Michael J. Rennie

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.

Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

We hypothesized that an acute bout of strenuous, non‐damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n= 8 and 6) of healthy young men were studied over 72 h after 1 h of one‐legged kicking exercise at 67% of maximum workload (Wmax). To label tissue proteins in muscle and tendon primed, constant infusions of [1‐13C]leucine or [1‐13C]valine and flooding doses of [15N] or [13C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h−1), muscle collagen (0.054% h−1), myofibrillar protein (0.121% h−1), and sarcoplasmic protein (0.134% h−1)). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF‐I and IGF binding proteins (IGFBP‐3 and IGFBP‐4) or procollagen type I N‐terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation.

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.

Human Muscle Protein Synthesis is Modulated by Extracellular, Not Intramuscular Amino Acid Availability: A Dose‐Response Study

To test the hypothesis that muscle protein synthesis (MPS) is regulated by the concentration of extracellular amino acids, we investigated the dose‐response relationship between the rate of human MPS and the concentrations of blood and intramuscular amino acids. We increased blood mixed amino acid concentrations by up to 240 % above basal levels by infusion of mixed amino acids (Aminosyn 15, 44‐261 mg kg−1 h−1) in 21 healthy subjects, (11 men 10 women, aged 29 ± 2 years) and measured the rate of incorporation of D5‐phenylalanine or D3‐leucine into muscle protein and blood and intramuscular amino acid concentrations. The relationship between the fold increase in MPS and blood essential amino acid concentration ([EAA], mM) was hyperbolic and fitted the equation MPS = (2.68 ×[EAA])/(1.51 +[EAA]) (P < 0.01). The pattern of stimulation of myofibrillar, sarcoplasmic and mitochondrial protein was similar. There was no clear relationship between the rate of MPS and the concentration of intramuscular EAAs; indeed, when MPS was increasing most rapidly, the concentration of intramuscular EAAs was below basal levels. We conclude that the rates of synthesis of all classes of muscle proteins are acutely regulated by the blood [EAA] over their normal diurnal range, but become saturated at high concentrations. We propose that the stimulation of protein synthesis depends on the sensing of the concentration of extracellular, rather than intramuscular EAAs.

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.

Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids.

1 The aim of this study was to describe the time course of the response of human muscle protein synthesis (MPS) to a square wave increase in availability of amino acids (AAs) in plasma. We investigated the responses of quadriceps MPS to a ≈1.7‐fold increase in plasma AA concentrations using an intravenous infusion of 162 mg (kg body weight)−1 h−1 of mixed AAs. MPS was estimated from D3‐leucine labelling in protein after a primed, constant intravenous infusion of D3‐ketoisocaproate, increased appropriately during AA infusion. 2 Muscle was separated into myofibrillar, sarcoplasmic and mitochondrial fractions. MPS, both of mixed muscle and of fractions, was estimated during a basal period (2.5 h) and at 0.5‐4 h intervals for 6 h of AA infusion. 3 Rates of mixed MPS were not significantly different from basal (0.076 ± 0.008 % h−1) in the first 0.5 h of AA infusion but then rose rapidly to a peak after 2 h of ≈2.8 times the basal value. Thereafter, rates declined rapidly to the basal value. All muscle fractions showed a similar pattern. 4 The results suggest that MPS responds rapidly to increased availability of AAs but is then inhibited, despite continued AA availability. These results suggest that the fed state accretion of muscle protein may be limited by a metabolic mechanism whenever the requirement for substrate for protein synthesis is exceeded.

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.

A positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle

During muscle‐protein wasting associated with injury and disease the distribution ratio of free glutamine between muscle and blood falls. In pursuing possible consequences of this, we investigated the relationship between the rate of muscle protein synthesis and intramuscular glutamine concentration, manipulated acutely in the isolated perfused rat hindquarter. Increasing perfusate glutamine from 0.67 to 5.0 mM caused a 200% increase in intracellular glutamine and a 66% increase in protein synthesis in the absence of insulin; in the presence of insulin a 30% increase in intramuscular glutamine was accompanied by an 80% increase in protein synthesis. Analysis of variance of the results confirmed the existence of positive relationships between intramuscular glutamine and protein synthesis in the presence or absence of insulin. Control of the size of the intramuscular free pool of glutamine may be important in determining the muscle protein mass.