2.35 ± 0.18, 2.76 ± 0.08, and 2.61 ± 0.17 g · min(-1) with MD, MD+F, and MD+F+P, respectively, although not significantly different. Peak CHO(EXO) rates with MD+F were significantly greater by 41.4% (p = 0.001) and 45.4% (p = 0.0001) compared with MD+F+P and MD, respectively (1.57 ± 0.22 g · min(-1), 1.11 ± 0.08 g · min(-1), and 1.08 ± 0.11 g · min(-1), respectively). Performance times were 2.2% and 5.0% faster with MD+F compared with MD+F+P and MD, respectively; however, they were not statistically significant. Ingestion of an MD-fructose-protein commercial sports beverage significantly reduced peak and mean CHO(EXO) rates compared with MD+F, but did not significantly influence CHOTOT. The addition of protein to an MD+F beverage did not enhance performance times.

An investigative study into the influence of a commercially available carbohydrate-protein-electrolyte beverage on short term repeated exercise performance.

BACKGROUND: The purpose of this study was to undertake an independent investigation into the effects of ingesting a carbohydrate-protein-electrolyte (CPE) beverage on repeated submaximal and time-trial cycling performance. METHODS: Sixteen recreationally trained males (height: 1.76 ± 0.07 m; weight: 70.05 ± 7.90 kg; VO2max: 49.69 ± 4.19 ml.kg-1.min-1) performed two exercise trials separated by 7 days. Each trial comprised two bouts of 90 minutes exercise separated by a 2 hour recovery period. Each bout comprised 45 minutes exercise on a cycle-ergometer at 60%VO2max (ST), followed immediately by a 45 minute performance test (PT). Participants were randomly assigned an 8% CPE beverage or colour/taste matched placebo (PL) prior to each trial. Participants consumed 100 ml of the assigned beverage every 10 minutes during each ST, and 500 ml at 0 and 60 minutes into recovery (total caloric delivery per trial: 617.6 kcal for CPE and12.8 kcal for PL). Mean power output (W), speed (km.hr-1) and distance covered (km) were assessed throughout both trials. Expired air was sampled at 10 minute intervals throughout ST. Blood glucose and lactate were assessed during ST and recovery. RESULTS: Distance covered during ST was significantly reduced with PL by 9.12% (20.18 ± 0.28 km in ST1 v 18.34 ± 0.36 km in ST2; P = 0.0001). With CPE, distance covered, power output and average speed were maintained between ST1 and ST2. Oxygen uptake was not significantly different between ST1 and ST2, or conditions. Respiratory exchange ratio (RER) values decreased from 0.98 ± 0.02 in ST1 to 0.91 ± 0.02 in ST2 for PL (P = 0.003), supporting reduced total carbohydrate oxidation rates (P = 0.007). Mean blood glucose was maintained in CPE across ST trials, and was significantly greater than PL in ST2 (4.77 ± 0.09 mmol.L-1 for CPE compared with 4.18 ± 0.06 mmol.L-1 for PL, P < 0.001). Mean distance during PT2 was 2.96 km (or 17.1%) further with CPE than PL (P = 0.003). Mean power significantly decreased across PT with PL (134.21 ± 4.79 W and 106.90 ± 3.25 W, respectively; P < 0.04). CONCLUSIONS: The use of a CPE beverage improves short-term repeated exercise and subsequent performance compared to PL. Higher rates of carbohydrate oxidation, maintenance of plasma glucose, and decreased levels of fatigue may be beneficial for secondary bouts of performance and faster recovery turnover.