Vastus Lateralis Muscle Samples Research Articles

Metals accumulation affects bone and muscle in osteoporotic patients: A pilot study

Osteoporosis is the most common bone disease, characterized by decreased bone mineral density (BMD) and often associated to decreased muscle mass and function. Metal exposure plays a role in the pathophysiology of osteoporosis and affects also muscle quality. The aim of this study was to assess the association between metal levels in bone and muscle samples and the degeneration of these tissues. A total of 58 subjects (30 male and 28 female) was enrolled and classified in osteoporotic (OP, n = 8), osteopenic (Ope, n = 30) and healthy (CTR, n = 20) subjects, according to BMD measures. Femoral head bone samples and vastus lateralis muscle samples were collected during hip arthroplasty surgeries. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis showed increased levels of Al, Cd and Pb in OP and Ope bone tissue compared to CTR subjects (p = 0.04, p = 0.005 and p = 0.01, respectively). Whereas, increased levels of Co, Cd and Pb were measured in OP and Ope muscle tissues, compared to CTRs (p < 0.001, p = 0.02 and p = 0.01, respectively). In addition, Al, Cd and Pb levels in bone and Cd and Co levels in muscle were negatively correlated with BMD. A negative association among Co, Cd, Cr and Hg levels and muscle fibers diameter was also observed in muscle tissues. This study assessed that metal exposure can affects bone and muscle tissue quality and may contribute to the onset and progression of musculoskeletal diseases such as osteoporosis. Therefore, it is important to implement metal exposure assessment and their impact on disease development, in order to manage and prevent metal accumulation effects on bone and muscle quality.

The Effect of Fava Bean (Vicia faba L.) Protein Ingestion on Myofibrillar Protein Synthesis at Rest and after Resistance Exercise in Healthy, Young Men and Women: A Randomised Control Trial.

The aim of the present study was to evaluate the effect of feeding fava bean (Vicia faba L.) protein (FBP) on resting and post-exercise myofibrillar fractional synthetic rate (myoFSR). In a parallel, double-blind, randomised control trial, sixteen young, healthy recreationally active adults (age = 25 (5) years, body mass = 70 (15) kg, stature = 1.72 (0.11) m, mean (SD)) ingested 0.33 g·kg−1 FBP (n = 8) or a negative control (CON, i.e., EAA-free mixture) (n = 8), immediately after a bout of unilateral knee-extensor resistance exercise. Plasma, saliva, and m. vastus lateralis muscle samples were obtained pre-ingestion and 3 h post-ingestion. MyoFSR was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography–pyrolysis–isotope ratio mass spectrometry (GC-Pyr-IRMS). Resistance exercise increased myoFSR (p = 0.012). However, ingestion of FBP did not evoke an increase in resting (FBP 29 [−5, 63] vs. CON 12 [−25, 49]%, p = 0.409, mean % change [95% CI]) or post-exercise (FBP 78 [33, 123]% vs. CON 58 [9, 107]%, p = 0.732) myoFSR. Ingestion of 0.33 g·kg−1 of FBP does not appear to enhance resting or post-exercise myoFSR in young, healthy, recreationally active adults.

Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice.

BackgroundMitochondrial oxidative phosphorylation (OxPhos) is a critical regulator of skeletal muscle mass and function. Although muscle atrophy due to mitochondrial dysfunction is closely associated with bone loss, the biological characteristics of the relationship between muscle and bone remain obscure. We showed that muscle atrophy caused by skeletal muscle‐specific CR6‐interacting factor 1 knockout (MKO) modulates the bone marrow (BM) inflammatory response, leading to low bone mass.MethodsMKO mice with lower muscle OxPhos were fed a normal chow or high‐fat diet and then evaluated for muscle mass and function, and bone mineral density. Immunophenotyping of BM immune cells was also performed. BM transcriptomic analysis was used to identify key factors regulating bone mass in MKO mice. To determine the effects of BM‐derived CXCL12 (C–X–C motif chemokine ligand 12) on regulation of bone homeostasis, a variety of BM niche‐resident cells were treated with recombinant CXCL12. Vastus lateralis muscle and BM immune cell samples from 14 patients with hip fracture were investigated to examine the association between muscle function and BM inflammation.ResultsMKO mice exhibited significant reductions in both muscle mass and expression of OxPhos subunits but increased transcription of mitochondrial stress response‐related genes in the extensor digitorum longus (P < 0.01). MKO mice showed a decline in grip strength and a higher drop rate in the wire hanging test (P < 0.01). Micro‐computed tomography and von Kossa staining revealed that MKO mice developed a low mass phenotype in cortical and trabecular bone (P < 0.01). Transcriptomic analysis of the BM revealed that mitochondrial stress responses in skeletal muscles induce an inflammatory response and adipogenesis in the BM and that the CXCL12–CXCR4 (C–X–C chemokine receptor 4) axis is important for T‐cell homing to the BM. Antagonism of CXCR4 attenuated BM inflammation and increased bone mass in MKO mice. In humans, patients with low body mass index (BMI = 17.2 ± 0.42 kg/m2) harboured a larger population of proinflammatory and cytotoxic senescent T‐cells in the BMI (P < 0.05) and showed reduced expression of OxPhos subunits in the vastus lateralis, compared with controls with a normal BMI (23.7 ± 0.88 kg/m2) (P < 0.01).ConclusionsDefects in muscle mitochondrial OxPhos promote BM inflammation in mice, leading to decreased bone mass. Muscle mitochondrial dysfunction is linked to BM inflammatory cytokine secretion via the CXCL12–CXCR4 signalling axis, which is critical for inducing low bone mass.

Mycoprotein ingestion stimulates protein synthesis rates to a greater extent than milk protein in rested and exercised skeletal muscle of healthy young men: a randomized controlled trial

Mycoprotein is a fungal-derived sustainable protein-rich food source, and its ingestion results in systemic amino acid and leucine concentrations similar to that following milk protein ingestion. We assessed the mixed skeletal muscle protein synthetic response to the ingestion of a single bolus of mycoprotein compared with a leucine-matched bolus of milk protein, in rested and exercised muscle of resistance-trained young men. Twenty resistance-trained healthy young males (age: 22±1 y, body mass: 82±2 kg, BMI: 25±1 kg·m-2) took part in a randomized, double-blind, parallel-group study. Participants received primed, continuous infusions of L-[ring-2H5]phenylalanine and ingested either 31 g (26.2 g protein: 2.5 g leucine) milk protein (MILK) or 70 g (31.5 g protein: 2.5 g leucine) mycoprotein (MYCO) following a bout of unilateral resistance-type exercise (contralateral leg acting as resting control). Blood and m. vastus lateralis muscle samples were collected before exercise and protein ingestion, and following a 4-h postprandial period to assess mixed muscle fractional protein synthetic rates (FSRs) and myocellular signaling in response to the protein beverages in resting and exercised muscle. Mixed muscle FSRs increased following MILK ingestion (from 0.036±0.008 to 0.052±0.006%·h-1 in rested, and 0.035±0.008 to 0.056±0.005%·h-1 in exercised muscle; P<0.01) but to a greater extent following MYCO ingestion (from 0.025±0.006 to 0.057±0.004%·h-1 in rested, and 0.024±0.007 to 0.072±0.005%·h-1 in exercised muscle; P<0.0001) (treatment×time interaction effect; P<0.05). Postprandial FSRs trended to be greater in MYCO compared with MILK (0.065±0.004 compared with 0.054±0.004%·h-1, respectively; P=0.093) and the postprandial rise in FSRs was greater in MYCO compared with MILK (Delta 0.040±0.006 compared with Delta 0.018±0.005%·h-1, respectively; P<0.01). The ingestion of a single bolus of mycoprotein stimulates resting and postexercise muscle protein synthesis rates, and to a greater extent than a leucine-matched bolus of milk protein, in resistance-trained young men. This trial was registered at clinicaltrials.gov as 660065600.

Proteomic analysis of the skeletal muscles from dysferlinopathy patients

Dysferlinopathy is an autosomal recessive disease caused by pathogenic variants in DYSF gene. We compared muscle protein extracts from dysferlinopathy patients and control subjects to identify new biomarkers of this myopathy. We reviewed the medical records from January 2002 to October 2016. Eight vastus lateralis muscle samples from five dysferlinopathy patients and three control subjects were selected. We separated proteins/peptides from all eight muscle protein extracts using two-dimensional electrophoresis (2DE). Data were acquired from liquid chromatography-mass spectrometry protein fragmentation patterns after comparing the spot volumes. Western blotting revealed total dysferlin loss in the dysferlinopathy patients but normal expression in the control subjects. 2DE indicated somewhat diverse protein constellations between the dysferlinopathy and control groups. Image analysis showed that 80 spots were differently expressed between two dysferlinopathy and one control samples. We selected 44 spots with consistently different volume between dysferlinopathy and control groups. Liquid chromatography-mass spectrometry indicated 26 differently expressed proteins. Western blotting revealed that creatine kinase M-type, carbonic anhydrase III (muscle specific) and desmin were significantly elevated in dysferlinopathy muscle. Additionally, four proteins (myosin light chain 1/3, skeletal muscle isoform; lamin A/C; ankyrin repeat domain 2; and eukaryotic translation initiation factor 5A-1) were inconsistently elevated in the dysferlinopathy samples. We confirmed the usefulness of the classic biomarker and have newly identified the altered expression of proteins in the skeletal muscles of dysferlinopathy patients.

Comparative transcriptome analysis of skeletal muscle in ADSSL1 myopathy

ADSSL1 myopathy was recently identified as the cause of muscular disorders in Korean patients with distal myopathy. We generated transcriptome profiles of muscles from control subjects and patients with ADSSL1 myopathy. In the present study, RNA sequencing was conducted with seven vastus lateralis muscle samples from four patients with ADSSL1 myopathy and three control subjects. The hierarchical clustering result revealed a separation between myopathy and control groups. A total of 1,260 transcripts were significantly differentially expressed (|fold change| ≥ 2, p < 0.05), with 740 upregulated transcripts and 520 downregulated transcripts in myopathy group. Eighteen transcripts that mapped to purine metabolism pathway were significantly differentially expressed between the two groups, with ten downregulated transcripts and eight upregulated transcripts in myopathy group. In particular, three genes involved in purine nucleotide cycle (ADSSL1, ADSL, and AMPD1) were significantly downregulated in myopathy group. Ten transcripts in glycolysis/gluconeogenesis pathway were also significantly differentially expressed. This is the first study on the altered expression of transcripts in muscle tissues from patients with ADSSL1 myopathy. Our results provide new insights into the pathogenesis of ADSSL1 myopathy.

Exercise and GLUT4 in human subcutaneous adipose tissue.

To examine the effect of acute and chronic exercise on adipose tissue GLUT4 expression, a total of 20 healthy, male subjects performed one of two studies. Ten subjects performed cycle ergometer exercise for 60 min at 73 ± 2% VO 2 peak and abdominal adipose tissue samples were obtained immediately before and after exercise and after 3 h of recovery. Another 10 subjects completed 10 days of exercise training, comprising a combination of six sessions of 60 min at 75% VO 2 peak and four sessions of 6 × 5 min at 90% VO 2 peak, separated by 3 min at 40% VO 2 peak. Abdominal adipose tissue and vastus lateralis muscle samples were obtained before training and 24 h after the last training session. A single bout of exercise did not change adipose tissue GLUT4 mRNA; however, there was a small, but significant, reduction in adipose tissue GLUT4 protein expression 3 h after exercise. There were no changes in adipose tissue GLUT4 or COX‐IV expression following exercise training. In contrast, skeletal muscle GLUT4 and COX‐IV were increased by 47% and 44%, respectively following exercise training. The exercise training‐induced increase in GLUT4 expression was similar in both type I and type IIa single muscle fibers. Our results indicate that neither a single exercise bout, nor 10 days of exercise training, increased adipose tissue GLUT4, in contrast with the increases observed in skeletal muscle GLUT4 expression.

High-Intensity Interval Training Improves Markers of Oxidative Metabolism in Skeletal Muscle of Individuals With Obesity and Insulin Resistance.

Background: The excess body fat characteristic of obesity is related to various metabolic alterations, which includes insulin resistance (IR). Among the non-pharmacological measures used to improve insulin sensitivity are aerobic physical training, such as high-intensity interval training (HIIT). This study investigated the effects of 8 weeks of HIIT on blood and skeletal muscle markers related to IR and oxidative metabolism in physically inactive individuals with obesity and compared the changes between insulin resistant and non-insulin resistant phenotypes.Methods: Initially to investigate the effect of obesity and IR in the analyzed parameters, insulin-sensitive eutrophic volunteers (CON; n = 9) and obese non-insulin (OB; n = 9) and insulin-resistant (OBR; n = 8) were enrolled. Volunteers with obesity completed 8 weeks of HIIT in a cycle ergometer. Venous blood and vastus lateralis muscle samples were obtained before and after the HIIT. Body composition and peak oxygen consumption (VO2peak) were estimated before and after HIIT.Results: HIIT reduced IR assessed by the homeostatic model assessment of insulin resistance (HOMA-IR) in OBR (4.4 ± 1.4 versus 4.1 ± 2.2 μU L−2), but not in OB (HOMA-IR 1.8 ± 0.5 versus 2.3 ± 1.0 μU L−2) volunteers. HIIT increased VO2peak with no change in body fat in both groups. In skeletal muscle, HIIT increased the phosphorylation of IRS (Tyr612), Akt (Ser473), and increased protein content of β-HAD and COX-IV in both groups. There was a reduction in ERK1/2 phosphorylation in OBR after HIIT.Conclusion: Eight weeks of HIIT increased the content of proteins related to oxidative metabolism in skeletal muscle of individuals with obesity, independent of changes total body fat.

PL - 039 Heat Shock Proteins in human single skeletal muscle fibres resist age associated alterations and differentially respond to high-intensity exercise training

Objective Heat shock proteins (HSPs) are ubiquitously expressed proteins that help preserve cellular homeostasis. Within mammalian skeletal muscle three of the better characterised HSPs are HSP72, HSP27 and αB-crystallin. Among other roles, these three HSPs are involved in regulation of muscle mass and function and may be of importance in ageing. HSP’s are fibre-type dependent in rat skeletal muscle and thus examining these proteins in humans should be completed on the single fibre level, particularly in ageing where maladaptations primarily occur in Type II fibres. High-Intensity Training (HIT) is a commonly used method to improve muscle health and function in the elderly, but HSP adaptability to training has not yet been investigated. &#x0D; Methods This study examined isolated single muscle fibre segments collected from freeze-dried vastus lateralis muscle samples from young (25 /- 3 year old) and older (70 /- 4 year old) healthy individuals. A further sample was collected from the older individuals following 12 weeks of HIT, where they performed 4 x 4 min @ ~90-95% of peak heart rate (HR), with 4 min active recovery at 50-60% peak HR&#x0D; Results Basal expression of HSP’s in skeletal muscle: HSP70 tended to be higher in Type I fibres compared to Type II in young adults (p=0.08) and was higher in Type I compared to Type II fibres of older adults (p=0.03). HSP27 abundance was higher in Type I fibres compared to Type II in young adults (p=0.01) and tended to be higher in Type I compared to Type II fibres in older adults (p=0.07). The abundance of αβ-crystallin was more abundant in Type I fibres compared to Type II in both young and older adults (p&lt;0.05). Preliminary data revealed that the abundance of pABCser59 and pHSP2782 displayed no fibre-type specific abundances in either young or older adults.&#x0D; Age effects on HSP’s: There was no difference in the abundance of HSP70, HSP27, ABC or pHSP2782 between young and older adults in either Type I or Type II fibres. There was an increase in the abundance of pABCser59 in Type I fibres in older adults compared to Type I fibres of young adults (p=0.03), with no difference in Type II fibres.&#x0D; Effects of HIT on HSP’s: HIT in the older individuals increased the abundance of HSP70 in Type I fibres (p&lt;0.01) but not Type II. HIT tended to decrease the abundance of HSP27 in Type I fibres (0.92±0.66, p=0.06) and tended to increase the abundance of αβ-crystallin in Type I fibres (1.03±1.51 p=0.07).&#x0D; Conclusions These results revealed that in healthy, older individuals, the basal levels of HSP27, ABC or pHSP2782 are not different to those in young adults in either Type I or Type II fibres. This could indicate that the muscle from the older individuals was not compromised. Interestingly, in response to HIT there were varying changes between these HSP’s, and of note these occurred only in Type I fibres. Given that during HIT Type II fibres would be activated to a greater extent, it appears that the recovery phases of the HIT were most responsive to HSPs.

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

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

Metabolomic Signatures of Insulin Resistance in Human Skeletal Muscle Are Exacerbated with Insulin Stimulation

Metabolomic profiling has identified signatures of insulin resistance in skeletal muscle; however, the direct effect of insulin stimulation on these metabolite signatures in humans remains largely unresolved. To address this, we investigated the effect of insulin stimulation on skeletal muscle metabolites in a well characterized cohort of humans spanning the spectrum of insulin sensitivity. Vastus lateralis muscle samples were obtained from endurance athletes, sedentary lean and obese adults, and individuals with type 2 diabetes (n=16,15,15,12, respectively) under basal conditions and 1-hour into a hyperinsulinemic-euglycemic clamp. Metabolomic analysis was performed using UPLC-MS/MS. Basal concentrations of TCA cycle intermediates, including citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate, were positively related to insulin sensitivity (steady-state clamp Rd glucose in mg/kg/min; all P&amp;lt;0.01). Most of these relationships strengthened during insulin stimulation, with the strongest relationships observed for malate (r2 = 0.453, P&amp;lt;0.01) and fumarate (r2 = 0.501, P&amp;lt;0.01) that reflected a blunted increase in TCA intermediates during insulin stimulation with insulin resistance. Insulin stimulation lowered most medium- and long-chain acyl-carnitines compared with basal levels; however, this effect was also attenuated as a function of insulin resistance, signaling impaired metabolic flexibility. Notably, basal levels of several acyl-carnitines, including C12:0, C14:0, C16:0 and C18:1, were increased in endurance athletes compared with other groups (all P&amp;lt;0.05). Several amino acids, including leucine, isoleucine and lysine, were negatively related to insulin sensitivity in the basal and insulin stimulated state (all P&amp;lt;0.01). Together these findings indicate that physiologic insulin stimulation exacerbates metabolic signatures of skeletal muscle insulin resistance in humans. Disclosure S.A. Newsom: None. L. Perreault: Advisory Panel; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Advisory Panel; Self; Merck &amp; Co., Inc.. Speaker's Bureau; Self; Merck &amp; Co., Inc., AstraZeneca, Janssen Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc.. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Advisory Panel; Self; Sanofi. Speaker's Bureau; Self; Sanofi. A. Kerege: None. K.A. Harrison: None. D.E. Kahn: None. T. Nemkov: None. A. D’Alessandro: None. B. Bergman: Research Support; Self; Eli Lilly and Company. Advisory Panel; Spouse/Partner; Novo Nordisk Inc., Merck &amp; Co., Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company.

Intensity-dependent gene expression after aerobic exercise in endurance-trained skeletal muscle.

We investigated acute exercise-induced gene expression in skeletal muscle adapted to aerobic training. Vastus lateralis muscle samples were taken in ten endurance-trained males prior to, and just after, 4 h, and 8 h after acute cycling sessions with different intensities, 70% and 50% . High-throughput RNA sequencing was applied in samples from two subjects to evaluate differentially expressed genes after intensive exercise (70% ), and then the changes in expression for selected genes were validated by quantitative PCR (qPCR). To define exercise-induced genes, we compared gene expression after acute exercise with different intensities, 70% and 50% , by qPCR. The transcriptome is dynamically changed during the first hours of recovery after intensive exercise (70% ). A computational approach revealed that the changes might be related to up- and down-regulation of the activity of transcription activators and repressors, respectively. The exercise increased expression of many genes encoding protein kinases, while genes encoding transcriptional regulators were both up- and down-regulated. Evaluation of the gene expression after exercise with different intensities revealed that some genes changed expression in an intensity-dependent manner, but others did not: the majority of genes encoding protein kinases, oxidative phosphorylation and activator protein (AP)-1-related genes significantly correlated with markers of exercise stress (power, blood lactate during exercise and post-exercise blood cortisol), while transcriptional repressors and circadian-related genes did not. Some of the changes in gene expression after exercise seemingly may be modulated by circadian rhythm.

Humanin skeletal muscle protein levels increase after resistance training in men with impaired glucose metabolism

Humanin (HN) is a mitochondrially encoded and secreted peptide linked to glucose metabolism and tissue protecting mechanisms. Plasma HN levels are lower in people with type 2 diabetes. Whether skeletal muscle HN expression is influenced by exercise is unknown. Therefore, the aim of this study was to investigate serum and skeletal muscle HN levels in men with impaired glucose control in response to 12 weeks of either resistance or aerobic (nordic walking) training.Men (n=55) with impaired glucose regulation (IGR) were recruited and randomly assigned to resistance training (RT), Nordic walking (NW) or control (CON). Subjects trained three times per week for twelve weeks with progressively increased intensity during the intervention period. Biopsies from the vastus lateralis muscle and venous blood samples were taken before and after the intervention. A 2‐hr oral glucose tolerance test (OGTT) with 75 g of glucose loading was performed before and after the intervention period. Skeletal muscle and serum HN protein levels and skeletal muscle gene expression of MT‐NRN2 (the 16S gene), MT‐NRN1 (the 12S gene) and HNM1 were analyzed.Skeletal muscle HN protein levels increased by 35 % after twelve weeks of resistance training (time × group interaction p=0.022). No significant interactions over time were seen in the NW or the C groups (ns). In the RT group, ΔHN serum protein concentration was correlated to the improvement of 2‐hr glucose loading test (R= 0.597, p=0.019). Twelve weeks of either RT or NW did not change mRNA levels of MT‐NRN2, MT‐NRN1 or of HNM1. There was a significant increase in citrate synthase activity in the RT group (1.8‐fold ± 0.47 p&lt;0.05, time × group interaction p=0.027).HN protein levels in skeletal muscle increase after regular resistance training in pre‐diabetic men. The significant correlation between ΔHN serum levels and improvements in the 2‐hr time point of the OGTT suggests a role for HN in the regulation of glucose metabolism. Given the preventative effect of exercise on diabetes type 2, the role of HN as a mitochondrially derived peptide and an exercise‐responsive mitokine warrants further investigation.Support or Funding InformationThe study was supported by grants from the Research Council for Physical Education and Sports, of the Finnish Ministry of Education, of Turku University of Applied Sciences R&amp;D program and from Karolinska Institutet, Department of Physiology &amp; Pharmacology.

Muscle Glycogen Depletion Following 75-km of Cycling Is Not Linked to Increased Muscle IL-6, IL-8, and MCP-1 mRNA Expression and Protein Content.

The cytokine response to heavy exertion varies widely for unknown reasons, and this study evaluated the relative importance of glycogen depletion, muscle damage, and stress hormone changes on blood and muscle cytokine measures. Cyclists (N = 20) participated in a 75-km cycling time trial (168 ± 26.0 min), with blood and vastus lateralis muscle samples collected before and after. Muscle glycogen decreased 77.2 ± 17.4%, muscle IL-6, IL-8, and MCP-1 mRNA increased 18.5 ± 2.8−, 45.3 ± 7.8−, and 8.25 ± 1.75-fold, and muscle IL-6, IL-8, and MCP-1 protein increased 70.5 ± 14.1%, 347 ± 68.1%, and 148 ± 21.3%, respectively (all, P < 0.001). Serum myoglobin and cortisol increased 32.1 ± 3.3 to 242 ± 48.3 mg/mL, and 295 ± 27.6 to 784 ± 63.5 nmol/L, respectively (both P < 0.001). Plasma IL-6, IL-8, and MCP-1 increased 0.42 ± 0.07 to 18.5 ± 3.8, 4.07 ± 0.37 to 17.0 ± 1.8, and 96.5 ± 3.7 to 240 ± 21.6 pg/mL, respectively (all P < 0.001). Increases in muscle IL-6, IL-8, and MCP-1 mRNA were unrelated to any of the outcome measures. Muscle glycogen depletion was related to change in plasma IL-6 (r = 0.462, P = 0.040), with change in myoglobin related to plasma IL-8 (r = 0.582, P = 0.007) and plasma MCP-1 (r = 0.457, P = 0.043), and muscle MCP-1 protein (r = 0.588, P = 0.017); cortisol was related to plasma IL-8 (r = 0.613, P = 0.004), muscle IL-8 protein (r = 0.681, P = 0.004), and plasma MCP-1 (r = 0.442, P = 0.050). In summary, this study showed that muscle IL-6, IL-8, and MCP-1 mRNA expression after 75-km cycling was unrelated to glycogen depletion and muscle damage, with change in muscle glycogen related to plasma IL-6, and changes in serum myoglobin and cortisol related to the chemotactic cytokines IL-8 and MCP-1.

Vitamin C and E supplementation prevents some of the cellular adaptations to endurance-training in humans

BackgroundIt is clear that reactive oxygen species (ROS) produced during skeletal muscle contraction have a regulatory role in skeletal muscle adaptation to endurance exercise. However, there is much controversy in the literature regarding whether attenuation of ROS by antioxidant supplementation can prevent these cellular adaptations. Therefore, the aim of this study was to determine whether vitamin C and E supplementation attenuates performance and cellular adaptations following acute endurance exercise and endurance training. MethodsA double-blinded, placebo-controlled randomized control trial was conducted in eleven healthy young males. Participants were matched for peak oxygen consumption (VO2peak) and randomly allocated to placebo or antioxidant (vitamin C (2×500mg/day) and E (400IU/day)) groups. Following a four-week supplement loading period, participants completed acute exercise (10×4min cycling at 90% VO2peak, 2min active recovery). Vastus lateralis muscle samples were collected pre-, immediately-post- and 3h-post-exercise. Participants then completed four weeks of training (3 days/week) using the aforementioned exercise protocol while continuing supplementation. Following exercise training, participants again completed an acute exercise bout with muscle biopsies. ResultsAcute exercise tended to increase skeletal muscle oxidative stress as measured by oxidized glutathione (GSSG) (P=0.058) and F2-isoprostanes (P=0.056), with no significant effect of supplementation. Acute exercise significantly increased mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), mitochondrial transcription factor A (TFAM) and PGC related coactivator (PRC), with no effect of supplementation. Following endurance training, supplementation did not prevent significantly increased VO2peak, skeletal muscle levels of citrate synthase activity or mRNA or protein abundance of cytochrome oxidase subunit 4 (COX IV) (P<0.05). However, following training, vitamin C and E supplementation significantly attenuated increased skeletal muscle superoxide dismutase (SOD) activity and protein abundance of SOD2 and TFAM. ConclusionFollowing acute exercise, supplementation with vitamin C and E did not attenuate skeletal muscle oxidative stress or increased gene expression of mitochondrial biogenesis markers. However, supplementation attenuated some (SOD, TFAM) of the increased skeletal muscle adaptations following training in healthy young men.

11β-hydroxysteroid dehydrogenase type 1 and age-associated muscle weakness in mice: implications for human ageing

BackgroundMuscle weakness is a key discriminatory feature of Cushing's syndrome, which is characterised by glucocorticoid excess. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive glucocorticoids to their active form (cortisone to cortisol in man, 11-dehydrocorticosterone [11DHC] to corticosterone [CORT] in mice). We hypothesised that chronic exposure of tissues to active glucocorticoids contributes to the sarcopenia of ageing, and that absence or inhibition of 11β-HSD1 modulates this effect. MethodsC2C12 murine myotubes were used to identify glucocorticoid-regulated genes involved in muscle atrophy and to assess the effect of selective 11β-HSD1 inhibition. Regulation of glucocorticoid excess and ageing was assessed in mice on a high-fat diet: for glucocorticoid excess, 6-week-old male wildtype (wt) mice treated with CORT (100 μg/mL), 11DHC (100 μg/mL), or vehicle via drinking water for 5 weeks; for ageing, young (26 weeks) and aged (112 weeks) wt and 11β-HSD1 knockout mice. Grip strength and weights of muscle tissue were assessed as markers of muscle function with muscle mRNA expression profiles completed by microfluidic arrays. Biopsy samples of vastus lateralis muscle were taken in a study of healthy human volunteers (55 women, 24 men). FindingsGlucocorticoids increased expression of 11β-HSD1 and key atrophy genes, Fbxo32 (p<0·01) and Mstn (p<0·05). Changes in Fbxo32 were attenuated by co-treatment with a selective 11β-HSD1 inhibitor (p<0·05). Tissue weights of quadriceps and tibialis anterior muscles were reduced in wt mice treated with active and inactive glucocorticoids (p<0·05), whereas 11β-HSD1 knockout mice treated with inactive glucocorticoids were protected. wt mice treated with glucocorticoids had reduced grip strength compared with controls (p<0·001), whereas 11β-HSD1 knockout mice were protected. CORT and 11DHC increased expression of Fbxo32, Trim63, Mstn, Foxo1, Gadd45a, eiF4bp, CHARNB, and Ncam1 in wt mice (p<0·01 vs wt control), and these changes were attenuated in 11β-HSD1 knockout mice. Old wt mice had reduced grip strength compared with young wt mice (p<0·05), and old 11β-HSD1 knockout mice had preservation of grip strength. Trim63 (p<0·01), Mstn (p<0·01), Gadd45a (p<0·0001), eiF4bp (p<0·05), CHARNB (p<0·001), and Ncam1 (p<0·05) were upregulated with age in wt mice, but not in 11β-HSD1 knockout mice. In the study of human volunteers, 11β-HSD1 expression was upregulated with age (<50 years vs >50 years, p=0·0007 in women), along with GADD45a (p=0·005 women, p=0·04 men) and PSMA2 (p=0·004 women), and PSMD4 (p=0·006 women, p=0·04 men). InterpretationWe have identified changes in skeletal muscle gene expression common to both glucocorticoid excess and ageing, that parallel changes in muscle function. Absence of 11β-HSD1 attenuates these changes and results in protection against muscle weakness. Human studies showed that 11β-HSD1 expression is increased with age in women, along with genes involved in proteolysis. Selective inhibition of 11β-HSD1 might offer a novel therapeutic target for the treatment of both glucocorticoid-induced myopathy and age-related sarcopenia. FundingEuropean Research Council.

Microbiopsies versus Bergström needle for skeletal muscle sampling: impact on maximal mitochondrial respiration rate

Microbiopsies are increasingly used as an alternative to the standard Bergström technique for skeletal muscle sampling. The potential impact of these two different procedures on mitochondrial respiration rate is unknown. The objective of this work was to compare microbiopsies versus Bergström procedure on mitochondrial respiration in skeletal muscle. 52 vastus lateralis muscle samples were obtained from 13 anesthetized pigs, either with a Bergström [6 gauges (G)] needle or with microbiopsy needles (12, 14, 18G). Maximal mitochondrial respiration (V GM-ADP) was assessed using an oxygraphic method on permeabilized fibers. The weight of the muscle samples and V GM-ADP decreased with the increasing gauge of the needles. A positive nonlinear relationship was observed between the weight of the muscle sample and the level of maximal mitochondrial respiration (r = 0.99, p < 0.05) and between needle size and maximal mitochondrial respiration (r = 0.99, p < 0.05). Microbiopsies give lower muscle sample weight and maximal rate of mitochondrial respiration compared to the standard Bergström needle.Therefore, the higher the gauge (i.e. the smaller the size) of the microbiopsy needle, the lower is the maximal rate of respiration. Microbiopsies of skeletal muscle underestimate the maximal mitochondrial respiration rate, and this finding needs to be highlighted for adequate interpretation and comparison with literature data.

Energy intake modulates intramuscular mTORC1 signaling and ubiquitin‐mediated proteolysis during energy deficit independent of dietary protein variations

Effects of variations in habitual and acute dietary protein consumption on intracellular regulators of human skeletal muscle mass during energy deficit (ED) were investigated. Using a randomized‐block design, 32 men and 7 women consumed diets providing protein at 0.8, 1.6, or 2.4 g·kg−1·d−1 for 31 days. A 10‐day weight maintenance (WM) period preceded 21 days of ED, during which energy intake was reduced to 60% of daily requirements. Phosphorylation of mTORC1 associated signaling proteins, protein ubiquitylation (Ub), and NFκB expression were assessed in vastus lateralis muscle samples obtained after an overnight fast (FAST) and again 120 min after consuming a mixed meal (480 kcal, 20 g protein; FED) on days 10 and 31. Independent of dietary protein level and feeding state, Ub was 25% higher (P&lt; 0.05) for ED versus WM. Energy state and feeding altered p70S6K1 phosphorylation, as FED‐ED levels were 26% lower than FED‐WM (energy‐by‐feeding, P&lt; 0.05). Overall, Akt and rpS6 phosphorylation were higher (P&lt; 0.05), and NFκB and Ub were lower (P&lt; 0.05), for FED versus FAST. These data suggest that ED may upregulate intramuscular ubiquitin‐mediated proteolysis, and attenuate p70S6K1 sensitivity to feeding, although energy consumption, independent of habitual protein intake, may confer skeletal muscle protection through concomitant stimulation of mTORC1 signaling and inhibition of Ub during ED.Supported by USAMRMC, EMU CHHS SSSA, and USDA ARS

Role of autophagy in COPD skeletal muscle dysfunction

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by parenchymal damage and irreversible airflow limitation. In addition to lung dysfunction, patients with COPD develop weight loss, malnutrition, poor exercise performance, and skeletal muscle atrophy. The latter has been attributed to an imbalance between muscle protein synthesis and protein degradation. Several reports have confirmed that enhanced protein degradation and atrophy of limb muscles of COPD patient is mediated in part through activation of the ubiquitin-proteasome pathway and that this activation is triggered by enhanced production of reactive oxygen species. Until recently, the importance of the autophagy-lysosome pathway in protein degradation of skeletal muscles has been largely ignored, however, recent evidence suggests that this pathway is actively involved in recycling of cytosolic proteins, organelles, and protein aggregates in normal skeletal muscles. The protective role of autophagy in the regulation of muscle mass has recently been uncovered in mice with muscle-specific suppression of autophagy. These mice develop severe muscle weakness, atrophy, and decreased muscle contractility. No information is yet available about the involvement of the autophagy in the regulation of skeletal muscle mass in COPD patients. Pilot experiments on vastus lateralis muscle samples suggest that the autophagy-lysosome system is induced in COPD patients compared with control subjects. In this review, we summarize recent progress related to molecular structure, regulation, and roles of the autophagy-lysosome pathway in normal and diseased skeletal muscles. We also speculate about regulation and functional importance of this system in skeletal muscle dysfunction in COPD patients.

The time course of the adaptations of human muscle proteome to bed rest and the underlying mechanisms

In order to get a comprehensive picture of the complex adaptations of human skeletal muscle to disuse and further the understanding of the underlying mechanisms, we participated in two bed rest campaigns, one lasting 35 days and one 24 days. In the first bed rest (BR) campaign, myofibrillar proteins, metabolic enzymes and antioxidant defence systems were found to be down-regulated both post-8 days and post-35 days BR by proteomic analysis of vastus lateralis muscle samples from nine subjects. Such profound alterations occurred early (post-8 days BR), before disuse atrophy developed, and persisted through BR (post-35 days BR). To understand the mechanisms underlying the protein adaptations observed, muscle biopsies from the second bed rest campaign (nine subjects) were used to evaluate the adaptations of master controllers of the balance between muscle protein breakdown and muscle protein synthesis (MuRF-1 and atrogin-1; Akt and p70S6K), of autophagy (Beclin-1, p62, LC3, bnip3, cathepsin-L), of expression of antioxidant defence systems (NRF2) and of energy metabolism (PGC-1α, SREBP-1, AMPK). The results indicate that: (i) redox imbalance and remodelling of muscle proteome occur early and persist through BR; (ii) impaired energy metabolism is an early and persistent phenomenon comprising both the oxidative and glycolytic one; (iii) although both major catabolic systems, ubiquitin proteasome and autophagy, could contribute to the progression of atrophy late into BR, a decreased protein synthesis cannot be ruled out; (iv) a decreased PGC-1α, with the concurrence of SREBP-1 up-regulation, is a likely trigger of metabolic impairment, whereas the AMPK pathway is unaltered.