Anabolic effects of oral leucine-rich protein with and without ß-hydroxybutyrate on muscle protein metabolism in a novel clinical model of systemic inflammation-a randomized crossover trial.

BACKGROUND: ß-lactoglobulin (BLG) stimulates muscle protein synthesis and ß-hydroxybutyrate (BHB) inhibits muscle breakdown. Whether combining the 2 can additively attenuate disease-induced muscle loss is unknown. OBJECTIVE: Based on previous observations of anticatabolic effects of protein and ketone bodies during inflammation, and using a novel model combining ongoing systemic inflammation, fasting, and immobilization, we tested whether the anticatabolic muscle response to oral amino acids is altered compared with control conditions, as well as whether coadministration of oral BHB and BLG further improves the muscle anabolic response. Muscle net balance (NBphe) was the primary outcome and intramyocellular signals were assessed. METHODS: In a randomized crossover design, 8 young men underwent either preconditioning with LPS (prestudy day: 1 ng/kg, study day: 0.5 ng/kg) combined with a 36-h fast and bed rest to mimic catabolic inflammatory disease (CAT) or an overnight fast (control [CTR]) prior to isocaloric nutritional interventions on 3 occasions separated by ∼6 wk (range 42 to 83 d). RESULTS: NBphe increased similarly upon all conditions (interaction P = 0.65). From comparable baseline rates, both Rdphe [muscle synthesis, median ratio (95% CI): 0.44 (0.23, 0.86) P = 0.017] and Raphe [muscle breakdown, median ratio (95% CI): 0.46 (0.27, 0.78) P = 0.005] decreased following BHB + BLG compared with BLG. BLG increased Rdphe more under CAT conditions compared with CTR (interaction P = 0.02). CAT increased inflammation, energy expenditure, and lipid oxidation and decreased Rdphe and anabolic signaling [mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E-binding protein 1 (4EPB1) phosphorylation]. CONCLUSION: In contrast to our initial hypothesis, NBphe increased similarly following BLG during CAT and CTR conditions; CAT however, specifically stimulated the BLG-mediated increase in protein synthesis, whereas BHB coadministration did not affect NBphe, but distinctly dampened the BLG-induced increase in muscle amino acid fluxes thereby liberating circulating amino acids for anabolic actions elsewhere.

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

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 TNFα was not different between groups, while IL1-receptor and TNFα-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.

No effect of anti-inflammatory medication on postprandial and postexercise muscle protein synthesis in elderly men with slightly elevated systemic inflammation.

BACKGROUND: Based on circulating C-reactive protein (CRP) levels, some individuals develop slightly increased inflammation as they age. In elderly inflamed rats, the muscle response to protein feeding is impaired, whereas it can be maintained by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). It is unknown whether this applies to elderly humans with increased inflammation. Thus, the muscle response to whey protein bolus ingestion with and without acute resistance exercise was compared between healthy elderly individuals and elderly individuals with slightly increased inflammation±NSAID treatment. METHODS: Twenty-four elderly men (>60years) were recruited. Of those, 14 displayed a slightly increased systemic inflammation (CRP>2mg/l) and were randomly assigned to NSAID (Ibuprofen 1800mg/day) or placebo treatment for 1week. The remaining 10 elderly individuals served as healthy controls (CRP<1mg/l). The muscle protein synthetic response was measured as the fractional synthetic rate (FSR) and p70S6K phosphorylation-to-total protein ratio. RESULTS: The basal myofibrillar FSR and the myofibrillar FSR responses to whey protein bolus ingestion with and without acute resistance exercise were maintained in inflamed elderly compared to healthy controls (p>0.05) and so was p70S6K phosphorylation. Moreover, NSAID treatment did not significantly improve the myofibrillar and connective tissue FSR responses or reduce the plasma CRP level in inflamed, elderly individuals (p>0.05). CONCLUSION: A slight increase in systemic inflammation does not affect the basal myofibrillar FSR or the myofibrillar FSR responses, which suggests that elderly individuals with slightly increased inflammation can benefit from protein ingestion and resistance exercise to stimulate muscle protein anabolism. Moreover, the NSAID treatment did not significantly affect the myofibrillar or connective tissue FSR responses to protein ingestion and acute resistance exercise.

Interactions between muscle stem cells, mesenchymal-derived cells and immune cells in muscle homeostasis, regeneration and disease.

Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells (also known as fibro-adipogenic progenitors (FAPs)) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise.

Alterations in molecular muscle mass regulators after 8 days immobilizing Special Forces mission.

In military operations, declined physical capacity can endanger the life of soldiers. During special support and reconnaissance (SSR) missions, Special Forces soldiers sustain 1-2 weeks full-body horizontal immobilization, which impairs muscle strength and performance. Adequate muscle mass and strength are necessary in combat or evacuation situations, which prompt for improved understanding of muscle mass modulation during SSR missions. To explore the molecular regulation of myofiber size during a simulated SSR operation, nine male Special Forces soldiers were biopsied in m. vastus lateralis pre and post 8 days immobilizing restricted prone position. After immobilization, total mammalian target of rapamycin protein was reduced by 42% (P < 0.05), whereas total and phosphorylated protein levels of Akt, ribosomal protein S6k, 4E-BP1, and glycogen synthase kinase3ß were unchanged. Messenger RNA (mRNA) levels of the atrogenes forkhead box O3 (FoxO3), atrogin1, and muscle ring finger protein1 (MuRF1) increased by 36%, 53%, and 71% (P < 0.01), MuRF1 protein by 51% (P = 0.05), whereas FoxO1 and peroxisome proliferator-activated receptor γ coactivator-1 ß mRNAs decreased by 29% and 40% (P < 0.01). In conclusion, occupational immobilization in Special Forces soldiers led to modulations in molecular muscle mass regulators during 8 days prone SSR mission, which likely contribute to muscle loss observed in such operations. The present data expand our knowledge of human muscle mass regulation during short-term immobilization.

Differential satellite cell density of type I and II fibres with lifelong endurance running in old men.

AIM: To investigate the influence of lifelong endurance running on the satellite cell pool of type I and type II fibres in healthy human skeletal muscle. METHODS: Muscle biopsies were collected from 15 healthy old trained men (O-Tr) who had been running 43 ± 16 (mean ± SD) kilometres a week for 28 ± 9 years. Twelve age-matched untrained men (O-Un) and a group of young trained and young untrained men were recruited for comparison. Frozen sections were immunohistochemically stained for Pax7, type I myosin and laminin, from which fibre area, the number of satellite cells, and the relationship between these variables were determined. RESULTS: In O-Un and O-Tr, type II fibres were smaller and contained fewer satellite cells than type I fibres. However, when expressed relative to fibre area, the difference in satellite cell content between fibre types was eliminated in O-Tr, but not O-Un. A strong positive relationship between fibre size and satellite cell content was detected in trained individuals. In line with a history of myofibre repair, a greater number of fibres with centrally located myonuclei were detected in O-Tr. CONCLUSION: Lifelong endurance training (i) does not deplete the satellite cell pool and (ii) is associated with a similar density of satellite cells in type I and II fibres despite a failure to preserve the equal fibre type distribution of satellite cells observed in young individuals. Taken together, these data reveal a differential regulation of satellite cell content between fibre types, in young and old healthy men with dramatically different training histories.

Life-long endurance exercise in humans: circulating levels of inflammatory markers and leg muscle size.

Human aging is associated with a loss of skeletal muscle and an increase in circulating inflammatory markers. It is unknown whether endurance training (Tr) can prevent these changes. Therefore we studied 15 old trained (O-Tr) healthy males and, for comparison, 12 old untrained (O-Un), 10 Young-Tr (Y-Tr) and 12 Young-Un (Y-Un). Quadriceps size, VO2 peak, CRP, IL-6, TNF-α and its receptors, suPAR, lipid profile, leucocytes and glucose homeostasis were measured. Tr was associated with an improved insulin profile (p<0.05), and lower leucocyte (p<0.05) and triglyceride levels (p<0.05), independent of age. Aging was associated with poorer glucose control (p<0.05), independent of training. The age-related changes in waist circumference, VO2 peak, cholesterol, LDL, leg muscle size, CRP and IL-6 were counteracted by physical activity (p<0.05). A significant increase in suPAR with age was observed (p<0.05). Most importantly, life-long endurance exercise was associated with a lower level of the inflammatory markers CRP and IL-6 (p<0.05), and with a greater thigh muscle area (p<0.05), compared to age-matched untrained counterparts. These findings in a limited group of individuals suggest that regular physical endurance activity may play a role in reducing some markers of systemic inflammation, even within the normal range, and in maintaining muscle mass with aging.

The heat shock protein response following eccentric exercise in human skeletal muscle is unaffected by local NSAID infusion.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed in relation to pain and injuries in skeletal muscle, but may adversely affect muscle adaptation probably via inhibition of prostaglandin synthesis. Induction of heat shock proteins (HSP) represents an important adaptive response in muscle subjected to stress, and in several cell types including cardiac myocytes prostaglandins are important in induction of the HSP response. This study aimed to determine the influence of NSAIDs on the HSP response to eccentric exercise in human skeletal muscle. Healthy males performed 200 maximal eccentric contractions with each leg with intramuscular infusion of the NSAID indomethacin or placebo. Biopsies were obtained from m. vastus lateralis before and after (5, 28 hrs and 8 days) the exercise bout from both legs (NSAID vs unblocked leg) and analysed for expression of the HSPs HSP70, HSP27 and αB-crystallin (mRNA and protein). NSAID did not affect the mRNA expression of any of the HSPs. Compared to pre values, the mRNA expression of all HSPs was increased; αB-crystallin, 3.6- and 5.4-fold; HSP70, 26- and 3.4-fold; and HSP27: 4.8- and 6.5-fold at 5 and 28 hrs post-exercise, respectively (all p < 0.008). Immunohistochemical stainings for αB-crystallin and HSP70 revealed increased staining in some samples but with no differences between legs. Changes in force-generating capacity correlated with both αB-crystallin and HSP70 mRNA and immunohistochemisty data. Increased expression of HSPs was observed on mRNA and protein level following eccentric exercise; however, this response was unaffected by local intramuscular infusion of NSAIDs.

Rehabilitation of muscle after injury - the role of anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed among athletes worldwide in relation to muscle injury and soreness. This review aims to provide an overview of studies investigating their effects on skeletal muscle, in particular the repair processes in injured muscle. Muscle injury occurs in diverse situations and the nature of muscle injuries varies significantly, complicating extrapolations between experimental models and "real life." Classical muscle strain injuries occur at the interphase between the muscle fibers and connective tissue, most often in the myotendinuous junction, whereas contusion or overload injury can damage both myofibers and intramuscular connective tissue. The role of NSAIDs in muscle repair is complicated by differences in injury models used, variables evaluated, and time point(s) selected for evaluations. While the temporal pattern of the influence of NSAIDs on muscle repair is difficult to settle on, it appears that a potential beneficial effect of NSAIDs in the early phase after injury is not maintained in the long term, or is even negated by a long-term repair deficit. At the cellular level, evidence exists for a negative influence of NSAIDs on the muscle stem cell population (satellite cells). At a structural level, it is known that muscle connective tissue undergoes significant remodeling during muscle regeneration, but the potential of NSAID exposure to alter this response in humans needs investigation.

Local NSAID infusion does not affect protein synthesis and gene expression in human muscle after eccentric exercise.

Unaccustomed exercise leads to satellite cell proliferation and increased skeletal muscle protein turnover. Several growth factors and cytokines may be involved in the adaptive responses. Non-steroidal anti-inflammatory drugs (NSAIDs) negatively affect muscle regeneration and adaptation in animal models, and inhibit the exercise-induced satellite cell proliferation and protein synthesis in humans. However, the cellular mechanisms eliciting these responses remain unknown. Eight healthy male volunteers performed 200 maximal eccentric contractions with each leg. To block prostaglandin synthesis locally in the skeletal muscle, indomethacin (NSAID) was infused for 7.5 h via microdialysis catheters into m. vastus lateralis of one leg. Protein synthesis was determined by the incorporation of 1,2-(13) C(2) leucine into muscle protein from 24 to 28 h post-exercise. Furthermore, mRNA expression of selected genes was measured in muscle biopsies (5 h and 8 days post-exercise) by real-time reverse transcriptase PCR. Myofibrillar and collagen protein synthesis were unaffected by the local NSAID infusion. Five hours post-exercise, the mRNA expression of cyclooxygenase-2 (COX2) was sixfold higher in the NSAID leg (P=0.016) compared with the unblocked leg. The expression of growth factors and matrix-related genes were unaffected by NSAID. Although NSAIDs inhibit the exercise-induced satellite cell proliferation, we observed only limited effects on gene expression, and on post-exercise protein synthesis.

Early osteoarthritis and microdialysis: a novel in vivo approach for measurements of biochemical markers in the perisynovium and intraarticularly.

The microdialysis technique was evaluated as a possible method to obtain local measurements of biochemical markers from knee joints with degenerative changes. Seven patients scheduled for arthroscopy of the knee due to minor to moderate degenerative changes had microdialysis catheters inserted under ultrasonographic guidance, intraarticularly and in the synovium-close tissue. Catheters were perfused at a rate of 2 µl/min for approximately 100 min with a Ringer solution containing radioactively labeled glucose, and the positions of the catheters were later visualized during arthroscopy. All intraarticular catheters and 6/7 subsynovial catheters were positioned correctly. Relative recovery (RR) was intraarticularly 0.64 ± 0.02 (mean ± SEM) and synovium-close 0.54 ± 0.06. Mean values of cartilage oligomeric matrix protein (COMP), aggrecan and glucosyl-galactosyl-pyridinoline in the intraarticular dialysates were 18.1 ± 7.0 U/l, 243.6 ± 108.6 ng/ml and 108.0 ± 29.0 pmol/ml, respectively. COMP and glucosyl-galactosyl-pyridinoline concentrations were significantly higher intraarticularly compared to perisynovial tissue (P < 0.05), whereas for aggrecan, no significant difference was found (P = 0.06). The microdialysis method can be used for intraarticular and subsynovial determination of metabolites in human knees at these sites. The present methodology displays a potential for future studies of simultaneous biochemical changes within and around joints.

Local NSAID infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise.

Despite the widespread consumption of nonsteroidal anti-inflammatory drugs (NSAIDs), the influence of these drugs on muscle satellite cells is not fully understood. The aim of the present study was to investigate the effect of a local NSAID infusion on satellite cells after unaccustomed eccentric exercise in vivo in human skeletal muscle. Eight young healthy males performed 200 maximal eccentric contractions with each leg. An NSAID was infused via a microdialysis catheter into the vastus lateralis muscle of one leg (NSAID leg) before, during, and for 4.5 h after exercise, with the other leg working as a control (unblocked leg). Muscle biopsies were collected before and 8 days after exercise. Changes in satellite cells and inflammatory cell numbers were investigated by immunohistochemistry. Satellite cells were identified using antibodies against neural cell adhesion molecule and Pax7. The number of Pax7(+) cells per myofiber was increased by 96% on day 8 after exercise in the unblocked leg (0.14 +/- 0.04, mean +/- SE) compared with the prevalue (0.07 +/- 0.02, P < 0.05), whereas the number of Pax7(+) cells was unchanged in the leg muscles exposed to the NSAID (0.07 +/- 0.01). The number of inflammatory cells (CD68(+) or CD16(+) cells) was not significantly increased in either of the legs 8 days after exercise and was unaffected by the NSAID. The main finding in the present study was that the NSAID infusion for 7.5 h during the exercise day suppressed the exercise-induced increase in the number of satellite cells 8 days after exercise. These results suggest that NSAIDs negatively affect satellite cell activity after unaccustomed eccentric exercise.