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Aim: High-resolution respirometry in human permeabilized muscle fibers is extensively used for analysis of mitochondrial adaptions to nutrition and exercise interventions, and is linked to athletic performance. However, the lack of standardization of experimental conditions limits quantitative inter- and intra-laboratory comparisons., Methods: In our study, an international team of investigators measured mitochondrial respiration of permeabilized muscle fibers obtained from three biopsies (vastus lateralis) from the same healthy volunteer to avoid inter-individual variability. High-resolution respirometry assays were performed together at the same laboratory to assess whether the heterogenity in published results are due to the effects of respiration media (MiR05 versus Z) with or without the myosin inhibitor blebbistatin at low- and high-oxygen regimes., Results: Our findings reveal significant differences between respiration media for OXPHOS and ETcapacities supported by NADH&succinate-linked substrates at different oxygen concentrations. Respiratory capacities were approximately 1.5-fold higher in MiR05 at high-oxygen regimes compared to medium Z near air saturation. The presence or absence of blebbistatin in human permeabilized muscle fiber preparations was without effect on oxygen flux., Conclusion: Our study constitutes a basis to harmonize and establish optimum experimental conditions for respirometric studies of permeabilized human skeletal muscle fibers to improve reproducibility., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

An attenuated ability to appropriately oxidize fat (metabolic inflexibility) has been associated with the development of obesity and type 2 diabetes. Previous studies have found that regular exercise training increases the body's ability to oxidize fat during exercise, but also shown that fat oxidation at the same relative and absolute exercise intensity is lower in old compared with young adults. Based on these studies we investigated the effect of training status on the whole-body peak fat oxidation rate (PFO) during exercise in young and middle-aged trained and untrained men. We hypothesized that aging was associated with decreased PFO, but regular exercise training could counteract this decline. 36 healthy non-overweight young and middle-aged men were recruited into a four groups: young (27 [24-30] yrs, (Mean [95% CI])) untrained (⩒O 2 peak: 47 [44-49] ml/min/kg), young (28 [26-30] yrs) trained (⩒O 2 peak: 64 [62-67] ml/min/kg), middle-aged (55 [53-57] yrs) untrained (⩒O 2 peak: 37 [32-42] ml/min/kg) and middle-aged (54 [51-57] yrs) trained (⩒O 2 peak: 55 [51-58] ml/min/kg). PFO was measured by indirect calorimetry while subjects performed a validated incremental exercise protocol on a cycle ergometer. Whole-body peak fat oxidation rate was higher in the young trained compared to young untrained subjects (0.70 [0.65-0.75] vs.0.45 [0.36-0.54] g/min, post-hoc: p  < 0.001); however, this training effect was attenuated in middle-aged trained and untrained subjects (0.44 [0.38-0.50] vs. 0.41 [0.35-0.47] g/min, post-hoc: p  = 0.83, respectively). In summary, these findings suggest that the training induced effects on whole-body fat oxidation found in young men may be attenuated in middle-aged men.

The aim of the present study was to investigate if peak fat oxidation rate (PFO) is related to Ironman performance in female athletes. Thirty-six female Ironman athletes (age: 34±1 yrs, [21-45 yrs.] SEM [Range]) with a BMI of 22.1±2.0 kg/m 2 [18.8-28.4 kg/m 2 ], a body fat percentage of 24.8±1.0% [9.0-37.0%] and a V̇O 2 peak of 53.0±1.3 ml/min/kg [36.5-70.5 ml/min/kg] were tested in the laboratory prior to the Ironman Copenhagen 2017. Race time ranged from 9:17:07 to 15:23:48 with mean race time being 11:57:26 h:min:s (717 min). By simple linear regression analyses we found associations between race time and P FO (r 2 =0.22, p<0.005), V̇O 2 peak (r 2 =0.65, p<0.0001) and the relative exercise intensity eliciting PFO (Fatmax) (r 2 =0.35, p=0.0001). Furthermore, associations were found between race time and body fat percentage (r 2 =0.44, p<0.0001) and age (r 2 =0.16, p<0.05). By means of multiple regression analysis, V̇O 2 peak was the only statistically significant variable explaining 64% of the variation in race time (adj. r 2 =0.64, p<0.005). In conclusion, these results demonstrate that PFO is not independently related to Ironman performance in a heterogeneous group of female athletes. Interestingly, V̇O 2 peak alone was able to predict 64% of the variation in Ironman race times., Competing Interests: The authors declare no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Skeletal muscle is a heterogeneous tissue and it is essential to know the methodological variation and reliability when measuring aspects of muscle function. We assessed the methodological and biological variation when measuring mitochondrial respiratory capacity (MRC), citrate synthase (CS) activity and myosin heavy chain (MHC) composition in muscle biopsies from nine healthy male participants, and in addition we assessed variation in MRC in isolated mitochondria and yeast suspension. We analysed MRC, CS activity and MHC composition in duplicates (intra-biopsy variation) to quantify the methodological variation, as well as the biological variation from multiple muscle biopsies (inter-biopsy variation) obtained at different sites of the same muscle. Three muscle biopsies (B1, B2 and B3) were obtained from each subject in m. vastus lateralis. Two of the biopsies were from the same leg and one from the other leg. For MRC, intra-biopsy coefficient of variation (CV) was 8.4% and inter-biopsy CV was 13.3%. For MHC type I, IIa and IIx intra-biopsy CV was 8.3, 6.0 and 22.3%, respectively. Inter-biopsy CV for these MHC types were 21.5, 15.4 and 42.0%, respectively. For CS activity intra-biopsy CV was 0.6% and inter-biopsy CV was 15.3%. No differences between B1, B2 and B3 were detected for MRC, CS activity or MHC composition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Adjuvant chemo‐ and radiotherapy cause cellular damage to tumorous and healthy dividing cells. Chemotherapy has been shown to cause mitochondrial respiratory dysfunction in non‐tumorous tissues, but the effects on human peripheral blood mononuclear cells (PBMCs) remain unknown. Aim: We aimed to investigate mitochondrial respiration of PBMCs before and after adjuvant chemo‐ and radiotherapy in postmenopausal patients with early breast cancer (EBC) and relate these to metabolic parameters of the patients. Methods: Twenty‐three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy with (n = 18) or without (n = 5) radiotherapy. Respiration (O2 flux per million PBMCs) was assessed by high‐resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics and mitochondrial DNA (mtDNA) content of PBMCs (mtDN relative to nuclear DNA) were furthermore assessed. Results: Respiration of intact and permeabilized PBMCs from EBC patients significantly increased with adjuvant chemo‐ and radiotherapy (p = 6 × 10−5 and p = 1 × 10−7, respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17–43% compared to before therapy initiation. Similarly, PBMC mtDNA content increased by 40% (p = 0.002). Leukocytes (p = 0.0001), hemoglobin (p = 0.0003), and HDL cholesterol (p = 0.003) concentrations decreased whereas triglyceride (p = 0.01) and LDL (p = 0.02) concentrations increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters or the mtDNA content of PBMCs correlated significantly with PBMC respiration. Conclusion: This study shows that mitochondrial respiration and mtDNA content in circulating PBMCs increase after adjuvant chemo‐ and radiotherapy in postmenopausal patients with EBC. Besides the increased mtDNA content, a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation, who may be less sensitive to chemotherapy, might influence the increased mitochondrial respiration observed iafter chemotherapy. [ABSTRACT FROM AUTHOR]

Introduction: Decreased fasting and oral glucose-stimulated incretin hormone concentrations following moderate-intensity continuous endurance training interventions have been reported in glucose-tolerant people, however results are conflicting. The effect of more time-efficient, very low-volume, high-intensity interval training (HIT) on circulating incretin hormone levels has never been studied. Materials and methods: Ten sedentary and overweight-to-obese participants (4 women and 6 men; age 43 ± 6 years (mean ± SD); BMI 30.2 ± 3.2 kg∙m−2; HbA1c 35 ± 5.1 mmol∙mol−1 (5.3 ± 0.3%); VO2max 30 ± 5 ml∙min−1∙kg−1) from the Copenhagen cohort of the METAPREDICT trial underwent 6 weeks of supervised low-volume HIT (3 sessions per week: 7 × 1 min at ∼100% VO2max separated by 1 min of active recovery). We measured glucose, insulin, C-peptide, glucagon, GLP-1 and GIP concentrations during a frequently sampled 75 g oral glucose tolerance test as well as VO2max and body composition before and after the intervention. Results: Training compliance was 100%. Relative VO2max improved after the intervention (median 2.69 ml∙min−1∙kg−1, IQR [0.43; 3.14], p = 0.037) while there were no significant effects on body weight and composition. No significant effects on oral glucose-stimulated glucose and hormone responses or estimates of insulin sensitivity and β-cell function were observed. Conclusion: Low-volume HIT improved aerobic fitness, but neither affected glucose tolerance nor oral glucose-stimulated incretin hormone responses in sedentary and overweight-to-obese people. Highlights Ten sedentary, overweight-to-obese, glucose-tolerant participants underwent 6 weeks of supervised, very low-volume HIT. Aerobic fitness improved. Fasting and oral glucose-stimulated incretin hormone concentrations were not affected. [ABSTRACT FROM AUTHOR]

The study aimed to determine the levels of skeletal muscle angiotensin‐converting enzyme 2 (ACE2, the SARS‐CoV‐2 receptor) protein expression in men and women and assess whether ACE2 expression in skeletal muscle is associated with cardiorespiratory fitness and adiposity. The level of ACE2 in vastus lateralis muscle biopsies collected in previous studies from 170 men (age: 19–65 years, weight: 56–137 kg, BMI: 23–44) and 69 women (age: 18–55 years, weight: 41–126 kg, BMI: 22–39) was analyzed in duplicate by western blot. VO2max was determined by ergospirometry and body composition by DXA. ACE2 protein expression was 1.8‐fold higher in women than men (p = 0.001, n = 239). This sex difference disappeared after accounting for the percentage of body fat (fat %), VO2max per kg of legs lean mass (VO2max‐LLM) and age (p = 0.47). Multiple regression analysis showed that the fat % (β = 0.47) is the main predictor of the variability in ACE2 protein expression in skeletal muscle, explaining 5.2% of the variance. VO2max‐LLM had also predictive value (β = 0.09). There was a significant fat % by VO2max‐LLM interaction, such that for subjects with low fat %, VO2max‐LLM was positively associated with ACE2 expression while as fat % increased the slope of the positive association between VO2max‐LLM and ACE2 was reduced. In conclusion, women express higher amounts of ACE2 in their skeletal muscles than men. This sexual dimorphism is mainly explained by sex differences in fat % and cardiorespiratory fitness. The percentage of body fat is the main predictor of the variability in ACE2 protein expression in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Context: The maximal fat oxidation rate (MFO) is higher in aerobically fit vs unfit young men, but this training-related increase in MFO is attenuated in middle-aged men. Further, it has also been found that unfit men with obesity may have an elevated MFO compared to unfit normal-weight men.Objective: Based hereupon, we aimed to investigate whether a fitness-related higher MFO were attenuated in middle-aged women compared to young women. Also, we aimed to investigate if unfit women with obesity have a higher MFO compared to unfit normal-weight women. We hypothesized that the training-related elevated MFO was attenuated in middle-aged women, but that unfit women with obesity would have an elevated MFO compared to unfit normal-weight women.Methods: We recruited 70 women stratified into 6 groups: young fit (n = 12), young unfit (n = 12) middle-aged fit (n = 12), middle-aged unfit (n = 12), unfit young women with obesity (n = 12), and unfit middle-aged women with obesity (n = 10). Body composition and resting blood samples were obtained and MFO was measured by a graded exercise test on a cycle ergometer via indirect calorimetry. Subsequently, a maximal exercise test was performed to establish peak oxygen uptake (V̇O2peak).Results: Young and middle-aged fit women had a higher MFO compared to age-matched unfit women, and young fit women had a higher MFO compared to fit middle-aged women. Unfit women with obesity, independent of age, had a higher MFO compared to their normal-weight and unfit counterparts.Conclusion: The training-related increase in MFO seems maintained in middle-aged women, and we find that unfit women with obesity, independent of age, have a higher MFO compared to unfit normal-weight women. [ABSTRACT FROM AUTHOR]

The peak fat oxidation rate (PFO) and the exercise intensity that elicits PFO (Fatmax) are associated with endurance performance during exercise primarily involving lower body musculature, but it remains elusive whether these associations are present during predominant upper body exercise. The aim was to investigate the relationship between PFO and Fatmax determined during a graded exercise test on a ski‐ergometer using double‐poling (GET‐DP) and performance in the long‐distance cross‐country skiing race, Vasaloppet. Forty‐three healthy men completed GET‐DP and Vasaloppet and were divided into two subgroups: recreational (RS, n = 35) and elite (ES, n = 8) skiers. Additionally, RS completed a cycle‐ergometer GET (GET‐Cycling) to elucidate whether the potential relationships were specific to exercise modality. PFO (r2 =.10, P =.044) and Fatmax (r2 =.26, P <.001) were correlated with performance; however, V˙O2peak was the only independent predictor of performance (adj. R2 =.36) across all participants. In ES, Fatmax was the only variable associated with performance (r2 =.54, P =.038). Within RS, DP V˙O2peak (r2 =.11, P =.047) and ski‐specific training background (r2 =.30, P =.001) were associated with performance. Between the two GETs, Fatmax (r2 =.20, P =.006) but not PFO (r2 =.07, P =.135) was correlated. Independent of exercise mode, neither PFO nor Fatmax were associated with performance in RS (P >.05). These findings suggest that prolonged endurance performance is related to PFO and Fatmax but foremost to V˙O2peak during predominant upper body exercise. Interestingly, Fatmax may be an important determinant of performance among ES. Among RS, DP V˙O2peak, and skiing experience appeared as performance predictors. Additionally, whole‐body fat oxidation seemed specifically coupled to exercise modality. [ABSTRACT FROM AUTHOR]

Introduction: High intensity interval training (HIIT) has shown to be as effective as moderate intensity endurance training to improve metabolic health. However, the current knowledge on the effect of HIIT in older individuals is limited and it is uncertain whether the adaptations are sex specific. The aim was to investigate effects of HIIT on mitochondrial respiratory capacity and mitochondrial content in older females and males. Methods: Twenty-two older sedentary males (n = 11) and females (n = 11) completed 6 weeks of supervised HIIT 3 days per week. The training consisted of 5 × 1 min cycling (124 ± 3% of max power output at session 2–6 and 135 ± 3% of max power output at session 7–20) interspersed by 1½ min recovery. Before the intervention and 72 h after last training session a muscle biopsy was obtained and mitochondrial respiratory capacity, citrate synthase activity and proteins involved in mitochondria metabolism were assessed. Furthermore, body composition and ⩒O2max were measured. Results: ⩒O2max increased and body fat percentage decreased after HIIT in both sexes (p < 0.05). In addition, CS activity and protein content of MnSOD and complex I-V increased in both sexes. Coupled and uncoupled mitochondrial respiratory capacity increased only in males. Mitochondrial respiratory capacity normalised to CS activity (intrinsic mitochondrial respiratory capacity) did not change following HIIT. Conclusion: HIIT induces favourable adaptions in skeletal muscle in older subjects by increasing mitochondrial content, which may help to maintain muscle oxidative capacity and slow down the process of sarcopenia associated with ageing. [ABSTRACT FROM AUTHOR]

Peak fat oxidation rate (PFO) and the intensity that elicits PFO (Fatmax) are commonly determined by a validated graded exercise test (GE) on a cycling ergometer with indirect calorimetry. However, for upper body exercise fat oxidation rates are not well elucidated and no protocol has been validated. Thus, our aim was to test validity and inter‐method reliability for determination of PFO and Fatmax in trained men using a GE protocol applying double poling on a ski‐ergometer. PFO and Fatmax were assessed during two identical GE tests (GE1 and GE2) and validated against separated short continuous exercise bouts (SCE) at 35%, 50%, and 65% of V̇O2peak on the ski‐ergometer in 10 endurance‐trained men (V̇O2peak: 65.1 ± 1.0 mL·min−1·kg−1, mean ± SEM). Between GE tests no differences were found in PFO (GE1: 0.42 ± 0.03; GE2: 0.45 ± 0.03 g·min−1, P = .256) or Fatmax (GE1: 41 ± 2%; GE2: 43 ± 3% of V̇O2peak, P = .457) and the intra‐individual coefficient of variation (CV) was 8 ± 2% and 11 ± 2% for PFO and Fatmax, respectively. Between GE and SCE tests, PFO (GEavg: 0.44 ± 0.03; SCE; 0.47 ± 0.06 g·min−1, P = .510) was not different, whereas a difference in Fatmax (GEavg: 42 ± 2%; SCE: 52 ± 4% of V̇O2peak, P = .030) was observed with a CV of 17 ± 4% and 15 ± 4% for PFO and Fatmax, respectively. In conclusion, GE has a high day‐to‐day reliability in determination of PFO and Fatmax in trained men, whereas it is unclear if PFO and Fatmax determined by GE reflect continuous exercise in general. [ABSTRACT FROM AUTHOR]

Sustaining a weight loss after a lifestyle intervention is challenging. The objective of the present study was to investigate if mitochondrial function is associated with the ability to maintain a weight loss. Sixty-eight former participants in an 11–12-week lifestyle intervention were recruited into 2 groups; weight loss maintenance (WLM; body mass index (BMI): 32 ± 1 kg/m2) and weight regain (WR; BMI: 43 ± 2 kg/m2) based on weight loss measured at a follow-up visit (WLM: 4.8 ± 0.4; WR: 7.6 ± 0.8 years after lifestyle intervention). Maximal oxygen consumption rate, physical activity level, and blood and muscle samples were obtained at the follow-up experiment. Mitochondrial respiratory capacity and reactive oxygen species (ROS) production were measured. Fasting blood samples were used to calculate glucose homeostasis index. WR had impaired glucose homeostasis and decreased maximal oxygen uptake and physical activity level compared with WLM. The decreased physical activity in WR was due to a lower activity level at vigorous and moderate intensities. Mitochondrial respiratory capacity and citrate synthase (CS) activity was higher in WLM, but intrinsic mitochondrial respiratory capacity (mitochondrial respiratory capacity corrected for mitochondrial content (CS activity)) was similar. ROS production was higher in WR compared with WLM, which was accompanied by a decreased content of antioxidant proteins in WR. Intrinsic mitochondrial respiratory capacity in skeletal muscle is not associated with the ability to maintain a long-term weight loss. WLM had a higher maximal oxygen uptake, physical activity level, mitochondrial respiratory capacity and CS activity compared with WR. The reduced glucose tolerance was concurrent with increased ROS production per mitochondria in WR, and could also be associated with the lower physical activity level in this group. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the relationship between maximal fat oxidation rate (MFO) measured during a progressive exercise test on a cycle ergometer and ultra-endurance performance. 61 male ironman athletes (age: 35 ± 1 yrs. [23-47 yrs.], with a BMI of 23.6 ± 0.3 kg/m² [20.0-30.1 kg/ m²], a body fat percentage of 16.7 ± 0.7 % [8.4-30.7 %] and a VO2peak of 58.7 ± 0.7 ml/min/kg [43.9-72.5 ml/m in/kg] SEM [Range]) were tested in the laboratory between 25 and 4 days prior to the ultra-endurance event, 2016 Ironman Copenhagen. Simple bivariate analyses revealed significant negative correlations between race time and MFO (r² = 0.12, p < 0.005) and VO2peak (r² = 0.45, p < 0.0001) and a positive correlation between race time and body fat percentage (r² = 0.27, p < 0.0001). MFO and VO2peak were not correlated. When the significant variables from the bivariate regression analyses were entered into the multiple regression models, VO2peak and MFO together explained 50 % of the variation observed in race time among the 61 Ironman athletes (adj R² = 0.50, p < 0.001). These results suggests that maximal fat oxidation rate exert an independent influence on ultra-endurance performance (> 9 h). Furthermore, we demonstrate that 50 % of the variation in Ironman triathlon race time can be explained by peak oxygen uptake and maximal fat oxidation. [ABSTRACT FROM AUTHOR]

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Actovegin, a deproteinized haemodialysate of calf blood, is suggested to have ergogenic properties, but this potential effect has never been investigated in human skeletal muscle. To investigate this purported ergogenic effect, we measured the mitochondrial respiratory capacity in permeabilized human skeletal muscle fibres acutely exposed to Actovegin in a low and in a high dose. We found that Actovegin, in the presence of complex I-linked substrates increased the oxidative phosphorylation (OXPHOS) capacity significantly in a concentration-dependent manner (19 ±3, 31 ± 4 and 45 ± 4 pmol/ mg/s). Maximal OXPHOS capacity with complex I and II-linked substrate was increased when the fibres were exposed to the high dose of Actovegin (62 ±6 and 77 ± 6 pmol/mg/s) (p<.05). The respiratory capacity of the electron transfer system as well as Vmax and Km were also increased in a concentration-dependent manner after Actovegin exposure (70 ± 6, 79 ±6 and 88 ± 7 pmol/mg/s; 13 ± 2, 25 ± 3 and 37 ± 4 pmol/mg/s; 0.08 ±0.02, 0.21 ± 0.03 and 0.36 ± 0.03 mM, respectively) (p<.05). In summary, we report for the first time that Actovegin has a marked effect on mitochondrial oxidative function in human skeletal muscle. Mitochondrial adaptations like this are also seen after a training program in human subjects. Whether this improvement translates into an ergogenic effect in athletes and thus reiterates the need to include Actovegin on the World Anti-Doping Agency's active list remains to be investigated. [ABSTRACT FROM AUTHOR]

Abstract: The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by the mitochondria in human skeletal muscle are used in many studies. There are several advantages of studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated whether the PBI shredder is useful in preparing a muscle homogenate for measurements of mitochondrial respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for preparing human skeletal muscle biopsies for respirometry. [Copyright &y& Elsevier]