Acute effects of a ketone monoester, whey protein, or their co-ingestion on mTOR trafficking and protein-protein co-localization in human skeletal muscle.

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their co-ingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their co-ingestion on mTOR-related protein-protein co-localization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2±4.1 y) ingested either: 1) 0.36 g ∙ kg-1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET+PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120- and 300-minutes in the postprandial period for immunofluorescence assessment of protein translocation and co-localization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P<0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) co-localization at 120-minutes vs. basal; however, the decrease was sustained at 300-minutes vs. basal (P<0.0001) only in KET+PRO. PRO and KET+PRO increased (Interaction: P<0.0001) mTOR-Rheb co-localization at 120-minutes vs. basal; however, KET+PRO resulted in a sustained increase in mTOR-Rheb co-localization at 300-minutes that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) co-localization at 120- and 300-minutes (Time: P=0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle.

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.

Trained Integrated Postexercise Myofibrillar Protein Synthesis Rates Correlate with Hypertrophy in Young Males and Females.

PURPOSE: Resistance training induces skeletal muscle hypertrophy via the summated effects of postexercise elevations in myofibrillar protein synthesis (MyoPS) that persist for up to 48 h, although research in females is currently lacking. MyoPS is regulated by mTOR translocation and colocalization; however, the effects of resistance training on these intracellular processes are unknown. We hypothesized that MyoPS would correlate with hypertrophy only after training in both sexes and would be associated with intracellular redistribution of mTOR. METHODS: Recreationally active males and females (n = 10 each) underwent 8 wk of whole-body resistance exercise three times a week. Fasted muscle biopsies were obtained immediately before (REST) and 24 and 48 h after acute resistance exercise in the untrained (UT) and trained (T) states to determine integrated MyoPS over 48 h (D2O ingestion) and intracellular mTOR colocalization (immunofluorescence microscopy). RESULTS: Training increased (P < 0.01) muscle strength (~20%-126%), muscle thickness (~8%-11%), and average fiber cross-sectional area (~15%-20%). MyoPS increased above REST in UT (P = 0.032) and T (P < 0.01), but to a greater extent in males (~23%; P = 0.023), and was positively (P < 0.01) associated with muscle thickness and fiber cross-sectional area at T only in both males and females. mTOR colocalization with the cell periphery increased (P < 0.01) in T, irrespective of sex or acute exercise. Training increased (P ≤ 0.043) total mTOR, LAMP2 (lysosomal marker), and their colocalization (P < 0.01), although their colocalization was greater in males at 24 and 48 h independent of training status (P < 0.01). CONCLUSIONS: MyoPS during prolonged recovery from exercise is greater in males but related to muscle hypertrophy regardless of sex only in the trained state, which may be underpinned by altered mTOR localization.

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.

Satellite cell and myonuclear accretion is related to training-induced skeletal muscle fiber hypertrophy in young males and females.

Satellite cells (SC) play an integral role in the recovery from skeletal muscle damage and supporting muscle hypertrophy. Acute resistance exercise typically elevates type I and type II SC content 24-96 h post exercise in healthy young males, although comparable research in females is lacking. We aimed to elucidate whether sex-based differences exist in fiber type-specific SC content after resistance exercise in the untrained (UT) and trained (T) states. Ten young males (23.0 ± 4.0 yr) and females (23.0 ± 4.8 yr) completed an acute bout of resistance exercise before and after 8 wk of whole body resistance training. Muscle biopsies were taken from the vastus lateralis immediately before and 24 and 48 h after each bout to determine SC and myonuclear content by immunohistochemistry. Males had greater SC associated with type II fibers (P ≤ 0.03). There was no effect of acute resistance exercise on SC content in either fiber type (P ≥ 0.58) for either sex; however, training increased SC in type II fibers (P < 0.01) irrespective of sex. The change in mean 0-48 h type II SC was positively correlated with muscle fiber hypertrophy in type II fibers (r = 0.47; P = 0.035). Furthermore, the change in myonuclei per fiber was positively correlated with type I and type II fiber hypertrophy (both r = 0.68; P < 0.01). Our results suggest that SC responses to acute and chronic resistance exercise are similar in males and females and that SC and myonuclear accretion is related to training-induced muscle fiber hypertrophy.NEW & NOTEWORTHY We demonstrate that training-induced increase in SC content in type II fibers and myonuclear content in type I and II fibers is similar between males and females. Furthermore, these changes are related to the extent of muscle fiber hypertrophy. Thus, SC and myonuclear accretion appear to contribute to muscle hypertrophy irrespective of sex, highlighting the importance of these muscle stem cells in human skeletal muscle growth.

Protein Timing Does Not Affect Next-Day Recovery of Strength or Power but May Enhance Aerobic Adaptations to Short-Term Variable Intensity Exercise Training in Recreationally Active Males: A Pilot Study.

Background: Variable intensity training (VIT) characteristic of stop-and-go team sport exercise may reduce performance capacity when performed on successive days but also represent a strategy to induce rapid training-induced increases in exercise capacity. Although post-exercise protein enhances muscle protein synthesis, the timing of protein ingestion following variable intensity training (VIT) on next-day recovery and short-term performance adaptation is unknown. Purpose: To determine if immediate (IMM) as compared to delayed (DEL) protein ingestion supports greater acute recovery of exercise performance during successive days of VIT and/or supports chronic training adaptations. Methods: Sixteen habitually active men performed 5 consecutive days of variable intensity training (VIT) in the evening prior to consuming a beverage providing carbohydrate and whey protein (IMM; 0.7 g and 0.3 g/kg, respectively) or carbohydrates alone (DEL; 1 g/kg) with the reciprocal beverage consumed the following morning. Performance was assessed before each VIT (recovery) and 2 days after the final VIT (adaptation). Results: Five consecutive days of VIT progressively decreased anaerobic peak power (~7%) and muscle strength (MVC; ~8%) with no impact of protein timing. Following 2 days of recovery, VIT increased maximal voluntary contraction and predicted VO2peak by ~10 and ~5%, respectively, with a moderate beneficial effect of IMM on predicted VO2peak (ES = 0.78). Conclusion: Successive days of simulated team sport exercise decreases markers of next-day performance capacity with no effect of protein timing on acute recovery. However, practical VIT increases muscle strength and aerobic capacity in as little as 5 days with the latter potentially enhanced by immediate post-exercise protein consumption.

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.

Leucine-enriched amino acids maintain peripheral mTOR-Rheb localization independent of myofibrillar protein synthesis and mTORC1 signaling postexercise.

Postexercise protein ingestion can elevate rates of myofibrillar protein synthesis (MyoPS), mTORC1 activity, and mTOR translocation/protein-protein interactions. However, it is unclear if leucine-enriched essential amino acids (LEAA) can similarly facilitate intracellular mTOR trafficking in humans after exercise. The purpose of this study was to determine the effect of postexercise LEAA (4 g total EAAs, 1.6 g leucine) on acute MyoPS and mTORC1 translocation and signaling. Recreationally active men performed lower-body resistance exercise (5 × 8-10 leg press and leg extension) to volitional failure. Following exercise participants consumed LEAA (n = 8) or an isocaloric carbohydrate drink (PLA; n = 10). MyoPS was measured over 1.5-4 h of recovery by oral pulse of l-[ring-2H5]-phenylalanine. Phosphorylation of proteins in the mTORC1 pathway were analyzed via immunoblotting and mTORC1-LAMP2/WGA/Rheb colocalization via immunofluorescence microscopy. There was no difference in MyoPS between groups (LEAA = 0.098 ± 0.01%/h; PL = 0.090 ± 0.01%/h; P > 0.05). Exercise increased (P < 0.05) rpS6Ser240/244(LEAA = 35.3-fold; PLA = 20.6-fold), mTORSer2448(LEAA = 1.8-fold; PLA = 1.2-fold) and 4EBP1Thr37/46(LEAA = 1.5-fold; PLA = 1.4-fold) phosphorylation irrespective of nutrition (P > 0.05). LAT1 and SNAT2 protein expression were not affected by exercise or nutrient ingestion. mTOR-LAMP2 colocalization was greater in LEAA preexercise and decreased following exercise and supplement ingestion (P < 0.05), yet was unchanged in PLA. mTOR-WGA (cell periphery marker) and mTOR-Rheb colocalization was greater in LEAA compared with PLA irrespective of time-point (P < 0.05). In conclusion, the postexercise consumption of 4 g of LEAA maintains mTOR in peripheral regions of muscle fibers, in closer proximity to its direct activator Rheb, during prolonged recovery independent of differences in MyoPS or mTORC1 signaling compared with PLA ingestion. This intracellular localization of mTOR may serve to "prime" the kinase for future anabolic stimuli.NEW & NOTEWORTHY This is the first study to investigate whether postexercise leucine-enriched amino acid (LEAA) ingestion elevates mTORC1 translocation and protein-protein interactions in human skeletal muscle. Here, we observed that although LEAA ingestion did not further elevate postexercise MyoPS or mTORC1 signaling compared with placebo, mTORC1 peripheral location and interaction with Rheb were maintained. This may serve to "prime" mTORC1 for subsequent anabolic stimuli.

Leucine-Enriched Essential Amino Acids Improve Recovery from Post-Exercise Muscle Damage Independent of Increases in Integrated Myofibrillar Protein Synthesis in Young Men.

BACKGROUND: Leucine-enriched essential amino acids (LEAAs) acutely enhance post-exercise myofibrillar protein synthesis (MyoPS), which has been suggested to be important for muscle repair and recovery. However, the ability of LEAAs to concurrently enhance MyoPS and muscle damage recovery in free-living humans has not been studied. METHODS: In a randomized, double-blind, placebo-controlled, parallel-group design, twenty recreationally active males consuming a controlled diet (1.2 g/kg/d of protein) were supplemented thrice daily with 4 g of LEAAs (containing 1.6 g leucine) or isocaloric placebo for four days following an acute bout of lower-body resistance exercise (RE). MyoPS at rest and integrated over 96 h of recovery was measured by D2O. Isometric and isokinetic torque, muscle soreness, Z-band streaming, muscle heat shock protein (HSP) 25 and 72, plasma creatine kinase (CK), and plasma interleukin-6 (IL-6) were measured over 96 h post-RE to assess various direct and indirect markers of muscle damage. RESULTS: Integrated MyoPS increased ~72% over 96 h after RE (p < 0.05), with no differences between groups (p = 0.98). Isometric, isokinetic, and total peak torque decreased ~21% by 48 h after RE (p < 0.05), whereas total peak torque was ~10% greater overall during recovery in LEAAs compared to placebo (p < 0.05). There were moderate to large effects for peak torque in favour of LEAAs. Muscle soreness increased during recovery with no statistical differences between groups but small to moderate effects in favour of LEAAs that correlated with changes in peak torque. Plasma CK, plasma IL-6, and muscle HSP25 increased after RE (p < 0.05) but were not significantly different between groups (p ≥ 0.13). Consistent with a trend toward attenuated Z-band streaming in LEAAs (p = 0.07), muscle HSP72 expression was lower (p < 0.05) during recovery in LEAAs compared with placebo. There were no correlations between MyoPS and any measures of muscle damage (p ≥ 0.37). CONCLUSION: Collectively, our data suggest that LEAAs moderately attenuated muscle damage without concomitant increases in integrated MyoPS in the days following an acute bout of resistance exercise in free-living recreationally active men.

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.

Protein to Maximize Whole-Body Anabolism in Resistance-trained Females after Exercise.

INTRODUCTION: Current athlete-specific protein recommendations are based almost exclusively on research in males. PURPOSE: Using the minimally invasive indicator amino acid oxidation technique, we determined the daily protein intake that maximizes whole-body protein synthesis (PS) and net protein balance (NB) after exercise in strength-trained females. METHODS: Eight resistance-trained females (23 ± 3.5 yr, 67.0 ± 7.7 kg, 163.3 ± 3.7 cm, 24.4% ± 6.9% body fat; mean ± SD) completed a 2-d controlled diet during the luteal phase before performing an acute bout of whole-body resistance exercise. During recovery, participants consumed eight hourly meals providing a randomized test protein intake (0.2-2.9 g·kg·d) as crystalline amino acids modeled after egg protein, with constant phenylalanine (30.5 mg·kg·d) and excess tyrosine (40.0 mg·kg·d) intakes. Steady-state whole-body phenylalanine rate of appearance (Ra), oxidation (Ox; the reciprocal of PS), and NB (PS - Ra) were determined from oral [C] phenylalanine ingestion. Total protein oxidation was estimated from the urinary urea-creatinine ratio (U/Cr). RESULTS: A mixed model biphase linear regression revealed a break point (i.e., estimated average requirement) of 1.49 ± 0.44 g·kg·d (mean ± 95% confidence interval) in Ox (r = 0.64) and 1.53 ± 0.32 g·kg·d in NB (r = 0.65), indicating a saturation in whole-body anabolism. U/Cr increased linearly with protein intake (r = 0.56, P < 0.01). CONCLUSIONS: Findings from this investigation indicate that the safe protein intake (upper 95% confidence interval) to maximize anabolism and minimize protein oxidation for strength-trained females during the early ~8-h postexercise recovery period is at the upper end of the recommendations of the American College of Sports Medicine for athletes (i.e., 1.2-2.0 g·kg·d).

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.

Translocation and protein complex co-localization of mTOR is associated with postprandial myofibrillar protein synthesis at rest and after endurance exercise.

Translocation and colocalization of mechanistic target of rapamycin complex 1 (mTORC1) with regulatory proteins represents a critical step in translation initiation of protein synthesis in vitro. However, mechanistic insight into the control of postprandial skeletal muscle protein synthesis rates at rest and after an acute bout of endurance exercise in humans is lacking. In crossover trials, eight endurance-trained men received primed-continuous infusions of L-[ring-2 H5 ]phenylalanine and consumed a mixed-macronutrient meal (18 g protein, 60 g carbohydrates, 17 g fat) at rest (REST) and after 60 min of treadmill running at 70% VO2peak (EX). Skeletal muscle biopsies were collected to measure changes in phosphorylation and colocalization in the mTORC1-pathway, in addition to rates of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis. MyoPS increased (P < 0.05) above fasted in REST (~2.1-fold) and EX (~twofold) during the 300 min postprandial period, with no corresponding changes in MitoPS (P > 0.05). TSC2/Rheb colocalization decreased below fasted at 60 and 300 min after feeding in REST and EX (P < 0.01). mTOR colocalization with Rheb increased above fasted at 60 and 300 min after feeding in REST and EX (P < 0.01), which was consistent with an increased phosphorylation 4E-BP1Thr37/46 and rpS6ser240/244 at 60 min. Our data suggest that MyoPS, but not MitoPS, is primarily nutrient responsive in trained young men at rest and after endurance exercise. The postprandial increase in MyoPS is associated with an increase in mTOR/Rheb colocalization and a reciprocal decrease in TSC2/Rheb colocalization and thus likely represent important regulatory events for in vivo skeletal muscle myofibrillar mRNA translation in humans.

Consumption of whole eggs promotes greater stimulation of postexercise muscle protein synthesis than consumption of isonitrogenous amounts of egg whites in young men.

Background: Protein in the diet is commonly ingested from whole foods that contain various macro- and micronutrients. However, the effect of consuming protein within its natural whole-food matrix on postprandial protein metabolism remains understudied in humans.Objective: We aimed to compare the whole-body and muscle protein metabolic responses after the consumption of whole eggs with egg whites during exercise recovery in young men.Design: In crossover trials, 10 resistance-trained men [aged 21 ± 1 y; 88 ± 3 kg; body fat: 16% ± 1% (means ± SEMs)] received primed continuous l-[ring-2H5]phenylalanine and l-[1-13C]leucine infusions and performed a single bout of resistance exercise. After exercise, participants consumed intrinsically l-[5,5,5-2H3]leucine-labeled whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat). Repeated blood and muscle biopsy samples were collected to assess whole-body leucine kinetics, intramuscular signaling, and myofibrillar protein synthesis.Results: Plasma appearance rates of protein-derived leucine were more rapid after the consumption of egg whites than after whole eggs (P = 0.01). Total plasma availability of leucine over the 300-min postprandial period was similar (P= 0.75) between the ingestion of whole eggs (68% ± 1%) and egg whites (66% ± 2%), with no difference in whole-body net leucine balance (P = 0.27). Both whole-egg and egg white conditions increased the phosphorylation of mammalian target of rapamycin complex 1, ribosomal protein S6 kinase 1, and eukaryotic translation initiation factor 4E-binding protein 1 during postexercise recovery (all P < 0.05). However, whole-egg ingestion increased the postexercise myofibrillar protein synthetic response to a greater extent than did the ingestion of egg whites (P= 0.04).Conclusions: We show that the ingestion of whole eggs immediately after resistance exercise resulted in greater stimulation of myofibrillar protein synthesis than did the ingestion of egg whites, despite being matched for protein content in young men. Our data indicate that the ingestion of nutrient- and protein-dense foods differentially stimulates muscle anabolism compared with protein-dense foods. This trial was registered at clinicaltrials.gov as NCT03117127.

Endurance Exercise Attenuates Postprandial Whole-Body Leucine Balance in Trained Men.

PURPOSE: Endurance exercise increases indices of small intestinal damage and leucine oxidation, which may attenuate dietary amino acid appearance and postprandial leucine balance during postexercise recovery. Therefore, the purpose of this study was to examine the effect of an acute bout of endurance exercise on postprandial leucine kinetics and net leucine balance. METHODS: In a crossover design, seven trained young men (age = 25.6 ± 2.3 yr; V˙O2peak = 61.4 ± 2.9 mL·kg·min; mean ± SEM) received a primed constant infusion of L-[1-C]leucine before and after ingesting a mixed macronutrient meal containing 18 g whole egg protein intrinsically labeled with L-[5,5,5-H3]leucine, 17 g fat, and 60 g carbohydrate at rest and after 60 min of treadmill running at 70% V˙O2peak. RESULTS: Plasma intestinal fatty acid binding protein concentrations and leucine oxidation both increased (P < 0.01) to peaks that were ~2.5-fold above baseline values during exercise with a concomitant decrease (P < 0.01) in nonoxidative leucine disposal. Meal ingestion attenuated (P < 0.01) endogenous leucine rates of appearance at rest and after exercise. There were no differences (both, P > 0.05) in dietary leucine appearance rates or in the amount of dietary protein-derived leucine that appeared into circulation over the 5-h postprandial period at rest and after exercise (62% ± 2% and 63% ± 2%, respectively). Leucine balance over the 5-h postprandial period was positive (P < 0.01) in both conditions but was negative (P < 0.01) during the exercise trial after accounting for exercise-induced leucine oxidation. CONCLUSIONS: We demonstrate that endurance exercise does not modulate dietary leucine availability from a mixed meal but attenuates postprandial whole-body leucine balance in trained young men.

Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study.

No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h) and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD)) performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey) or an energy-matched placebo (CHO) immediately post-exercise (0 h), and again the following morning (~10 h of recovery). A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest). Participants ingested [15N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES) = 0.61, PRO vs. CHO) during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036) but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO), which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP), maximal strength (MVC), peak and mean power, and countermovement jump performance (CMJ) at 0 h (all P < 0.05 vs. Pre). At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56), mean power (ES = 0.49), and CMJ variables (ES: 0.27-0.49) in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76), REP (ES = 0.44), and peak power (ES = 0.55). In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of exercise performance after a strenuous bout of resistance exercise.