The effect of young and old ex vivo human serum on cellular protein synthesis and growth in an in vitro model of aging.

In vitro models of muscle aging are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from four old (72 ± 1 yr) and four young (26 ± 3 yr) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1 h and conditioned with human serum (10%) for 4 h or 24 h. After 4 h, C2C12 cells were treated with 5 mM leucine for 30 min. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways were measured via Western blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared with young serum before leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K, and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.

Protein Source and Quality for Skeletal Muscle Anabolism in Young and Older Adults: A Systematic Review and Meta-Analysis.

BACKGROUND: There is much debate regarding the source/quality of dietary proteins in supporting indices of skeletal muscle anabolism. OBJECTIVE: We performed a systematic review and meta-analysis to determine the effect of protein source/quality on acute muscle protein synthesis (MPS) and changes in lean body mass (LBM) and strength, when combined with resistance exercise (RE). METHODS: A systematic search of the literature was conducted to identify studies that compared the effects of ≥2 dose-matched, predominantly isolated protein sources of varying "quality." Three separate models were employed as follows: 1) protein feeding alone on MPS, 2) protein feeding combined with a bout of RE on MPS, and 3) protein feeding combined with longer-term resistance exercise training (RET) on LBM and strength. Further subgroup analyses were performed to compare the effects of protein source/quality between young and older adults. A total of 27 studies in young (18-35 y) and older (≥60 y) adults were included. RESULTS: Analysis revealed an effect favoring higher-quality protein for postprandial MPS at rest [mean difference (MD): 0.014%/h; 95% CI: 0.006, 0.021; P < 0.001] and following RE (MD: 0.022%/h; 95% CI: 0.014, 0.030; P < 0.00001) in young (model 1: 0.016%/h; 95% CI: -0.004, 0.036; P = 0.12; model 2: 0.030%/h; 95% CI: 0.015, 0.045; P < 0.0001) and older (model 1: 0.012%/h; 95% CI: 0.006, 0.018; P < 0.001; model 2: 0.014%/h; 95% CI: 0.007, 0.021; P < 0.001) adults. However, although higher protein quality was associated with superior strength gains with RET [standardized mean difference (SMD): 0.24 kg; 95% CI: 0.02, 0.45; P = 0.03)], no effect was observed on changes to LBM (SMD: 0.05 kg; 95% CI: -0.16, 0.25; P = 0.65). CONCLUSIONS: The current review suggests that protein quality may provide a small but significant impact on indices of muscle protein anabolism in young and older adults. However, further research is warranted to elucidate the importance of protein source/quality on musculoskeletal aging, particularly in situations of low protein intake.

Dietary isoleucine content defines the metabolic and molecular response to a Western diet.

The amino acid composition of the diet has recently emerged as a critical regulator of metabolic health. Consumption of the branched-chain amino acid isoleucine is positively correlated with body mass index in humans, and reducing dietary levels of isoleucine rapidly improves the metabolic health of diet-induced obese male C57BL/6J mice. However, it is unknown how sex, strain, and dietary isoleucine intake may interact to impact the response to a Western Diet (WD). Here, we find that although the magnitude of the effect varies by sex and strain, reducing dietary levels of isoleucine protects C57BL/6J and DBA/2J mice of both sexes from the deleterious metabolic effects of a WD, while increasing dietary levels of isoleucine impairs aspects of metabolic health. Despite broadly positive responses across all sexes and strains to reduced isoleucine, the molecular response of each sex and strain is highly distinctive. Using a multi-omics approach, we identify a core sex- and strain- independent molecular response to dietary isoleucine, and identify mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype. Intriguingly, the metabolic effects of reduced isoleucine in mice are not associated with FGF21 - and we find that in humans plasma FGF21 levels are likewise not associated with dietary levels of isoleucine. Finally, we find that foods contain a range of isoleucine levels, and that consumption of dietary isoleucine is lower in humans with healthy eating habits. Our results demonstrate that the dietary level of isoleucine is critical in the metabolic and molecular response to a WD, and suggest that lowering dietary levels of isoleucine may be an innovative and translatable strategy to protect from the negative metabolic consequences of a WD.

Myofibrillar Protein Synthesis and Acute Intracellular Signaling with Elastic Band Resistance Exercise in Young and Older Men.

PURPOSE: Resistance exercise training (RET) attenuates age-related muscle and strength loss ("sarcopenia"). However, compared with machine-based RET, the efficacy of cost-effective, accessible elastic band RET (EB-RET) for muscle adaptive remodeling lacks supporting mechanistic evidence. METHODS: Eight young (YM; 24 ± 4 yr) and eight older (OM; 68 ± 6 yr) untrained males consumed an oral stable isotope tracer (D 2 O) combined with serial vastus lateralis muscle biopsies to measure integrated myofibrillar protein synthesis (iMyoPS) and regulatory signaling over ~48 h before (habitual) and after an acute bout of EB-RET (6 × 12 repetitions at ~70% of one-repetition maximum). iMyoPS was determined via gas chromatography-pyrolysis-isotope ratio mass spectroscopy and regulatory signaling expression by immunoblot. RESULTS: Habitual iMyoPS did not differ between YM and OM (1.62% ± 0.21% vs 1.43% ± 0.47%·d -1 , respectively, P = 0.128). There was a significant increase in iMyoPS after EB-RET in YM (2.23% ± 0.69%·d -1 , P = 0.02), but not OM (1.75% ± 0.54%·d -1 , P = 0.30). EB-RET increased the phosphorylation of key anabolic signaling proteins similarly in YM and OM at 1 h postexercise, including p-IRS-1 Ser636/639 , p-Akt Ser473 , p-4EBP-1 Thr37/46 , p-P70S6K Thr389 , and p-RPS6 Ser240/244 , whereas p-TSC2 Thr1462 and p-mTOR Ser2448 increased only in YM (all P < 0.05). There were no differences in the expression of amino acid transporters/sensors or proteolytic markers after EB-RET. CONCLUSIONS: iMyoPS was elevated after EB-RET in YM but not OM. However, the increase in acute anabolic signaling with EB-RET was largely similar between groups. In conclusion, the capacity for EB-RET to stimulate iMyoPS may be impaired in older age. Further work may be necessary to optimize prescriptive programming in YM and OM.

Greater myofibrillar protein synthesis following weight-bearing activity in obese old compared with non-obese old and young individuals.

The mechanisms through which obesity impacts age-related muscle mass regulation are unclear. In the present study, rates of integrated myofibrillar protein synthesis (iMyoPS) were measured over 48-h prior-to and following a 45-min treadmill walk in 10 older-obese (O-OB, body fat[%]: 33 ± 3%), 10 older-non-obese (O-NO, 20 ± 3%), and 15 younger-non-obese (Y-NO, 13 ± 5%) individuals. Surface electromyography was used to determine thigh muscle "activation". Quadriceps cross-sectional area (CSA), volume, and intramuscular thigh fat fraction (ITFF) were measured by magnetic resonance imaging. Quadriceps maximal voluntary contraction (MVC) was measured by dynamometry. Quadriceps CSA and volume were greater (muscle volume, Y-NO: 1182 ± 232 cm3; O-NO: 869 ± 155 cm3; O-OB: 881 ± 212 cm3, P < 0.01) and ITFF significantly lower (m. vastus lateralis, Y-NO: 3.0 ± 1.0%; O-NO: 4.0 ± 0.9%; O-OB: 9.1 ± 2.6%, P ≤ 0.03) in Y-NO compared with O-NO and O-OB, with no difference between O-NO and O-OB in quadriceps CSA and volume. ITFF was significantly higher in O-OB compared with O-NO. Relative MVC was lower in O-OB compared with Y-NO and O-NO (Y-NO: 5.5 ± 1.6 n·m/kg-1; O-NO: 3.9 ± 1.0 n·m/kg-1; O-OB: 2.9 ± 1.1 n·m/kg-1, P < 0.0001). Thigh muscle "activation" during the treadmill walk was greater in O-OB compared with Y-NO and O-NO (Y-NO: 30.5 ± 13.5%; O-NO: 35.8 ± 19.7%; O-OB: 68.3 ± 32.3%, P < 0.01). Habitual iMyoPS did not differ between groups, whereas iMyoPS was significantly elevated over 48-h post-walk in O-OB (+ 38.6 ± 1.2%·day-1, P < 0.01) but not Y-NO or O-NO (+ 11.4 ± 1.1%·day-1 and + 17.1 ± 1.1%·day-1, respectively, both P > 0.271). Equivalent muscle mass in O-OB may be explained by the muscle anabolic response to weight-bearing activity, whereas the age-related decline in indices of muscle quality appears to be exacerbated in O-OB and warrants further exploration.

No effect of five days of bed rest or short-term resistance exercise prehabilitation on markers of skeletal muscle mitochondrial content and dynamics in older adults.

Bed rest (BR) results in significant impairments in skeletal muscle metabolism. Mitochondrial metabolism is reportedly highly sensitive to disuse, with dysregulated fission-fusion events and impaired oxidative function previously reported. The effects of clinically relevant short-term BR (≤5 days) on mitochondrial protein expression are presently unclear, as are the effects of exercise prehabilitation as a potential counteractive intervention. The present study examined the effects of a 5-day period of BR and short-term resistance exercise prehabilitation (ST-REP) on mitochondrial-protein content. Ten older men (71 ± 4 years) underwent 5 days of BR, completing four sessions of high-volume unilateral resistance exercise prehabilitation over 7 days beforehand. Muscle biopsies were obtained from the vastus lateralis in the non-exercised control and exercised legs, both pre- and post-prehabilitation and pre- and post-BR, to determine changes in citrate synthase enzyme activity and the expression of key proteins in the mitochondrial electron transport chain and molecular regulators of fission-fusion dynamics, biosynthesis, and mitophagy. We observed no significant effect of either BR or ST-REP on citrate synthase protein content, enzyme activity, or ETC complex I-V protein content. Moreover, we observed no significant changes in markers of mitochondrial fission and fusion (p-DRP1S616 , p-DRP1S637 , p-DRP1S616/S637 ratio, p-MFFS146 , Mitofillin, OPA1, or MFN2 (p > 0.05 for all). Finally, we observed no differences in markers of biosynthesis (p-AMPKT172 , p-ACCS79 , PGC1a, TFAM) or mitophagy-related signaling (ULK-1, BNIP3/NIX, LC3B I/II) (p > 0.05 for all). In contrast to previous longer-term periods of musculoskeletal disuse (i.e., 7-14 days), a clinically relevant, 5-day period of BR resulted in no significant perturbation in muscle mitochondrial protein signaling in healthy older adults, with no effect of ST-REP in the week prior to BR. Accordingly, disuse-induced muscle atrophy may precede alterations in mitochondrial content.

Short-term step reduction reduces citrate synthase activity without altering skeletal muscle markers of oxidative metabolism or insulin-mediated signaling in young males.

Mitochondria are critical to skeletal muscle contractile function and metabolic health. Short-term periods of step reduction (SR) are associated with alterations in muscle protein turnover and mass. However, the effects of SR on mitochondrial metabolism/muscle oxidative metabolism and insulin-mediated signaling are unclear. We tested the hypothesis that the total and/or phosphorylated protein content of key skeletal muscle markers of mitochondrial/oxidative metabolism, and insulin-mediated signaling would be altered over 7 days of SR in young healthy males. Eleven, healthy, recreationally active males (means ± SE, age: 22 ± 1 yr, BMI: 23.4 ± 0.7 kg·m2) underwent a 7-day period of SR. Immediately before and following SR, fasted-state muscle biopsy samples were acquired and analyzed for the assessment of total and phosphorylated protein content of key markers of mitochondrial/oxidative metabolism and insulin-mediated signaling. Daily step count was significantly reduced during the SR intervention (13,054 ± 833 to 1,192 ± 99 steps·day-1, P < 0.001). Following SR, there was a significant decline in maximal citrate synthase activity (fold change: 0.94 ± 0.08, P < 0.05) and a significant increase in the protein content of p-glycogen synthase (P-GSS641; fold change: 1.47 ± 0.14, P < 0.05). No significant differences were observed in the total or phosphorylated protein content of other key markers of insulin-mediated signaling, oxidative metabolism, mitochondrial function, or mitochondrial dynamics (all P > 0.05). These results suggest that short-term SR reduces the maximal activity of citrate synthase, a marker of mitochondrial content, without altering the total or phosphorylated protein content of key markers of skeletal muscle mitochondrial metabolism and insulin signaling in young healthy males.NEW & NOTEWORTHY Short-term (7 day) step reduction reduces the activity of citrate synthase without altering the total or phosphorylated protein content of key markers of skeletal muscle mitochondrial metabolism and insulin signaling in young healthy males.

Nutritional Strategies to Offset Disuse-Induced Skeletal Muscle Atrophy and Anabolic Resistance in Older Adults: From Whole-Foods to Isolated Ingredients.

Preserving skeletal muscle mass and functional capacity is essential for healthy ageing. Transient periods of disuse and/or inactivity in combination with sub-optimal dietary intake have been shown to accelerate the age-related loss of muscle mass and strength, predisposing to disability and metabolic disease. Mechanisms underlying disuse and/or inactivity-related muscle deterioration in the older adults, whilst multifaceted, ultimately manifest in an imbalance between rates of muscle protein synthesis and breakdown, resulting in net muscle loss. To date, the most potent intervention to mitigate disuse-induced muscle deterioration is mechanical loading in the form of resistance exercise. However, the feasibility of older individuals performing resistance exercise during disuse and inactivity has been questioned, particularly as illness and injury may affect adherence and safety, as well as accessibility to appropriate equipment and physical therapists. Therefore, optimising nutritional intake during disuse events, through the introduction of protein-rich whole-foods, isolated proteins and nutrient compounds with purported pro-anabolic and anti-catabolic properties could offset impairments in muscle protein turnover and, ultimately, the degree of muscle atrophy and recovery upon re-ambulation. The current review therefore aims to provide an overview of nutritional countermeasures to disuse atrophy and anabolic resistance in older individuals.

Quadriceps muscle electromyography activity during physical activities and resistance exercise modes in younger and older adults.

BACKGROUND: Understanding the root cause of the age-related impairment in muscle adaptive remodelling with resistance exercise training (RET) and developing pragmatic and accessible resistance exercise for older adults, are essential research directives. METHODS: We sought to determine whether indices of quadriceps muscle EMG activity in response to different modes of RET and activities of daily living (ADL), differed between 15 healthy younger (25 ± 3 years) and 15 older (70 ± 5 years) adults. On four separate days, participants completed a maximal voluntary contraction (MVC) of the knee extensors, followed by a 15 m walking task, stair climbing task (i.e. ADL) and lower-limb RET through body-weight squats (BW-RET) and seated knee extensions on a machine (MN-RET) or via elastic bands (EB-RET). Surface quadriceps electromyography (EMG) was measured throughout all tasks to provide indirect estimates of changes in muscle activity. RESULTS: MVC was significantly greater in young vs. older adults (Young: 256 ± 72 vs. Old: 137 ± 48 N·m, P < 0.001). EMG activity during all exercise tasks was significantly higher in older vs. younger adults when expressed relative to maximal EMG achieved during MVC (P < 0.01, for all). In addition, relative quadriceps muscle EMG activity was significantly greater in EB-RET (Young: 20.3 ± 8.7 vs. Old: 37.0 ± 10.7%) and MN-RET (Young: 22.9 ± 10.3, vs. Old: 37.8 ± 10.8%) compared with BW-RET (Young: 8.6 ± 2.9 vs. Old: 27.0 ± 9.3%), in young and older adults (P < 0.001). However, there was no significant difference in quadriceps EMG between EB-RET and MN-RET (P > 0.05). CONCLUSIONS: In conclusion, relative quadriceps muscle EMG activity was higher across a range of activities/exercise modes in older vs. younger adults. The similar quadriceps muscle EMG activity between EB-RET and MN-RET provides a platform for detailed investigation of the neuromuscular and muscle metabolic responses to such pragmatic forms of RET to strengthen the evidence-base for this mode of RET as a potential countermeasure to sarcopenia.

High-dose leucine supplementation does not prevent muscle atrophy or strength loss over 7 days of immobilization in healthy young males.

BACKGROUND: Unavoidable periods of disuse lead to muscle atrophy and functional decline. Preventing such declines can reduce the risk of re-injury and improve recovery of normal physiological functioning. OBJECTIVES: We aimed to determine the effectiveness of high-dose leucine supplementation on muscle morphology and strength during 7 d of unilateral lower-limb immobilization, and the role of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis in disuse atrophy. METHODS: Sixteen healthy males (mean ± SEM age: 23 ± 1 y) underwent 7 d of unilateral lower-limb immobilization, with thrice-daily leucine (LEU; n = 8) or placebo (PLA; n = 8) supplementation (15 g/d). Before and after immobilization, muscle strength and compartmental tissue composition were assessed. A primed continuous infusion of l-[ring-13C6]-phenylalanine with serial muscle biopsies was used to determine postabsorptive and postprandial (20 g milk protein) MyoPS and MitoPS, fiber morphology, markers of protein turnover, and mitochondrial function between the control leg (CTL) and the immobilized leg (IMB). RESULTS: Leg fat-free mass was reduced in IMB (mean ± SEM: -3.6% ± 0.5%; P = 0.030) but not CTL with no difference between supplementation groups. Isometric knee extensor strength declined to a greater extent in IMB (-27.9% ± 4.4%) than in CTL (-14.3% ± 4.4%; P = 0.043) with no difference between groups. In response to 20 g milk protein, postprandial MyoPS rates were significantly lower in IMB than in CTL (-22% ± 4%; P < 0.01) in both LEU and PLA. Postabsorptive MyoPS rates did not differ between legs or groups. Postabsorptive MitoPS rates were significantly lower in IMB than in CTL (-14% ± 5%; P < 0.01) and postprandial MitoPS rates significantly declined in response to 20 g milk protein ingestion (CTL: -10% ± 8%; IMB: -15% ± 10%; P = 0.039), with no differences between legs or groups. There were no significant differences in measures of mitochondrial respiration between legs, but peroxisome proliferator-activated receptor γ coactivator 1-α and oxidative phosphorylation complex II and III were significantly lower in IMB than in CTL (P < 0.05), with no differences between groups. CONCLUSIONS: High-dose leucine supplementation (15 g/d) does not appear to attenuate any functional declines associated with 7 d of limb immobilization in young, healthy males.This trial was registered at clinicaltrials.gov as NCT03762278.