The influence of prolonged strength training upon muscle and fat in healthy and chronically diseased older adults.

BACKGROUND: Physical muscle function and brain hippocampus size declines with age, accelerating after the age of 60. Strength training over a few months improves physical function, but less is known about how long-term strength training affects physical function and hippocampus volume. Therefore, we aimed to investigate the effect of 1-year strength training of two different intensities upon muscle mass, function, and hippocampus volume in retirement-age individuals. METHODS: In this multidisciplinary randomized controlled trial (clinicaltrials.gov: NCT02123641), participants were allocated to either a) supervised, heavy resistance training (HRT, n = 149, 3/wk), b) moderate intensity resistance training (MIT, n = 154, 3/wk) or c) non-exercise activities (CON, n = 148). 451 participants were randomized (62-70 yrs., women 61%, ≈80% with a chronic medical disease) and 419 were included in the intention-to-treat analysis (n = 143, 144 and 132; HRT, MIT and CON). Changes in muscle power (primary outcome), strength and size, physical function, body composition, hippocampus volume and physical/mental well-being were analyzed. FINDINGS: Of the participants (HRT + MIT), 83% completed training at least 2/week. Leg extensor power was unchanged in all groups, but strength training had a positive effect on isometric knee extensor strength in both groups, whereas an increased muscle mass, cross-sectional area of vastus lateralis muscle, a decreased whole-body fat percentage, visceral fat content and an improved mental health (SF-36) occurred in HRT only. Further, chair-stand performance improved in all groups, whereas hippocampus volume decreased in all groups over time with no influence of strength training. INTERPRETATION: Together, the results indicate that leg extensor power did not respond to long-term supervised strength training, but this type of training in a mixed group of healthy and chronically diseased elderly individuals can be implemented with good compliance and induces consistent changes in physiological parameters of muscle strength, muscle mass and abdominal fat.

Phenylalanine stable isotope tracer labeling of cow milk and meat and human experimental applications to study dietary protein-derived amino acid availability.

BACKGROUND & AIMS: Availability of dietary protein-derived amino acids (AA) is an important determinant for their utilization in metabolism and for protein synthesis. Intrinsic labeling of protein is the only method to directly trace availability and utilization. The purpose of the present study was to produce labeled milk and meat proteins and investigate how dietary protein-derived AA availability is affected by the protein-meal matrix. METHODS: Four lactating cows were infused with L-[ring-d5]phenylalanine and one with L-[15N]phenylalanine for 72 h. Milk was collected, and three of the [d5]phenylalanine cows were subsequently slaughtered. Two human studies were performed to explore plasma AA availability properties utilizing the labeled proteins. One study compared the intake of whey protein either alone or together with carbohydrates-fat food-matrix. The other study compared the intake of meat hydrolysate with minced beef. Cow blood, milk, meat and human blood samples were collected and analyzed by mass spectrometry. RESULTS: Whey and caseinate acquired label to 15-20 mol percent excess (MPE), and the meat proteins reached 0.41-0.73 MPE. The [d5]phenylalanine appeared fast in plasma and peaked 30 min after whey protein alone and meat hydrolysate intake, whereas whey protein with a food-matrix and the meat minced beef postponed the [d5]phenylalanine peak until 2 and 1 h, respectively. CONCLUSIONS: Phenylalanine stable isotope-labeled milk and meat were produced and proved a valuable tool to investigate AA absorption characteristics. Dietary protein in food-matrices showed delayed postprandial plasma AA availability as compared to whey protein alone and meat hydrolysate.

An exploration of the methods to determine the protein-specific synthesis and breakdown rates in vivo in humans.

The present study explores the methods to determine human in vivo protein-specific myofibrillar and collagenous connective tissue protein fractional synthesis and breakdown rates. We found that in human myofibrillar proteins, the protein-bound tracer disappearance method to determine the protein fractional breakdown rate (FBR) (via 2 H2 O ingestion, endogenous labeling of 2 H-alanine that is incorporated into proteins, and FBR quantified by its disappearance from these proteins) has a comparable intrasubject reproducibility (range: 0.09-53.5%) as the established direct-essential amino acid, here L-ring-13 C6 -phenylalanine, incorporation method to determine the muscle protein fractional synthesis rate (FSR) (range: 2.8-56.2%). Further, the determination of the protein breakdown in a protein structure with complex post-translational processing and maturation, exemplified by human tendon tissue, was not achieved in this experimentation, but more investigation is encouraged to reveal the possibility. Finally, we found that muscle protein FBR measured with an essential amino acid tracer prelabeling is inappropriate presumably because of significant and prolonged intracellular recycling, which also may become a significant limitation for determination of the myofibrillar FSR when repeated infusion trials are completed in the same participants.

Investigating circadian clock gene expression in human tendon biopsies from acute exercise and immobilization studies.

PURPOSE: The discovery of musculoskeletal tissues, including muscle, tendons, and cartilage, as peripheral circadian clocks strongly implicates their role in tissue-specific homeostasis. Age-related dampening and misalignment of the tendon circadian rhythm and its outputs may be responsible for the decline in tendon homeostasis. It is unknown which entrainment signals are responsible for the synchronization of the tendon clock to the light-dark cycle. METHODS: We sought to examine any changes in the expression levels of core clock genes (BMAL1, CLOCK, PER2, CRY1, and NR1D1) in healthy human patellar tendon biopsies obtained from three different intervention studies: increased physical activity (leg kicks for 1 h) in young, reduced activity (2 weeks immobilization of one leg) in young, and in old tendons. RESULTS: The expression level of clock genes in human tendon in vivo was very low and a high variation between individuals was found. We were thus unable to detect any differences in core clock gene expression neither after acute exercise nor immobilization. CONCLUSIONS: We are unable to find evidence for an effect of exercise or immobilization on circadian clock gene expression in human tendon samples.

Even effect of milk protein and carbohydrate intake but no further effect of heavy resistance exercise on myofibrillar protein synthesis in older men.

PURPOSE: The responsiveness of older individuals' skeletal muscle to anabolic strategies may be impaired. However, direct comparisons within the same experimental setting are sparse. The aim of this study was to assess the resting and post-resistance exercise muscle protein synthesis rates in response to two types of milk protein and carbohydrate using a unilateral exercise leg model. METHODS: Twenty-seven older (69 ± 1 year, mean ± SE) men were randomly assigned one of three groups: Whey hydrolysate (WH), caseinate (CAS), or carbohydrate (CHO). By applying stable isotope tracer techniques (L-[15N]phenylalanine), the fasted-rested (basal) myofibrillar fractional synthesis rate (FSR) was measured. Hereafter, FSR was measured in the postprandial phase (0.45 g nutrient/kg LBM) in both legs, one rested (fed-rest) and one exercised (10 × 8 reps at 70% 1RM; fed-exercise). In addition, the activity of p70S6K and venous plasma insulin, phenylalanine, and leucine concentrations were measured. RESULTS: Insulin, phenylalanine, and leucine concentrations differed markedly after intake of the different study drinks. The basal FSR in WH, CAS, and CHO were 0.027 ± 0.003, 0.030 ± 0.003, and 0.030 ± 0.004%/h, the fed-rested FSR were 0.043 ± 0.004, 0.045 ± 0.003, and 0.035 ± 0.004%/h, and the fed-exercised FSR were 0.041 ± 0.004, 0.043 ± 0.004, and 0.034 ± 0.004%/h, respectively. No significant differences were observed at any state between the groups. Fed-rested- and fed-exercised FSR were higher than basal (P < 0.001). 3 h after exercise and feeding, no significant group differences were detected in the activity of p70S6K. CONCLUSIONS: Milk protein and carbohydrate supplementation stimulate myofibrillar protein synthesis in older men, with no further effect of heavy resistance exercise within 0-3 h post exercise.

Reproducibility of incremental maximal cycle ergometer tests in healthy recreationally active subjects.

PURPOSE: Testing of the ventilatory threshold (VT) and maximal oxygen uptake (VO2 peak) is relevant for the evaluation of a range of training studies, clinical trials and cross-sectional studies. Due to a possible learning effect, a familiarization test is often performed to increase test reproducibility. However, limited research has investigated this learning effect and reproducibility of maximal exercise testing. The most appropriate ways to determine VT and VO2 peak are not clear, and this study evaluated two approaches (V-slope and a combined method) for the determination of VT and five time-averaging intervals (60, 30, 15, 10 and 5 s) for the determination of VO2 peak to compare test results and reproducibility. METHODS: Thirteen recreational triathletes completed three identical incremental maximal cycle ergometer tests. The initial workload was 75 and 100 watt (W) for women and men, respectively, and the workload was increased by 4 W/10 s thereafter. No familiarization test was performed. RESULTS: VO2 peak increased significantly as the time-averaging interval became shorter (e.g. 5-s interval 48·7 versus 60-s interval 44·8 ml O2  kg-1  min-1 ; overall P<0·001). All test results were similar for the three test rounds, indicating that repeated testing was not associated with any learning effect. The different VT measuring methods (CV 7·6 versus 7·7%, P = 0·58) and VO2 peak time-averaging intervals (CV 3·7-4·4%, P = 0·99) did not influence test reliability. CONCLUSIONS: The reproducibility of VT and VO2 peak was not affected by measuring methods and time-averaging intervals. However, the time-averaging intervals significantly affect the absolute VO2 peak values. Furthermore, no learning effect of maximal cycle ergometer testing was observed.

Tendon collagen synthesis declines with immobilization in elderly humans: no effect of anti-inflammatory medication.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used as pain killers during periods of unloading caused by traumatic occurrences or diseases. However, it is unknown how tendon protein turnover and mechanical properties respond to unloading and subsequent reloading in elderly humans, and whether NSAID treatment would affect the tendon adaptations during such periods. Thus we studied human patellar tendon protein synthesis and mechanical properties during immobilization and subsequent rehabilitating resistance training and the influence of NSAIDs upon these parameters. Nineteen men (range 60-80 yr) were randomly assigned to NSAIDs (ibuprofen 1,200 mg/day; Ibu) or placebo (Plc). One lower limb was immobilized in a cast for 2 wk and retrained for 6 wk. Tendon collagen protein synthesis, mechanical properties, size, expression of genes related to collagen turnover and remodeling, and signal intensity (from magnetic resonance imaging) were investigated. Tendon collagen synthesis decreased (P < 0.001), whereas tendon mechanical properties and size were generally unchanged with immobilization, and NSAIDs did not influence this. Matrix metalloproteinase-2 mRNA tended to increase (P < 0.1) after immobilization in both groups, whereas scleraxis mRNA decreased with inactivity in the Plc group only (P < 0.05). In elderly human tendons, collagen protein synthesis decreased after 2 wk of immobilization, whereas tendon stiffness and modulus were only marginally reduced, and NSAIDs had no influence upon this. This indicates an importance of mechanical loading for maintenance of tendon collagen turnover. However, reduced collagen production induced by short-term unloading may only marginally affect tendon mechanical properties in elderly individuals. NEW & NOTEWORTHY: In elderly humans, 2 wk of inactivity reduces tendon collagen protein synthesis, while tendon stiffness and modulus are only marginally reduced, and NSAID treatment does not affect this. This indicates that mechanical loading is important for maintenance of tendon collagen turnover and that changes in collagen turnover induced by short-term immobilization may only have minor impact on the internal structures that are essential for mechanical properties in elderly tendons.

Physical activity as intervention for age-related loss of muscle mass and function: protocol for a randomised controlled trial (the LISA study).

INTRODUCTION: Physical and cognitive function decline with age, accelerating during the 6th decade. Loss of muscle power (force×velocity product) is a dominant physical determinant for loss of functional ability, especially if the lower extremities are affected. Muscle strength training is known to maintain or even improve muscle power as well as physical function in older adults, but the optimal type of training for beneficial long-term training effects over several years is unknown. Moreover, the impact of muscle strength training on cognitive function and brain structure remains speculative. The primary aim of this randomised controlled trial is to compare the efficacy of two different 1 year strength training regimens on immediate and long-lasting improvements in muscle power in retirement-age individuals. Secondary aims are to evaluate the effect on muscle strength, muscle mass, physical and cognitive function, mental well-being, health-related quality of life and brain morphology. METHODS AND ANALYSIS: The study includes 450 home-dwelling men and women (62-70 years). Participants are randomly allocated to (1) 1 year of supervised, centre-based heavy resistance training, (2) home-based moderate intensity resistance training or (3) habitual physical activity (control). Changes in primary (leg extensor power) and secondary outcomes are analysed according to the intention to treat principle and per protocol at 1, 2, 4, 7 and 10 years. ETHICS AND DISSEMINATION: The study is expected to generate new insights into training-induced promotion of functional ability and independency after retirement and will help to formulate national recommendations regarding physical activity schemes for the growing population of older individuals in western societies. Results will be published in scientific peer-reviewed journals, in PhD theses and at public meetings. The study is approved by the Regional Ethical Committee (Capital Region, Copenhagen, Denmark, number H-3-2014-017). TRIAL REGISTRATION NUMBER: NCT02123641.

Effects of eHealth physical activity encouragement in adolescents with complex congenital heart disease: The PReVaiL randomized clinical trial.

OBJECTIVE: To assess benefit and harms of adding an eHealth intervention to health education and individual counseling in adolescents with congenital heart disease. DESIGN: Randomized clinical trial. SETTING: Denmark. PATIENTS: A total of 158 adolescents aged 13-16years with no physical activity restrictions after repaired complex congenital heart disease. INTERVENTIONS: PReVaiL consisted of individually tailored eHealth encouragement physical activity for 52weeks. All patients received 45min of group-based health education and 15min of individual counseling involving patients' parents. OUTCOMES: The primary outcome was maximal oxygen uptake (VO2 peak) at 52weeks after randomization. The secondary outcome was physical activity. Exploratory outcomes were generic and disease-specific questionnaires. RESULTS: In the intervention group, 58 patients (72%) completed the final test, but of those, only 46 (57%) fulfilled the compliance criteria of using the eHealth application for at least 2 consecutive weeks. In the control group, 61 patients (79%) completed both exercise tests. Adjusted for baseline values, the difference between the intervention group and the control group in mean VO2 peak at 1year was -0.65ml·kg(-1)·min(-1) (95% CI -2.66 to 1.36). Between-group differences at 1year in physical activity, generic health-related quality of life, and disease-specific quality of life were not statistically significant. CONCLUSIONS: Adding a tailored eHealth intervention to health education and individual counseling did not affect outcomes among adolescents with congenital heart disease. Our results do not support the use of this eHealth intervention in adolescents with complex congenital heart disease. TRIAL REGISTRATION: Clinical trials.gov identifier: NCT01189981.

Preserved skeletal muscle protein anabolic response to acute exercise and protein intake in well-treated rheumatoid arthritis patients.

INTRODUCTION: Rheumatoid arthritis (RA) is often associated with diminished muscle mass, reflecting an imbalance between protein synthesis and protein breakdown. To investigate the anabolic potential of both exercise and nutritional protein intake we investigated the muscle protein synthesis rate and anabolic signaling response in patients with RA compared to healthy controls. METHODS: Thirteen RA patients (age range 34-84 years; diagnosed for 1-32 years, median 8 years) were individually matched with 13 healthy controls for gender, age, BMI and activity level (CON). Plasma levels of C-reactive protein (CRP), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay (ELISA) in resting blood samples obtained on two separate days. Skeletal muscle myofibrillar and connective tissue protein fractional synthesis rate (FSR) was measured by incorporation of the amino acid (13)C6-phenylalanine tracer in the overnight fasted state for 3 hours (BASAL) and 3 hours after intake of whey protein (0.5 g/kg lean body mass) alone (PROT, 3 hrs) and in combination with knee-extensor exercise (EX) with one leg (8 × 10 reps at 70 % of 1RM; PROT + EX, 3 hrs). Expression of genes related to inflammatory signaling, myogenesis and muscle growth/atrophy were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: CRP was significantly higher in the RA patients (2.25 (0.50) mg/l) than in controls (1.07 (0.25) mg/l; p = 0.038) and so was TNF-α (RA 1.18 (0.30) pg/ml vs. CON 0.64 (0.07) pg/ml; p = 0.008). Muscle myofibrillar protein synthesis in both RA patients and CON increased in response to PROT and PROT + EX, and even more with PROT + EX (p < 0.001), with no difference between groups (p > 0.05). The gene expression response was largely similar in RA vs. CON, however, expression of the genes coding for TNF-α, myogenin and HGF1 were more responsive to exercise in RA patients than in CON. CONCLUSIONS: The study demonstrates that muscle protein synthesis rate and muscle gene expression can be stimulated by protein intake alone and in combination with physical exercise in patients with well-treated RA to a similar extent as in healthy individuals. This indicates that moderately inflamed RA patients have maintained their muscle anabolic responsiveness to physical activity and protein intake.

Health-related fitness profiles in adolescents with complex congenital heart disease.

PURPOSE: This study investigates whether subgroups of different health-related fitness (HrF) profiles exist among girls and boys with complex congenital heart disease (ConHD) and how these are associated with lifestyle behaviors. METHODS: We measured the cardiorespiratory fitness, muscle strength, and body composition of 158 adolescents aged 13-16 years with previous surgery for a complex ConHD. Data on lifestyle behaviors were collected concomitantly between October 2010 and April 2013. A cluster analysis was conducted to identify profiles with similar HrF. For comparisons between clusters, multivariate analyses of covariance were used to test the differences in lifestyle behaviors. RESULTS: Three distinct profiles were formed: (1) Robust (43, 27%; 20 girls and 23 boys); (2) Moderately Robust (85, 54%; 37 girls and 48 boys); and (3) Less robust (30, 19%; 9 girls and 21 boys). The participants in the Robust clusters reported leading a physically active lifestyle and participants in the Less robust cluster reported leading a sedentary lifestyle. Diagnoses were evenly distributed between clusters. CONCLUSIONS: The cluster analysis attributed some of the variability in cardiorespiratory fitness among adolescents with complex ConHD to lifestyle behaviors and physical activity. Profiling of HrF offers a valuable new option in the management of person-centered health promotion.

The single-biopsy approach in determining protein synthesis in human slow-turning-over tissue: use of flood-primed, continuous infusion of amino acid tracers.

Muscle protein synthesis (MPS) rate is determined conventionally by obtaining two or more tissue biopsies during a primed, continuous infusion of a stable isotopically labeled amino acid. The purpose of the present study was to test whether tracer priming given as a flooding dose, thereby securing an instantaneous labeling of the tissue pools of free tracee amino acids, followed by a continuous infusion of the same tracer to maintain tracer isotopic steady state, could be used to determine the MPS rate over a prolonged period of time by obtaining only a single tissue biopsy. We showed that the tracer from the flood prime appeared immediately in the muscle free pool of amino acids and that this abundance could be kept constant by a subsequent continuous infusion of the tracer. When using phenylalanine as tracer, the flood-primed, continuous infusion protocol does not stimulate the MPS rate per se. In conclusion, the flood-primed, continuous infusion protocol using phenylalanine as tracer can validly be used to measure the protein synthesis rate in human in vivo experiments by obtaining only a single tissue biopsy after a prolonged infusion period.

Muscle and tendon connective tissue adaptation to unloading, exercise and NSAID.

The extracellular matrix network of skeletal muscle and tendon connective tissue is primarily composed of collagen and connects the muscle contractile protein to the bones in the human body. The mechanical properties of the connective tissue are important for the effectiveness of which the muscle force is transformed into movement. Periods of unloading and exercise affect the synthesis rate of connective tissue collagen protein, whereas only sparse information exits regarding collagen protein degradation. It is likely, though, that changes in both collagen protein synthesis and degradation are required for remodeling of the connective tissue internal structure that ultimately results in altered mechanical properties of the connective tissue. Both unloading and exercise lead to increased production of growth factors and inflammatory mediators that are involved in connective tissue remodeling. Despite the fact that non-steroidal anti-inflammatory drugs seem to inhibit the healing process of connective tissue and the stimulating effect of exercise on connective tissue protein synthesis, these drugs are often consumed in relation to connective tissue injury and soreness. However, the potential effect of non-steroidal anti-inflammatory drugs on connective tissue needs further investigation.

Effect of acute exercise on patella tendon protein synthesis and gene expression.

Evidence suggests that habitual loading can result in patellar tendon hypertrophy, especially at the proximal and distal parts of the patellar tendon. The underlying protein kinetic changes and its regulation remains controversial and human data, investigating this topic, are limited. The present study investigated how acute exercise affects growth factor production and collagen fractional synthetic rate in patellar tendon tissue from patients undergoing an anterior cruciate ligament reconstruction operation. The operation was performed by use of the bone-patellar tendon-bone method under spinal anesthesia. Twelve subjects were randomized to one of two groups: a control group or an exercise group (1-hr unilateral knee-extension 67% of Wmax 24 hours before operation). Two hours before the anterior cruciate ligament operation a flooding-dose of L-[1-(13)C]proline was given. Tissue from the most proximal part of the patellar tendon was obtained during the operation. Tendon collagen fractional synthetic rate and mRNA concentrations of TGF-ß-1, CTGF, and type I and III collagen were measured. CTGF and type I collagen expression were higher in the exercise group compared to the control group (p < 0.05). Type III collagen expression (p = 0.11), TGF-ß-1 expression (p = 0.34), and collagen fractional synthetic rate (p = 0.26) did not differ between groups. Although the expression of CTGF and type I collagen were higher, the patellar tendon collagen fractional synthetic rate was not correspondingly higher after exercise. The elevated CTGF expression in the exercise group indicates that the TGF-beta pathway could be an important link between mechanical loading and stimulation of tendon tissue type I collagen expression.

Influence of amino acids, dietary protein, and physical activity on muscle mass development in humans.

Ingestion of protein is crucial for maintenance of a variety of body functions and within the scope of this review we will specifically focus on the regulation of skeletal muscle mass. A quantitative limitation exists as to how much muscle protein the body can synthesize in response to protein intake. Ingestion of excess protein exerts an unwanted load to the body and therefore, it is important to find the least amount of protein that provides the maximal hypertrophic stimulus. Hence, research has focused on revealing the relationship between protein intake (dose) and its resulting stimulation of muscle protein synthesis (response). In addition to the protein amount, the protein digestibility and, hence, the availability of its constituent amino acids is decisive for the response. In this regard, recent studies have provided in-depth knowledge about the time-course of the muscle protein synthetic response dependent on the characteristics of the protein ingested. The effect of protein intake on muscle protein accretion can further be stimulated by prior exercise training. In the ageing population, physical training may counteract the development of "anabolic resistance" and restore the beneficial effect of protein feeding. Presently, our knowledge is based on measures obtained in standardized experimental settings or during long-term intervention periods. However, to improve coherence between these types of data and to further improve our knowledge of the effects of protein ingestion, other investigative approaches than those presently used are requested.

The anabolic potential of dietary protein intake on skeletal muscle is prolonged by prior light-load exercise.

BACKGROUND & AIMS: Hyperaminoacidemia stimulates myofibrillar fractional synthesis rate (myoFSR) transiently in resting skeletal muscle. We investigated whether light-load resistance exercise can extent this responsiveness. METHODS: Ten healthy males exercised one leg with a light-load resistance-like exercise at 16% of 1 repetition maximum and received oral protein boluses every hour for a 10-h period. Their myoFSR was determined by [1-(13)C]-leucine incorporation. Muscle biopsies were obtained from the resting (REST) and exercised (EXC) muscles every 2.5-h in the protein-fed period. RESULTS: Protein feeding significantly elevated plasma leucine and essential amino acids by an average of 39 ± 9% (mean ± SEM) and 20 ± 4%, respectively, compared to the basal concentrations: 197 ± 12 µmol L(-1) and 854 ± 35 µmol L(-1), respectively. The myoFSR was similar in EXC and REST muscles in the first 8 h (all time intervals p > 0.05). After 8 h the myoFSR dropped in the REST muscle to 0.041 ± 0.005%·h(-1), which was 65 ± 5% of the rate in EXC leg at the same time point (0.062 ± 0.004%·h(-1)) and 80 ± 14% of the level in REST leg from 0.5 to 8 h (0.056 ± 0.005%·h(-1)) (interaction p < 0.05). CONCLUSIONS: Compared to rest, light-load exercise prolonged the stimulatory effect of dietary protein on muscle biosynthesis providing perspectives for a muscle restorative effect in clinical settings where strenuous activity is intolerable.

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 3ß (GSK3ß) 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, GSK3ß, 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.