Jakob Agergaard

Whey and casein labeled with l-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1.

Age-related differences in lean mass, protein synthesis and skeletal muscle markers of proteolysis after bed rest and exercise rehabilitation.

Five days of bed rest resulted in a reduction in leg lean mass and strength in older adults. After bed rest, older (but not younger) adults had reduced amino acid‐induced anabolic sensitivity (blunted muscle protein synthesis; MPS) and enhanced markers associated with the ubiquitin proteasome and autophagy–lysosomal systems (increase in molecular markers related to muscle proteolysis). Younger adults did not lose leg lean mass (via DXA) after 5 days of bed rest despite blunted amino acid‐induced mTORC1 signalling and increased skeletal muscle REDD1, REDD2 and MURF1 mRNA expression. Exercise rehabilitation restored bed rest‐induced deficits in lean mass, strength, nutrient‐induced protein anabolism (protein synthesis and mTORC1 signalling) and select muscle proteolytic markers in older adults.

Activated Protein Synthesis and Suppressed Protein Breakdown Signaling in Skeletal Muscle of Critically Ill Patients

Background Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls. Methodology/Principal Findings ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2 = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated. Conclusions/Significance We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

Myogenic, matrix and growth factor mRNA expression in human skeletal muscle: effect of contraction intensity and feeding

Introduction: We examined short‐term (3‐hour) and long‐term (12‐week) training effects after heavy load [HL; 70% 1RM] and light load (LL; 16% 1RM) exercise. Methods: mRNA expression of genes involved in skeletal muscle remodeling were analyzed and muscle activity (EMG measurements) was measured. Results: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle‐fiber‐type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance exercise. No major differences were seen in atrophy‐related genes between HL and LL resistance exercise. No changes were seen over 12‐week training for any of the targets. Conclusions: Resistance exercise at LL and HL elevated the expression of genes involved in skeletal muscle hypertrophy, although the greatest response was from HL. However, no long‐term effect from either LL or HL resistance exercise was seen on basal levels of the mRNA targets. Muscle Nerve 47: 748–759, 2013

Skeletal muscle morphology and regulatory signalling in endurance-trained and sedentary individuals: The influence of ageing

ABSTRACT Muscle mass in humans is inversely associated with circulating levels of inflammatory cytokines, but the interaction between ageing and training on muscle composition and the intra‐muscular signalling behind inflammation and contractile protein synthesis and degradation is unknown. We studied 15 healthy life‐long endurance runners, 12 age‐matched untrained controls, 10 young trained and 12 young untrained individuals. Thigh muscle composition was investigated by magnetic resonance imaging (MRI), where non‐contractile intramuscular tissue (NCIT) area (fat and connective tissue) was found to be greater in older but lower in trained individuals. Subcutaneous adipose tissue was also lower in trained individuals but was not affected by age. In vastus lateralis biopsies, no influence of age or training was found on levels of endomysial collagen, determined by Sirius Red and Collagen III staining, whereas perimysial organisation tended to be more complex in older individuals. No clear difference with training was seen on intramuscular inflammatory signalling, whereas lower protein levels of NFkB subunits p105, p50 and p65 were observed with ageing. Gene expression of IL6 and TNF&agr; was not different between groups, while IL1‐receptor and TNF&agr;‐receptor1 levels were lower with age. Myostatin mRNA was lower in older and trained groups, while expression of MuRF1 was lower in trained individuals and FoxO3 expression was greater in aged groups. The association of increased muscle NCIT with age‐associated muscle loss in humans is not accompanied by any major alterations in intramuscular signalling for inflammation, but rather by direct regulatory factors for protein synthesis and proteolysis in skeletal muscle. HighlightsWe analysed thigh muscle quality and inflammatory signalling in young and old men.Older muscle had a greater content of non‐contractile intramuscular tissue (NCIT).The skeletal muscle of endurance trained vs. untrained men contained less NCIT.Muscle perimysium tended to be more ramified in older individuals.Differences were observed in signalling for protein turnover but not inflammation.

GH receptor blocker administration and muscle-tendon collagen synthesis in humans

CONTEXT The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis stimulates collagen synthesis in tendon and skeletal muscle, but no studies have investigated the effect of reducing IGF-I on collagen synthesis in healthy humans. OBJECTIVE We hypothesised, that a GH blockade would decrease IGF-I and collagen synthesis in the connective tissue of skeletal muscle and tendon. DESIGN The study was randomised and double blinded. PARTICIPANTS 20 healthy young males completed the study. INTERVENTION The participants were randomised to 2 weeks of GH receptor blocker supplementation (pegvisomant, 5 mg/day, n=9) or placebo (n=11). MAIN OUTCOME MEASURES Serum levels of GH, IGF-I and IGF-binding protein 3 (IGFBP-3) were measured before and after pegvisomant/placebo supplementation. Fractional synthesis rates (FSR) for collagen and myofibrillar protein were determined with stable isotopes in tendon and muscle, and mRNA for collagen (COL1A1 and COL3A1) as well as IGF-I isoforms (Ea and Ec) were measured in skeletal muscle. RESULTS Pegvisomant decreased serum IGF-I by 20% (p<0.01) and serum IGFBP-3 by 10% (p<0.05). Pegvisomant supplementation had no effect on collagen synthesis in tendon and skeletal muscle, nor was muscle myofibrillar protein synthesis affected. Similarly, pegvisomant supplementation had no effect on mRNA expression of IGF-I and collagen in skeletal muscle. CONCLUSION GH receptor blocker administration in healthy humans resulted in a moderate decrease in serum IGF-I. Collagen synthesis in tendon and skeletal muscle, as well as skeletal muscle IGF-I and collagen mRNA expression, was unaffected by GH receptor blocker supplementation.

Skeletal Muscle Ras-Related GTP Binding B mRNA and Protein Expression Is Increased after Essential Amino Acid Ingestion in Healthy Humans

Essential amino acids (EAAs) are potent stimulators of mechanistic target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis. However, regulators upstream of mTORC1 that are responsive to EAA availability are not well described, especially in human skeletal muscle. The purpose of this study was to determine changes in leucyl-tRNA synthetase (LARS/LARS) and Ras-related GTP binding B (RAGB/RAGB) mRNA and protein expression in healthy human skeletal muscle after acute EAA ingestion. Muscle biopsies sampled from the vastus lateralis were obtained from 13 young adults (7 males, 6 females; aged 22.9 ± 0.9 y; body mass index 21.7 ± 0.9 kg/m(2)) in the fasting state (baseline) and 1 and 3 h after EAA (13 g; 2.4 g of Leu) ingestion. Real-time quantitative polymerase chain reaction and Western blotting were used to determine changes in LARS/LARS and RAGB/RAGB mRNA and protein expression, respectively. Stable isotope tracers and gas chromatography mass spectrometry were used to determine Leu intracellular concentrations and muscle protein synthesis. EAA ingestion increased RAGB/RAGB mRNA (∼60%) and protein (∼100%) abundance in adult skeletal muscle (P ≤ 0.05). EAAs also increased muscle Leu concentrations (∼130%), mTOR phosphorylation (∼30%), and muscle protein synthesis (∼50%; P ≤ 0.05) but did not alter muscle LARS/LARS abundance (P > 0.05). We conclude that acute EAA ingestion is capable of increasing RAGB expression in human skeletal muscle. Future work is needed to determine whether this adaptive response is important to promote muscle protein anabolism in humans. This trial was registered at clinicaltrials.gov as NCT01669590.

No additional effect of different types of physical activity on 10-hour muscle protein synthesis in elderly men on a controlled energy- and protein-sufficient diet.

PURPOSE The elderly lose skeletal muscle mass with age, which may be detrimental for function and quality of life. Both inactivity and heavy resistance exercise are known to have marked but opposite effects upon muscle mass. However, the potential effects of daily physical activity upon muscle protein synthesis (MPS) are less investigated. The aim of this study was to determine the effects of daily physical activities upon MPS in elderly individuals. METHODS A total of 24 elderly men (70±1year) were recruited and randomly assigned: inactivity in form of bed-rest (IA), daily physical activities (DA), or heavy resistance exercise (RE). All groups undertook a normal eating routine containing carbohydrates (52 E%), fat (32 E%), and protein (16 E%). Ingestion of labeled milk protein ([1-(13)C]leucine-labeled whey and caseinate) served to maintain tracer enrichment for determination of 10-hour myofibrillar protein fractional synthesis rates (FSR), and typical prerequisites for calculating FSR were fulfilled. Physical activities were monitored, and venous blood and muscle biopsies collected. RESULTS Physical activity was highest in the DA compared to both the IA and RE groups. Nutrient ingestion increased insulin, leucine, and phenylalanine plasma concentrations in all groups. [1-(13)C]leucine enrichment was stable throughout the 10-hour FSR period. Myofibrillar protein FSR were similar for IA, DA, and RE groups, 0.055±0.003%/h, 0.058±0.006%/h, and 0.065±0.008%/h, respectively (means±SE, P=0.44). CONCLUSIONS In elderly males, inactivity, daily activities, and resistance exercise interventions result in equal 10-hour, whole day MPS during an energy- and protein-sufficient diet regimen.

Skeletal muscle morphology, protein synthesis and gene expression in Ehlers Danlos Syndrome

Patients with Ehlers-Danlos syndrome (EDS) are known to have genetically impaired connective tissue and skeletal muscle symptoms in form of pain, fatigue, and cramps; however earlier studies have not been able to link these symptoms to morphological muscle changes. We obtained skeletal muscle biopsies in patients with classic EDS [cEDS; n = 5 (Denmark)+ 8 (The Netherlands)] and vascular EDS (vEDS; n = 3) and analyzed muscle fiber morphology and content (Western blotting and muscle fiber type/area distributions) and muscle mRNA expression and protein synthesis rate (RT-PCR and stable isotope technique). The cEDS patients did not differ from healthy controls (n = 7-11) with regard to muscle fiber type/area, myosin/α-actin ratio, muscle protein synthesis rate, or mRNA expression. In contrast, the vEDS patients demonstrated higher expression of matrix proteins compared with cEDS patients (fibronectin and MMP-2). The cEDS patients had surprisingly normal muscle morphology and protein synthesis, whereas vEDS patients demonstrated higher mRNA expression for extracellular matrix remodeling in skeletal musculature compared with cEDS patients.NEW & NOTEWORTHY This study is the first of its kind to systematically investigate muscle biopsies from Ehlers-Danlos patients, focusing on muscle structure and function. These patients suffer from severe muscle symptoms, but in our study they show surprisingly normal muscle findings, which points toward indirect muscle symptoms originating from the surrounding connective tissue. These findings have basal physiological importance and implications for future physiotherapeutic treatment options for these patients.