Small peptides: could they have a big role in metabolism and the response to exercise?

Exercise is a powerful non-pharmacological intervention for the treatment and prevention of numerous chronic diseases. Contracting skeletal muscles provoke widespread perturbations in numerous cells, tissues and organs, which stimulate multiple integrated adaptations that ultimately contribute to the many health benefits associated with regular exercise. Despite much research, the molecular mechanisms driving such changes are not completely resolved. Technological advancements beginning in the early 1960s have opened new avenues to explore the mechanisms responsible for the many beneficial adaptations to exercise. This has led to increased research into the role of small peptides (<100 amino acids) and mitochondrially derived peptides in metabolism and disease, including those coded within small open reading frames (sORFs; coding sequences that encode small peptides). Recently, it has been hypothesized that sORF-encoded mitochondrially derived peptides and other small peptides play significant roles as exercise-sensitive peptides in exercise-induced physiological adaptation. In this review, we highlight the discovery of mitochondrially derived peptides and newly discovered small peptides involved in metabolism, with a specific emphasis on their functions in exercise-induced adaptations and the prevention of metabolic diseases. In light of the few studies available, we also present data on how both single exercise sessions and exercise training affect expression of sORF-encoded mitochondrially derived peptides. Finally, we outline numerous research questions that await investigation regarding the roles of mitochondrially derived peptides in metabolism and prevention of various diseases, in addition to their roles in exercise-induced physiological adaptations, for future studies.

Interpretation of exercise-induced changes in human skeletal muscle mRNA expression depends on the timing of the post-exercise biopsies.

BACKGROUND: Exercise elicits a range of adaptive responses in skeletal muscle, which include changes in mRNA expression. To better understand the health benefits of exercise training, it is important to investigate the underlying molecular mechanisms of skeletal muscle adaptation to exercise. However, most studies have assessed the molecular events at only a few time-points within a short time frame post-exercise, and the variations of gene expression kinetics have not been addressed systematically. METHODS: We assessed the mRNA expression of 23 gene isoforms implicated in the adaptive response to exercise at six time-points (0, 3, 9, 24, 48, and 72 h post exercise) over a 3-day period following a single session of high-intensity interval exercise. RESULTS: The temporal patterns of target gene expression were highly variable and the expression of mRNA transcripts detected was largely dependent on the timing of muscle sampling. The largest fold change in mRNA expression of each tested target gene was observed between 3 and 72 h post-exercise. DISCUSSION AND CONCLUSIONS: Our findings highlight an important gap in knowledge regarding the molecular response to exercise, where the use of limited time-points within a short period post-exercise has led to an incomplete understanding of the molecular response to exercise. Muscle sampling timing for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.

Aerobic-interval exercise with blood flow restriction potentiates early markers of metabolic health in man.

AIM: This study examined whether aerobic-interval exercise with blood flow restriction (BFR) potentiates early markers of metabolic health compared to exercise with systemic hypoxia or normoxia in man. METHODS: In a randomized-crossover fashion, eight healthy men completed nine 2-minute running bouts at 105% of their lactate threshold on three occasions separated by one week, either with BFR (BFR-trial), systemic hypoxia (HYP-trial) or normoxia (control; CON-trial). Near-infrared spectroscopy was used to assess the muscle level of hypoxia. A muscle biopsy was collected at rest and 3 hours after exercise to quantify genes involved in cholesterol synthesis (PGC-1α2), glucose disposal (GLUT4) and capillary growth (HIF-1α; VEGFA), as well as mitochondrial respiration (PGC-1α2/3), uncoupling (UCP3) and expansion (p53; COXIV-1/2; CS; AMPKα1/2). RESULTS: The muscle level of hypoxia was matched between the BFR-trial and HYP-trial (~90%; P > .05), which was greater than the CON-trial (~70%; P < .05). PGC-1α2 increased most in the BFR-trial (16-fold vs CON-trial; 11-fold vs HYP-trial; P < .05). GLUT4 and VEGFA selectively increased by 2.0 and 3.4-fold, respectively in BFR-trial (P < .05), which was greater than CON-trial (1.2 and 1.3 fold) and HYP-trial (1.2 and 1.8 fold; P < .05). UCP3 increased more in BFR-trial than the HYP-trial (4.3 vs 1.6 fold), but was not different between BFR-trial and CON-trial (2.1 fold) or between CON-trial and HYP-trial (P > .05). No trial differences were evident for other genes (P > .05). CONCLUSION: Independent of the muscle level of hypoxia, BFR-exercise potentiates early markers of metabolic health associated with the regulation of cholesterol production and glucose homeostasis in man.

Factors Influencing AMPK Activation During Cycling Exercise: A Pooled Analysis and Meta-Regression.

BACKGROUND: The 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that is activated by increases in the cellular AMP/adenosine diphosphate:adenosine triphosphate (ADP:ATP) ratios and plays a key role in metabolic adaptations to endurance training. The degree of AMPK activation during exercise can be influenced by many factors that impact on cellular energetics, including exercise intensity, exercise duration, muscle glycogen, fitness level, and nutrient availability. However, the relative importance of these factors for inducing AMPK activation remains unclear, and robust relationships between exercise-related variables and indices of AMPK activation have not been established. OBJECTIVES: The purpose of this analysis was to (1) investigate correlations between factors influencing AMPK activation and the magnitude of change in AMPK activity during cycling exercise, (2) investigate correlations between commonly reported measures of AMPK activation (AMPK-α2 activity, phosphorylated (p)-AMPK, and p-acetyl coenzyme A carboxylase (p-ACC), and (3) formulate linear regression models to determine the most important factors for AMPK activation during exercise. METHODS: Data were pooled from 89 studies, including 982 participants (93.8% male, maximal oxygen consumption [[Formula: see text]] 51.9 ± 7.8 mL kg-1 min-1). Pearson's correlation analysis was performed to determine relationships between effect sizes for each of the primary outcome markers (AMPK-α2 activity, p-AMPK, p-ACC) and factors purported to influence AMPK signaling (muscle glycogen, carbohydrate ingestion, exercise duration and intensity, fitness level, and muscle metabolites). General linear mixed-effect models were used to examine which factors influenced AMPK activation. RESULTS: Significant correlations (r = 0.19-0.55, p < .05) with AMPK activity were found between end-exercise muscle glycogen, exercise intensity, and muscle metabolites phosphocreatine, creatine, and free ADP. All markers of AMPK activation were significantly correlated, with the strongest relationship between AMPK-α2 activity and p-AMPK (r = 0.56, p < 0.001). The most important predictors of AMPK activation were the muscle metabolites and exercise intensity. CONCLUSION: Muscle glycogen, fitness level, exercise intensity, and exercise duration each influence AMPK activity during exercise when all other factors are held constant. However, disrupting cellular energy charge is the most influential factor for AMPK activation during endurance exercise.

High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content.

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings.

Revisiting the contribution of mitochondrial biology to the pathophysiology of skeletal muscle insulin resistance.

While the etiology of type 2 diabetes is multifaceted, the induction of insulin resistance in skeletal muscle is a key phenomenon, and impairments in insulin signaling in this tissue directly contribute to hyperglycemia. Despite the lack of clarity regarding the specific mechanisms whereby insulin signaling is impaired, the key role of a high lipid environment within skeletal muscle has been recognized for decades. Many of the proposed mechanisms leading to the attenuation of insulin signaling - namely the accumulation of reactive lipids and the pathological production of reactive oxygen species (ROS), appear to rely on this high lipid environment. Mitochondrial biology is a central component to these processes, as these organelles are almost exclusively responsible for the oxidation and metabolism of lipids within skeletal muscle and are a primary source of ROS production. Classic studies have suggested that reductions in skeletal muscle mitochondrial content and/or function contribute to lipid-induced insulin resistance; however, in recent years the role of mitochondria in the pathophysiology of insulin resistance has been gradually re-evaluated to consider the biological effects of alterations in mitochondrial content. In this respect, while reductions in mitochondrial content are not required for the induction of insulin resistance, mechanisms that increase mitochondrial content are thought to enhance mitochondrial substrate sensitivity and submaximal adenosine diphosphate (ADP) kinetics. Thus, this review will describe the central role of a high lipid environment in the pathophysiology of insulin resistance, and present both classic and contemporary views of how mitochondrial biology contributes to insulin resistance in skeletal muscle.

Fifteen days of moderate normobaric hypoxia does not affect mitochondrial function, and related genes and proteins, in healthy men.

PURPOSE: To investigate within the one study potential molecular and cellular changes associated with mitochondrial biogenesis following 15 days of exposure to moderate hypoxia. METHODS: Eight males underwent a muscle biopsy before and after 15 days of hypoxia exposure (FiO2 = 0.140-0.154; ~ 2500-3200 m) in a hypoxic hotel. Mitochondrial respiration, citrate synthase (CS) activity, and the content of genes and proteins associated with mitochondrial biogenesis were investigated. RESULTS: Our main findings were the absence of significant changes in the mean values of CS activity, mitochondrial respiration in permeabilised fibers, or the content of genes and proteins associated with mitochondrial biogenesis, after 15 days of moderate normobaric hypoxia. CONCLUSION: Our data provide evidence that 15 days of moderate normobaric hypoxia have negligible influence on skeletal muscle mitochondrial content and function, or genes and proteins content associated with mitochondrial biogenesis, in young recreationally active males. However, the increase in mitochondrial protease LON content after hypoxia exposure suggests the possibility of adaptations to optimise respiratory chain function under conditions of reduced O2 availability.

Resistance training upregulates skeletal muscle Na+, K+-ATPase content, with elevations in both α1 and α2, but not ß isoforms.

PURPOSE: The Na+, K+-ATPase (NKA) is important in regulating trans-membrane ion gradients, cellular excitability and muscle function. We investigated the effects of resistance training in healthy young adults on the adaptability of NKA content and of the specific α and ß isoforms in human skeletal muscle. METHODS: Twenty-one healthy young males (22.9 ± 4.6 year; 1.80 ± 0.70 m, 85.1 ± 17.8 kg, mean ± SD) underwent 7 weeks of resistance training, training three times per week (RT, n = 16) or control (CON, n = 5). The training program was effective with a 39% gain in leg press muscle strength (p = 0.001). A resting vastus lateralis muscle biopsy was taken before and following RT or CON and assayed for NKA content ([3H]ouabain binding site content) and NKA isoform (α1, α2, ß1, ß2) abundances. RESULTS: After RT, each of NKA content (12%, 311 ± 76 vs 349 ± 76 pmol g wet weight-1, p = 0.01), NKA α1 (32%, p = 0.01) and α2 (10%, p < 0.01) isoforms were increased, whereas ß1 (p = 0.18) and ß2 (p = 0.22) isoforms were unchanged. NKA content and isoform abundances were unchanged during CON. CONCLUSIONS: Resistance training increased muscle NKA content through upregulation of both α1 and α2 isoforms, which were independent of ß isoform changes. In animal models, modulations in α1 and α2 isoform abundances in skeletal muscle may affect fatigue resistance during exercise, muscle hypertrophy and strength. Whether similar in-vivo functional benefits of these NKA isoform adaptations occurs in human muscle with resistance training remains to be determined.

ADOR2A C Allele Carriers Exhibit Ergogenic Responses to Caffeine Supplementation.

Caffeine's ergogenic effects on exercise performance are generally explained by its ability to bind to adenosine receptors. ADORA2A is the gene that encodes A2A subtypes of adenosine receptors. It has been suggested that ADORA2A gene polymorphisms may be responsible for the inter-individual variations in the effects of caffeine on exercise performance. In the only study that explored the influence of variation in ADORA2A-in this case, a common polymorphism (rs5751876)-on the ergogenic effects of caffeine on exercise performance, C allele carriers were identified as "non-responders" to caffeine. To explore if C allele carriers are true "non-responders" to the ergogenic effects of caffeine, in this randomized, double-blind study, we examined the acute effects of caffeine ingestion among a sample consisting exclusively of ADORA2A C allele carriers. Twenty resistance-trained men identified as ADORA2A C allele carriers (CC/CT genotype) were tested on two occasions, following the ingestion of caffeine (3 mg/kg) and a placebo. Exercise performance was evaluated with movement velocity, power output, and muscle endurance during the bench press exercise, countermovement jump height, and power output during a Wingate test. Out of the 25 analyzed variables, caffeine was ergogenic in 21 (effect size range: 0.14 to 0.96). In conclusion, ADORA2A (rs5751876) C allele carriers exhibited ergogenic responses to caffeine ingestion, with the magnitude of improvements similar to what was previously reported in the literature among samples that were not genotype-specific. Therefore, individuals with the CT/CC genotype may still consider supplementing with caffeine for acute improvements in performance.

Cycling with blood flow restriction improves performance and muscle K+ regulation and alters the effect of anti-oxidant infusion in humans.

KEY POINTS: Training with blood flow restriction (BFR) is a well-recognized strategy for promoting muscle hypertrophy and strength. However, its potential to enhance muscle function during sustained, intense exercise remains largely unexplored. In the present study, we report that interval training with BFR augments improvements in performance and reduces net K+ release from contracting muscles during high-intensity exercise in active men. A better K+ regulation after BFR-training is associated with an elevated blood flow to exercising muscles and altered muscle anti-oxidant function, as indicated by a higher reduced to oxidized glutathione (GSH:GSSG) ratio, compared to control, as well as an increased thigh net K+ release during intense exercise with concomitant anti-oxidant infusion. Training with BFR also invoked fibre type-specific adaptations in the abundance of Na+ ,K+ -ATPase isoforms (α1 , ß1 , phospholemman/FXYD1). Thus, BFR-training enhances performance and K+ regulation during intense exercise, which may be a result of adaptations in anti-oxidant function, blood flow and Na+ ,K+ -ATPase-isoform abundance at the fibre-type level. ABSTRACT: We examined whether blood flow restriction (BFR) augments training-induced improvements in K+ regulation and performance during intense exercise in men, and also whether these adaptations are associated with an altered muscle anti-oxidant function, blood flow and/or with fibre type-dependent changes in Na+ ,K+ -ATPase-isoform abundance. Ten recreationally-active men (25 ± 4 years, 49.7 ± 5.3 mL kg-1  min-1 ) performed 6 weeks of interval cycling, where one leg trained without BFR (control; CON-leg) and the other trained with BFR (BFR-leg, pressure: ∼180 mmHg). Before and after training, femoral arterial and venous K+ concentrations and artery blood flow were measured during single-leg knee-extensor exercise at 25% (Ex1) and 90% of thigh incremental peak power (Ex2) with i.v. infusion of N-acetylcysteine (NAC) or placebo (saline) and a resting muscle biopsy was collected. After training, performance increased more in BFR-leg (23%) than in CON-leg (12%, P < 0.05), whereas K+ release during Ex2 was attenuated only from BFR-leg (P < 0.05). The muscle GSH:GSSG ratio at rest and blood flow during exercise was higher in BFR-leg than in CON-leg after training (P < 0.05). After training, NAC increased resting muscle GSH concentration and thigh net K+ release during Ex2 only in BFR-leg (P < 0.05), whereas the abundance of Na+ ,K+ -ATPase-isoform α1 in type II (51%), ß1 in type I (33%), and FXYD1 in type I (108%) and type II (60%) fibres was higher in BFR-leg than in CON-leg (P < 0.05). Thus, training with BFR elicited greater improvements in performance and reduced thigh K+ release during intense exercise, which were associated with adaptations in muscle anti-oxidant function, blood flow and Na+ ,K+ -ATPase-isoform abundance at the fibre-type level.

A physiological drop in pH decreases mitochondrial respiration, and HDAC and Akt signaling, in L6 myocytes.

Exercise stimulates mitochondrial biogenesis and increases mitochondrial respiratory function and content. However, during high-intensity exercise muscle pH can decrease below pH 6.8 with a concomitant increase in lactate concentration. This drop in muscle pH is associated with reduced exercise-induced mitochondrial biogenesis, while increased lactate may act as a signaling molecule to affect mitochondrial biogenesis. Therefore, in this study we wished to determine the impact of altering pH and lactate concentration in L6 myotubes on genes and proteins known to be involved in mitochondrial biogenesis. We also examined mitochondrial respiration in response to these perturbations. Differentiated L6 myotubes were exposed to normal (pH 7.5)-, low (pH 7.0)-, or high (pH 8.0)-pH media with and without 20 mM sodium l-lactate for 1 and 6 h. Low pH and 20 mM sodium l-lactate resulted in decreased Akt (Ser473) and AMPK (T172) phosphorylation at 1 h compared with controls, while at 6 h the nuclear localization of histone deacetylase 5 (HDAC5) was decreased. When the pH was increased both Akt (Ser473) and AMPK (T172) phosphorylation was increased at 1 h. Overall increased lactate decreased the nuclear content of HDAC5 at 6 h. Exposure to both high- and low-pH media decreased basal mitochondrial respiration, ATP turnover, and maximum mitochondrial respiratory capacity. These data indicate that muscle pH affects several metabolic signaling pathways, including those required for mitochondrial function.

The Effects of Acute and Chronic Sprint-Interval Training on Cytokine Responses Are Independent of Prior Caffeine Intake.

We examined the effect of acute and chronic sprint interval training (SIT), with or without prior caffeine intake, on levels of exercise-induced inflammatory plasma cytokines [interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α]. Twenty physically-active men ingested either a placebo (n = 10) or caffeine (n = 10) 1 h before each SIT session(13-s × 30-s sprint/15 s of rest) during six training sessions (2 weeks). The early (before, immediately after, and 45 min after the exercise) and late (24 and 48 h after the exercise) cytokine and creatine kinase (CK) responses were analyzed for the first and last training sessions. Plasma IL-6 and IL-10 peaked 45 min after the exercise, and then returned to basal values within 24 h (p < 0.05) in both groups on both occasions (p > 0.05). On both occasions, and for both groups, plasma TNF-α increased from rest to immediately after the exercise and then decreased at 45 min before reaching values at or below basal levels 48 h after the exercise (p < 0.05). Serum CK increased from rest to 24 and 48 h post-exercise in the first training session (p < 0.05), but did not alter in the last training session for the PLA group (p > 0.05). Serum CK was unchanged in both the first and last training sessions for the CAF group (p > 0.05). Two weeks of SIT induced a late decrease in the IL-6/IL-10 ratio (p < 0.05) regardless of caffeine intake, suggesting an improved overall inflammatory status after training. In conclusion, a single session of SIT induces muscle damage that seems to be mitigated by caffeine intake. Two weeks of SIT improves the late SIT-induced muscle damage and inflammatory status, which seems to be independent of caffeine intake.

Cold-water immersion after training sessions: effects on fiber type-specific adaptations in muscle K+ transport proteins to sprint-interval training in men.

Effects of regular use of cold-water immersion (CWI) on fiber type-specific adaptations in muscle K+ transport proteins to intense training, along with their relationship to changes in mRNA levels after the first training session, were investigated in humans. Nineteen recreationally active men (24 ± 6 yr, 79.5 ± 10.8 kg, 44.6 ± 5.8 ml·kg-1·min-1) completed six weeks of sprint-interval cycling, either without (passive rest; CON) or with training sessions followed by CWI (15 min at 10°C; COLD). Muscle biopsies were obtained before and after training to determine abundance of Na+, K+-ATPase isoforms (α1-3, ß1-3) and phospholemman (FXYD1) and after recovery treatments (+0 h and +3 h) on the first day of training to measure mRNA content. Training increased ( P < 0.05) the abundance of α1 and ß3 in both fiber types and ß1 in type-II fibers and decreased FXYD1 in type-I fibers, whereas α2 and α3 abundance was not altered by training ( P > 0.05). CWI after each session did not influence responses to training ( P > 0.05). However, α2 mRNA increased after the first session in COLD (+0 h, P < 0.05) but not in CON ( P > 0.05). In both conditions, α1 and ß3 mRNA increased (+3 h; P < 0.05) and ß2 mRNA decreased (+3 h; P < 0.05), whereas α3, ß1, and FXYD1 mRNA remained unchanged ( P > 0.05) after the first session. In summary, Na+,K+-ATPase isoforms are differently regulated in type I and II muscle fibers by sprint-interval training in humans, which, for most isoforms, do not associate with changes in mRNA levels after the first training session. CWI neither impairs nor improves protein adaptations to intense training of importance for muscle K+ regulation. NEW & NOTEWORTHY Although cold-water immersion (CWI) after training and competition has become a routine for many athletes, limited published evidence exists regarding its impact on training adaptation. Here, we show that CWI can be performed regularly without impairing training-induced adaptations at the fiber-type level important for muscle K+ handling. Furthermore, sprint-interval training invoked fiber type-specific adaptations in K+ transport proteins, which may explain the dissociated responses of whole-muscle protein levels and K+ transport function to training previously reported.

Principles of Exercise Prescription, and How They Influence Exercise-Induced Changes of Transcription Factors and Other Regulators of Mitochondrial Biogenesis.

Physical inactivity represents the fourth leading risk factor for mortality, and it has been linked with a series of chronic disorders, the treatment of which absorbs ~ 85% of healthcare costs in developed countries. Conversely, physical activity promotes many health benefits; endurance exercise in particular represents a powerful stimulus to induce mitochondrial biogenesis, and it is routinely used to prevent and treat chronic metabolic disorders linked with sub-optimal mitochondrial characteristics. Given the importance of maintaining a healthy mitochondrial pool, it is vital to better characterize how manipulating the endurance exercise dose affects cellular mechanisms of exercise-induced mitochondrial biogenesis. Herein, we propose a definition of mitochondrial biogenesis and the techniques available to assess it, and we emphasize the importance of standardizing biopsy timing and the determination of relative exercise intensity when comparing different studies. We report an intensity-dependent regulation of exercise-induced increases in nuclear peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content, nuclear phosphorylation of p53 (serine 15), and PGC-1α messenger RNA (mRNA), as well as training-induced increases in PGC-1α and p53 protein content. Despite evidence that PGC-1α protein content plateaus within a few exercise sessions, we demonstrate that greater training volumes induce further increases in PGC-1α (and p53) protein content, and that short-term reductions in training volume decrease the content of both proteins, suggesting training volume is still a factor affecting training-induced mitochondrial biogenesis. Finally, training-induced changes in mitochondrial transcription factor A (TFAM) protein content are regulated in a training volume-dependent manner and have been linked with training-induced changes in mitochondrial content.

The gene SMART study: method, study design, and preliminary findings.

The gene SMART (genes and the Skeletal Muscle Adaptive Response to Training) Study aims to identify genetic variants that predict the response to both a single session of High-Intensity Interval Exercise (HIIE) and to four weeks of High-Intensity Interval Training (HIIT). While the training and testing centre is located at Victoria University, Melbourne, three other centres have been launched at Bond University, Queensland University of Technology, Australia, and the University of Brighton, UK. Currently 39 participants have already completed the study and the overall aim is to recruit 200 moderately-trained, healthy Caucasians participants (all males 18-45 y, BMI < 30). Participants will undergo exercise testing and exercise training by an identical exercise program. Dietary habits will be assessed by questionnaire and dietitian consultation. Activity history is assessed by questionnaire and current activity level is assessed by an activity monitor. Skeletal muscle biopsies and blood samples will be collected before, immediately after and 3 h post HIIE, with the fourth resting biopsy and blood sample taken after four weeks of supervised HIIT (3 training sessions per week). Each session consists of eight to fourteen 2-min intervals performed at the pre-training lactate threshold (LT) power plus 40 to 70% of the difference between pre-training lactate threshold (LT) and peak aerobic power (Wpeak). A number of muscle and blood analyses will be performed, including (but not limited to) genotyping, mitochondrial respiration, transcriptomics, protein expression analyses, and enzyme activity. The participants serve as their own controls. Even though the gene SMART study is tightly controlled, our preliminary findings still indicate considerable individual variability in both performance (in-vivo) and muscle (in-situ) adaptations to similar training. More participants are required to allow us to better investigate potential underlying genetic and molecular mechanisms responsible for this individual variability.

Cold-water immersion following sprint interval training does not alter endurance signaling pathways or training adaptations in human skeletal muscle.

We investigated the underlying molecular mechanisms by which postexercise cold-water immersion (CWI) may alter key markers of mitochondrial biogenesis following both a single session and 6 wk of sprint interval training (SIT). Nineteen men performed a single SIT session, followed by one of two 15-min recovery conditions: cold-water immersion (10°C) or a passive room temperature control (23°C). Sixteen of these participants also completed 6 wk of SIT, each session followed immediately by their designated recovery condition. Four muscle biopsies were obtained in total, three during the single SIT session (preexercise, postrecovery, and 3 h postrecovery) and one 48 h after the last SIT session. After a single SIT session, phosphorylated (p-)AMPK, p-p38 MAPK, p-p53, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA were all increased (P < 0.05). Postexercise CWI had no effect on these responses. Consistent with the lack of a response after a single session, regular postexercise CWI had no effect on PGC-1α or p53 protein content. Six weeks of SIT increased peak aerobic power, maximal oxygen consumption, maximal uncoupled respiration (complexes I and II), and 2-km time trial performance (P < 0.05). However, regular CWI had no effect on changes in these markers, consistent with the lack of response in the markers of mitochondrial biogenesis. Although these observations suggest that CWI is not detrimental to endurance adaptations following 6 wk of SIT, they question whether postexercise CWI is an effective strategy to promote mitochondrial biogenesis and improvements in endurance performance.

Sprint-interval but not continuous exercise increases PGC-1α protein content and p53 phosphorylation in nuclear fractions of human skeletal muscle.

Sprint interval training has been reported to induce similar or greater mitochondrial adaptations to continuous training. However, there is limited knowledge about the effects of different exercise types on the early molecular events regulating mitochondrial biogenesis. Therefore, we compared the effects of continuous and sprint interval exercise on key regulatory proteins linked to mitochondrial biogenesis in subcellular fractions of human skeletal muscle. Nineteen men, performed either 24 min of moderate-intensity continuous cycling at 63% of WPeak (CE), or 4 × 30-s "all-out" cycling sprints (SIE). Muscle samples (vastus lateralis) were collected pre-, immediately (+0 h) and 3 (+3 h) hours post-exercise. Nuclear p53 and PHF20 protein content increased at +0 h, with no difference between groups. Nuclear p53 phosphorylation and PGC-1α protein content increased at +0 h after SIE, but not CE. We demonstrate an exercise-induced increase in nuclear p53 protein content, an event that may relate to greater p53 stability - as also suggested by increased PHF20 protein content. Increased nuclear p53 phosphorylation and PGC-1α protein content immediately following SIE but not CE suggests these may represent important early molecular events in the exercise-induced response to exercise, and that SIE is a time-efficient and possibly superior option than CE to promote these adaptations.

Rest interval duration does not influence adaptations in acid/base transport proteins following 10 wk of sprint-interval training in active women.

The removal of protons (H+) produced during intense exercise is important for skeletal muscle function, yet it remains unclear how best to structure exercise training to improve muscle pH regulation. We investigated whether 4 wk of work-matched sprint-interval trining (SIT), performed 3 days/wk, with either 1 (Rest-1; n = 7) or 5 (Rest-5; n = 7) min of rest between sprints, influenced adaptations in acid/base transport protein content, nonbicarbonate muscle buffer capacity (ßmin vitro), and exercise capacity in active women. Following 1 wk of posttesting, comprising a biopsy, a repeated-sprint ability (RSA) test, and a graded-exercise test, maintenance of adaptations was then studied by reducing SIT volume to 1 day/wk for a further 5 wk. After 4 wk of SIT, there was increased protein abundance of monocarboxylate transporter (MCT)-1, sodium/hydrogen exchanger (NHE)-1, and carbonic anhydrase (CA) XIV for both groups, but rest interval duration did not influence the adaptive response. In contrast, greater improvements in total work performed during the RSA test after 4 wk of SIT were evident for Rest-5 compared with Rest-1 (effect size: 0.51; 90% confidence limits ±0.37), whereas both groups had similarly modest improvements in V̇o2peak When training volume was reduced to 1 day/wk, enhanced acid/base transport protein abundance was maintained, although NHE1 content increased further for Rest-5 only. Finally, our data support intracellular lactate as a signaling molecule for inducing MCT1 expression, but neither lactate nor H+ accumulation appears to be important signaling factors in MCT4 regulation.

Distinct protein and mRNA kinetics of skeletal muscle proton transporters following exercise can influence interpretation of adaptations to training.

NEW FINDINGS: What is the central question of this study? Following a training intervention, how is the interpretation of adaptations in skeletal muscle H+ transporters influenced by biopsy timing in the context of individual protein and mRNA kinetics after the final exercise bout? What is the main finding and its importance? We show that distinct postexercise protein and mRNA kinetics for monocarboxylate transporter 1/4 and sodium-hydrogen exchanger 1 indicate that timing of a single end-point biopsy after a training intervention can influence the inferences made. Furthermore, we found the intrasubject, intersample variability of the muscle buffer capacity titration assay to be greater than the typical training effect. In order to gain a better understanding of training-induced adaptations in skeletal muscle pH regulation, in this study we measured protein and mRNA kinetics of proton (H+ ) transporters for 72 h following a bout of high-intensity interval exercise (HIIE), conducted after 4 weeks of similar training. We also assayed muscle buffer capacity (ßm) by a titration technique (ßmin vitro ) over the same period. Sixteen active men cycled for seven bouts of 2 min at ∼80% of peak aerobic power, interspersed with 1 min rest. Compared with the first 9 h postexercise, monocarboxylate transporter (MCT) 1 protein content was ∼1.3-fold greater 24-72 h post-HIIE, whereas there was no such change in MCT4 protein content. Conversely, MCT1 and MCT4 mRNA expression progressively decreased 9-72 h post-HIIE. Sodium-hydrogen exchanger 1 (NHE1) protein content was lower 9 h post-HIIE (∼0.8-fold) compared with every other postexercise time point, but NHE1 mRNA expression was 2.2 to 2.9-fold greater 24-72 h post-HIIE, compared with the first 24 h post-HIIE. Furthermore, we determined the intrasubject, intersample variability (11.5%) of ßmin vitro for resting samples taken on consecutive days to be greater than the typical training effect (mean 6%; 95% confidence limits ±4%). In conclusion, the delay in steady-state protein turnover should inform biopsy timing in studies investigating the response to training of the H+ transport proteins, whereas the temporal resolution provided by single time points has been shown to be of limited epistemological value for their corresponding mRNA expression. Finally, our data cast doubt on the ecological validity of the ßmin vitro assay for measuring true changes in ßm.

Mitochondrial adaptations to high-volume exercise training are rapidly reversed after a reduction in training volume in human skeletal muscle.

Increased mitochondrial content and respiration have both been reported after exercise training. However, no study has directly compared how different training volumes influence mitochondrial respiration and markers of mitochondrial biogenesis. Ten healthy men performed high-intensity interval cycling during 3 consecutive training phases; 4 wk of normal-volume training (NVT; 3/wk), followed by 20 d of high-volume training (HVT; 2/d) and 2 wk of reduced-volume training (RVT; 5 sessions). Resting biopsy samples (vastus lateralis) were obtained at baseline and after each phase. No mitochondrial parameter changed after NVT. After HVT, mitochondrial respiration and citrate synthase activity (∼40-50%), as well as the protein content of electron transport system (ETS) subunits (∼10-40%), and that of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), NRF1, mitochondrial transcription factor A (TFAM), PHF20, and p53 (∼65-170%) all increased compared to baseline; mitochondrial specific respiration remained unchanged. After RVT, all the mitochondrial parameters measured except citrate synthase activity (∼36% above initial) were not significantly different compared to baseline (all P > 0.05). Our findings demonstrate that training volume is an important determinant of training-induced mitochondrial adaptations and highlight the rapid reversibility of human skeletal muscle to a reduction in training volume.-Granata, C., Oliveira, R. S. F., Little, J. P., Renner, K., Bishop, D. J. Mitochondrial adaptations to high-volume exercise training are rapidly reversed after a reduction in training volume in human skeletal muscle.