Underpinning the Food Matrix Regulation of Postexercise Myofibrillar Protein Synthesis by Comparing Salmon Ingestion With the Sum of Its Isolated Nutrients in Healthy Young Adults.

BACKGROUND: Protein is most commonly consumed as whole foods as opposed to single nutrients. However, the food matrix regulation of the postprandial muscle protein synthetic response has received little attention. OBJECTIVES: The purpose of this study was to assess the effects of eating salmon (SAL) and of ingesting the same nutrients as an isolated mixture of crystalline amino acids and fish oil (ISO) on the stimulation of postexercise myofibrillar protein synthesis (MPS) and whole-body leucine oxidation rates in healthy young adults. METHODS: Ten recreationally active adults (24 ± 4 y; 5 men, 5 women) performed an acute bout of resistance exercise, followed by the ingestion of SAL or ISO in a crossover fashion. Blood, breath, and muscle biopsies were collected at rest and after exercise during primed continuous infusions of L-[ring-2H5]phenylalanine and L-[1-13C]leucine. All data are presented as means ± SD and/or mean differences (95% CIs). RESULTS: Postprandial essential amino acid (EAA) concentrations peaked earlier (P = 0.024) in the ISO group than those in the SAL group. Postprandial leucine oxidation rates increased over time (P < 0.001) and peaked earlier in the ISO group (1.239 ± 0.321 nmol/kg/min; 63 ± 25 min) than those in the SAL group (1.230 ± 0.561 nmol/kg/min; 105 ± 20 min; P = 0.003). MPS rates for SAL (0.056 ± 0.022 %/h; P = 0.001) and ISO (0.046 ± 0.025 %/h; P = 0.025) were greater than the basal rates (0.020 ± 0.011 %/h) during the 0- to 5-h recovery period, with no differences between conditions (P = 0.308). CONCLUSION: We showed that the postexercise ingestion of SAL or ISO stimulate postexercise MPS rates with no differences between the conditions. Thus, our results indicate that ingesting protein from SAL as a whole-food matrix is similarly anabolic to ISO in healthy young adults. This trial was registered at www. CLINICALTRIALS: gov as NCT03870165.

Greater plasma essential amino acids and lower 3-methylhistidine with higher protein intake during endurance training: a randomised control trial.

Endurance exercise alters amino acid (AA) metabolism that necessitates greater AA intake in the post exercise recovery period to support recovery. Thus, daily AA ingestion during a period of endurance training may affect the metabolically active plasma free AA pool, which is otherwise maintained during periods of inadequate protein intake by the breakdown of skeletal muscle proteins. Nine endurance-trained males completed a 4-day running protocol (20 km, 5 km, 10 km and 20 km on days 1-4, respectively) on three occasions with a controlled diet providing different protein intakes [0.94(LOW), 1.20(MOD) or 1.83gprotein kgbody mass-1 day-1 (HIGH)]. Urine collected over 24 h on day-4 and plasma collected after an overnight fast on day-5 were analyzed for free AA (plasma) and 3-methylhistidine (3MH; plasma and urine), a marker of myofibrillar protein breakdown. There was an effect of protein intake (HIGH > MOD/LOW; P < 0.05) on fasted plasma essential AA, branched chain AA and 3MH but no effect on 24-h urinary 3-MH excretion. Consuming a previously determined optimal daily protein intake of 1.83 g kg-1 day-1 during endurance training maintains fasted plasma free AA and may attenuate myofibrillar protein catabolism, although this latter effect was not detected in 24-h urinary excretion. The maintenance of the metabolically active free plasma AA pool may support greater recovery from exercise and contribute to the previously determined greater whole-body net protein balance in this athletic population. TRN: NCT02801344 (June 15, 2016).

Leucine ingestion promotes mTOR translocation to the periphery and enhances total and peripheral RPS6 phosphorylation in human skeletal muscle.

The activation of the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, by anabolic stimuli (such as muscle contraction or essential amino acids) involves its translocation to the cell periphery. Leucine is generally considered the most anabolic of amino acids for its ability to independently modulate muscle protein synthesis. However, it is currently unknown if free leucine impacts region-specific mTORC1-mediated phosphorylation events and protein-protein interactions. In this clinical trial (NCT03952884; registered May 16, 2019), we used immunofluorescence methods to investigate the role of dietary leucine on the postprandial regulation of mTORC1 and ribosomal protein S6 (RPS6), an important downstream readout of mTORC1 activity. Eight young, healthy, recreationally active males (n = 8; 23 ± 3 yrs) ingested 2 g of leucine with vastus lateralis biopsies collected at baseline, 30, 60, and 180 min postprandial. Leucine promoted mTOR translocation to the periphery (~ 18-29%; p ≤ 0.012) and enhanced mTOR localization with the lysosome (~ 16%; both p = 0.049) at 30 and 60 min post-feeding. p-RPS6Ser240/244 staining intensity, a readout of mTORC1 activity, was significantly elevated at all postprandial timepoints in both the total fiber (~ 14-30%; p ≤ 0.032) and peripheral regions (~ 16-33%; p ≤ 0.014). Additionally, total and peripheral p-RPS6Ser240/244 staining intensity at 60 min was positively correlated (r = 0.74, p = 0.036; r = 0.80, p = 0.016, respectively) with rates of myofibrillar protein synthesis over 180 min. The ability of leucine to activate mTORC1 in peripheral regions favors an enhanced rate of MPS, as this is the intracellular space thought to be replete with the cellular machinery that facilitates this anabolic process.

Estimation of skeletal muscle mass in 4-year-old children using the D3-creatine dilution method.

BACKGROUND: Given limited experience in applying the creatine-(methyl-D3) (D3Cr) dilution method to measure skeletal muscle mass (SMM) in young children, the feasibility of deployment in a fielding setting and performance of the method was assessed in a cohort of 4-year-old children in Dhaka, Bangladesh. METHODS: Following D3Cr oral dose (10 mg) administration, single fasting urine samples were collected at 2-4 days (n = 100). Twenty-four-hour post-dose collections and serial spot urine samples on days 2, 3 and 4 were obtained in a subset of participants (n = 10). Urinary creatine, creatinine, D3Cr and D3-creatinine enrichment were analyzed by liquid chromatography-tandem mass spectrometry. Appendicular lean mass (ALM) was measured by dual-energy x-ray absorptiometry and grip strength was measured by a hand-held dynamometer. RESULTS: SMM was measured successfully in 91% of participants, and there were no adverse events. Mean ± SD SMM was greater than ALM (4.5 ± 0.4 and 3.2 ± 0.6 kg, respectively). Precision of SMM was low (intraclass correlation = 0.20; 95% CI: 0.02, 0.75; n = 10). Grip strength was not associated with SMM in multivariable analysis (0.004 kg per 100 g of SMM; 95% CI: -0.031, 0.038; n = 91). CONCLUSIONS: The D3Cr dilution method was feasible in a community setting. However, high within-child variability in SMM estimates suggests the need for further optimization of this approach. IMPACT: The D3-creatine (D3Cr) stable isotope dilution method was considered a feasible method for the estimation of skeletal muscle mass (SMM) in young children in a community setting and was well accepted among participants. SMM was weakly associated with both dual-energy x-ray absorptiometry-derived values of appendicular lean mass and grip strength. High within-child variability in estimated values of SMM suggests that further optimization of the D3Cr stable isotope dilution method is required prior to implementation in community research settings.

RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli.

Following anabolic stimuli (mechanical loading and/or amino acid provision), the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or before translocation (i.e., in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25 g/kg protein and 0.75 g/kg carbohydrate) alone [n = 7; 23 ± 5 yr; 76.8 ± 3.6 kg; and 13.6 ± 3.8% body fat (BF), FED] or following a whole body resistance exercise bout (n = 7; 22 ± 2 yr; 78.1 ± 3.6 kg; and 12.2 ± 4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300 min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r = 0.76, P < 0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120 min (∼54% and ∼138%, respectively, P < 0.05) but was greater in EXFED at both poststimuli time points (P < 0.05). The peripheral-to-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120 min irrespective of stimulus (P = 0.006) before returning to PRE at 300 min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

Identification of underexplored mesenchymal and vascular-related cell populations in human skeletal muscle.

Skeletal muscle repair and maintenance are directly and indirectly supported by interstitial cell populations such as vascular cells and fibro-adipogenic progenitors (FAPs), a subset of which express Twist2 and possess direct myogenic potential. Furthermore, work in rodents has highlighted the potential of pericytes to act as progenitor cells, giving rise to muscle cells and transdifferentiating into endothelial cells. However, less is understood about these populations in human skeletal muscle. Here, we performed single-cell RNA sequencing (scRNAseq) on ∼2,000 cells isolated from the human semitendinosus muscle of young individuals. This demonstrated the presence of a vascular-related cell type that expressed pericyte and pan-endothelial genes that we localized to large blood vessels within skeletal muscle cross sections and termed endothelial-like pericytes (ELPCs). RNA velocity analysis indicated that ELPCs may represent a "transition state" between endothelial cells and pericytes. Analysis of published scRNAseq data sets revealed evidence for ELPCs in trunk and heart musculature, which showed transcriptional similarity. In addition, we identified a subset of FAPs expressing TWIST2 mRNA and protein. Human TWIST2-expressing cells were anatomically and transcriptionally comparable to mouse Twist2 cells as they were restricted to the myofiber interstitium, expressed fibrogenic genes but lacked satellite cell markers, and colocalized with the FAPs marker PDGFRα in human muscle cross sections. Taken together, these results highlight the complexity of stromal cells residing in human skeletal muscle and support the utility of scRNAseq for discovery and characterization of poorly described cell populations.

Challenges of rapamycin repurposing as a potential therapeutic candidate for COVID-19: implications for skeletal muscle metabolic health in older persons.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that has spread worldwide, resulting in over 6 million deaths as of March 2022. Older people have been disproportionately affected by the disease, as they have a greater risk of hospitalization, are more vulnerable to severe infection, and have higher mortality than younger patients. Although effective vaccines have been rapidly developed and administered globally, several clinical trials are ongoing to repurpose existing drugs to combat severe infection. One such drug, rapamycin, is currently under study for this purpose, given its immunosuppressant effects that are mediated by its inhibition of the mechanistic target of rapamycin (mTOR), a master regulator of cell growth. Consistent with this premise, acute rapamycin administration in young healthy humans blocks or attenuates mTOR and its downstream effectors, leading to the inhibition of muscle protein synthesis (MPS). Skeletal muscle mass declines when MPS is chronically lower than muscle protein breakdown. This is consequential for older people who are more susceptible to anabolic resistance (i.e., the blunting of MPS) due to reduced activity, sedentariness, or bed rest such as that associated with COVID-19 hospitalization, and who have also demonstrated a delayed or blunted ability to regain inactivity-induced muscle loss. The lack of studies investigating rapamycin administration on skeletal muscle in older people, and the emergence of effective antiviral medications against severe infection, may indicate the reduced relevance of drug repurposing for present or future pandemics.

Early resistance training-mediated stimulation of daily muscle protein synthetic responses to higher habitual protein intake in middle-aged adults.

KEY POINTS: The ingestion of protein potentiates the stimulation of myofibrillar protein synthesis rates after an acute bout of resistance exercise. Protein supplementation (eating above the protein Recommended Dietary Allowance) during resistance training has been shown to maximize lean mass and strength gains in healthy young and older adults. Here, contractile, oxidative, and structural protein synthesis were assessed in skeletal muscle in response to a moderate or higher protein diet during the early adaptive phase of resistance training in middle-aged adults. The stimulation of myofibrillar, mitochondrial or collagen protein synthesis rates during 0-3 weeks of resistance training is not further enhanced by a higher protein diet. These results show that moderate protein diets are sufficient to support the skeletal muscle adaptive response during the early phase of a resistance training programme. ABSTRACT: Protein ingestion augments muscle protein synthesis (MPS) rates acutely after resistance exercise and can offset age-related loss in muscle mass. Skeletal muscle contains a variety of protein pools, such as myofibrillar (contractile), mitochondrial (substrate oxidation), and collagen (structural support) proteins, and the sensitivity to nutrition and exercise seems to be dependent on the major protein fraction studied. However, it is unknown how free-living conditions with high dietary protein density and habitual resistance exercise mediates muscle protein subfraction synthesis. Therefore, we investigated the effect of moderate (MOD: 1.06 ± 0.22 g kg-1  day-1 ) or high (HIGH: 1.55 ± 0.25 g kg-1  day-1 ) protein intake on daily MPS rates within the myofibrillar (MyoPS), mitochondrial (MitoPS) and collagen (CPS) protein fractions in middle-aged men and women (n = 20, 47 ± 1 years, BMI 28 ± 1 kg m-2 ) during the early phase (0-3 weeks) of a dietary counselling-controlled resistance training programme. Participants were loaded with deuterated water, followed by daily maintenance doses throughout the intervention. Muscle biopsies were collected at baseline and after weeks 1, 2 and 3. MyoPS in the HIGH condition remained constant (P = 1.000), but MOD decreased over time (P = 0.023). MitoPS decreased after 0-3 weeks when compared to 0-1 week (P = 0.010) with no effects of protein intake (P = 0.827). A similar decline with no difference between groups (P = 0.323) was also observed for CPS (P = 0.007). Our results demonstrated that additional protein intake above moderate amounts does not potentiate the stimulation of longer-term MPS responses during the early stage of resistance training adaptations in middle-aged adults.

A muscle-centric view of time-restricted feeding for older adults.

PURPOSE OF REVIEW: The practice of time-restricted feeding (TRF) has received fervent interest in recent years as a strategy to mitigate obesity and metabolic disease. We sought to review the implications of TRF for skeletal muscle health and function in aging. RECENT FINDINGS: TRF has high adherence and can promote body weight loss in older populations. Body weight reductions favor fat mass in the young, however, there is also the potential for undesirable losses in lean mass. There is currently no evidence to support TRF for skeletal muscle function and metabolism in older persons, and only tentative findings in the young. With a narrow eating window of 6-8 h and a prolonged fasting period to minimize daily insulin exposure, TRF may contradict recommended dietary practices for optimizing skeletal muscle anabolism in older people. SUMMARY: TRF might represent a promising intervention to address obesity and its associated metabolic diseases, however, at present there is insufficient evidence for optimizing skeletal muscle mass or health in older individuals. Further research is needed to: (1) ascertain the impact of TRF on body composition, skeletal muscle anabolism, and autophagy in aging, and; (2) delineate the potentially myoprotective roles of dietary protein and exercise within the framework of TRF in older persons.

Incorporation of Dietary Amino Acids Into Myofibrillar and Sarcoplasmic Proteins in Free-Living Adults Is Influenced by Sex, Resistance Exercise, and Training Status.

BACKGROUND: Acute exercise increases the incorporation of dietary amino acids into de novo myofibrillar proteins after a single meal in controlled laboratory studies in males. It is unclear whether this extends to free-living settings or is influenced by training or sex. OBJECTIVES: We determined the effects of exercise, training status, and sex on 24-hour free-living dietary phenylalanine incorporation into skeletal muscle proteins. METHODS: In a parallel group design, recreationally active males (mean ± SD age, 23 ± 3 years;  BMI. 23.4 ± 2.9 kg/m2; n = 10) and females (age 24 ± 5 years;  BMI, 23.1 ± 3.9 kg/m2; n = 9) underwent 8 weeks of whole-body resistance exercise 3 times a week. Controlled diets containing 1.6 g/kg-1/d-1 (amino acids modelled after egg), enriched to 10% with [13C6] or [2H5]phenylalanine, were consumed before and after an acute bout of resistance exercise. Fasted muscle biopsies were obtained before [untrained, pre-exercise condition (REST ] and 24 hours after an acute bout of resistance exercise in untrained (UT) and trained (T) states to determine dietary phenylalanine incorporation into myofibrillar (ΔMyo) and sarcoplasmic (ΔSarc) proteins, intracellular mechanistic target of rapamycin (mTOR) colocalization with ulex europaeus agglutinin-1 (UEA-1; capillary marker; immunofluorescence), and amino acid transporter expression (Western blotting). RESULTS: The ΔMyo values were ∼62% greater (P < 0.01) in females than males at REST. The ΔMyo values increased above REST by ∼51% during UT and ∼30% in T (both P < 0.01) in males, remained unchanged in females during UT, and were ∼33% lower at T when compared to UT (P = 0.013). Irrespective of sex, ΔMyo and ΔSarc were decreased at T compared to UT (P ≤ 0.026). Resistance training increased mTOR colocalization with UEA-1 (P = 0.004), while L amino acid transporter 1, which was greater in males (P < 0.01), and sodium-coupled neutral amino acid transporter 2 protein expression were not affected by acute exercise (P ≥ 0.33) or training (P ≥ 0.45). CONCLUSIONS: The exercise-induced incorporation of dietary phenylalanine into myofibrillar and sarcoplasmic proteins is attenuated after training regardless of sex, suggesting a reduced reliance on dietary amino acids for postexercise skeletal muscle remodeling in the T state.

More than just a garbage can: emerging roles of the lysosome as an anabolic organelle in skeletal muscle.

Skeletal muscle is a highly plastic tissue capable of remodeling in response to a range of physiological stimuli, including nutrients and exercise. Historically, the lysosome has been considered an essentially catabolic organelle contributing to autophagy, phagocytosis, and exo-/endocytosis in skeletal muscle. However, recent evidence has emerged of several anabolic roles for the lysosome, including the requirement for autophagy in skeletal muscle mass maintenance, the discovery of the lysosome as an intracellular signaling hub for mechanistic target of rapamycin complex 1 (mTORC1) activation, and the importance of transcription factor EB/lysosomal biogenesis-related signaling in the regulation of mTORC1-mediated protein synthesis. We, therefore, propose that the lysosome is an understudied organelle with the potential to underpin the skeletal muscle adaptive response to anabolic stimuli. Within this review, we describe the molecular regulation of lysosome biogenesis and detail the emerging anabolic roles of the lysosome in skeletal muscle with particular emphasis on how these roles may mediate adaptations to chronic resistance exercise. Furthermore, given the well-established role of amino acids to support muscle protein remodeling, we describe how dietary proteins "labeled" with stable isotopes could provide a complementary research tool to better understand how lysosomal biogenesis, autophagy regulation, and/or mTORC1-lysosomal repositioning can mediate the intracellular usage of dietary amino acids in response to anabolic stimuli. Finally, we provide avenues for future research with the aim of elucidating how the regulation of this important organelle could mediate skeletal muscle anabolism.

Protein Intake to Maximize Whole-Body Anabolism during Postexercise Recovery in Resistance-Trained Men with High Habitual Intakes is Severalfold Greater than the Current Recommended Dietary Allowance.

BACKGROUND: Dietary protein supports resistance exercise-induced anabolism primarily via the stimulation of protein synthesis rates. The indicator amino acid oxidation (IAAO) technique provides a noninvasive estimate of the protein intake that maximizes whole-body protein synthesis rates and net protein balance. OBJECTIVE: We utilized IAAO to determine the maximal anabolic response to postexercise protein ingestion in resistance-trained men. METHODS: Seven resistance-trained men (mean ± SD age 24 ± 3 y; weight 80 ± 9 kg; 11 ± 5% body fat; habitual protein intake 2.3 ± 0.6 g·kg-1·d-1) performed a bout of whole-body resistance exercise prior to ingesting hourly mixed meals, which provided a variable amount of protein (0.20-3.00 g·kg-1·d-1) as crystalline amino acids modeled after egg protein. Steady-state protein kinetics were modeled with oral l-[1-13C]-phenylalanine. Breath and urine samples were taken at isotopic steady state to determine phenylalanine flux (PheRa), phenylalanine excretion (F13CO2; reciprocal of protein synthesis), and net balance (protein synthesis - PheRa). Total amino acid oxidation was estimated from the ratio of urinary urea and creatinine. RESULTS: Mixed model biphasic linear regression revealed a plateau in F13CO2 (mean: 2.00; 95% CI: 1.62, 2.38 g protein·kg-1·d-1) (r2 = 0.64; P Ë‚ 0.01) and in net balance (mean: 2.01; 95% CI: 1.44, 2.57 g protein·kg-1·d-1) (r2 = 0.63; P Ë‚ 0.01). Ratios of urinary urea and creatinine concentrations increased linearly (r = 0.84; P Ë‚ 0.01) across the range of protein intakes. CONCLUSIONS: A breakpoint protein intake of ∼2.0 g·kg-1·d-1, which maximized whole-body anabolism in resistance-trained men after exercise, is greater than previous IAAO-derived estimates for nonexercising men and is at the upper range of current general protein recommendations for athletes. The capacity to enhance whole-body net balance may be greater than previously suggested to maximize muscle protein synthesis in resistance-trained athletes accustomed to a high habitual protein intake. This trial was registered at clinicaltrials.gov as NCT03696264.

Molecular regulation of human skeletal muscle protein synthesis in response to exercise and nutrients: a compass for overcoming age-related anabolic resistance.

Skeletal muscle mass, a strong predictor of longevity and health in humans, is determined by the balance of two cellular processes, muscle protein synthesis (MPS) and muscle protein breakdown. MPS seems to be particularly sensitive to changes in mechanical load and/or nutritional status; therefore, much research has focused on understanding the molecular mechanisms that underpin this cellular process. Furthermore, older individuals display an attenuated MPS response to anabolic stimuli, termed anabolic resistance, which has a negative impact on muscle mass and function, as well as quality of life. Therefore, an understanding of which, if any, molecular mechanisms contribute to anabolic resistance of MPS is of vital importance in formulation of therapeutic interventions for such populations. This review summarizes the current knowledge of the mechanisms that underpin MPS, which are broadly divided into mechanistic target of rapamycin complex 1 (mTORC1)-dependent, mTORC1-independent, and ribosomal biogenesis-related, and describes the evidence that shows how they are regulated by anabolic stimuli (exercise and/or nutrition) in healthy human skeletal muscle. This review also summarizes evidence regarding which of these mechanisms may be implicated in age-related skeletal muscle anabolic resistance and provides recommendations for future avenues of research that can expand our knowledge of this area.

Protein Metabolism in Active Youth: Not Just Little Adults.

Understanding how exercise and dietary protein alter the turnover and synthesis of body proteins in youth can provide guidelines for the optimal development of lean mass. This review hypothesizes that active youth obtain similar anabolic benefits from exercise and dietary protein as adults, but the requirement for amino acids to support growth renders them more sensitive to these nutrients.

Nutrition for Special Populations: Young, Female, and Masters Athletes.

Adolescent, female, and masters athletes have unique nutritional requirements as a consequence of undertaking daily training and competition in addition to the specific demands of age- and gender-related physiological changes. Dietary education and recommendations for these special population athletes require a focus on eating for long-term health, with special consideration given to "at-risk" dietary patterns and nutrients (e.g., sustained restricted eating, low calcium, vitamin D and/or iron intakes relative to requirements). Recent research highlighting strategies to address age-related changes in protein metabolism and the development of tools to assist in the management of Relative Energy Deficiency in Sport are of particular relevance to special population athletes. Whenever possible, special population athletes should be encouraged to meet their nutrient needs by the consumption of whole foods rather than supplements. The recommendation of dietary supplements (particularly to young athletes) overemphasizes their ability to manipulate performance in comparison with other training/dietary strategies.

Whole egg, but not egg white, ingestion induces mTOR colocalization with the lysosome after resistance exercise.

We have recently demonstrated that whole egg ingestion induces a greater muscle protein synthetic (MPS) response when compared with isonitrogenous egg white ingestion after resistance exercise in young men. Our aim was to determine whether whole egg or egg white ingestion differentially influenced colocalization of key regulators of mechanistic target of rapamycin complex 1 (mTORC1) as means to explain our previously observed divergent postexercise MPS response. In crossover trials, 10 healthy resistance-trained men (21 ± 1 yr; 88 ± 3 kg; body fat: 16 ± 1%; means ± SE) completed lower body resistance exercise before ingesting whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat). Muscle biopsies were obtained before exercise and at 120 and 300 min after egg ingestion to assess, by immunofluorescence, protein colocalization of key anabolic signaling molecules. After resistance exercise, tuberous sclerosis 2-Ras homolog enriched in brain (Rheb) colocalization decreased ( P < 0.01) at 120 and 300 min after whole egg and egg white ingestion with concomitant increases ( P < 0.01) in mTOR-Rheb colocalization. After resistance exercise, mTOR-lysosome-associated membrane protein 2 (LAMP2) colocalization significantly increased at 120 and 300 min only after whole egg ingestion ( P < 0.01), and mTOR-LAMP2 colocalization correlated with rates of MPS at rest and after exercise ( r = 0.40, P < 0.05). We demonstrated that the greater postexercise MPS response with whole egg ingestion is related in part to an enhanced recruitment of mTORC1-Rheb complexes to the lysosome during recovery. These data suggest nonprotein dietary factors influence the postexercise regulation of mRNA translation in human skeletal muscle.

Branched-Chain Amino Acids Are the Primary Limiting Amino Acids in the Diets of Endurance-Trained Men after a Bout of Prolonged Exercise.

Background: The indicator amino acid oxidation (IAAO) method estimates the protein intake required to maximize whole-body protein synthesis and identify the daily protein requirement in a variety of populations. However, it is unclear whether the greater requirements for endurance athletes previously determined by the IAAO reflect an increased demand for all or only some amino acids. Objective: The aim of this study was to determine the primary rate-limiting amino acids in endurance-trained athletes after prolonged exercise, by measuring the oxidation of ingested [1-13C]phenylalanine in response to variable amino acid intake. Methods: Five endurance-trained men (means ± SDs: age, 26 ± 7 y; body weight, 66.9 ± 9.5 kg; maximal oxygen consumption, 63.3 ± 4.3 mL · kg-1 · min-1) performed 5 trials that involved 2 d of controlled diet (1.4 g protein · kg-1 · d-1) and running (10 km on day 1 and 5 km on day 2) prior to performing an acute bout of endurance exercise (20-km treadmill run) on day 3. During recovery on day 3, participants consumed test diets as 8 isocaloric hourly meals providing sufficient energy and carbohydrate but a variable amino acid intake. The test diets, consumed in random order, were deficient (BASE: 0.8 g · kg-1 · d-1) and sufficient (SUF; 1.75 g · kg-1 · d-1) amino acid diets modeled after egg protein, and BASE supplemented with branched-chain amino acids (BCAA diet; 1.03 g · kg-1 · d-1), essential amino acids (EAA diet; 1.23 g · kg-1 · d-1), or nonessential amino acids (NEAA diet; 1.75 g · kg-1 · d-1). Whole-body phenylalanine flux (Q), 13CO2 excretion (F13CO2), and phenylalanine oxidation (OX) were determined according to standard IAAO methodology. Results: There was no effect of amino acid intake on Q (P = 0.43). F13CO2 was significantly (all P < 0.01) lower than BASE for the BCAA (∼32%), EAA (∼31%), and SUF (∼36%) diet treatments. F13CO2 for the NEAA diet was ∼18% lower than for BASE (P < 0.05) but ∼28% greater than for SUF (P < 0.05). OX was similarly decreased (∼24-41%) in all conditions compared with BASE (all P < 0.05). Conclusion: Our results suggest that the BCAAs may be the primary rate-liming amino acids in the greater daily protein requirement of endurance trained men. This trial was registered at clinicaltrials.gov as NCT02628249.

Protein Intake at Breakfast Promotes a Positive Whole-Body Protein Balance in a Dose-Response Manner in Healthy Children: A Randomized Trial.

Background: Protein ingestion promotes whole-body net protein balance (NB) in children, which is a prerequisite for growth. Determining how much protein is required at breakfast to promote a positive NB, which may be negative after the traditional overnight fast in children, has yet to be determined. Objective: We determined the impact of incremental doses of milk protein at breakfast as well as the impact of daily dietary protein distribution on NB in children. Methods: A total of 28 children [14 boys, 14 girls; age range: 7-11 y; body mass index (mean ± SD, in kg/m2): 16.0 ± 1.9] completed 2 intervention trials. During the breakfast meal, participants consumed an isoenergetic beverage with different amounts of protein (0, 7, 14, or 21 g for Groups A-D, respectively) and [15N]-glycine to measure whole body protein metabolism. Whole-body nitrogen turnover, protein synthesis (PS), protein breakdown, and NB were measured over 9 and 24 h. Results: Following an overnight fast, children were in negative NB (-64.5 mg · kg-1 · h-1). Protein ingestion at breakfast induced a stepwise increase in NB over 9 h [Groups A (6.2 mg · kg-1 · h-1) < B (27.9 mg · kg-1 · h-1) < C (46.9 mg · kg-1 · h-1) < D (66.0 mg · kg-1 · h-1)] with all conditions different from each other (all P < 0.01). PS was 42% greater in Group D than in Group A over 9 h (P < 0.05). Conclusions: Consuming ≥7 g of the total daily protein intake at breakfast attenuates the observed overnight protein losses in children during the subsequent 9 h following breakfast consumption. The dose-dependent increase in NB over a daytime fed period, inclusive of breakfast and lunch, highlights the importance of breakfast protein intake on acute anabolism in healthy active children. This trial was registered at clinicaltrials.gov as NCT02465151.

Nutritionally non-essential amino acids are dispensable for whole-body protein synthesis after exercise in endurance athletes with an adequate essential amino acid intake.

The increased protein requirement of endurance athletes may be related to the need to replace exercise-induced oxidative losses, especially of the branched-chain amino acids (BCAA). However, it is unknown if non-essential amino acids (NEAA) influence the requirement for essential amino acids (EAA) during post-exercise recovery. Seven endurance-trained males ran 20 km prior to consuming [13C]phenylalanine, sufficient energy, and: (1) deficient protein (BASE); (2) BASE supplemented with sufficient BCAA (BCAAsup); (3) an equivalent EAA intake as BCAA (LowEAA), and; (4) sufficient EAA intake (HighEAA). [13C]Phenylalanine oxidation (the reciprocal of protein synthesis) for BCAAsup and HighEAA (0.54 ± 0.15, 0.49 ± 0.11 µmol kg-1 h-1; Mean ± SD) were significantly lower than BASE (0.74 ± 0.14 µmol kg-1 h-1; P < 0.01 for both) and LowEAA (0.70 ± 0.11 µmol kg-1 h-1; P < 0.05 and 0.01, respectively). Our results suggest that exogenous NEAA are dispensable for whole-body protein synthesis during recovery from endurance exercise provided sufficient EAA are consumed. Endurance athletes who may be at risk of not meeting their elevated protein requirements should prioritize the intake of EAA-enriched foods and/or supplements.