Effects of high versus standard essential amino acid intakes on whole-body protein turnover and mixed muscle protein synthesis during energy deficit: A randomized, crossover study

Open AccessPublished:July 21, 2020DOI:https://doi.org/10.1016/j.clnu.2020.07.019

      Summary

      Background & aims

      Consuming 0.10–0.14 g essential amino acids (EAA)/kg/dose (0.25–0.30 g protein/kg/dose) maximally stimulates muscle protein synthesis (MPS) during energy balance. Whether consuming EAA beyond that amount enhances MPS and whole-body anabolism following energy deficit is unknown. The aims of this study were to determine the effects of standard and high EAA ingestion on mixed MPS and whole-body protein turnover following energy deficit.

      Design

      Nineteen males (mean ± SD; 23 ± 5 y; 25.4 ± 2.7 kg/m2) completed a randomized, double-blind crossover study consisting of two, 5-d energy deficits (−30 ± 4% of total energy requirements), separated by 14-d. Following each energy deficit, mixed MPS and whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and post-resistance exercise (RE) using primed, constant L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Beverages providing standard (0.1 g/kg, 7.87 ± 0.87 g) or high (0.3 g/kg, 23.5 ± 2.54 g) EAA were consumed post-RE. Circulating EAA were measured.

      Results

      Postabsorptive mixed MPS (%/h) at rest was not different (P = 0.67) between treatments. Independent of EAA, postprandial mixed MPS at rest (standard EAA, 0.055 ± 0.01; high EAA, 0.061 ± 0.02) and post-RE (standard EAA, 0.055 ± 0.01; high EAA, 0.065 ± 0.02) were greater than postabsorptive mixed MPS at rest (P = 0.02 and P = 0.01, respectively). Change in (Δ postabsorptive) whole-body (g/180 min) PS and PB was greater for high than standard EAA [mean treatment difference (95% CI), 3.4 (2.3, 4.4); P = 0.001 and −15.6 (−17.8, −13.5); P = 0.001, respectively]. NET was more positive for high than standard EAA [19.0 (17.3, 20.7); P = 0.001]. EAA concentrations were greater in high than standard EAA (P = 0.001).

      Conclusions

      These data demonstrate that high compared to standard EAA ingestion enhances whole-body protein status during underfeeding. However, the effects of consuming high and standard EAA on mixed MPS are the same during energy deficit.

      Clinical trial registry

      Keywords

      Abbreviations:

      EAA (essential amino acids), MPB (muscle protein breakdown), MPS (muscle protein synthesis), RE (resistance exercise), TTR (tracer-to-tracee), PS (whole-body protein synthesis), PB (whole-body protein breakdown), NET (whole-body net protein balance)

      1. Introduction

      In non-obese, young adults, negative energy balance generally elicits muscle mass loss in proportion to the magnitude and duration of the energy deficit incurred [
      • Murphy N.E.
      • Carrigan C.T.
      • Philip Karl J.
      • Pasiakos S.M.
      • Margolis L.M.
      Threshold of energy deficit and lower-body performance declines in military personnel: a meta-regression.
      ,
      • Friedl K.E.
      • Moore R.J.
      • Martinez-Lopez L.E.
      • Vogel J.A.
      • Askew E.W.
      • Marchitelli L.J.
      • et al.
      Lower limit of body fat in healthy active men.
      ]. Consuming dietary protein high in essential amino acids (EAA, i.e., the anabolic component of protein [
      • Volpi E.
      • Ferrando A.A.
      • Yeckel C.W.
      • Tipton K.D.
      • Wolfe R.R.
      Exogenous amino acids stimulate net muscle protein synthesis in the elderly.
      ]), as part of a higher protein diet (>0.8 g/kg/d but <35% total energy), may attenuate muscle loss during energy deficit, in part, by optimizing muscle protein synthesis (MPS) [
      • Carbone J.W.
      • McClung J.P.
      • Pasiakos S.M.
      Recent advances in the characterization of skeletal muscle and whole-body protein responses to dietary protein and exercise during negative energy balance.
      ]. During energy balance, Witard et al. [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ] demonstrated that resting and post-resistance exercise (RE) myofibrillar MPS were maximally stimulated after consuming 20 g (~10 g EAA) of whey protein. Others have reported similar plateaus in MPS in response to varying EAA amounts when examined as components of other high quality intact proteins [
      • Miller S.L.
      • Tipton K.D.
      • Chinkes D.L.
      • Wolf S.E.
      • Wolfe R.R.
      Independent and combined effects of amino acids and glucose after resistance exercise.
      ,
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      Aging is associated with diminished accretion of muscle proteins after the ingestion of a small bolus of essential amino acids.
      ,
      • Tipton K.D.
      • Gurkin B.E.
      • Matin S.
      • Wolfe R.R.
      Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers.
      ,
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]. These results have led to the widely accepted notion that 0.25–0.30 g protein/kg per dose (i.e., 0.10–0.14 g EAA/kg per dose) maximally stimulates MPS in young adults, particularly when combined with the mechanical stress of RE. Moreover, these findings form the basis for post-exercise protein recommendations supported by internationally recognized exercise science and nutrition organizations [
      • Thomas D.T.
      • Erdman K.A.
      • Burke L.M.
      American College of Sports Medicine Joint Position Statement. Nutrition and Athletic Performance.
      ,
      • Pasiakos S.M.
      • Austin K.G.
      • Lieberman H.R.
      • Askew E.W.
      Efficacy and safety of protein supplements for U.S. Armed Forces personnel: consensus statement.
      ,
      • Jager R.
      • Kerksick C.M.
      • Campbell B.I.
      • Cribb P.J.
      • Wells S.D.
      • Skwiat T.M.
      • et al.
      International Society of Sports Nutrition Position Stand: protein and exercise.
      ]. Although these recommendations are scientifically sound, they focus solely on muscle and were conducted during energy balance. The effect of EAA intakes above 0.10–0.14 g/kg per dose on MPS during energy deficit is undetermined.
      Quantifying whole-body protein turnover responses to EAA feeding during energy deficit may indicate the potential protein requirements needed to prevent disruptions in whole-body protein status induced by the catabolic stress of underfeeding. In addition, relying solely on MPS to assess the efficacy of dietary protein or EAA interventions during energy deficit may underestimate the total body anabolic response, which is determined by the balance between protein synthesis and breakdown [
      • Deutz N.E.
      • Wolfe R.R.
      Is there a maximal anabolic response to protein intake with a meal?.
      ]. Whole-body protein synthesis (PS) may reach a maximum in response to increasing amounts of EAA. However, whole-body net protein balance (NET; PS – whole-body protein breakdown, PB) may increase further if the rise in EAA concentrations subsequent to increasing EAA intake induce a reduction in PB [
      • Wolfe R.R.
      • Miller S.L.
      Amino acid availability controls muscle protein metabolism.
      ]. This aspect of protein status in response to energy deficit and EAA intake is only evident when whole-body protein turnover is measured in addition to MPS. To the best of our knowledge, no studies have examined the effects of ingesting varying amounts of EAA on simultaneous measures of MPS and whole-body protein turnover during moderate (~−30% of total energy requirements), short-term (5-d) energy deficit.
      As such, the objective of the current study was to determine the effects of ingesting free-form EAA beyond 0.10–0.14 g/kg in a single dose on resting and post-RE mixed MPS and whole-body protein turnover following a moderate, short-term energy deficit. Our intent was to expand the current literature and to use the information from this study to help inform and optimize dietary strategies for strenuous, catabolic military operations. Our primary hypotheses were that following energy deficit 1) postprandial mixed MPS would be greater following high versus standard EAA ingestion and compared to postabsorptive mixed MPS rates, 2) postprandial post-RE mixed MPS would be stimulated to a greater degree versus postprandial resting mixed MPS, and 3) NET would be more positive after ingesting high versus standard EAA, due primarily to a greater reduction in PB.

      2. Methods

      2.1 Participants

      Participants were eligible for the study if they were healthy (without evidence of chronic illness or injury), young (18–35 y), non-obese (body mass index, <30.0 kg/m2), RE trained (≥2 session/week for previous 6 months) adults and willing to refrain from nonsteroidal anti-inflammatory medications, alcohol, nicotine products, caffeine, and dietary supplements throughout the study. Additional exclusion criteria included having any of the following: injuries that compromise exercise capability; metabolic, cardiovascular, or gastrointestinal disorders (e.g., kidney disease, diabetes, cardiovascular disease); abnormal or problematic blood clotting; lidocaine allergies; condition of alcoholism or substance abuse; blood donation within 8-wk of beginning the study; adherence to a special diet (i.e., vegetarian diet or weight loss diet); or unwillingness or inability to consume study diets and foods. Inclusion and exclusion criteria were assessed using a background history questionnaire which included lifestyle and physical activity questions and confirmed during a general medical clearance conducted by USARIEM medical oversight. Twenty-two participants provided informed, written consent and were enrolled in the study (Fig. 1). One participant was withdrawn before data collection because he no longer met inclusion criteria and two participants withdrew after the first infusion study due to personal reasons. Thus, nineteen participants completed all study procedures, had complete data sets, and were included in the final analyses. This study was approved by the U.S. Army Medical Research and Development Command Institutional Review Board and registered at www.clinicaltrials.gov (NCT03372928). Investigators adhered to the policies for protection of human subjects as prescribed in the U.S. Department of Defense Instruction 3216.02, and the research was conducted in adherence with the provisions of 32 CFR Part 219.

      2.2 Experimental design

      This randomized, double-blind crossover study consisted of two, 5-d periods of controlled, diet-induced energy deficit (−30% of total energy requirements), separated by a 14-d washout (Fig. 2). At the end of each energy deficit period, participants completed a stable isotope infusion study to determine mixed MPS and whole-body PS, PB, and NET in response to standard (0.10 g/kg/dose) or high (0.30 g/kg/dose) EAA intakes at rest and post-RE. To limit the potential confounding effects of pre-study diet, and to be consistent with our previous research [
      • Pasiakos S.M.
      • McClung H.L.
      • Margolis L.M.
      • Murphy N.E.
      • Lin G.G.
      • Hydren J.R.
      • et al.
      Human muscle protein synthetic responses during weight-bearing and non-weight-bearing exercise: a comparative study of exercise modes and recovery nutrition.
      ,
      • Pasiakos S.M.
      • McClung H.L.
      • McClung J.P.
      • Margolis L.M.
      • Andersen N.E.
      • Cloutier G.J.
      • et al.
      Leucine-enriched essential amino acid supplementation during moderate steady state exercise enhances postexercise muscle protein synthesis.
      ,
      • Kim I.Y.
      • Schutzler S.
      • Schrader A.
      • Spencer H.J.
      • Azhar G.
      • Ferrando A.A.
      • et al.
      The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.
      ], volunteers were provided an individualized 3-d run-in, weight-maintaining diet, followed immediately by the 5-d, 30% energy deficit diet. Routine exercise was prohibited during each diet intervention to limit the effects of previous exercise on protein turnover [
      • Phillips S.M.
      • Tipton K.D.
      • Aarsland A.
      • Wolf S.E.
      • Wolfe R.R.
      Mixed muscle protein synthesis and breakdown following resistance exercise in humans.
      ]. To avoid bias, a random numbers generator was used to create the EAA treatment (high versus standard) randomization. All volunteers and study staff were blinded to the EAA treatments, with the exception of a designated staff member who developed the treatment code and prepared the treatments.
      Fig. 2
      Fig. 2Schematic of the infusion study. Muscle biopsy and blood samples were used in combination with primed, constant infusions of L-[2H5]-phenylalanine and L-[2H2]-tyrosine to determine the effects of high versus standard essential amino acid (EAA) amounts on mixed muscle protein synthesis and whole-body protein turnover at rest and post-resistance exercise (unilateral leg model) during energy deficit. Postabsorptive denotes the fasting state (i.e., overnight, ≥8-h) and postprandial denotes the fed state (i.e., after the intake of the high or standard EAA beverage). EAA, essential amino acid.

      2.3 Anthropometrics

      Height was measured in duplicate to the nearest 0.1 cm using a stadiometer (Seritex, Inc., Carlstadt, NJ, USA) at baseline. Fasted (overnight, ≥8-h), nude body weights were measured using a digital scale (Taylor Precision Products, Oak Brook, IL, USA) to the nearest 0.1 kg after voiding at baseline, daily throughout each intervention, and every third day during the washout periods. Fat mass and lean body mass [total mass − (fat mass + bone mass)] were determined using dual energy X-ray absorptiometry (DXA; DPX-IQ; GE Lunar Corp., Madison, WI, USA) at baseline and on the first day of the second, 8-d diet intervention, both after a ≥8-h overnight fast and void.

      2.4 Diet intervention

      Pre-study dietary intake and physical activity levels were determined using 3-d diet and activity records (2 weekdays, 1 weekend day). Total daily energy requirements for the 3-d run-in diets were individualized per participant using the average of the Harris-Benedict [
      • Harris J.A.
      • Benedict F.G.
      A biometric study of basal metabolism in man.
      ] and Mifflin-St Jeor [
      • Mifflin M.D.
      • St Jeor S.T.
      • Hill L.A.
      • Scott B.J.
      • Daugherty S.A.
      • Koh Y.O.
      A new predictive equation for resting energy expenditure in healthy individuals.
      ] equations, multiplied by 0.3 to account for activities of daily living and diet-induced thermogenesis. Registered Dietitians developed menus (Food Processor SQL, v.11.3.2; ESHA Research, Salem, OR, USA) consisting primarily of military combat rations (Meal, Ready-to-Eat; menu 37; Ameriqual, Evansville, IN, USA), supplemented with commercial products (e.g., prepared frozen foods, yogurt, snack foods). Dietary protein was provided at 1.6 g/kg/d, carbohydrate comprised 50–55% of total energy, and fat provided the remaining energy. The 30% energy deficit was induced by reducing carbohydrate and fat intake, while maintaining protein intake at 1.6 g/kg/d. All foods and beverages were weighed to the nearest 0.1 g and distributed to the participants at the start of each run-in and energy deficit diet. Participants were instructed to consume all provided foods and beverages and return the empty packaging. Any uneaten foods or beverages were weighed and documented. Water was allowed ad libitum. During the washout period, participants were instructed to return to their pre-study dietary habits and physical activity patterns, which were recorded every third day using 24-h diet and activity records.

      2.5 Stable isotope infusion studies

      Stable isotope infusion studies were performed the morning (≥8-h overnight fast) after completing each 5-d energy deficit to determine mixed MPS and whole-body protein turnover (Fig. 2). Intravenous catheters were placed, one in the antecubital space for the continuous isotope infusions, and the second in the contralateral dorsal hand vein (or forearm) for serial blood draws. The dorsal hand vein was kept warm using heating pads to reflect arterialized blood [
      • Abumrad N.N.
      • Rabin D.
      • Diamond M.P.
      • Lacy W.W.
      Use of a heated superficial hand vein as an alternative site for the measurement of amino acid concentrations and for the study of glucose and alanine kinetics in man.
      ]. After collecting the baseline blood sample, primed, constant infusions of L-[ring-2H5]-phenylalanine and L-[3,3-2H2]-tyrosine were started and maintained for the next 420 min. A priming dose of L-[ring-2H4]-tyrosine was also administered at the same time to achieve isotopic equilibrium of L-[ring-2H4] tyrosine enrichment derived from L-[ring-2H5]-phenylalanine. All isotopes were purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and the preparations were constituted by a licensed pharmacist and certified sterile and pyrogen free (Johnson Compounding and Wellness, Waltham, MA, USA).
      Vastus lateralis muscle biopsies were collected on both legs during the infusion study. The non-dominant leg was used to assess resting MPS in the postabsorptive and postprandial state during both infusion studies (i.e., standard and high EAA). The first muscle biopsy on the resting leg was performed at 120-min. Participants then rested until they performed a high volume, single leg-RE bout using the dominant leg (same leg for each infusion study) at 195-min as previously described [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. The exercise bout consisted of 8 sets of 10 repetitions of both leg press and leg extension at 80% of pre-determined one repetition maximum. Participants rested 1.5-min between sets and had approximately 45-min to complete the entire exercise bout. If the participant was unable to complete the prescribed workload, the weight was reduced by 4.5 kg until the 8 sets of each exercise were completed [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ]. The exercise bouts were matched between infusion studies. Within 5-min of completing the exercise bout (240-min), muscle biopsies were taken from the exercised and rested legs. Immediately after the biopsies, volunteers consumed either standard (0.10 g/kg/dose) or high (0.30 g/kg/dose) EAA beverages (REAAL Brazilian Berry; Twinlab Consolidated Corporation, Boca Raton, FL dissolved in 500 mL of water and consumed as a bolus, Table 1). The amino acid profile of the product was confirmed by chemical analysis (Eurofins Food Integrity and Innovation, Madison, WI). The EAA formulation of the product used in this study includes arginine because under catabolic stress it is a conditionally essential amino acid [
      • Morris Jr., S.M.
      Arginine: beyond protein.
      ]. EAA were provided relative to body mass to be consistent with overall dietary protein recommendations [
      • Otten J.J.
      • Hellwig J.P.
      • Meyers L.D.
      Dietary reference intakes: the essential guide to nutrient requirements.
      ] and recent work from Moore [
      • Moore D.R.
      Maximizing post-exercise anabolism: the case for relative protein intakes.
      ]. The EAA beverages were spiked with L-[ring-2H5]-phenylalanine to 4% of the total phenylalanine content to limit potential disturbances in isotopic steady-state [
      • Burd N.A.
      • West D.W.
      • Rerecich T.
      • Prior T.
      • Baker S.K.
      • Phillips S.M.
      Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans.
      ]. After the biopsies and EAA ingestion, participants rested for the next 180-min, during which time blood samples were collected, ending with final muscle biopsies on the rested and exercised leg at 420-min (Fig. 2). The postprandial duration was chosen based on previous evidence demonstrating maximal MPS stimulation occurs within 3-h after consuming a 15 g bolus of free-form EAA [
      • Mitchell W.K.
      • Phillips B.E.
      • Williams J.P.
      • Rankin D.
      • Lund J.N.
      • Smith K.
      • et al.
      A dose- rather than delivery profile-dependent mechanism regulates the “muscle-full” effect in response to oral essential amino acid intake in young men.
      ]. Five biopsies were collected from each participant per infusion study; two through one incision on the exercised leg and three through one incision on the rested leg. For each subsequent biopsy, the biopsy needle was angled away from the previous sampling location by ~5 cm to reduce the chance of sampling from a pre-biopsied area and to avoid local inflammation [
      • Volpi E.
      • Chinkes D.L.
      • Rasmussen B.B.
      Sequential muscle biopsies during a 6-h tracer infusion do not affect human mixed muscle protein synthesis and muscle phenylalanine kinetics.
      ,
      • Carbone J.W.
      • Margolis L.M.
      • McClung J.P.
      • Cao J.J.
      • Murphy N.E.
      • Sauter E.R.
      • et al.
      Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.
      ,
      • Drummond M.J.
      • Fry C.S.
      • Glynn E.L.
      • Timmerman K.L.
      • Dickinson J.M.
      • Walker D.K.
      • et al.
      Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise.
      ]. New incisions were made for the second infusion study ~3–5 cm from the first infusion study incisions [
      • Volpi E.
      • Chinkes D.L.
      • Rasmussen B.B.
      Sequential muscle biopsies during a 6-h tracer infusion do not affect human mixed muscle protein synthesis and muscle phenylalanine kinetics.
      ].
      Table 1Amino acid composition of the essential amino acid beverages.
      Values are means ± SD. Listed values correspond to the average g/kg dose used for each treatment. The amino acid profile of the product was confirmed by chemical analysis (Eurofins Food Integrity and Innovation, Madison, WI).
      High EAAStandard EAA
      g/kgg/kg
      Alanine0.54 ± 0.060.18 ± 0.02
      Arginine
      The EAA formulation of the product used in this study includes arginine because under catabolic stress it is a conditionally essential amino acid. EAA, essential amino acid.
      2.20 ± 0.240.74 ± 0.08
      Aspartic acid0.00 ± 0.000.00 ± 0.00
      Cysteine0.00 ± 0.000.00 ± 0.00
      Glutamic acid0.00 ± 0.000.00 ± 0.00
      Glycine0.00 ± 0.000.00 ± 0.00
      Histidine0.40 ± 0.040.13 ± 0.01
      Isoleucine2.34 ± 0.250.78 ± 0.09
      Leucine8.44 ± 0.912.82 ± 0.31
      Lysine3.15 ± 0.341.06 ± 0.12
      Methionine0.63 ± 0.070.21 ± 0.02
      Phenylalanine1.37 ± 0.150.46 ± 0.05
      Proline0.00 ± 0.000.00 ± 0.00
      Serine0.00 ± 0.000.00 ± 0.00
      Threonine1.95 ± 0.270.65 ± 0.07
      Tryptophan0.00 ± 0.000.00 ± 0.00
      Tyrosine0.00 ± 0.000.00 ± 0.00
      Valine2.48 ± 0.270.83 ± 0.09
      Total EAA23.50 ± 2.547.87 ± 0.87
      a Values are means ± SD. Listed values correspond to the average g/kg dose used for each treatment. The amino acid profile of the product was confirmed by chemical analysis (Eurofins Food Integrity and Innovation, Madison, WI).
      b The EAA formulation of the product used in this study includes arginine because under catabolic stress it is a conditionally essential amino acid. EAA, essential amino acid.

      2.6 Analytical procedures

      Plasma was precipitated with 125 μL of 10% sulfosalicylic acid (SSA), centrifuged, and the supernatant was used to determine EAA concentrations using the internal standard technique [
      • de Betue C.T.
      • Joosten K.F.
      • Deutz N.E.
      • Vreugdenhil A.C.
      • van Waardenburg D.A.
      Arginine appearance and nitric oxide synthesis in critically ill infants can be increased with a protein-energy-enriched enteral formula.
      ]. Phenylalanine and tyrosine enrichments were measured using the tert-butyldimethylsilyl derivative and gas chromatography-mass spectrometry (models 7890A/5975; Agilent Technologies, Santa Clara, CA) [
      • Paddon-Jones D.
      • Sheffield-Moore M.
      • Zhang X.J.
      • Volpi E.
      • Wolf S.E.
      • Aarsland A.
      • et al.
      Amino acid ingestion improves muscle protein synthesis in the young and elderly.
      ,
      • Volpi E.
      • Mittendorfer B.
      • Wolf S.E.
      • Wolfe R.R.
      Oral amino acids stimulate muscle protein anabolism in the elderly despite higher first-pass splanchnic extraction.
      ]. Ions of mass-to-charge ratio of 234, 235, and 239 for phenylalanine and of 466, 467, 468, and 470 for tyrosine were monitored with electron impact ionization and selective ion monitoring. Serum insulin concentrations were measured using a Siemens Immulite 2000XPI (Siemens Medical Solutions USA, Inc., Malvern, PA).
      Muscle samples were weighed and tissue proteins were precipitated with 0.5 mL of 4% SSA. Samples were then homogenized, centrifuged, and the muscle pellet (bound protein) was washed, dried, and hydrolyzed in 0.5 mL of 6 N HCl at 105 °C for 24-h. Mixed muscle-bound protein enrichments were determined as described above for plasma enrichments.

      2.7 Mixed MPS and whole-body PS, PB, and NET calculations

      The precursor-product model was used to determine mixed MPS (i.e., fractional synthetic rate) [
      • Baumann P.Q.
      • Stirewalt W.S.
      • O'Rourke B.D.
      • Howard D.
      • Nair K.S.
      Precursor pools of protein synthesis: a stable isotope study in a swine model.
      ]:
      Mixed MPS (%/h) = [(EBP2 − EBP1)/(Ep)] × 60 × 100


      where EBP1 and EBP2 are the enrichments of bound L-[ring-2H5]-phenylalanine in muscle collected in the postabsorptive (rested leg only, 240-min − 120-min) and postprandial (rested and exercised legs, 420-min − 240-min) state. The precursor enrichment (Ep) is the calculated area under the curve (AUC) for L-[ring-2H5]-phenylalanine enrichment in the plasma extracellular pool for the postabsorptive (120-min to 240-min) and postprandial (240-min to 420-min) state since it more accurately reflects the blood perturbations and is consistent with previous work by Witard et al. [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ], which used a nearly identical study design. Factors 60 and 100 were used to express mixed MPS as percent per hour.
      Whole-body PS and PB rates were calculated based on the determinations of the rate of appearance (Ra) into the plasma of phenylalanine and tyrosine and the fractional Ra of endogenous tyrosine converted from phenylalanine [
      • Kim I.Y.
      • Schutzler S.
      • Schrader A.
      • Spencer H.J.
      • Azhar G.
      • Ferrando A.A.
      • et al.
      The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.
      ,
      • Wolfe R.R.
      • Chinkes D.L.
      Isotope tracers in metabolic research.
      ]. The phenylalanine (Phe) and tyrosine (Tyr) plasma enrichment area AUC were calculated in the postabsorptive (start to 240-min) and postprandial state (240-min to 420-min) (Fig. 3). Whole-body protein turnover was calculated by dividing kinetic values of phenylalanine by its fractional contribution to protein. For calculations of whole-body PB rate, contribution from tracers infused were subtracted from total Ra. The following equations were used to calculate whole-body PS, PB, and NET [
      • Kim I.Y.
      • Schutzler S.
      • Schrader A.
      • Spencer H.J.
      • Azhar G.
      • Ferrando A.A.
      • et al.
      The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.
      ]:
      Total plasma Ra = F/E


      Fractional Ra of Tyr from Phe = ETyr M+4/EPhe M+5


      Phe hydroxylation = fractional Ra of Tyr from Phe × Ra Tyr


      PS = [(Ra Phe − Phe hydroxylation) × 25]


      Exogenous Ra Phe = (Ingested Phe × digestibility) − Phe hydroxylation above basal


      PB = [(Total Ra Phe − Exogenous Ra Phe) × 25]


      NET = PS − PB


      where E is enrichment of respective tracers at plateau and expressed as tracer-to-tracee ratio (TTR) or mole percent excess (MPE), calculated as TTR/(TTR + 1). TTR was used for calculations of PB, whereas MPE was used for calculations of PS. F is respective tracer infusion rate into a venous side: FPhe for phenylalanine tracer. ETyr M+4 and EPhe M+5 are plasma enrichments of tyrosine and phenylalanine tracers at M + 4 and M + 5 relative to M + 0, respectively. In the fed state, fractional Ra of Tyr from Phe was divided by 0.8 to account for hepatic dilution [
      • Reeds P.J.
      • Hachey D.L.
      • Patterson B.W.
      • Motil K.J.
      • Klein P.D.
      VLDL apolipoprotein B-100, a potential indicator of the isotopic labeling of the hepatic protein synthetic precursor pool in humans: studies with multiple stable isotopically labeled amino acids.
      ]. The correction factor of 25 is for conversion of phenylalanine values to total protein based on the assumption that the contribution of phenylalanine to skeletal muscle protein is 4% (100/4 = 25) [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Wolfe R.R.
      Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle.
      ]. Phe is the amount of exogenous phenylalanine (g) that appeared in circulation, which was calculated as total amount of Phe provided (in the postprandial period only), based on the assumption that 100% of the ingested Phe was absorbed. Phe hydroxylation is the Ra of tyrosine derived via endogenous hydroxylation of phenylalanine.
      Fig. 3
      Fig. 3Mean ± SD (n = 19). Stable-isotope enrichments during the infusion studies used to examine high and standard essential amino acid (EAA) intakes.

      2.8 Statistical analysis

      Statistical power and sample size were determined by those used in previous studies examining myofibrillar MPS [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ,
      • Macnaughton L.S.
      • Wardle S.L.
      • Witard O.C.
      • McGlory C.
      • Hamilton D.L.
      • Jeromson S.
      • et al.
      The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.
      ]. An expected mean difference of 0.015%/h in both resting and post-RE MPS between the standard and high EAA treatments, a SD of 0.02%/h, and an α of 0.05, to provide 80% power were used to detect differences with a minimum of 16 participants. While we intended to complete data collection on 20 participants, attrition resulted in 19 completing all testing procedures which increased statistical power and accounted for the potential that variability was greater than previously reported and expected [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ,
      • Macnaughton L.S.
      • Wardle S.L.
      • Witard O.C.
      • McGlory C.
      • Hamilton D.L.
      • Jeromson S.
      • et al.
      The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.
      ]. Nineteen participants also provided greater than 90% power to detect differences between the standard and high EAA treatments on post-RE NET based on an expected mean difference of 18.9 g/180 min between the standard and high EAA treatments, a SD of 2.0 g/180 min, and an α of 0.05 [
      • Kim I.Y.
      • Schutzler S.
      • Schrader A.
      • Spencer H.J.
      • Azhar G.
      • Ferrando A.A.
      • et al.
      The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.
      ]. The primary outcomes for this study were mixed MPS (i.e., postabsorptive versus postprandial, postprandial rest versus postprandial post-RE) and whole-body protein turnover (i.e., postprandial versus postabsorptive) responses to high and standard EAA ingestion, whereas secondary outcomes were EAA, leucine, phenylalanine, tyrosine, and insulin concentrations over time and incremental area under the curve (iAUC) after high and standard EAA ingestion. Statistical analyses were performed with IBM SPSS software (version 26; IBM Corp. Armonk, NY, USA). Linear mixed models, with participant treated as a random effect, were used to determine the effects of EAA treatment (standard and high), condition (postabsorptive rest, postprandial rest, and postprandial post-RE), and their interaction (treatment-by-condition) on mixed MPS and whole-body protein kinetics. A linear mixed model, with participant treated as a random effect, was used to determine the effects of EAA amount (standard and high), condition (postabsorptive and postprandial), and their interaction (treatment-by-time) on tyrosine hydroxylation. Linear mixed models, with participant treated as a random effect, were used to determine the effects of EAA amount (standard and high), time (min), and their interaction (treatment-by-time) on EAA, leucine, phenylalanine, tyrosine, and insulin concentrations. If main or interaction effects were significant, Bonferroni post hoc comparisons were used to limit Type-1 error with multiple comparisons. Paired t-tests were used to determine body composition (i.e., baseline and first day of the second, 8-d diet intervention), body mass (i.e., change during each 5-d energy deficit and day of infusion studies), and changes in whole-body PS, PB, and NET (i.e., postprandial/post-RE − postabsorptive). EAA, leucine, phenylalanine, tyrosine, and insulin were also calculated using iAUC [
      • Pruessner J.C.
      • Kirschbaum C.
      • Meinlschmid G.
      • Hellhammer D.H.
      Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change.
      ]. Paired t-tests were used to evaluate whether postprandial/post-RE iAUC differed between standard and high EAA. Trial order effects were examined for MPS using a linear mixed model and for whole-body PS, PB, NET using paired t-tests and confirmed no carryover effects occurred. Significance was set at P < 0.05 and data are presented as means ± SD.

      3. Results

      Participants were young (23 ± 5 y), normal weight (79.7 ± 9.2 kg, 176.4 ± 6.5 cm, 25.4 ± 2.7 kg/m2) males, habitually consuming diets (i.e., pre-study) providing 2392 ± 574 kcal/d (protein, 1.4 ± 0.5 g/kg/d; carbohydrate, 42 ± 7% total energy; fat, 36 ± 7% total energy). Body mass loss in response to the energy deficits was the same between the standard (0.74 ± 0.73 kg) and high EAA treatments (0.88 ± 0.65 kg, P = 0.44). Body mass on the day of the infusion studies was the same (standard EAA: 78.4 ± 8.8 kg and high EAA: 78.6 ± 9.1 kg, P = 0.40). Lean body mass remained constant (P = 0.16) at baseline (59.8 ± 4.5 kg) and following the washout period (59.4 ± 4.9 kg), whereas fat mass was higher following the washout period [all bracketed data are mean difference between EAA treatments (95% CI), −0.45 kg, (−0.85, −0.06), P = 0.03; Table 2]. Dietary intake and the magnitude of energy deficit incurred were not different between the standard and high EAA diet interventions (each variable, P > 0.05; Table 3).
      Table 2Body composition at baseline and start of second diet intervention.
      Values are means ± SD (n = 19). Body composition measured using dual energy X-ray absorptiometry a baseline and following the washout period.
      BaselineStart of second diet interventionP-value
      Total body mass80.0 ± 8.980.0 ± 8.8P = 0.90
      Lean body mass59.7 ± 4.559.3 ± 4.9P = 0.16
      Fat mass17.0 ± 5.9
      Indicates difference (P = 0.001) between baseline and start of second diet intervention for each variable detected using paired samples t-tests. EAA, essential amino acid.
      17.4 ± 5.7P = 0.03
      a Values are means ± SD (n = 19). Body composition measured using dual energy X-ray absorptiometry a baseline and following the washout period.
      b Indicates difference (P = 0.001) between baseline and start of second diet intervention for each variable detected using paired samples t-tests. EAA, essential amino acid.
      Table 3Dietary intake during the controlled feeding periods prior to the single-day infusion studies of high versus standard EAA intakes on mixed-muscle protein synthesis and whole-body protein turnover during a randomized, cross-over study.
      Values are means ± SD (n = 19). Dietary intake was directly measured during each 3-d run-in and 5-d energy deficit diet. EAA, essential amino acid.
      High EAA

      Run-in
      Standard EAA

      Run-in
      High EAA

      Energy deficit
      Standard EAA

      Energy deficit
      Absolute intake (kcal/d or g/d)
      Energy2427.8 ± 177.32396.8 ± 158.51686.1 ± 135.11681.7 ± 140.7
      Protein129.8 ± 13.9129.3 ± 11.9128.8 ± 13.2129.0 ± 13.2
      Carbohydrate326.2 ± 22.5322.5 ± 20.8200.9 ± 20.9199.8 ± 17.3
      Fat71.7 ± 5.270.3 ± 4.443.6 ± 3.043.4 ± 4.3
      Relative intake (kcal/kg/d or g/kg/d)
      Energy30.6 ± 1.630.2 ± 1.821.5 ± 1.721.4 ± 1.6
      Protein1.6 ± 0.11.6 ± 0.11.6 ± 0.11.6 ± 0.1
      Carbohydrate4.1 ± 0.34.1 ± 0.32.6 ± 0.32.5 ± 0.3
      Fat0.9 ± 0.10.9 ± 0.10.6 ± 0.10.6 ± 0.1
      Energy intake (%)
      Protein21 ± 122 ± 131 ± 231 ± 2
      Carbohydrate54 ± 154 ± 148 ± 248 ± 2
      Fat27 ± 126 ± 123 ± 123 ± 1
      Energy deficit (%)
      Energy0 ± 00 ± 030.4 ± 4.129.9 ± 3.9
      a Values are means ± SD (n = 19). Dietary intake was directly measured during each 3-d run-in and 5-d energy deficit diet. EAA, essential amino acid.

      3.1 Mixed MPS and whole-body protein turnover

      Postabsorptive mixed MPS at rest was not different (P = 0.67) between standard and high EAA (Fig. 4). In the postprandial state, and independent of EAA amount, mixed MPS at rest and post-RE were greater than postabsorptive mixed MPS at rest (P = 0.02 and P = 0.01, respectively, Fig. 4). Postprandial mixed MPS at rest and post-RE were not different (P = 0.33 and P = 0.13, respectively).
      Fig. 4
      Fig. 4Mean ± SD (n = 19). Mixed muscle protein synthesis at rest and post-resistance exercise (unilateral leg model) after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting), postprandial (i.e., after ingesting the standard and high EAA beverages), rest (i.e., no exercise), and post-resistance exercise (i.e., unilateral leg resistance exercise). Data were analyzed using linear mixed models, different lowercase letters indicate difference between postabsorptive rest and postprandial rest or postprandial post-resistance exercise, P = 0.02 and P = 0.01, respectively. EAA, essential amino acid.
      Postabsorptive whole-body PS, PB, and NET were not different (P = 0.95, P = 0.80, and P = 0.59, respectively) between standard and high EAA (Fig. 5A–C). A treatment-by-condition interaction (P = 0.02) was observed for whole-body PS such that postprandial PS for high EAA was greater than postabsorptive PS (P = 0.001) and postprandial PS for standard EAA [3.3 g/180 min (1.3, 5.3); P = 0.001, Fig. 5A]. Postprandial whole-body PB was lower than postabsorptive whole-body PB in both treatments (treatment-by-condition, P = 0.001) and in the postprandial state the reduction in PB was greater in the high versus standard EAA [−15.3 g/180 min (−17.7, −12.9); P = 0.001, Fig. 5B]. A treatment-by-condition interaction (P = 0.001) was observed for NET such that postprandial versus postabsorptive NET was increased in both treatments, but the increase was greater in high versus standard EAA [18.6 g/180 min (17.2, 20.0); P = 0.001, Fig. 5C]. There was a treatment-by-condition interaction (P = 0.001) for tyrosine hydroxylation such that postprandial hydroxylation for standard EAA was lower than high EAA [−0.041 μmol/kg/min (−0.05, −0.03); P = 0.001, Supplementary Fig. 1].
      Fig. 5
      Fig. 5A: Mean ± SD (n = 19). Postabsorptive and postprandial whole-body protein synthesis after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting) and postprandial (i.e., after ingesting the standard and high EAA beverages). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard within the same condition (P = 0.001) and different lowercase letters indicate post hoc difference within the same EAA amount (P = 0.001). EAA, essential amino acid. B: Mean ± SD (n = 19). Postabsorptive and postprandial whole-body protein breakdown after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting) and postprandial (i.e., after ingesting the standard and high EAA beverages). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard within the same condition (P = 0.001) and different lowercase letters indicate post hoc difference within the same EAA amount (P = 0.001). EAA, essential amino acid. C: Mean ± SD (n = 19). Postabsorptive and postprandial whole-body protein net balance after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting) and postprandial (i.e., after ingesting the standard and high EAA beverages). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard within the same condition (P = 0.001) and different lowercase letters indicate post hoc difference within the same EAA amount (P = 0.001). EAA, essential amino acid. D: Mean ± SD (n = 19). Change in whole-body protein turnover after high versus standard essential amino acid (EAA) intake during energy deficit. Data were analyzed using paired t-tests, ∗indicates difference between high versus standard EAA within each parameter of whole-body protein turnover (P < 0.01). EAA, essential amino acid.
      Changes in whole-body PS and PB were greater for high than standard EAA [3.4 g/180 min (2.3, 4.4); P = 0.001 and −15.6 g/180 min (−17.8, −13.5); P = 0.001, respectively, Fig. 5D]. The resulting change in NET was more positive for high than standard EAA [19.0 g/180 min (17.3, 20.7); P = 0.001, Fig. 5D].

      3.2 EAA and insulin concentrations

      A treatment-by-time interaction (all, P = 0.001) was observed for the EAA, leucine, phenylalanine, and tyrosine such that concentrations increased over time and peak concentrations were greater for high versus standard EAA (Fig. 6A–D). The iAUCs for EAA, leucine, and phenylalanine concentrations for high were also greater than standard EAA (all, P = 0.001, Table 4). Independent of treatment, insulin concentrations increased before returning to baseline (main effect time, P = 0.001; Fig. 6E). Insulin and tyrosine concentration iAUCs did not differ between treatments (P = 0.57 and P = 0.17, respectively, Table 4).
      Fig. 6
      Fig. 6A: Mean ± SD (n = 19). Plasma essential amino acid (EAA) concentrations after high versus standard EAA intake during energy deficit. Postabsorptive (i.e., fasting). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard and different lowercase letters indicate post hoc difference between time points within the same EAA amount (P < 0.05). EAA, essential amino acid. B: Mean ± SD (n = 19). Plasma leucine concentrations after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard and different lowercase letters indicate post hoc difference between time points within the same EAA amount (P < 0.05). EAA, essential amino acid. C: Mean ± SD (n = 19). Plasma phenylalanine concentrations after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting). Data were analyzed using linear mixed models, ∗indicates post hoc difference between high and standard and different lowercase letters indicate post hoc difference between time points within the same EAA amount (P < 0.05). EAA, essential amino acid. D: Mean ± SD (n = 19). Plasma tyrosine concentrations after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting). Data were analyzed using linear mixed models. ∗indicates post hoc difference between high and standard and different lowercase letters indicate post hoc difference between time points within the same EAA amount (P < 0.05). Different lowercase letters indicate post hoc difference between time points, independent of EAA amount (P < 0.05). EAA, essential amino acid. E: Mean ± SD (n = 19). Plasma insulin concentrations after high versus standard essential amino acid (EAA) intake during energy deficit. Postabsorptive (i.e., fasting). Data were analyzed using linear mixed models, different lowercase letters indicate post hoc difference between time points, independent of EAA amount (P < 0.05). EAA, essential amino acid.
      Table 4Plasma amino acid and insulin concentrations presented as incremental area under the curve following high versus standard EAA intakes during a randomized, cross-over study.
      Values are means ± SD (n = 19). Incremental area under the curve for plasma amino acid and plasma insulin concentrations measured during the infusion studies.
      High EAAStandard EAAP-Value
      Incremental area under curve
      EAA (μmol/L/240 min)236,745 ± 47,328
      Indicates difference (P = 0.001) between high and standard EAA treatments for each variable detected using paired samples t-tests. EAA, essential amino acid.
      74,752 ± 32,580P = 0.001
      Leucine (μmol/L/240 min)97,951 ± 23,713
      Indicates difference (P = 0.001) between high and standard EAA treatments for each variable detected using paired samples t-tests. EAA, essential amino acid.
      34,967 ± 10,781P = 0.001
      Phenylalanine (μmol/L/240 min)6795 ± 2129
      Indicates difference (P = 0.001) between high and standard EAA treatments for each variable detected using paired samples t-tests. EAA, essential amino acid.
      2653 ± 1445P = 0.001
      Tyrosine (μmol/L/240 min)586 ± 1061125 ± 1255P = 0.19
      Insulin (pmol/L/240 min)7396 ± 53878755 ± 7315P = 0.50
      a Values are means ± SD (n = 19). Incremental area under the curve for plasma amino acid and plasma insulin concentrations measured during the infusion studies.
      b Indicates difference (P = 0.001) between high and standard EAA treatments for each variable detected using paired samples t-tests. EAA, essential amino acid.

      4. Discussion

      We studied the effects of standard (0.10 g/kg/dose) and high (0.30 g/kg/dose) free-form EAA ingestion on resting and post-RE mixed MPS and whole-body protein turnover during a moderate, 5-d energy deficit. Our work demonstrated that whole-body NET was greater for high compared to standard EAA. The improvement in NET was due to both a greater increase in whole-body PS and reduction in whole-body PB after ingesting the high EAA dose compared to the standard EAA dose. The reduction in PB was independent of circulating insulin concentrations, as these were not different between treatments. Despite greater increases in circulating EAA concentrations following the high versus standard EAA dose, resting and post-RE mixed MPS were equally stimulated across a 3-h postprandial period. These results suggest that the primary purpose of EAA ingestion during short-term, moderate energy deficit is likely to meet whole-body EAA requirements (i.e., splanchnic PS), and that routing of precursors for MPS is of secondary physiological importance. The enhanced whole-body protein status in the postprandial state suggests greater EAA doses may offset whole-body protein losses induced by the catabolic stress of underfeeding in non-obese, young adults [
      • Margolis L.M.
      • Murphy N.E.
      • Martini S.
      • Spitz M.G.
      • Thrane I.
      • McGraw S.M.
      • et al.
      Effects of winter military training on energy balance, whole-body protein balance, muscle damage, soreness, and physical performance.
      ,
      • Berryman C.E.
      • Sepowitz J.J.
      • McClung H.L.
      • Lieberman H.R.
      • Farina E.K.
      • McClung J.P.
      • et al.
      Supplementing an energy adequate, higher protein diet with protein does not enhance fat-free mass restoration after short-term severe negative energy balance.
      ]. However, there is likely no mixed muscle-specific advantage of increasing EAA intake, in the free-form, above the current recommendations of 0.10–0.14 g EAA/kg per dose under energy deficient conditions.
      The most significant finding from our study was the greater enhancement of NET in response to higher EAA ingestion during moderate, short-term energy deficit. Although a large amount of dietary EAA are directed towards the periphery, splanchnic proteins have a higher turnover rate, particularly in circumstances of catabolic stress, and require a relatively greater amount of EAA [
      • Kim I.Y.
      • Deutz N.E.P.
      • Wolfe R.R.
      Update on maximal anabolic response to dietary protein.
      ,
      • Raj D.S.
      • Moseley P.
      • Dominic E.A.
      • Onime A.
      • Tzamaloukas A.H.
      • Boyd A.
      • et al.
      Interleukin-6 modulates hepatic and muscle protein synthesis during hemodialysis.
      ,
      • Carraro F.
      • Hartl W.H.
      • Stuart C.A.
      • Layman D.K.
      • Jahoor F.
      • Wolfe R.R.
      Whole body and plasma protein synthesis in exercise and recovery in human subjects.
      ]. The current findings confirm our hypothesis and indicate that enhanced NET was attributed to a greater suppression of PB and a greater stimulation of PS. Circulating EAA concentrations increased 3-fold after the high EAA dose, resulting in concentrations nearly 60% greater than the standard EAA dose. The greater EAA concentrations for high were sustained for more than 2 h, compared to less than 1 h for the standard EAA dose. These findings are in agreement with the premise that EAA ingestion elevates circulating EAA above basal concentrations, which in turn stimulates MPS until a saturation point is reached. Further increases in EAA availability impart no additional MPS-specific advantage; rather the effects are directed to the improvement of NET at the whole-body level. The greater NET with the higher dose of EAA may be attributed to a reduced reliance on PB-derived precursor amino acids to support PS in non-muscle tissue [
      • Kim I.Y.
      • Deutz N.E.P.
      • Wolfe R.R.
      Update on maximal anabolic response to dietary protein.
      ]. These findings are also supported by evidence indicating that amino acids inhibit PB in a dose-dependent manner in the splanchnic region independent of insulin effects [
      • Nygren J.
      • Nair K.S.
      Differential regulation of protein dynamics in splanchnic and skeletal muscle beds by insulin and amino acids in healthy human subjects.
      ]. Increased PS due to greater EAA availability may also reduce the proportion of ingested amino acids directed towards the oxidation of carbon skeletons [
      • Kim I.Y.
      • Deutz N.E.P.
      • Wolfe R.R.
      Update on maximal anabolic response to dietary protein.
      ].
      We also hypothesized that consuming greater than 0.10 g EAA/kg/dose would optimize mixed MPS during energy deficit. However, our results indicate that 0.10 g EAA/kg/dose, or ~8 g EAA, was sufficient to stimulate resting and post-RE mixed MPS during moderate (30% total energy), short-term (5-d) energy deficit. In contrast, Areta et al. [
      • Areta J.L.
      • Burke L.M.
      • Camera D.M.
      • West D.W.
      • Crawshay S.
      • Moore D.R.
      • et al.
      Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.
      ], demonstrated that consuming 15 g (~7.5 g EAA) and 30 g (~15 g EAA) of whey protein after RE during an energy deficit (–30% of total energy requirements) stimulated myofibrillar MPS in a dose-dependent manner. Differences between our data and those reported by Areta [
      • Areta J.L.
      • Burke L.M.
      • Camera D.M.
      • West D.W.
      • Crawshay S.
      • Moore D.R.
      • et al.
      Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.
      ] are more than likely due to the study design, statistical analyses, and small differences in training status between study participant pools [
      • Damas F.
      • Phillips S.
      • Vechin F.C.
      • Ugrinowitsch C.
      A review of resistance training-induced changes in skeletal muscle protein synthesis and their contribution to hypertrophy.
      ], as the magnitude of change in post-exercise MPS responses are similar between studies. In our study, the post-RE changes in MPS were 0.008%/h and 0.019%/h for 0.10 g EAA/kg and 0.30 g EAA/kg intakes, respectively. In the Areta study [
      • Areta J.L.
      • Burke L.M.
      • Camera D.M.
      • West D.W.
      • Crawshay S.
      • Moore D.R.
      • et al.
      Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.
      ], post-RE MPS increased approximately 0.011%/h and 0.019%/h following 15 g and 30 g of whey protein, respectively. The difference in MPS response between the higher and lower doses of whey in the Areta study was also comparable to the numerical difference we report in response to standard and high EAA (0.008 versus 0.009%/h). Regarding statistical analyses, our model accounted for exercise (rest versus exercise), feeding state (postabsorptive versus postprandial), and amino acid dose effects (0.10 versus 0.30 g EAA/kg) while controlling for variance by treating participants as random effects. Whereas, Areta et al., did not include a postprandial rest versus exercise comparison in their design and therefore used a one-way repeated-measures ANOVA.
      The lack of difference in postprandial MPS responses between the resting and exercised legs in our study may also be considered at odds with a recent study by Hector et al. [
      • Hector A.J.
      • McGlory C.
      • Damas F.
      • Mazara N.
      • Baker S.K.
      • Phillips S.M.
      Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.
      ], who reported that postabsorptive mixed MPS 48-h after completing unilateral RE was greater in the exercised compared to the resting leg in young, overweight males exposed to a 10 d, 40% energy deficit. However, direct comparison of the Hector study to ours is difficult given differences in study population, study design, and the lack of a postprandial mixed MPS measure in the Hector study. Nevertheless, we report findings consistent with those of several other studies [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ,
      • Moore D.R.
      • Churchward-Venne T.A.
      • Witard O.
      • Breen L.
      • Burd N.A.
      • Tipton K.D.
      • et al.
      Protein ingestion to stimulate myofibrillar protein synthesis requires greater relative protein intakes in healthy older versus younger men.
      ,
      • Cuthbertson D.
      • Smith K.
      • Babraj J.
      • Leese G.
      • Waddell T.
      • Atherton P.
      • et al.
      Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle.
      ]. Witard et al. [
      • Witard O.C.
      • Jackman S.R.
      • Breen L.
      • Smith K.
      • Selby A.
      • Tipton K.D.
      Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise.
      ], demonstrated no difference in myofibrillar MPS between 20 g whey protein (~10 g EAA) and 40 g whey protein (~20 g EAA) in young adults. Likewise, the saturation of mixed MPS induced by ~8 g of free-form EAA in our study is in agreement with the myofibrillar and sarcoplasmic MPS response to 10 g of free-form EAA reported by Cuthbertson et al. [
      • Cuthbertson D.
      • Smith K.
      • Babraj J.
      • Leese G.
      • Waddell T.
      • Atherton P.
      • et al.
      Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle.
      ] and the mixed MPS response to 20 g (~9 g EAA) of egg protein reported by Moore et al. [
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ]. Taken together, mixed MPS appears to be optimally stimulated in young subjects after ingesting 8–10 g EAA, an amount delivered in 20–30 g of high-quality protein, regardless of whether an individual is in a state of energy balance or moderate energy deficit.
      A novel aspect of our work is the integration of MPS and whole-body protein turnover measures to comprehensively assess body protein status during the catabolic stress induced by energy deficit in non-obese, healthy individuals. As catabolic stress increases, so does the reliance on whole-body PB to supply the amino acid precursors needed to sustain acute-phase protein synthesis, wound healing, immune function, and energy production [
      • Biolo G.
      • Fleming R.Y.
      • Maggi S.P.
      • Nguyen T.T.
      • Herndon D.N.
      • Wolfe R.R.
      Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients.
      ,
      • Owen O.E.
      • Smalley K.J.
      • D'Alessio D.A.
      • Mozzoli M.A.
      • Dawson E.K.
      Protein, fat, and carbohydrate requirements during starvation: anaplerosis and cataplerosis.
      ]. Thus, whole-body protein status becomes more negative as the magnitude and duration of energy deficit increases. The efficacy of countermeasures to offset negative body protein status induced by catabolic stress should therefore be evaluated on the basis of their combined effects on MPS and whole-body protein status. If the two EAA doses in the current study were only evaluated on the basis of MPS effects, then they would be considered equally effective. Yet whole-body protein balance was more positive following the high versus standard EAA ingestion, suggesting a protective effect of the higher EAA intervention on whole-body protein balance. We contend that as catabolic stress increases, integrated measures to evaluate whole-body protein status are necessary to understand the state of body protein health. Our findings are particularly relevant to military personnel conducting strenuous operations during which providing supplemental protein consistent with current recommendations (0.10–0.14 g EAA/kg/dose or 0.25–0.30 g high-quality protein/kg/dose) does not prevent whole-body and muscle protein loss [
      • Margolis L.M.
      • Murphy N.E.
      • Martini S.
      • Gundersen Y.
      • Castellani J.W.
      • Karl J.P.
      • et al.
      Effects of supplemental energy on protein balance during 4-d arctic military training.
      ,
      • Berryman C.E.
      • Young A.J.
      • Karl J.P.
      • Kenefick R.W.
      • Margolis L.M.
      • Cole R.E.
      • et al.
      Severe negative energy balance during 21 d at high altitude decreases fat-free mass regardless of dietary protein intake: a randomized controlled trial.
      ]. Our findings demonstrating the whole-body protein advantage of high versus standard EAA consumption provide the basis for the development of new dietary strategies necessary to enhance whole-body protein balance during military operations that elicit the catabolic stress of underfeeding.
      The dietary control, large sample size, and standardized exercise model used in the current study strengthen its findings. However, there are limitations that should be acknowledged. Our experimental design did not include an energy balance comparator, which would have provided the ability to isolate the effects of energy status on postabsorptive and postprandial whole-body protein turnover and mixed MPS responses to standard and high EAA intake. However, the addition of an energy balance comparator was not logistically feasible. The methodological inability to quantify changes in net muscle protein balance by measuring muscle protein breakdown (MPB) in the postprandial state may also limit the extension of our data, particularly in the context of the marked suppression of whole-body PB we demonstrated in response to high versus standard EAA intake. However, there are no reliable data to suggest that moderate energy deficit would have any effect on postabsorptive and postprandial measures of MPB. For example, although examined in overweight individuals in the postabsorptive resting state, Hector et al. [
      • Hector A.J.
      • McGlory C.
      • Damas F.
      • Mazara N.
      • Baker S.K.
      • Phillips S.M.
      Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.
      ], reported no difference in MPB in young, males undergoing 10 d of 40% energy deficit while consuming lower (1.2 g/kg/d) and higher (2.4 g/kg/d) protein diets and performing unilateral RE. There are also inherent limitations of whole-body protein turnover measures and their inability to account for intracellular recycling of amino acids which results in a systematic underestimation of PS and PB. Importantly, PS and PB are underestimated to the same extent, so there is no effect on calculated protein gain or loss. Intra and inter-individual variability in MPS and the inherent risk of making a Type I error due to the multiple comparisons may also be considered as limitations. However, the variability in MPS in our study is consistent with previous reports [
      • Smith G.I.
      • Patterson B.W.
      • Mittendorfer B.
      Human muscle protein turnover--why is it so variable?.
      ] and we applied the conservative Bonferroni correction to limit Type 1 error.
      In conclusion, higher EAA intake enhanced NET during moderate energy deficit, through an increase in PS and an attenuation of PB. We also demonstrated that mixed MPS is likely optimally stimulated at rest and in response to resistance exercise after consuming EAA in amounts consistent with current sport nutrition, muscle-centric recommendations [
      • Miller S.L.
      • Tipton K.D.
      • Chinkes D.L.
      • Wolf S.E.
      • Wolfe R.R.
      Independent and combined effects of amino acids and glucose after resistance exercise.
      ,
      • Katsanos C.S.
      • Kobayashi H.
      • Sheffield-Moore M.
      • Aarsland A.
      • Wolfe R.R.
      Aging is associated with diminished accretion of muscle proteins after the ingestion of a small bolus of essential amino acids.
      ,
      • Tipton K.D.
      • Gurkin B.E.
      • Matin S.
      • Wolfe R.R.
      Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers.
      ,
      • Moore D.R.
      • Robinson M.J.
      • Fry J.L.
      • Tang J.E.
      • Glover E.I.
      • Wilkinson S.B.
      • et al.
      Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men.
      ,
      • Thomas D.T.
      • Erdman K.A.
      • Burke L.M.
      American College of Sports Medicine Joint Position Statement. Nutrition and Athletic Performance.
      ,
      • Pasiakos S.M.
      • Austin K.G.
      • Lieberman H.R.
      • Askew E.W.
      Efficacy and safety of protein supplements for U.S. Armed Forces personnel: consensus statement.
      ,
      • Jager R.
      • Kerksick C.M.
      • Campbell B.I.
      • Cribb P.J.
      • Wells S.D.
      • Skwiat T.M.
      • et al.
      International Society of Sports Nutrition Position Stand: protein and exercise.
      ]. Taken together, these results suggest that higher EAA doses are necessary to optimize both muscle and whole-body protein status during the catabolic stress of underfeeding.

      Disclaimers

      The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations.

      Sources of support

      U.S. Army Medical Research and Development Command. The study sponsor had no role in study design or data collection, analysis, and interpretation; writing the report, nor the decision to submit the report for publication.

      Data sharing

      Data described in the manuscript, code book, and analytic code will be made available upon request.

      Author contributions

      The authors' responsibilities were as follows – AAF and SMP: designed the research; JAG, DDC, AHM, EEH, CTC, NEM, MAW, and LMM: conducted the research; JAG, DDC, RRW, and AAF: analyzed the data; JAG, DDC, LMM, JWC, RRW, AAF, and SMP: interpreted the data; JAG and SMP: wrote the manuscript; SMP: had primary responsibility for the final content; and all authors read and approved the final manuscript.

      Conflicts of interest

      JAG, DDC, AHM, EEH, CTC, NEM, MAW, LMM, JWC, and SMP have no conflicts of interest associated with this research. The EAA formula used in this research is UAMS patent entitled “Essential Amino Acid Supplementation for Recovery of Muscle Strength and Function during Rehabilitation.” US Patent (9,364,463 B2; UAMS). Inventors are AAF and RRW. Neither AAF nor RRW were involved in data collection or analyses and were blinded to all data until final consolidation by JAG and SMP.

      Acknowledgments

      The authors thank the individuals that participated in this study.

      Appendix A. Supplementary data

      The following are the supplementary data to this article:

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