Beta2-Adrenergic Stimulation Induces Resistance Training-Like Adaptations in Human Skeletal Muscle: Potential Role of KLHL41.

Skeletal muscle mass plays a pivotal role in metabolic function, but conditions such as bed rest or injury often render resistance training impractical. The beta2-adrenergic receptor has been highlighted as a potential target to promote muscle hypertrophy and treat atrophic conditions. Here, we investigate the proteomic changes associated with beta2-adrenergic-mediated muscle hypertrophy, using resistance training as a hypertrophic comparator. We utilize MS-based proteomics to map skeletal muscle proteome remodeling in response to beta2-adrenergic stimulation or resistance training as well as cell model validation. We report that beta2-adrenergic stimulation mimics multiple features of resistance training in proteome-wide remodeling, comprising systematic upregulation of ribosomal subunits and concomitant downregulation of mitochondrial proteins. Approximately 20% of proteins were regulated in both conditions, comprising proteins involved in steroid metabolism (AKR1C1, AKR1C2, AKRC1C3), protein-folding (SERPINB1), and extracellular matrix organization (COL1A1, COL1A2). Among overall most significantly upregulated proteins were kelch-like family members (KLHL) 40 and 41. In follow-up experiments, we identify KLHL41 as having novel implications for beta2-adrenergic-mediated muscle hypertrophy. Treating C2C12 cells with beta2-agonist for 96 h increased myotube diameter by 48% (p < 0.001). This anabolic effect was abolished by prior knockdown of KLHL41. Using siRNA, KLHL41 abundance was decreased by 60%, and the anabolic response to beta2-agonist was diminished (+ 15%, i.e., greater in the presence of KLHL41, knock-down × treatment: p = 0.004). In conclusion, protein-wide remodeling induced by beta2-adrenergic stimulation mimics multiple features of resistance training, and thus the beta2-adrenergic receptor may be a target with therapeutic potential in the treatment of muscle wasting conditions without imposing mechanical load.

Effect of fenofibrate on residual beta cell function in adults and adolescents with newly diagnosed type 1 diabetes: a randomised clinical trial.

AIMS/HYPOTHESIS: Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, shows some promise in alleviating beta cell stress and preserving beta cell function in preclinical studies of type 1 diabetes. The aim of this phase 2, placebo-controlled, double-blinded, randomised clinical trial was to investigate the efficacy and safety of fenofibrate in adults and adolescents with newly diagnosed type 1 diabetes. METHODS: We enrolled 58 individuals (aged 16 to 40 years old) with newly diagnosed type 1 diabetes and randomised them to daily oral treatment with fenofibrate 160 mg or placebo for 52 weeks (in a block design with a block size of 4, assigned in a 1:1 ratio). Our primary outcome was change in beta cell function after 52 weeks of treatment, assessed by AUC for C-peptide levels following a 2 h mixed-meal tolerance test. Secondary outcomes included glycaemic control (assessed by HbA1c and continuous glucose monitoring), daily insulin use, and proinsulin/C-peptide (PI/C) ratio as a marker of beta cell stress. We assessed outcome measures before and after 4, 12, 26 and 52 weeks of treatment. Blinding was maintained for participants, their healthcare providers and all staff involved in handling outcome samples and assessment. RESULTS: The statistical analyses for the primary outcome included 56 participants (n=27 in the fenofibrate group, after two withdrawals, and n=29 in the placebo group). We found no significant differences between the groups in either 2 h C-peptide levels (mean difference of 0.08 nmol/l [95% CI -0.05, 0.23]), insulin use or glycaemic control after 52 weeks of treatment. On the contrary, the fenofibrate group showed a higher PI/C ratio at week 52 compared with placebo (mean difference of 0.024 [95% CI 0.000, 0.048], p<0.05). Blood lipidome analysis revealed that fenofibrate repressed pathways involved in sphingolipid metabolism and signalling at week 52 compared with placebo. The 52 week intervention evoked few adverse events and no serious adverse events. Follow-up in vitro experiments in human pancreatic islets demonstrated a stress-inducing effect of fenofibrate. CONCLUSIONS/INTERPRETATION: Contrary to the beneficial effects of fenofibrate found in preclinical studies, this longitudinal, randomised, placebo-controlled trial does not support the use of fenofibrate for preserving beta cell function in individuals with newly diagnosed type 1 diabetes. TRIAL REGISTRATION: EudraCT number: 2019-004434-41 FUNDING: This study was funded by the Sehested Hansens Foundation.

Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans.

Preclinical models suggest mitochondria-derived oxidative stress as an underlying cause of insulin resistance. However, it remains unknown whether this pathophysiological mechanism is conserved in humans. Here, we used an invasive in vivo mechanistic approach to interrogate muscle insulin action while selectively manipulating the mitochondrial redox state in humans. To this end, we conducted insulin clamp studies combining intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mitoquinone). Under lipid overload, selective modulation of mitochondrial redox state by mitoquinone enhanced insulin-stimulated glucose uptake in skeletal muscle. Mechanistically, mitoquinone did not affect canonical insulin signaling but augmented insulin-stimulated glucose transporter type 4 (GLUT4) translocation while reducing the mitochondrial oxidative burden under lipid oversupply. Complementary ex vivo studies in human muscle fibers exposed to high intracellular lipid levels revealed that mitoquinone improves features of mitochondrial bioenergetics, including diminished mitochondrial H2O2 emission. These findings provide translational and mechanistic evidence implicating mitochondrial oxidants in the development of lipid-induced muscle insulin resistance in humans.

Beta2-adrenergic agonist salbutamol exhibits enantioselective disposition in skeletal muscle of lean young men following oral administration.

Salbutamol is a common short-acting beta2-adrenergic agonist used in treatment of asthma and exercise-induced bronchoconstriction but also possesses anabolic and metabolic actions in skeletal muscle. As a chiral compound, salbutamol is a racemic 1:1 mixture of two enantiomers, (R)-salbutamol and (S)-salbutamol, which exhibit divergent pharmacokinetic and pharmacodynamic actions. Despite salbutamol being available for decades, information on the enantioselective disposition of salbutamol enantiomers in human skeletal muscle is absent. In this study, we determined concentrations of (R)-salbutamol and (S)-salbutamol by UHPLC-MS/MS in arterial plasma and vastus lateralis muscle samples from 12 lean young men 2½ and 7 h following ingestion of 24 mg oral salbutamol. Mean (range) arterial plasma concentrations were 10-fold higher (p < 0.001) for (S)-salbutamol than (R)-salbutamol, being 33(9-62) and 49(30-84) ng·mL-1 for (S)-salbutamol and 4 (1-6) and 4 (2-5) ng·mL-1 for (R)-salbutamol 2½ and 7 h following administration, respectively, reflecting faster elimination of the (R)-enantiomer. Mean (range) muscle concentrations were higher (p < 0.001) for (S)-salbutamol than (R)-salbutamol 2½ h (0.17 [0.1-0.26] vs. 0.04 [0.02-0.06]) and 7 h (0.31 [0.21-0.46] vs. 0.06 [0.04-0.12] ng·mgd.w. -1) after administration. However, muscle:plasma partition coefficient was two-fold higher (p < 0.001) for (R)-salbutamol than (S)-salbutamol 7 h following administration. These observations demonstrate that oral salbutamol exhibits enantioselective disposition in systemic circulation and muscle favoring the (S)-enantiomer but with higher relative partitioning of the (R)-enantiomer in skeletal muscle. Furthermore, the concentration-time profiles of salbutamol enantiomers are different in skeletal muscle and systemic circulation following oral ingestion. These findings have implications for the application of chiral switch (R)-salbutamol in doping control.

Intensified training augments cardiac function, but not blood volume, in male youth elite ice hockey team players.

While it is well-established that a period of interval training performed at near maximal effort, such as speed endurance training (SET), enhances intense exercise performance in well-trained individuals, less is known about its effect on cardiac morphology and function as well as blood volume. To investigate this, we subjected 12 Under-20 Danish national team ice hockey players (age 18 ± 1 years, mean ± SD) to 4 weeks of SET, consisting of 6-10 × 20 s skating bouts at maximal effort interspersed by 2 min of recovery conducted three times weekly. This was followed by 4 weeks of regular training (follow-up). We assessed resting cardiac function and dimensions using transthoracic echocardiography and quantified total blood volume with the carbon monoxide rebreathing technique at three time points: before SET, after SET and after the follow-up period. After SET, stroke volume had increased by 10 (2-18) mL (mean (95% CI)), left atrial end-diastolic volume by 10 (3-17) mL, and circumferential strain improved by 0.9%-points (1.7-0.1) (all P < 0.05). At follow-up, circumferential strain and left atrial end-diastolic volume were reverted to baseline levels, while stroke volume remained elevated. Blood volume and morphological parameters for the left ventricle, including mass and end-diastolic volume, did not change during the study. In conclusion, our findings demonstrate that a brief period of SET elicits beneficial central cardiac adaptations in elite ice hockey players independent of changes in blood volume.

Beta2-agonist impairs muscle insulin sensitivity in persons with insulin resistance.

CONTEXT: Given the promising effects of prolonged treatment with beta2-agonist on insulin sensitivity in animals and non-diabetic individuals, the beta2-adrenergic receptor has been proposed as a target to counter peripheral insulin resistance. On the other hand, rodent studies also reveal that beta2-agonists acutely impair insulin action, posing a potential caveat for their use in treating insulin resistance. OBJECTIVE: To assess the impact of beta2-agonist on muscle insulin action and glucose metabolism and identify the underlying mechanism(s) in 10 insulin-resistant subjects. METHODS AND PARTICIPANTS: In a cross-over design, we assessed the effect of beta2-agonist on insulin-stimulated muscle glucose uptake during a 3-h hyperinsulinemic isoglycemic clamp with and without intralipid infusion in 10 insulin-resistant overweight subjects. Two hours into the clamp, we infused beta2-agonist. We collected muscle biopsies before, two hours into and by the end of the clamp and analyzed them using metabolomic and lipidomic techniques. RESULTS: We establish that beta2-agonist, independently from and additively to intralipid, impairs insulin-stimulated muscle glucose uptake via different mechanisms. In combination, beta2-agonist and intralipid nearly eliminates insulin-dependent muscle glucose uptake. While both beta2-agonist and intralipid elevated muscle glucose-6-phosphate, only intralipid caused accumulation of downstream muscle glycolytic intermediates, whereas beta2-agonist attenuated incorporation of glucose into glycogen. CONCLUSIONS: Our findings suggest that beta2-agonist inhibits glycogenesis while intralipid inhibits glycolysis in skeletal muscle of insulin-resistant individuals. These results should be addressed in future treatment of insulin resistance with beta2-agonist.

Low-Volume Speed Endurance Training with Reduced Volume Improves Short-Term Exercise Performance in Highly Trained Cyclists.

PURPOSE: We investigated the effects of low- and high-volume speed endurance training (SET), with a reduced training volume, on sprint ability, short- and long-term exercise capacity, muscle mitochondrial properties, ion transport proteins, and maximal enzyme activity in highly trained athletes. METHODS: Highly trained male cyclists (maximal oxygen consumption (V̇O 2max ): 68.3 ± 5.0 mL·min -1 ·kg -1 , n = 24) completed 6 wk of either low (SET-L; 6 × 30-s intervals, n = 8) or high (SET-H; 12 × 30-s intervals, n = 8) volume SET twice per week with a 30% reduction in training volume. A control group (CON; n = 8) maintained their training. Exercise performance was evaluated by i) 6-s sprinting, ii) a 4-min time trial, and iii) a 60-min preload at 60% V̇O 2max followed by a 20-min time trial. A biopsy of m. vastus lateralis was collected before and after the training intervention. RESULTS: In SET-L, 4-min time trial performance was improved ( P < 0.05) by 3.8%, with no change in SET-H and CON. Sprint ability, prolonged endurance exercise capacity, V̇O 2max , muscle mitochondrial respiratory capacity, maximal citrate synthase activity, fiber type-specific mitochondrial proteins (complexes I-V), and phosphofructokinase (PFK) content did not change in any of the groups. In SET-H, maximal activity of muscle PFK and abundance of Na + -K + pump-subunit α 1 , α 2 , ß 1 , and phospholemman (FXYD1) were 20%, 50%, 19%, 24%, and 42% higher ( P < 0.05), respectively after compared with before the intervention, with no changes in SET-L or CON. CONCLUSIONS: Low SET volume combined with a reduced aerobic low- and moderate-intensity training volume does improve short-duration intense exercise performance and maintain sprinting ability, V̇O 2max , endurance exercise performance, and muscle oxidative capacity, whereas, high volume of SET seems necessary to upregulate muscle ion transporter content and maximal PFK activity in highly trained cyclists.

No additive effect of creatine, caffeine, and sodium bicarbonate on intense exercise performance in endurance-trained individuals.

BACKGROUND: Athletes commonly use creatine, caffeine, and sodium bicarbonate for performance enhancement. While their isolated effects are well-described, less is known about their potential additive effects. METHODS: Following a baseline trial, we randomized 12 endurance-trained males (age: 25 ± 5 years, VO2max: 56.7 ± 4.6 mL kg-1 min-1; mean ± SD) and 11 females (age: 25 ± 3 years, VO2max: 50.2 ± 3.4 mL kg-1 min-1) to 5 days of creatine monohydrate (0.3 g kg-1 per day) or placebo loading, followed by a daily maintenance dose (0.04 g kg-1) throughout the study. After the loading period, subjects completed four trials in randomized order where they ingested caffeine (3 mg kg-1), sodium bicarbonate (0.3 g kg-1), placebo, or both caffeine and sodium bicarbonate before a maximal voluntary contraction (MVC), 15-s sprint, and 6-min time trial. RESULTS: Compared to placebo, mean power output during 15-s sprint was higher following loading with creatine than placebo (+34 W, 95% CI: 10 to 58, p = 0.008), but with no additional effect of caffeine (+10 W, 95% CI: -7 to 24, p = 0.156) or sodium bicarbonate (+5 W, 95% CI: -4 to 13, p = 0.397). Mean power output during 6-min time trial was higher with caffeine (+12 W, 95% CI: 5 to 18, p = 0.001) and caffeine + sodium bicarbonate (+8 W, 95% CI: 0 to 15, p = 0.038), whereas sodium bicarbonate (-1 W, 95% CI: -7 to 6, p = 0.851) and creatine (-6 W, 95% CI: -15 to 4, p = 0.250) had no effects. CONCLUSION: While creatine and caffeine can enhance sprint- and time trial performance, respectively, these effects do not seem additive. Therefore, supplementing with either creatine or caffeine appears sufficient to enhance sprint or short intense exercise performance.

Beyond bronchodilation: Illuminating the performance benefits of inhaled beta2 -agonists in sports.

Given the prevalent use of inhaled beta2 -agonists in sports, there is an ongoing debate as to whether they enhance athletic performance. Over the last decades, inhaled beta2 -agonists have been claimed not to enhance performance with little consideration of dose or exercise modality. In contrast, orally administered beta2 -agonists are perceived as being performance enhancing, predominantly on muscle strength and sprint ability, but can also induce muscle hypertrophy and slow-to-fast fiber phenotypic switching. But because inhaled beta2 -agonists are more efficient to achieve high systemic concentrations than oral delivery relative to dose, it follows that the inhaled route has the potential to enhance performance too. The question is at which inhaled doses such effects occur. While supratherapeutic doses of inhaled beta2 -agonists enhance muscle strength and short intense exercise performance, effects at low therapeutic doses are less apparent. However, even high therapeutic inhaled doses of commonly used beta2 -agonists have been shown to induce muscle hypertrophy and to enhance sprint performance. This is concerning from an anti-doping perspective. In this paper, we raise awareness of the circumstances under which inhaled beta2 -agonists can constitute a performance-enhancing benefit.

Asthma and exercise-induced bronchoconstriction in athletes: Diagnosis, treatment, and anti-doping challenges.

Athletes often experience lower airway dysfunction, such as asthma and exercise-induced bronchoconstriction (EIB), which affects more than half the athletes in some sports, not least in endurance sports. Symptoms include coughing, wheezing, and breathlessness, alongside airway narrowing, hyperresponsiveness, and inflammation. Early diagnosis and management are essential. Not only because untreated or poorly managed asthma and EIB potentially affects competition performance and training, but also because untreated airway inflammation can result in airway epithelial damage, remodeling, and fibrosis. Asthma and EIB do not hinder performance, as advancements in treatment strategies have made it possible for affected athletes to compete at the highest level. However, practitioners and athletes must ensure that the treatment complies with general guidelines and anti-doping regulations to prevent the risk of a doping sanction because of inadvertently exceeding specified dosing limits. In this review, we describe considerations and challenges in diagnosing and managing athletes with asthma and EIB. We also discuss challenges facing athletes with asthma and EIB, while also being subject to anti-doping regulations.

Importance of training volume during intensified training in elite cyclists: Maintained vs. reduced volume at moderate intensity.

INTRODUCTION: Male elite cyclists (average VO2 -max: 71 mL/min/kg, n = 18) completed 7 weeks of high-intensity interval training (HIT) (3×/week; 4-min and 30-s intervals) during the competitive part of the season. The influence of a maintained or lowered total training volume combined with HIT was evaluated in a two-group design. Weekly moderate-intensity training was lowered by ~33% (~5 h) (LOW, n = 8) or maintained at normal volume (NOR, n = 10). Endurance performance and fatigue resistance were evaluated via 400 kcal time-trials (~20 min) commenced either with or without prior completion of a 120-min preload (including repeated 20-s sprints to simulate physiologic demands during road races). RESULTS: Time-trial performance without preload was improved after the intervention (p = 0.006) with a 3% increase in LOW (p = 0.04) and a 2% increase in NOR (p = 0.07). Preloaded time-trial was not significantly improved (p = 0.19). In the preload, average power during repeated sprinting increased by 6% in LOW (p < 0.01) and fatigue resistance in sprinting (start vs end of preload) was improved (p < 0.05) in both groups. Blood lactate during the preload was lowered (p < 0.001) solely in NOR. Measures of oxidative enzyme activity remained unchanged, whereas the glycolytic enzyme PFK increased by 22% for LOW (p = 0.02). CONCLUSION: The present study demonstrates that elite cyclists can benefit from intensified training during the competitive season both with maintained and lowered training volume at moderate intensity. In addition to benchmarking the effects of such training in ecological elite settings, the results also indicate how some performance and physiological parameters may interact with training volume.

Inhaled beta2 -agonist, formoterol, enhances intense exercise performance, and sprint ability in elite cyclists.

PURPOSE: Many athletes use long-acting beta2 -agonist formoterol in treatment of asthma. However, studies in non-athlete cohorts demonstrate that inhaled formoterol can enhance sprint performance calling into question whether its use in competitive sports should be restricted. We investigated whether formoterol at upper recommended inhaled doses (54 µg) would enhance sprint ability and intense exercise performance in elite cyclists. METHODS: Twenty-one male cyclists (V̇O2max : 70.4 ± 4.3 mL × min-1 × kg-1 , mean ± SD) completed two 6-s all-out sprints followed by 4-min all-out cycling after inhaling either 54 µg formoterol or placebo. We also assessed cyclists' leg muscle mass by dual-energy X-ray absorptiometry and muscle fiber type distribution of vastus lateralis biopsies. RESULTS: Peak and mean power output during the 6-s sprint was 32 W (95% CI, 19-44 W, p < 0.001) and 36 W (95% CI, 24-48 W, p < 0.001) higher with formoterol than placebo, corresponding to an enhancing effect of around 3%. Power output during 4-min all-out cycling was 9 W (95% CI, 2-16 W, p = 0.01) greater with formoterol than placebo, corresponding to an enhancing effect of 2.3%. Performance changes in response to formoterol were unrelated to cyclists' VO2max and leg lean mass, whereas muscle fiber Type I distribution correlated with change in sprinting peak power in response to formoterol (r2 = 0.314, p = 0.012). CONCLUSION: Our findings demonstrate that an inhaled one-off dose of 54 µg formoterol has a performance-enhancing potential on sprint ability and short intense performance in elite male cyclists, which is irrespective of training status but partly related to muscle fiber type distribution for sprint ability.

Inhaled salbutamol induces leanness in well-trained healthy females but not males during a period of endurance training: a randomised controlled trial.

Introduction: Many athletes use short-acting inhaled ß2-agonists multiple times weekly during training sessions to prevent exercise-induced bronchoconstriction, but it is unclear if treatment impairs training outcomes. Herein, we investigated performance adaptations in well-trained females and males training with prior inhalation of salbutamol. Methods: 19 females and 21 males with maximal oxygen uptake (V'O2max) of 50.5±3.3 and 57.9±4.9 mL·min-1·kg-1, respectively, participated in this double-blinded, placebo-controlled, parallel-group study. We randomised participants to placebo or salbutamol inhalation (800-1600 µg·training day-1) for 6 weeks of combined endurance (1× per week) and high-intensity interval training (2× per week). We assessed participants' body composition, V'O2max and muscle contractile function, and collected vastus lateralis muscle biopsies. Results: Salbutamol induced a sex-specific loss of whole-body fat mass (sex×treatment: p=0.048) where only salbutamol-treated females had a fat mass reduction compared to placebo (-0.8 kg at 6 weeks; 95% CI: -0.5 to -1.6; p=0.039). Furthermore, salbutamol-treated females exhibited a repartitioning effect, lowering fat mass while gaining lean mass (p=0.011), which was not apparent for males (p=0.303). Salbutamol negatively impacted V'O2max in both sexes (treatment main effect: p=0.014) due to a blunted increase in V'O2max during the initial 4 weeks of the intervention. Quadriceps contractile strength was impaired in salbutamol-treated females (-39 N·m; 95% CI: -61 to -17; p=0.002) compared to placebo at 6 weeks. Muscle electron transport chain complex I-V abundance increased with salbutamol (treatment main effect: p=0.035), while content of SERCAI, ß2-adrenoceptor and desmin remained unchanged. Conclusion: Inhaled salbutamol appears to be an effective repartitioning agent in females but may impair aerobic and strength-related training outcomes.

The effect of aerobic exercise training on asthma control in postmenopausal women (ATOM): a randomized controlled pilot study.

Objective: To evaluate if high-intensity interval training three times weekly for 12 weeks improves asthma control in overweight, postmenopausal women with uncontrolled, late-onset asthma. Methods: The reported study is a randomized clinical pilot study (www.clinicaltrials.gov; NCT03747211) that compared 12 weeks of high-intensity interval training (spinning) with usual care. The five-question Asthma Control Questionnaire (ACQ-5) was used as primary outcome. Secondary measures included systemic inflammation and inflammation of the airways, body composition, and cardiac function during exercise. Results: We included 12 women with asthma (mean age 65 years (SD 6); mean body mass index 30 kg/m2 (SD 2)) from whom eight were randomized to exercise and four to control. Baseline ACQ-5 was 1.95 (SD 0.53) in the control group and 2.03 (0.54) in the exercise group. Patients had a mean blood eosinophil level of 0.16 × 109cells/L (SD 0.07) and a mean fraction of exhaled nitric oxide of 23 ppb (SD 25). Mixed models showed that participants in the exercise group reduced their ACQ-5 by 0.55 points (95%CI -1.10 to -0.00; P = 0.08) compared with the control group. The exercise group significantly reduced their mean body fat percentage (-2.7%; 95%CI -4.5 to -0.8; P = 0.02), fat mass (-2.8 kg; 95%CI -5.1 to -0.4; P = 0.044) and android fat mass (-0.33 kg; 95%CI -0.60- -0.06; P = 0.038). In analyses of cardiac measures, we saw no significant effects on right ventricular function (fractional area change), diastolic function or left ventricular function. Conclusions: Although changes in ACQ-5 were slightly insignificant, these preliminary findings indicate that aerobic exercise training can be used as a means to improve asthma control in overweight, postmenopausal women with asthma.

Effect of High-Intensity Interval Training on Inhaled Corticosteroid Dose in Asthma Patients: A Randomized Controlled Trial.

BACKGROUND: Inhaled corticosteroids (ICS) are the cornerstone of asthma treatment. However, ICS has side effects, and dose reduction is recommended when possible. Physical exercise improves asthma control, but it is unknown whether it reduces the reliance on ICS. OBJECTIVE: To assess whether supervised high-intensity interval training reduces the need for ICS in untrained asthma patients. METHODS: An assessor-blinded single-center randomized controlled trial, Copenhagen, Denmark. One hundred fifty untrained ICS-treated adults with symptomatic asthma were randomly assigned (2:1) to 6 months of supervised exercise 3 times weekly or a lifestyle as usual control group. Every second month, a clinical algorithm based on symptom control was applied in both groups to adjust ICS dose. Primary outcome was the proportion who had their ICS dose reduced by 25% or more after 6 months. Secondary outcomes included actual ICS dosage in micrograms per day. RESULTS: Between October 2017 and December 2019, 102 patients were allocated to exercise intervention (86% completed) and 48 to the control (85% completed). At the 6-month visit, 63% versus 50% met the primary outcome in the exercise and control groups, respectively (adjusted risk difference 9.6% [95% CI -3.8 to 18.8]; P = .15). Daily ICS dose was reduced in favor of the exercise group, with a mean difference of -234 µg (95% CI -391 to -77; P = .0037), corresponding to a 24% reduction from baseline. This effect was sustained at 12 months. The intervention was safe and well tolerated. CONCLUSIONS: Six months of regular exercise results in reduction in daily ICS dose without compromising asthma control.

Inhaled formoterol impairs aerobic exercise capacity in endurance-trained individuals: a randomised controlled trial.

Background: The 2022 Global Initiative for Asthma guidelines emphasise the inhaled long-acting ß2-agonist formoterol as part of the first treatment step, and therefore formoterol use among athletes will probably increase. However, prolonged supratherapeutic use of inhaled ß2-agonists impairs training outcomes in moderately trained men. We investigated whether inhaled formoterol, at therapeutic doses, imposes detrimental effects in endurance-trained individuals of both sexes. Methods: 51 endurance-trained participants (31 male, 20 female; mean±sd maximal oxygen consumption (V̇ O2 max) 62±6 mL·min-1·kg bw-1 and 52±5 mL·min-1·kg bw-1, respectively) inhaled formoterol (24 µg; n=26) or placebo (n=25) twice daily for 6 weeks. At baseline and follow-up, we assessed V̇ O2 max and incremental exercise performance during a bike-ergometer ramp-test; body composition by dual-energy X-ray absorptiometry; muscle oxidative capacity by high-resolution mitochondrial respirometry, enzymatic activity assays and immunoblotting; intravascular volumes by carbon monoxide rebreathing; and cardiac left ventricle mass and function by echocardiography. Results: Compared to placebo, formoterol increased lean body mass by 0.7 kg (95% CI 0.2-1.2 kg; treatment×trial p=0.022), but decreased V̇ O2 max by 5% (treatment×trial p=0.013) and incremental exercise performance by 3% (treatment×trial p<0.001). In addition, formoterol lowered muscle citrate synthase activity by 15% (treatment×trial p=0.063), mitochondrial complex II and III content (treatment×trial p=0.028 and p=0.007, respectively), and maximal mitochondrial respiration through complexes I and I+II by 14% and 16% (treatment×trial p=0.044 and p=0.017, respectively). No apparent changes were observed in cardiac parameters and intravascular blood volumes. All effects were sex-independent. Conclusion: Our findings demonstrate that inhaled therapeutic doses of formoterol impair aerobic exercise capacity in endurance-trained individuals, which is in part related to impaired muscle mitochondrial oxidative capacity. Thus, if low-dose formoterol fails to control respiratory symptoms in asthmatic athletes, physicians may consider alternative treatment options.

Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes.

BACKGROUND: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. OBJECTIVE: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. METHODS: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 µg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (Feno) with a cutoff of 25 parts per billion. RESULTS: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with Feno-high and Feno-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with Feno-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, -0.42; P = .04), and in those with Feno-low asthma, it correlated with the density in the airway smooth muscle (ρ, -0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. CONCLUSIONS: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with Feno-high asthma and with airway smooth muscle mast cells in patients with Feno-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.

High-Intensity Training Represses FXYD5 and Glycosylates Na,K-ATPase in Type II Muscle Fibres, Which Are Linked with Improved Muscle K+ Handling and Performance.

Na+/K+ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, ß, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and ß1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAß1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K+ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min-1, p < 0.01) while also increasing cumulated leg K+ reuptake 0-3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAß1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = -0.53, p < 0.05). NKAα2 and ß1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAß1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K+ shifts and enhance muscle performance during intense exercise.

Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase.

Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAß1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

Urine concentrations of vilanterol and its metabolites, GSK932009 and GW630200, after inhalation of therapeutic and supratherapeutic doses.

The 2023 Prohibited List issued by the World Anti-Doping Agency (WADA) permits athletes to inhale the beta2 -agonist vilanterol at a standard dose of 25 µg daily. However, given limited data on urine pharmacokinetics, vilanterol has no urinary threshold or decision limit to discriminate therapeutic from supratherapeutic use. We investigated urine concentrations of vilanterol and its main metabolites GSK932009 and GW630200 over 0-72 h following inhalation of therapeutic (25 µg) or supratherapeutic (100 µg) doses and repeat-dose administration for 7 days of 25 or 100 µg·day-1 in 25 trained men and women. Vilanterol administration was followed by 1 h of exercise. GW630200 urine concentrations were low and insufficient for threshold purposes, and while GSK932009 had higher urine concentrations, it could not discriminate between therapeutic and supratherapeutic use. Mean (range) maximum urine concentrations of parent vilanterol were 1.2 (0.2-4.1) and 6.2 (1.4-14.3) ng·ml-1 for single-dose 25 and 100 µg vilanterol, respectively, and 2.0 (0.3-4.8) and 22.4 (6.4-42.1) ng·ml-1 for repeat-dose 25 and 100 µg·day-1 vilanterol. In 333 samples collected 6 h post-administration and considering WADA TD2022DL, a 3.1 ng·ml-1 vilanterol cut-off showed 30% sensitivity in detecting supratherapeutic use at 100 µg versus therapeutic use at 25 µg. Considering inter- and intra-individual variability and guard bands in doping analysis, a 6 ng·ml-1 decision limit, which could be shifted upwards in samples with specific gravity >1.018, appears sufficiently high to minimize risk of samples exceeding the decision limit after therapeutic use of vilanterol, while demonstrating the ability to detect supratherapeutic use at 100 µg.