Philip J. Atherton

Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle

The nature of the deficit underlying age‐related muscle wasting remains controversial. To test whether it could be due to a poor anabolic response to dietary amino acids, we measured the rates of myofibrillar and sarcoplasmic muscle protein synthesis (MPS) in 44 healthy young and old men, of similar body build, after ingesting different amounts of essential amino acids (EAA). Basal rates of MPS were indistinguishable, but the elderly showed less anabolic sensitivity and responsiveness of MPS to EAA, possibly due to decreased intramuscular expression, and activation (phosphorylation) after EAA, of amino acid sensing/signaling proteins (mammalian target of rapamycin, mTOR; p70 S6 kinase, or p70S6k; eukaryotic initiation factor [eIF]4BP‐1; and eIF2B). The effects were independent of insulin signaling since plasma insulin was clamped at basal values. Associated with the anabolic deficits were marked increases in NFκB, the inflammation‐associated transcription factor. These results demonstrate first, EAA stimulate MPS independently of increased insulin availability; second, in the elderly, a deficit in MPS in the basal state is unlikely; and third, the decreased sensitivity and responsiveness of MPS to EAA, associated with decrements in the expression and activation of components of anabolic signaling pathways, are probably major contributors to the failure of muscle maintenance in the elderly. Countermeasures to maximize muscle maintenance should target these deficits.

Age-related differences in the dose–response relationship of muscle protein synthesis to resistance exercise in young and old men

We investigated how myofibrillar protein synthesis (MPS) and muscle anabolic signalling were affected by resistance exercise at 20–90% of 1 repetition maximum (1 RM) in two groups (25 each) of post‐absorptive, healthy, young (24 ± 6 years) and old (70 ± 5 years) men with identical body mass indices (24 ± 2 kg m−2). We hypothesized that, in response to exercise, anabolic signalling molecule phosphorylation and MPS would be modified in a dose‐dependant fashion, but to a lesser extent in older men. Vastus lateralis muscle was sampled before, immediately after, and 1, 2 and 4 h post‐exercise. MPS was measured by incorporation of [1,2‐13C] leucine (gas chromatography–combustion–mass spectrometry using plasma [1,2‐13C]α‐ketoisocaparoate as surrogate precursor); the phosphorylation of p70 ribosomal S6 kinase (p70s6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was measured using Western analysis with anti‐phosphoantibodies. In each group, there was a sigmoidal dose–response relationship between MPS at 1–2 h post‐exercise and exercise intensity, which was blunted (P < 0.05) in the older men. At all intensities, MPS fell in both groups to near‐basal values by 2–4 h post‐exercise. The phosphorylation of p70s6K and 4EBP1 at 60–90% 1 RM was blunted in older men. At 1 h post‐exercise at 60–90% 1 RM, p70s6K phosphorylation predicted the rate of MPS at 1–2 h post‐exercise in the young but not in the old. The results suggest that in the post‐absorptive state: (i) MPS is dose dependant on intensity rising to a plateau at 60–90% 1 RM; (ii) older men show anabolic resistance of signalling and MPS to resistance exercise.

Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.

Resistance (RE) and endurance (EE) exercise stimulate mixed skeletal muscle protein synthesis. The phenotypes induced by RE (myofibrillar protein accretion) and EE (mitochondrial expansion) training must result from differential stimulation of myofibrillar and mitochondrial protein synthesis. We measured the synthetic rates of myofibrillar and mitochondrial proteins and the activation of signalling proteins (Akt–mTOR–p70S6K) at rest and after an acute bout of RE or EE in the untrained state and after 10 weeks of RE or EE training in young healthy men. While untrained, RE stimulated both myofibrillar and mitochondrial protein synthesis, 67% and 69% (P < 0.02), respectively. After training, only myofibrillar protein synthesis increased with RE (36%, P= 0.05). EE stimulated mitochondrial protein synthesis in both the untrained, 154%, and trained, 105% (both P < 0.05), but not myofibrillar protein synthesis. Acute RE and EE increased the phosphorylation of proteins in the Akt–mTOR–p70S6K pathway with comparatively minor differences between two exercise stimuli. Phosphorylation of Akt–mTOR–p70S6K proteins was increased after 10 weeks of RE training but not by EE training. Chronic RE or EE training modifies the protein synthetic response of functional protein fractions, with a shift toward exercise phenotype‐specific responses, without an obvious explanatory change in the phosphorylation of regulatory signalling pathway proteins.

Selective activation of AMPK-PGC-1α or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation

Endurance training induces a partial fast‐to‐slow muscle phenotype transformation and mitochondrial biogenesis but no growth. In contrast, resistance training mainly stimulates muscle protein synthesis resulting in hypertrophy. The aim of this study was to identify signaling events that may mediate the specific adaptations to these types of exercise. Isolated rat muscles were electrically stimulated with either high frequency (HFS; 6×10 repetitions of 3 s‐bursts at 100 Hz to mimic resistance training) or low frequency (LFS; 3 h at 10 Hz to mimic endurance training). HFS significantly increased myofibrillar and sarcoplasmic protein synthesis 3 h after stimulation 5.3‐ and 2.7‐fold, respectively. LFS had no significant effect on protein synthesis 3 h after stimulation but increased UCP3 mRNA 11.7‐fold, whereas HFS had no significant effect on UCP3 mRNA. Only LFS increased AMPK phosphorylation significantly at Thr172 by ∼2‐fold and increased PGC‐1α protein to 1.3 times of control. LFS had no effect on PKB phosphorylation but reduced TSC2 phosphorylation at Thr1462 and deactivated translational regulators. In contrast, HFS acutely increased phosphorylation of PKB at Ser473 5.3‐fold and the phosphorylation of TSC2, mTOR, GSK‐3β at PKB‐sensitive sites. HFS also caused a prolonged activation of the translational regulators p70 S6k, 4E‐BP1, eIF‐2B, and eEF2. These data suggest that a specific signaling response to LFS is a specific activation of the AMPK‐PGC‐1α signaling pathway which may explain some endurance training adaptations. HFS selectively activates the PKB‐TSC2‐mTOR cascade causing a prolonged activation of translational regulators, which is consistent with increased protein synthesis and muscle growth. We term this behavior the “AMPK‐PKB switch.” We hypothesize that the AMPK‐PKB switch is a mechanism that partially mediates specific adaptations to endurance and resistance training, respectively.

Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle.

We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-13C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d5-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB (P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser473 and p70S6k Thr389 increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser2448, 4E-BP1 Thr37/46, or GSK3β Ser9 and decreased that of eEF2 Thr56, higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.

Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review.

Changing demographics make it ever more important to understand the modifiable risk factors for disability and loss of independence with advancing age. For more than two decades there has been increasing interest in the role of sarcopenia, the age-related loss of muscle or lean mass, in curtailing active and healthy aging. There is now evidence to suggest that lack of strength, or dynapenia, is a more constant factor in compromised wellbeing in old age and it is apparent that the decline in muscle mass and the decline in strength can take quite different trajectories. This demands recognition of the concept of muscle quality; that is the force generating per capacity per unit cross-sectional area (CSA). An understanding of the impact of aging on skeletal muscle will require attention to both the changes in muscle size and the changes in muscle quality. The aim of this review is to present current knowledge of the decline in human muscle mass and strength with advancing age and the associated risk to health and survival and to review the underlying changes in muscle characteristics and the etiology of sarcopenia. Cross-sectional studies comparing young (18–45 years) and old (>65 years) samples show dramatic variation based on the technique used and population studied. The median of values of rate of loss reported across studies is 0.47% per year in men and 0.37% per year in women. Longitudinal studies show that in people aged 75 years, muscle mass is lost at a rate of 0.64–0.70% per year in women and 0.80–00.98% per year in men. Strength is lost more rapidly. Longitudinal studies show that at age 75 years, strength is lost at a rate of 3–4% per year in men and 2.5–3% per year in women. Studies that assessed changes in mass and strength in the same sample report a loss of strength 2–5 times faster than loss of mass. Loss of strength is a more consistent risk for disability and death than is loss of muscle mass.

Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial

BACKGROUND Loss of muscle mass with aging is a major public health concern. Omega-3 (n-3) fatty acids stimulate protein anabolism in animals and might therefore be useful for the treatment of sarcopenia. However, the effect of omega-3 fatty acids on human protein metabolism is unknown. OBJECTIVE The objective of this study was to evaluate the effect of omega-3 fatty acid supplementation on the rate of muscle protein synthesis in older adults. DESIGN Sixteen healthy, older adults were randomly assigned to receive either omega-3 fatty acids or corn oil for 8 wk. The rate of muscle protein synthesis and the phosphorylation of key elements of the anabolic signaling pathway were evaluated before and after supplementation during basal, postabsorptive conditions and during a hyperaminoacidemic-hyperinsulinemic clamp. RESULTS Corn oil supplementation had no effect on the muscle protein synthesis rate and the extent of anabolic signaling element phosphorylation in muscle. Omega-3 fatty acid supplementation had no effect on the basal rate of muscle protein synthesis (mean ± SEM: 0.051 ± 0.005%/h compared with 0.053 ± 0.008%/h before and after supplementation, respectively; P = 0.80) but augmented the hyperaminoacidemia-hyperinsulinemia-induced increase in the rate of muscle protein synthesis (from 0.009 ± 0.005%/h above basal values to 0.031 ± 0.003%/h above basal values; P < 0.01), which was accompanied by greater increases in muscle mTOR(Ser2448) (P = 0.08) and p70s6k(Thr389) (P < 0.01) phosphorylation. CONCLUSION Omega-3 fatty acids stimulate muscle protein synthesis in older adults and may be useful for the prevention and treatment of sarcopenia. This trial was registered at clinical trials.gov as NCT00794079.

Low-load high volume resistance exercise stimulates muscle protein synthesis more than high-load low volume resistance exercise in young men.

Background We aimed to determine the effect of resistance exercise intensity (% 1 repetition maximum—1RM) and volume on muscle protein synthesis, anabolic signaling, and myogenic gene expression. Methodology/Principal Findings Fifteen men (21±1 years; BMI = 24.1±0.8 kg/m2) performed 4 sets of unilateral leg extension exercise at different exercise loads and/or volumes: 90% of repetition maximum (1RM) until volitional failure (90FAIL), 30% 1RM work-matched to 90%FAIL (30WM), or 30% 1RM performed until volitional failure (30FAIL). Infusion of [ring-13C6] phenylalanine with biopsies was used to measure rates of mixed (MIX), myofibrillar (MYO), and sarcoplasmic (SARC) protein synthesis at rest, and 4 h and 24 h after exercise. Exercise at 30WM induced a significant increase above rest in MIX (121%) and MYO (87%) protein synthesis at 4 h post-exercise and but at 24 h in the MIX only. The increase in the rate of protein synthesis in MIX and MYO at 4 h post-exercise with 90FAIL and 30FAIL was greater than 30WM, with no difference between these conditions; however, MYO remained elevated (199%) above rest at 24 h only in 30FAIL. There was a significant increase in AktSer473 at 24h in all conditions (P = 0.023) and mTORSer2448 phosphorylation at 4 h post-exercise (P = 0.025). Phosporylation of Erk1/2Tyr202/204, p70S6KThr389, and 4E-BP1Thr37/46 increased significantly (P<0.05) only in the 30FAIL condition at 4 h post-exercise, whereas, 4E-BP1Thr37/46 phosphorylation was greater 24 h after exercise than at rest in both 90FAIL (237%) and 30FAIL (312%) conditions. Pax7 mRNA expression increased at 24 h post-exercise (P = 0.02) regardless of condition. The mRNA expression of MyoD and myogenin were consistently elevated in the 30FAIL condition. Conclusions/Significance These results suggest that low-load high volume resistance exercise is more effective in inducing acute muscle anabolism than high-load low volume or work matched resistance exercise modes.

Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling

BACKGROUND We previously showed that human muscle protein synthesis (MPS) increased during infusion of amino acids (AAs) and peaked at ≈120 min before returning to baseline rates, despite elevated plasma AA concentrations. OBJECTIVE We tested whether a protein meal elicited a similar response and whether signaling responses that regulate messenger RNA translation matched MPS changes. DESIGN Eight postabsorptive healthy men (≈21 y of age) were studied during 8.5 h of primed continuous infusion of [1,2-¹³C₂]leucine with intermittent quadriceps biopsies for determination of MPS and anabolic signaling. After 2.5 h, subjects consumed 48 g whey protein. RESULTS At 45-90 min after oral protein bolus, mean (± SEM) myofibrillar protein synthesis increased from 0.03 ± 0.003% to 0.10 ± 0.01%/h; thereafter, myofibrillar protein synthesis returned to baseline rates even though plasma essential AA (EAA) concentrations remained elevated (+130% at 120 min, +80% at 180 min). The activity of protein kinase B (PKB) and phosphorylation of eukaryotic initiation factor 4G preceded the rise of MPS and increases in phosphorylation of ribosomal protein kinase S6 (S6K1), and 4E-binding protein 1 (4EBP1) was superimposable with MPS responses until 90 min. However, although MPS decreased thereafter, all signals, with the exception of PKB activity (which mirrored insulin responses), remained elevated, which echoed the slowly declining plasma EAA profile. The phosphorylation of eukaryotic initiation factor 2α increased only at 180 min. Thus, discordance existed between MPS and the mammalian target of rapamycin complex 1 (mTORC1) and signaling (ie, S6K1 and 4EBP1 phosphorylation). CONCLUSIONS We confirm our previous findings that MPS responses to AAs are transient, even with oral protein bolus. However, changes in MPS only reflect elevated mTORC1 signaling during the upswing in MPS.

Is irisin a human exercise gene

Arising from P. Boström et al. 481, 463–468 (2012)10.1038/nature10777Boström et al. report that exercise training induces the expression of the FNDC5 gene in human muscle, producing irisin, which can convert white fat into brown fat, so enhancing metabolic uncoupling and hence caloric expenditure, and propose that this is a new health promoting hormone. This assertion is based on experimental evidence that exogenous FNDC5 induces uncoupling protein 1 (UCP1) expression in white subcutaneous adipocytes; overexpression of FNDC5 in liver (elevating systemic irisin) prevents diet-induced weight gain and metabolic dysfunction and stimulates oxygen consumption in mice; and FNDC5 mRNA expression levels double after exercise training in eight human skeletal muscle samples. However, the UCP1 induction was lower than observed during Brite formation or the level associated with an improved diabetes profile in humans. Here we demonstrate that muscle FNDC5 induction occurs only in a minority of subjects—whereas all types of exercise training programmes, in the vast majority of people, yield some gain in cardiovascular or metabolic health, in our analysis of ∼200 subjects muscle FNDC5 was increased only in highly active elderly subjects, whereas FNDC5 expression was unrelated to metabolic status, which casts doubt over the general relevance of skeletal muscle FNDC5 to human health.