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Insulin deficiency in type 1 diabetes (T1D) leads to an impairment of glucose metabolism and mitochondrial function. Actovegin is a hemodialysate of calf blood, which has been shown to enhance glucose uptake and cell metabolism in healthy human skeletal muscle. The objectives of this study were to determine the effects of Actovegin on skeletal muscle mitochondrial respiration and functional aerobic capacity in a T1D mouse model. Effects on the expression of mitochondrial proteins, body mass, and food and water consumption were also investigated. Streptozotocin-induced T1D male C57B1/6 mice (aged 3–4 months) were randomized to an Actovegin group and a control group. Every third day, the Actovegin and control groups were injected intraperitoneally with (0.1 mL) Actovegin and (0.1 mL) physiological salt solution, respectively. Oxidative phosphorylation (OXPHOS) capacity of the vastus lateralis muscle was measured by high resolution respirometry in addition to the expression levels of the mitochondrial complexes as well as voltage-dependent anion channel. Functional aerobic capacity was measured using a rodent treadmill protocol. Body mass and food and water consumption were also measured. After 13 days, in comparison to the control group, the Actovegin group demonstrated a significantly higher skeletal muscle mitochondrial respiratory capacity in an ADP-restricted and ADP-stimulated environment. The Actovegin group displayed a significantly lesser decline in functional aerobic capacity and baseline body mass after 13 days. There were no significant differences in food or water consumption between groups. Actovegin could act as an effective agent for facilitating glucose metabolism and improving OXPHOS capacity and functional aerobic capacity in T1D. Further investigation is warranted to establish Actovegin's potential as an alternative therapeutic drug for T1D. [ABSTRACT FROM AUTHOR]

This review provides an overview of the evidence regarding mtDNA and valid biomarkers for assessing mitochondrial adaptions. Mitochondria are small organelles that exist in almost all cells throughout the human body. As the only organelle, mitochondria contain their own DNA, mitochondrial DNA (mtDNA). mtDNA-encoded polypeptides are subunits of the enzyme complexes in the electron transport chain (ETC) that are responsible for production of ATP to the cells. mtDNA is frequently used as a biomarker for mitochondrial content, since changes in mitochondrial volume are thought to induce similar changes in mtDNA. However, some exercise studies have challenged this "gene-dosage theory", and have indicated that changes in mitochondrial content can adapt without changes in mtDNA. Thus, the aim of this scoping review was to summarize the studies that used mtDNA as a biomarker for mitochondrial adaptions and address the question as to whether changes in mitochondrial content, induce changes in mtDNA in response to aerobic exercise in the healthy skeletal muscle. The literature was searched in PubMed and Embase. Eligibility criteria included: interventional study design, aerobic exercise, mtDNA measurements reported pre- and postintervention for the healthy skeletal muscle and English language. Overall, 1585 studies were identified. Nine studies were included for analysis. Eight out of the nine studies showed proof of increased oxidative capacity, six found improvements in mitochondrial volume, content and/or improved mitochondrial enzyme activity and seven studies did not find evidence of change in mtDNA copy number. In conclusion, the findings imply that mitochondrial adaptions, as a response to aerobic exercise, can occur without a change in mtDNA copy number. [ABSTRACT FROM AUTHOR]

Myalgia and new-onset of type 2 diabetes have been associated with statin treatment, which both could be linked to reduced coenzyme Q10 (CoQ10) in skeletal muscle and impaired mitochondrial function. Supplementation with CoQ10 focusing on levels of CoQ10 in skeletal muscle and mitochondrial function has not been investigated in patients treated with statins. To investigate whether concomitant administration of CoQ10 with statins increases the muscle CoQ10 levels and improves the mitochondrial function, and if changes in muscle CoQ10 levels correlate with changes in the intensity of myalgia. 37 men and women in simvastatin therapy with and without myalgia were randomized to receive 400 mg CoQ10 daily or matched placebo tablets for eight weeks. Muscle CoQ10 levels, mitochondrial respiratory capacity, mitochondrial content (using citrate synthase activity as a biomarker), and production of reactive oxygen species were measured before and after CoQ10 supplementation, and intensity of myalgia was determined using the 10 cm visual analogue scale. Muscle CoQ10 content and mitochondrial function were unaltered by CoQ10 supplementation. Individual changes in muscle CoQ10 levels were not correlated with changes in intensity of myalgia. CoQ10 supplementation had no effect on muscle CoQ10 levels or mitochondrial function and did not affect symptoms of myalgia. [ABSTRACT FROM AUTHOR]

Skeletal muscles play an essential role in whole-body glucose homeostasis. They are a key organ system engaged in the development of insulin resistance, and also a crucial tissue mediating the beneficial metabolic effects of physical activity. However, molecular mechanisms underlying both these processes in skeletal muscle remain unclear. The aim of our study was to compare metabolomic profiles in skeletal muscle of patients at different stages of dysglycemia, from normoglycemia through prediabetes to T2D, and its changes under a mixed-mode (strength and endurance) exercise intervention. We performed targeted metabolomics comprising several major metabolite classes, including amino acids, biogenic amines and lipid subgroups in skeletal muscles of male patients. Dysglycemic groups differed significantly at baseline in lysophosphatidylcholines, phosphatidylcholines, sphingomyelins, glutamine, ornithine, and carnosine. Following the exercise intervention, we detected significant changes in lipids and metabolites related to lipid metabolism, including in ceramides and acylcarnitines. With their larger and more significant change over the intervention and among dysglycemic groups, these findings suggest that lipid species may play a predominant role in both the pathogenesis of type 2 diabetes and its protection by exercise. Simultaneously, we demonstrated that amino acid metabolism, especially glutamate dysregulation, is correlated to the development of insulin resistance and parallels disturbances in lipid metabolites. [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]

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ. [ABSTRACT FROM AUTHOR]

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations. [ABSTRACT FROM AUTHOR]

Key points: This is the first long‐term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology.NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle.NR supplementation does not affect NAD metabolite concentrations in skeletal muscle.Respiration, distribution and quantity of muscle mitochondria are unaffected by NR.NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle‐aged, obese, insulin‐resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo‐controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high‐resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady‐state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin‐resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people. Key points: This is the first long‐term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology.NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle.NR supplementation does not affect NAD metabolite concentrations in skeletal muscle.Respiration, distribution and quantity of muscle mitochondria are unaffected by NR.NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. [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]

Elite endurance athletes possess a high capacity for whole‐body maximal fat oxidation (MFO). The aim was to investigate the determinants of a high MFO in endurance athletes. The hypotheses were that augmented MFO in endurance athletes is related to concomitantly increments of skeletal muscle mitochondrial volume density (MitoVD) and mitochondrial fatty acid oxidation (FAOp), that is, quantitative mitochondrial adaptations as well as intrinsic FAOp per mitochondria, that is, qualitative adaptations. Eight competitive male cross‐country skiers and eight untrained controls were compared in the study. A graded exercise test was performed to determine MFO, the intensity where MFO occurs (FatMax), and V˙O2Max. Skeletal muscle biopsies were obtained to determine MitoVD (electron microscopy), FAOp, and OXPHOSp (high‐resolution respirometry). The following were higher (P < 0.05) in endurance athletes compared to controls: MFO (mean [95% confidence intervals]) (0.60 g/min [0.50‐0.70] vs 0.32 [0.24‐0.39]), FatMax (46% V˙O2Max [44‐47] vs 35 [34‐37]), V˙O2Max (71 mL/min/kg [69‐72] vs 48 [47‐49]), MitoVD (7.8% [7.2‐8.5] vs 6.0 [5.3‐6.8]), FAOp (34 pmol/s/mg muscle ww [27‐40] vs 21 [17‐25]), and OXPHOSp(108 pmol/s/mg muscle ww [104‐112] vs 69 [68‐71]). Intrinsic FAOp (4.0 pmol/s/mg muscle w.w/MitoVD [2.7‐5.3] vs 3.3 [2.7‐3.9]) and OXPHOSp(14 pmol/s/mg muscle ww/MitoVD [13‐15] vs 11 [10‐13]) were, however, similar in the endurance athletes and untrained controls. MFO and MitoVD correlated (r2 = 0.504, P < 0.05) in the endurance athletes. A strong correlation between MitoVD and MFO suggests that expansion of MitoVD might be rate‐limiting for MFO in the endurance athletes. In contrast, intrinsic mitochondrial changes were not associated with augmented MFO. [ABSTRACT FROM AUTHOR]

Abstract: Bed rest leads to impaired glucose tolerance. Whether this is linked to maladaptation's in skeletal muscle mitochondrial function and in particular to the level of reactive oxygen species (ROS) is at present unknown. The aim of this longitudinal study was to quantify skeletal muscle mitochondrial function (respiratory capacity and ROS production) together with glucose tolerance after 4 days of strict bed rest in healthy young male subjects (n = 14). Mitochondrial function was determined in permeabilized muscle fibers using high‐resolution respirometry and fluorometry, mitochondrial content (citrate synthase [CS] activity) and antioxidant protein expression levels were assessed in parallel to this. Glucose tolerance was determined by means of oral glucose tolerance tests. Intrinsic mitochondrial respiratory capacity was augmented after the bed rest period (CI + IIP: 0.43 ± 0.12 vs. 0.55 ± 0.14 [pmol/sec/mg]/CS activity), due to a decreased CS activity (158 ± 39 vs. 129 ± 25 mU/mg dw.). No differences were observed in ROS production (per mg of tissue or when normalized to CS activity). Furthermore, the protein content for catalase was increased while superoxide dismutase and glutathione peroxidase remained unaffected. These findings were accompanied by an impaired glucose tolerance after the bed rest period (Matsuda index: 12 ± 6 vs. 9 ± 5). The change in intrinsic mitochondrial respiratory capacity could be an early indication in the development of impaired glucose tolerance. The increased catalase protein content might explain that no change was seen in ROS production after 4 days of bed rest. Whether these findings can be extrapolated to lifestyle‐dependent decrements in physical activity and the development of type‐2‐diabetes remains unknown. [ABSTRACT FROM AUTHOR]

Abstract: The effect of high‐intensity training (HIT) on mitochondrial ADP sensitivity and respiratory capacity was investigated in human skeletal muscle and subcutaneous adipose tissue (SAT). Twelve men and women underwent 6 weeks of HIT (7 × 1 min at app. 100% of maximal oxygen uptake (VO2max)). Mitochondrial respiration was measured in permeabilized muscle fibers and in abdominal SAT. Mitochondrial ADP sensitivity was determined using Michaelis Menten enzyme kinetics. VO2max, body composition and citrate synthase (CS) activity (skeletal muscle) and mtDNA (SAT) were measured before and after training. VO2max increased from 2.6 ± 0.2 to 2.8 ± 0.2 L O2/min (P = 0.011) accompanied by a decreased mitochondrial ADP sensitivity in skeletal muscle (Km: 0.14 ± 0.02 to 0.29 ± 0.03 mmol/L ADP (P = 0.002)), with no changes in SAT (Km: 0.12 ± 0.02 to 0.16 ± 0.05 mmol/L ADP; P = 0.186), following training. Mitochondrial respiratory capacity increased in skeletal muscle from 57 ± 4 to 67 ± 4 pmol O2·mg−1·sec−1 (P < 0.001), but decreased with training in SAT from 1.3 ± 0.1 to 1.0 ± 0.1 pmol O2·mg−1·sec−1 (P < 0.001). CS activity increased (P = 0.027) and mtDNA was unchanged following training. Intrinsic mitochondrial respiratory capacity was unchanged in skeletal muscle, but increased in SAT after HIT. In summary, our results demonstrate that mitochondrial adaptations to HIT in skeletal muscle are comparable to adaptations to endurance training, with an increased mitochondrial respiratory capacity and CS activity. However, mitochondria in SAT adapts differently compared to skeletal muscle mitochondria, where mitochondrial respiratory capacity decreased and mtDNA remained unchanged after HIT. [ABSTRACT FROM AUTHOR]

Background. A prevalent side-effect of simvastatin is attenuated glucose homeostasis. The underlying mechanism is unknown, but impaired lipid metabolism may provide the link. The aim of this study was to investigate whether simvastatin-treated patients had a lower capacity to oxidize lipids and reduced expression of the major proteins regulating lipid uptake, synthesis, lipolysis, and storage in skeletal muscle than matched controls. Materials and Methods. Ten men were treated with simvastatin (HbA1c: 5.7 ± 0.1%), and 10 healthy men (HbA1c: 5.2 ± 0.1%) underwent an oral glucose tolerance test and a muscle biopsy was obtained. Fat oxidation rates were measured at rest and during exercise. Western blotting was used to assess protein content. Results. Patients treated with simvastatin had impaired glucose tolerance compared with control subjects, but fat oxidation at rest and during exercise was compatible. Skeletal muscle protein content of CD36, lipoprotein lipase (LPL), and diacylglycerol acyltransferase (DGAT) 1 were lower, and DGAT 2 tended to be lower in patients treated with simvastatin. Conclusions. Patients treated with simvastatin had a reduced capacity to synthesize FA and diacylglycerol (DAG) into triacylglycerol in skeletal muscle compared to matched controls. Decreased lipid synthesis capacity may lead to accumulation of lipotoxic intermediates (FA and DAG) and hence impair glucose tolerance. [ABSTRACT FROM AUTHOR]

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]

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s-1·mg-1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g-1·min-1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s-1·mg-1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production. [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]

Abstract: It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present. [Copyright &y& Elsevier]

Objectives: Glucose tolerance and skeletal muscle coenzyme Q10 (Q10) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9). Background: A prevalent side effect of statin therapy is muscle pain, and yet the basic mechanism behind it remains unknown. We hypothesize that a statin-induced reduction in muscle Q10 may attenuate mitochondrial OXPHOS capacity, which may be an underlying mechanism. Methods: Plasma glucose and insulin concentrations were measured during an oral glucose tolerance test. Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers by high-resolution respirometry in a cross-sectional design. Mitochondrial content (estimated by citrate synthase [CS] activity, cardiolipin content, and voltage-dependent anion channel [VDAC] content) as well as Q10 content was determined. Results: Simvastatin-treated patients had an impaired glucose tolerance and displayed a decreased insulin sensitivity index. Regarding mitochondrial studies, Q10 content was reduced (p = 0.05), whereas mitochondrial content was similar between the groups. OXPHOS capacity was comparable between groups when complex I– and complex II–linked substrates were used alone, but when complex I + II–linked substrates were used (eliciting convergent electron input into the Q intersection [maximal ex vivo OXPHOS capacity]), a decreased (p < 0.01) capacity was observed in the patients compared with the control subjects. Conclusions: These simvastatin-treated patients were glucose intolerant. A decreased Q10 content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment. [Copyright &y& Elsevier]

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5'AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α2 (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice. We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes, but its contribution to the early stages of dysglycemia remains poorly understood. By collecting a high-resolution stage-based spectrum of dysglycemia, our study fills this gap by evaluating derangement in both the function and quantity of mitochondria. We sampled mitochondria in skeletal muscle and subcutaneous adipose tissues of subjects with progressive advancement of dysglycemia under a three-month exercise intervention. Methods: We measured clinical metabolic parameters and gathered skeletal muscle and adipose tissue biopsies before and after the three-month exercise intervention. We then assayed the number of mitochondria via citrate synthase (CS) activity and functional parameters with high-resolution respirometry. Results: In muscle, there were no differences in mitochondrial quantity or function at baseline between normoglycemics and prediabetics. However, the intervention caused improvement in CS activity, implying an increase in mitochondrial quantity. By contrast in adipose tissue, baseline differences in CS activity were present, with the lowest CS activity coincident with impaired fasting glucose and impaired glucose tolerance (IFG + IGT). Finally, CS activity, but few of the functional metrics, improved under the intervention. Conclusions: We show that in prediabetes, no differences in the function or amount of mitochondria (measured by CS activity) in skeletal muscle are apparent, but in adipose tissue of subjects with IFG + IGT, a significantly reduced activity of CS was observed. Finally, metabolic improvements under the exercise correlate to improvements in the amount, rather than function, of mitochondria in both tissues. [ABSTRACT FROM AUTHOR]

Aging decreases skeletal muscle mass and strength, but aerobic and resistance exercise training maintains skeletal muscle function. NAD+ is a coenzyme for ATP production and a required substrate for enzymes regulating cellular homeostasis. In skeletal muscle, NAD+ is mainly generated by the NAD+ salvage pathway in which nicotinamide phosphoribosyltransferase (NAMPT) is rate‐limiting. NAMPT decreases with age in human skeletal muscle, and aerobic exercise training increases NAMPT levels in young men. However, whether distinct modes of exercise training increase NAMPT levels in both young and old people is unknown. We assessed the effects of 12 weeks of aerobic and resistance exercise training on skeletal muscle abundance of NAMPT, nicotinamide riboside kinase 2 (NRK2), and nicotinamide mononucleotide adenylyltransferase (NMNAT) 1 and 3 in young (≤35 years) and older (≥55 years) individuals. NAMPT in skeletal muscle correlated negatively with age (r2 = 0.297, P < 0.001, n = 57), and VO2peak was the best predictor of NAMPT levels. Moreover, aerobic exercise training increased NAMPT abundance 12% and 28% in young and older individuals, respectively, whereas resistance exercise training increased NAMPT abundance 25% and 30% in young and in older individuals, respectively. None of the other proteins changed with exercise training. In a separate cohort of young and old people, levels of NAMPT, NRK1, and NMNAT1/2 in abdominal subcutaneous adipose tissue were not affected by either age or 6 weeks of high‐intensity interval training. Collectively, exercise training reverses the age‐dependent decline in skeletal muscle NAMPT abundance, and our findings highlight the value of exercise training in ameliorating age‐associated deterioration of skeletal muscle function. [ABSTRACT FROM AUTHOR]

Aim: To examine the effect of high‐intensity interval training (HIIT) on glucose clearance rates in skeletal muscle and explore the mechanism within the muscle. Methods: Ten males with type 2 diabetes mellitus (T2DM) and ten matched healthy subjects performed 2 weeks of one‐legged HIIT (total of eight sessions, each comprised of 10 × 1 minute ergometer bicycle exercise at >80% of maximal heart rate, interspersed with one min of rest). Insulin sensitivity was assessed by an isoglycaemic, hyperinsulinaemic clamp combined with arteriovenous leg balance technique of the trained (T) and the untrained (UT) leg and muscle biopsies of both legs. Results: Insulin‐stimulated glucose clearance in T legs was ~30% higher compared with UT legs in both groups due to increased blood flow in T vs UT legs and maintained glucose extraction. With each training session, muscle glycogen content decreased only in the training leg, and after the training, glycogen synthase and citrate synthase activities were higher in T vs UT legs. No major changes occurred in the expression of proteins in the insulin signalling cascade. Mitochondrial respiratory capacity was similar in T2DM and healthy subjects, and unchanged by HIIT. Conclusion: HIIT improves skeletal muscle insulin sensitivity. With HIIT, the skeletal muscle of patients with T2DM becomes just as insulin sensitive as untrained muscle in healthy subjects. The mechanism includes oscillations in muscle glycogen stores and a maintained ability to extract glucose from the blood in the face of increased blood flow in the trained leg. [ABSTRACT FROM AUTHOR]

Ageing, a sedentary lifestyle, and obesity are associated with increased oxidative stress, while regular exercise is associated with an increased antioxidant capacity in trained skeletal muscles. Whether a higher aerobic fitness is associated with increased expression of antioxidant enzymes and their regulatory factors in skeletal muscle remains unknown. Although oestrogens could promote a higher antioxidant capacity in females, it remains unknown whether a sex dimorphism exists in humans regarding the antioxidant capacity of skeletal muscle. Thus, the aim was to determine the protein expression levels of the antioxidant enzymes SOD1, SOD2, catalase and glutathione reductase (GR) and their regulatory factors Nrf2 and Keap1 in 189 volunteers (120 males and 69 females) to establish whether sex differences exist and how age, VO 2 max and adiposity influence these. For this purpose, vastus lateralis muscle biopsies were obtained in all participants under resting and unstressed conditions. No significant sex differences in Nrf2, Keap1, SOD1, SOD2, catalase and GR protein expression levels were observed after accounting for VO 2 max, age and adiposity differences. Multiple regression analysis indicates that the VO 2 max in mL.kg LLM−1.min−1can be predicted from the levels of SOD2, Total Nrf2 and Keap1 (R = 0.58, P < 0.001), with SOD2 being the main predictor explaining 28 % of variance in VO 2 max, while Nrf2 and Keap1 explained each around 3 % of the variance. SOD1 protein expression increased with ageing in the whole group after accounting for differences in VO 2 max and body fat percentage. Overweight and obesity were associated with increased pSer40-Nrf2, pSer40-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels after accounting for differences in age and VO 2 max. Overall, at the population level, higher aerobic fitness is associated with increased basal expression of muscle antioxidant enzymes, which may explain some of the benefits of regular exercise. [Display omitted] • It remains unknown how aerobic fitness, adiposity, age and sex may influence antioxidant enzymes in human skeletal muscle. • Here we show that SOD2 is the main antioxidant enzyme associated with VO 2 max. • In young and middle-aged humans, there is no sex-dimorphism in antioxidant enzymes expression in skeletal muscle or their regulatory factors. • SOD1 protein expression is increased with ageing after accounting for differences in VO2max and body fat percentage. • Overweight and obesity are associated with increased pSer40-Nrf2, pSer40-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels. [ABSTRACT FROM AUTHOR]

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin. [ABSTRACT FROM AUTHOR]

Obesity has been associated with increased production of reactive oxygen species (ROS), which may be involved in the development of cardiovascular disease and type 2 diabetes (T2D). Endurance exercise lowers ROS production and increases antioxidant capacity in muscle cells, but it is currently unknown whether high intensity interval training (HIT) elicits the same effects. Twelve sedentary obese subjects at risk of developing T2D took part in a six-week intervention, performing three HIT sessions per week (five 1-min sets of high-intensity cycling (125% of VO 2 peak), with 90 s recovery in between sets). Muscle biopsies were obtained for assessment of ROS production (H 2 O 2 emission), mitochondrial respiratory capacity, and antioxidant protein levels before and after the intervention. H 2 O 2 emission decreased 60.4% after the intervention (Succinate 3 mmol･l−1), concurrent with a 35.1% increase in protein levels of the antioxidant manganese superoxide dismutase (MnSOD) and a trend towards increased levels of the antioxidant catalase (p = 0.06, 72.9%). These findings were accompanied by a 19% increased mitochondrial respiratory capacity (CI + II), a 6.9% increased VO 2 peak and a 1.7% lower body fat percentage. These effects were achieved after just 15 min of high-intensity work and 40 min of total time spent per week. Overall, this suggests that a relatively small amount of HIT is sufficient to induce beneficial effects on ROS production and antioxidant status in muscle cells, which may lower oxidative stress and potentially protect against the development of cardiovascular disease. [Display omitted] • HIT reduces H 2 O 2 emission in muscle cells of obese adults in risk of developing T2D. • HIT leads to increased protein levels of antioxidant enzymes in skeletal muscle cells. • HIT improves mitochondrial respiratory capacity and general health parameters. • The effects occur after just 40 min of weekly time commitment for 6 weeks. [ABSTRACT FROM AUTHOR]

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole‐body energy metabolism and body temperature has not been resolved. Here, we show that T3‐induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα1) in skeletal muscle, but that T3‐mediated elevation in body temperature is achieved in the absence of muscle‐TRα1. In slow‐twitch soleus muscle, loss‐of‐function of TRα1 (TRαHSACre) alters the fiber‐type composition toward a more oxidative phenotype. The change in fiber‐type composition, however, does not influence the running capacity or motivation to run. RNA‐sequencing of soleus muscle from WT mice and TRαHSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα1‐linked control of whole‐body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3‐stimulated increase in whole‐body energy expenditure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]