Metabolic stress-dependent regulation of the mitochondrial biogenic molecular response to high-intensity exercise in human skeletal muscle.

KEY POINTS: Low-volume high-intensity exercise training promotes muscle mitochondrial adaptations that resemble those associated with high-volume moderate-intensity exercise training. These training-induced mitochondrial adaptations stem from the cumulative effects of transient transcriptional responses to each acute exercise bout. However, whether metabolic stress is a key mediator of the acute molecular responses to high-intensity exercise is still incompletely understood. Here we show that, by comparing different work-matched low-volume high-intensity exercise protocols, more marked metabolic perturbations were associated with enhanced mitochondrial biogenesis-related muscle mRNA responses. Furthermore, when compared with high-volume moderate-intensity exercise, only the low-volume high-intensity exercise eliciting severe metabolic stress compensated for reduced exercise volume in the induction of mitochondrial biogenic mRNA responses. The present results, besides improving our understanding of the mechanisms mediating exercise-induced mitochondrial biogenesis, may have implications for applied and clinical research that adopts exercise as a means to increase muscle mitochondrial content and function in healthy or diseased individuals. ABSTRACT: The aim of the present study was to examine the impact of exercise-induced metabolic stress on regulation of the molecular responses promoting skeletal muscle mitochondrial biogenesis. Twelve endurance-trained men performed three cycling exercise protocols characterized by different metabolic profiles in a randomized, counter-balanced order. Specifically, two work-matched low-volume supramaximal-intensity intermittent regimes, consisting of repeated-sprint (RS) and speed endurance (SE) exercise, were employed and compared with a high-volume continuous moderate-intensity exercise (CM) protocol. Vastus lateralis muscle samples were obtained before, immediately after, and 3 h after exercise. SE produced the most marked metabolic perturbations as evidenced by the greatest changes in muscle lactate and pH, concomitantly with higher post-exercise plasma adrenaline levels in comparison with RS and CM. Exercise-induced phosphorylation of CaMKII and p38 MAPK was greater in SE than in RS and CM. The exercise-induced PGC-1α mRNA response was higher in SE and CM than in RS, with no difference between SE and CM. Muscle NRF-2, TFAM, MFN2, DRP1 and SOD2 mRNA content was elevated to the same extent by SE and CM, while RS had no effect on these mRNAs. The exercise-induced HSP72 mRNA response was larger in SE than in RS and CM. Thus, the present results suggest that, for a given exercise volume, the initial events associated with mitochondrial biogenesis are modulated by metabolic stress. In addition, high-intensity exercise seems to compensate for reduced exercise volume in the induction of mitochondrial biogenic molecular responses only when the intense exercise elicits marked metabolic perturbations.

Hypertrophic effect of inhaled beta2 -agonist with and without concurrent exercise training: A randomized controlled trial.

Due to a high prevalence of asthma and exercise-induced bronchoconstriction in elite athletes, there is a high use of beta2 -adrenoceptor agonists (beta2 -agonists) in the athletic population. While anabolic in rodents, no study has been able to detect hypertrophy in humans after chronic beta2 -agonist inhalation. We investigated whether inhaled beta2 -agonist, terbutaline, alters body composition and metabolic rate with and without concurrent exercise training in healthy young men. Sixty-seven participants completed a 4-week intervention of daily terbutaline (8 × 0.5 mg) or placebo treatment without concurrent training (habitual; n = 23), with resistance (n = 23) or endurance (n = 21) training 3 times weekly. Before and after the interventions, participant's body composition was determined by dual-energy X-ray absorptiometry and resting metabolic rate and substrate oxidation by indirect calorimetry. Terbutaline increased lean body mass by 1.03 kg (95% CI 0.29-1.76; P < .05) and 1.04 kg (95% CI 0.16-1.93; P < .05) compared to placebo in the habitual and resistance training group, respectively, but had no effect compared to placebo in the endurance training group [-0.56 kg (95% CI -1.74-0.62; P > .05)]. Fat mass, bone mineral content, and resting metabolic rate did not change differently between treatments with the intervention. Daily inhalation of terbutaline in near-therapeutic doses induces skeletal muscle growth. This observation should be a concern for antidoping authorities.

Two-week inhalation of budesonide increases muscle Na,K ATPase content but not endurance in response to terbutaline in men.

While chronic systemic administration of glucocorticoids increases muscle Na+ ,K+ ATPase content, such effect is unexplored after therapeutic inhalation. We investigated the effect of therapeutic inhalation of the glucocorticoid budesonide on Na+ ,K+ ATPase content of skeletal muscle in men. Ten healthy trained subjects, aged 23 ± 4 years (mean ± 95% CI), participated in the study. Before and after 2 weeks of daily inhalation of budesonide (1.6 mg/day), a biopsy was taken from the vastus lateralis muscle for measurement of Na+ ,K+ ATPase content and blood samples were drawn for determination of plasma budesonide, cortisol, and K+ . Subjects' performance during cycling to fatigue at 90% of incremental peak power output (iPPO) was measured in response to 4 mg inhaled terbutaline to maximally stimulate Na+ ,K+ ATPase activity. Plasma concentrations of budesonide rose to 5.0 ± 1.6 nM with the intervention, whereas no changes were observed in plasma cortisol. Muscle Na+ ,K+ ATPase content increased (P ≤ 0.01) by 46 ± 34 pmol/(g wet wt) (17% increase) with the intervention. Cycling performance at 90% of iPPO did not change (P = 0.21) with the intervention (203 vs 214 s) in response to terbutaline. The present observations show that therapeutic inhalation of glucocorticoids increases muscle Na+ ,K+ ATPase content, but does not enhance high-intensity cycling endurance in response to terbutaline.

Influence of exercise in normal and hot ambient conditions on the pharmacokinetics of inhaled terbutaline in trained men.

This study investigated the pharmacokinetics of inhaled terbutaline at rest and after exercise in normal and hot ambient conditions with respect to doping analysis. Thirteen trained young men participated in the study. Urine and blood samples were collected after inhalation of 4 mg terbutaline during three trials: exercise in hot ambient conditions (30-35 °C) (EXH), exercise in normal ambient conditions (20-25 °C) (EX), and rest (20-25 °C) (R). Exercise consisted of 130 min at various intensities. Adjustment of urine concentrations of terbutaline to a specific gravity (USG) of 1.02 g/mL was compared with no adjustment. Area under the serum concentration-time curve within the first 6 h was higher for EX (27 ± 3 ng/mL/h) (P ≤ 0.01) and EXH (25 ± 4 ng/mL/h) (P ≤ 0.05) than for R (20 ± 3 ng/mL/h). When unadjusted for USG, urine concentrations of terbutaline after 4 h were different in the order EXH > EX > R (P ≤ 0.01). When unadjusted for USG, urine concentrations of terbutaline were 299 ± 151 ng/mL higher (P ≤ 0.001) after 4 h compared with adjusted concentrations in EXH. Excretion rate of terbutaline was higher (P ≤ 0.001) for EX than for EXH and R within the first 0-1½ h. In conclusion, EXHs results in higher urine concentrations of terbutaline. This should be considered when evaluating doping cases of terbutaline.

Effects of acute and 2-week administration of oral salbutamol on exercise performance and muscle strength in athletes.

Our objective was to investigate effects of acute and 2-week administration of oral salbutamol on repeated sprint ability, exercise performance, and muscle strength in elite endurance athletes. Twenty male elite athletes [VO2max: 69.4 ± 1.8 (Mean ± SE) mL/min/kg], aged 25.9 ± 1.4 years, were included in a randomized, double-blinded and placebo-controlled parallel study. At baseline, after acute administration, and again after 2-week administration of the study drugs (8 mg salbutamol or placebo), subjects' maximal voluntary contraction (MVC) of m. quadriceps and isometric endurance of m. deltoideus were measured, followed by three repeated Wingate tests. Exercise performance at 110% of VO2max was determined on a bike ergometer. Acute administration of salbutamol increased peak power during first Wingate test by 4.1 ± 1.7% (P < 0.05). Two-week administration of salbutamol increased (P < 0.05) peak power during first and second Wingate test by 6.4 ± 2.0 and 4.2 ± 1.0%. Neither acute nor 2-week administration of salbutamol had any effect on MVC, exercise performance at 110% of VO2max or on isometric endurance. No differences were observed in the placebo group. In conclusion, salbutamol benefits athletes' sprint ability. Thus, the present study supports the restriction of oral salbutamol in competitive sports.

ß2-adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+-K+-ATPase Vmax in trained men.

The aim of the present study was to examine the effect of ß2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca(2+) release and uptake, and Na(+)-K(+)-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where ß2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (V̇O2, max ). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P < 0.05) with terbutaline than placebo in EXP1 and EXP2, respectively. Furthermore, peak twitch force was 11 ± 7% higher (P < 0.01) with terbutaline than placebo at non-fatigue. After sprints, MVC declined (P < 0.05) to the same levels with terbutaline as placebo, whereas peak twitch force was lower (P < 0.05) and half-relaxation time was prolonged (P < 0.05) with terbutaline. Rates of SR Ca(2+) release and uptake at 400 nm [Ca(2+)] were 15 ± 5 and 14 ± 5% (P < 0.05) higher, respectively, with terbutaline than placebo at non-fatigue, but declined (P < 0.05) to similar levels at time of fatigue. Na(+)-K(+)-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by ß2-adrenergic stimulation is associated with enhanced rate of Ca(2+) release in humans. While ß2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue.

Combined inhalation of beta2 -agonists improves swim ergometer sprint performance but not high-intensity swim performance.

There is a high prevalence of asthma and airway hyperresponsiveness (AHR) in elite athletes, which leads to a major use of beta2 -agonists. In a randomized double-blinded crossover study, we investigated the effects of combined inhalation of beta2 -agonists (salbutamol, formoterol, and salmeterol), in permitted doses within the World Anti-Doping Agency 2013 prohibited list, in elite swimmers with (AHR, n = 13) or without (non-AHR, n = 17) AHR. Maximal voluntary isometric contraction of m. quadriceps (MVC), sprint performance on a swim ergometer and performance in an exhaustive swim test at 110% of VO2max were determined. Venous plasma interleukin-6 (IL-6) and interleukin-8 (IL-8) were measured post-exercise. No improvement was observed in the exhaustive swim test, but swim ergometer sprint time was improved (P < 0.05) in both groups from 57 ± 1.7 to 56 ± 1.8 s in AHR and 58.3 ± 1 to 57.4 ± 1 s in non-AHR. MVC and post-exercise plasma IL-6 increased (P < 0.05) with beta2 -agonists in both groups, whereas IL-8 only increased in AHR. In summary, inhalation of beta2 -agonists, in permitted doses, did not improve swim performance in elite swimmers. However, swim ergometer sprint performance and MVC were increased, which should be considered when making future anti-doping regulations.

Urine and serum concentrations of inhaled and oral terbutaline.

We examined urine and serum concentrations after therapeutic use of single and repetitive doses of inhaled and supratherapeutic oral use of terbutaline. We compared the concentrations in 10 asthmatics and 10 healthy subjects in an open-label, cross-over study with 2 mg inhaled and 10 mg oral terbutaline on 2 study days. Further, 10 healthy subjects were administrated 1 mg inhaled terbutaline in 4 repetive doses with total 4 mg. Blood samples were collected at baseline and during 6 h after the first inhalations. Urine samples were collected at baseline and during 12 h after the first inhalations. Median (IQR) urine concentrations peaked in the period 0-4 h after inhalation with Cmax 472 (324) ng/mL in asthmatics and 661 (517) ng/mL in healthy subjects, and 4-8 h after oral use with Cmax 666 (877) ng/mL in asthmatic and 402 (663) ng/mL in healthy subjects. In conclusion we found no significant differences in urine and serum concentrations between asthmatic and healthy subjects. We compared urine and serum concentrations after therapeutic inhaled doses and supratherapeutic oral doses and observed significant statistical differences in both groups but found it impossible to distinguish between therapeutic and prohibited use based on doping tests with urine and blood samples.

Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Feasibility of high-intensity training in asthma.

Background: High-intensity interval training is an effective and popular training regime but its feasibility in untrained adults with asthma is insufficiently described. Objective: The randomized controlled trial 'EFFORT Asthma' explored the effects of behavioural interventions including high-intensity interval training on clinical outcomes in nonobese sedentary adults with asthma. In this article we present a sub analysis of data aiming to evaluate if patients' pre-intervention levels of asthma control, FEV1, airway inflammation and airway hyperresponsiveness (AHR) predicted their training response to the high-intensity interval training program, measured as increase in maximal oxygen consumption (VO2max). Design: We used data from the EFFORT Asthma Study. Of the 36 patients randomized to the 8-week exercise intervention consisting of high-intensity training three times per week, 29 patients (45% females) completed the study and were included in this data analysis. Pre-intervention assessment included the asthma control questionnaire (ACQ), spirometry, fractional exhaled nitric oxide (FeNO) and AHR to mannitol. VO2 max was measured during an incremental cycle test. Results: The majority of included patients had partly or uncontrolled asthma reflected by a mean (SD) ACQ at 1.7 (0.6). Median (IQR) FeNO was 28.5 (23.8) ppb and 75% had a positive mannitol test indicating AHR. The association between patients' training response measured as increase in VO2max and pre-intervention ACQ scores was not statistically significant (p = 0.49). Likewise, the association between patients' increase in VO2max and FeNO as well as AHR was not statistically significant (p = 0.80 and p = 0.58). Conclusions: Included asthma patients could adhere to the high-intensity interval protocol and improve their VO2max regardless of pre-intervention levels of asthma control, airway inflammation and AHR.