Anna Selby

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.

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.

Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion

We tested the hypothesis that increasing blood amino acid (AA) availability would counter the physical inactivity‐induced reduction in muscle protein synthesis. We determined how 14 days of unilateral knee immobilization affected quadriceps myofibrillar protein synthesis (MPS) in young healthy subjects (10 men, 2 women, 21 ± 1 years; 80.2 ± 4.0 kg, mean ±s.e.m.) in the post‐absorptive state and after infusing AA (10% Primene) at low or high doses (43 and 261 mg kg−1 h−1). Muscle cross‐sectional area (MRI) and peak isometric torque declined in the immobilized leg (−5.0 ± 1.2% and −25 ± 3%, respectively, both P < 0.005), but were unchanged (all P > 0.6) in the non‐immobilized leg. Immobilization induced a 27% decline in the rate of post‐absorptive MPS (immobilized, 0.027 ± 0.003: non‐immobilized, 0.037 ± 0.003% h−1; P < 0.001). Regardless of dose, AA infusion stimulated a greater rise in MPS in the non‐immobilized legs; at 4 h MPS was greater by +54 ± 12% with low dose and +68 ± 17% with high dose AA infusion (both P < 0.001). There was some evidence of delayed responsiveness of phosphorylation of Akt to high doses of AA and p70S6k at both doses but no marked differences in that of mTOR, GSK3β or eEF2. Phosphorylation of focal adhesion kinase (Tyr576/577) was reduced (P < 0.05) with immobilization. We observed no change in polyubiquitinated protein content after immobilization. We confirm that 14 days of immobilization reduces MPS in the post‐absorptive state and this diminution is reduced but not abolished by increased provision of AA, even at high rates. The immobilization‐induced decline in post‐absorptive MPS with the ‘anabolic resistance’ to amino acids can account for much of immobilization‐induced muscle atrophy.

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.

The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse.

We hypothesized that rates of myofibrillar and patellar tendon collagen synthesis would fall over time during disuse, the changes being accompanied in muscle by decreases in focal adhesion kinase (FAK) phosphorylation and in gene expression for proteolytic enzymes. We studied nine men (22 ± 4 years, BMI 24 ± 3 kg m−2 (means ±s.d.) who underwent unilateral lower leg suspension for 23 days; five were studied between 0 and 10 days and four between 10 and 21 days. Muscle and tendon biopsies were taken in the postabsorptive state at days 0, 10 and 21 for measurement of protein synthesis, gene expression and protein phosphorylation. Muscle cross‐sectional area decreased by 5.2% at 14 days and 10.0% (both P < 0.001), at 23 days, i.e. 0.5% day−1, whereas tendon dimensions were constant. Rates of myofibrillar protein synthesis fell (P < 0.01) from 0.047% h−1 at day 0 to 0.022% h−1 at 10 days without further changes. Tendon collagen synthetic rates also fell (P < 0.01), from 0.052 to 0.023% h−1 at 10 days and then to 0.010% h−1 at 21 days. FAK phosphorylation decreased 30% (P < 0.01) at 10 days. No changes occurred in the amounts/phosphorylation of PKB–P70s6k–mTOR pathway components. Expression of mRNA for MuRF‐1 increased ∼3‐fold at 10 days without changes in MAFbx or tripeptidyl peptidase II mRNA, but all decreased between 10 and 21 days. Thus, both myofibrillar and tendon protein synthetic rates show progressive decreases during 21 days of disuse; in muscle, this is accompanied by decreased phosphorylation of FAK, with no marked increases in genes for proteolytic enzymes.

Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise

BACKGROUND The intake of whey, compared with casein and soy protein intakes, stimulates a greater acute response of muscle protein synthesis (MPS) to protein ingestion in rested and exercised muscle. OBJECTIVE We characterized the dose-response relation of postabsorptive rates of myofibrillar MPS to increasing amounts of whey protein at rest and after exercise in resistance-trained, young men. DESIGN Volunteers (n = 48) consumed a standardized, high-protein (0.54 g/kg body mass) breakfast. Three hours later, a bout of unilateral exercise (8 × 10 leg presses and leg extensions; 80% one-repetition maximum) was performed. Volunteers ingested 0, 10, 20, or 40 g whey protein isolate immediately (~10 min) after exercise. Postabsorptive rates of myofibrillar MPS and whole-body rates of phenylalanine oxidation and urea production were measured over a 4-h postdrink period by continuous tracer infusion of labeled [(13)C6] phenylalanine and [(15)N2] urea. RESULTS Myofibrillar MPS (mean ± SD) increased (P < 0.05) above 0 g whey protein (0.041 ± 0.015%/h) by 49% and 56% with the ingestion of 20 and 40 g whey protein, respectively, whereas no additional stimulation was observed with 10 g whey protein (P > 0.05). Rates of phenylalanine oxidation and urea production increased with the ingestion of 40 g whey protein. CONCLUSIONS A 20-g dose of whey protein is sufficient for the maximal stimulation of postabsorptive rates of myofibrillar MPS in rested and exercised muscle of ~80-kg resistance-trained, young men. A dose of whey protein >20 g stimulates amino acid oxidation and ureagenesis. This trial was registered at http://www.isrctn.org/ as ISRCTN92528122.

Blunting of insulin inhibition of proteolysis in legs of older subjects may contribute to age-related sarcopenia

BACKGROUND Reduced postprandial muscle proteolysis is mainly due to increased insulin availability. Whether rates of proteolysis in response to low physiologic doses of insulin are affected by aging is unknown. OBJECTIVES We tested the hypothesis that suppression of leg protein breakdown (LPB) by insulin is blunted in older subjects, together with blunted activation of Akt-protein kinase B (PKB). DESIGN Groups of 8 young [mean (+/-SD) age: 24.5 +/- 1.8 y] and older (65.0 +/- 1.3 y) participants were studied during euglycemic (5 mmol/L), isoaminoacidemic (blood leucine approximately 120 micromol/L) clamp procedures at plasma insulin concentrations of approximately 5 and approximately 15 microIU/mL for 1.5 h. Leg amino acid balance, whole-leg protein turnover (as dilution of amino acid tracers), and muscle protein synthesis were measured with D(5)-phenylalanine and [1,2-(13)C(2)]leucine. The kinase activity of muscle Akt-PKB and the extent of phosphorylation of signaling proteins associated with the mTOR (mammalian target of rapamycin) pathway were measured before and after the clamp procedures. RESULTS Basal LPB rates were not different between groups (66 +/- 11 compared with 51 +/- 10 nmol leucine x 100 mL leg(-1) x min(-1) and 30 +/- 5 compared with 24 +/- 4 nmol phenylalanine x 100 mL leg(-1) x min(-1) in young and older groups, respectively). However, although insulin at approximately 15 microIU/mL lowered LPB by 47% in the young subjects (P < 0.05) and abolished the negative leg amino acid balance, this caused only a 12% fall (P > 0.05) in the older group. Akt-PKB activity mirrored decreases in LPB. No differences were seen in muscle protein synthesis or associated anabolic signaling phosphoproteins. CONCLUSIONS At moderate availability, the effect of insulin on LPB is diminished in older human beings, and this effect may be mediated through blunted Akt-PKB activation.

Facts, noise and wishful thinking: muscle protein turnover in aging and human disuse atrophy

Surprisingly little is known about the mechanisms of muscle atrophy with aging and disuse in human beings, in contrast to rodents, from which much has been extrapolated to explain the human condition. However, this extrapolation is likely unwarranted because the time course, extent of wasting, muscle fiber involvement and alterations of muscle protein turnover are all quite different in rodent and human muscle. Furthermore, there is little evidence that static indices of protein turnover represent dynamic changes and may be misleading. With disuse there are reductions in the rate of muscle protein synthesis (MPS) large enough to explain the atrophic loss of muscle protein without a concomitant increase in proteolysis. In aging, there is no evidence that there are marked alterations in basal muscle protein turnover in healthy individuals but instead the ability to maintain muscle after feeding is compromised. This anabolic resistance is evident with physical inactivity, which exacerbates the inability to maintain muscle mass with aging. The main conclusion of this review is that in uncomplicated, non‐inflammatory disuse atrophy, the facilitative change causing loss of muscle mass is a depression of MPS, exacerbated by anabolic resistance during feeding, with possible adaptive depressions, rather than increases, of muscle proteolysis.

Architectural, functional and molecular responses to concentric and eccentric loading in human skeletal muscle.

We investigated architectural, functional and molecular responses of human skeletal muscle to concentric (CON) or eccentric (ECC) resistance training (RT).

The influence of carbohydrate–protein co‐ingestion following endurance exercise on myofibrillar and mitochondrial protein synthesis

Non‐technical summary  A single bout of exercise stimulates the production of new muscle proteins. Furthermore, ingesting protein in close proximity to exercise enhances the metabolic response. Long‐term exercise training promotes muscle adaptation, and the mode of exercise performed determines the type of proteins that are made. To date, the types of proteins that are made when protein is ingested after endurance exercise are not known. We report that when well‐trained male cyclists ingest protein with a carbohydrate drink after a high‐intensity ride, production of proteins responsible for muscle contraction is increased. Proteins responsible for aerobic energy production are not responsive to protein feeding. Furthermore, specific signals within the muscle that control protein synthesis are responsive to protein ingestion, providing a potential mechanism to underpin our primary findings. These results suggest that protein feeding after intense endurance exercise may be important in maintaining the structural quality and power generating capacity of the muscle.