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Ingested protein dose response of muscle and albumin
protein synthesis after resistance exercise in young men1–3
Daniel R Moore, Meghann J Robinson, Jessica L Fry, Jason E Tang, Elisa I Glover, Sarah B Wilkinson, Todd Prior,
Mark A Tarnopolsky, and Stuart M Phillips
ABSTRACT
Background: The anabolic effect of resistance exercise is enhanced
by the provision of dietary protein.
Objectives: We aimed to determine the ingested protein dose response
of muscle (MPS) and albumin protein synthesis (APS) after
resistance exercise. In addition, we measured the phosphorylation of
candidate signaling proteins thought to regulate acute changes in
MPS.
Design: Six healthy young men reported to the laboratory on 5
separate occasions to perform an intense bout of leg-based resistance
exercise. After exercise, participants consumed, in a randomized
order, drinks containing 0, 5, 10, 20, or 40 g of whole egg
protein. Protein synthesis and whole-body leucine oxidation were
measured over 4 h after exercise by a primed constant infusion of
[1-13C]leucine.
Results: MPS displayed a dose response to dietary protein ingestion
and was maximally stimulated at 20 g. The phosphorylation of
ribosomal protein S6 kinase (Thr389), ribosomal protein S6 (Ser240/244),
and the e-subunit of eukaryotic initiation factor 2B (Ser539) were
unaffected by protein ingestion. APS increased in a dose-dependent
manner and also reached a plateau at 20 g of ingested protein. Leucine
oxidation was significantly increased after 20 and 40 g of protein were
ingested.
Conclusions: Ingestion of 20 g of intact protein is sufficient to
maximally stimulate MPS and APS after resistance exercise. Phosphorylation
of candidate signaling proteins was not enhanced with
any dose of protein ingested, which suggested that the stimulation
of MPS after resistance exercise may be related to amino acid
availability. Finally, dietary protein consumed after exercise in excess
of the rate at which it can be incorporated into tissue protein
stimulates irreversible oxidation. Am J Clin Nutr 2009;89:1–8.
INTRODUCTION
The provision of an exogenous source ofamino acids, likely only
the essential amino acids (EAAs), enhances the anabolic effect of
resistance exercise on muscle (1–4). Muscle protein synthesis
(MPS), not breakdown, is the more responsive variable to a variety
of anabolic stimuli in healthy individuals (5) and is stimulated in
a dose-dependent manner with increasing amounts of ingested
EAAs at rest (6, 7). Data from a series of studies suggest that
a similar dose response of protein synthesis to amino acids exists
after resistance exercise (2, 8, 9). No study to date has directly
measured a dose-response relation between ingested whole protein
and muscle protein synthetic rates after resistance exercise.
The acute synthesis of muscle proteins is primarily regulated at
the level of messenger RNA translation via the activation of
a variety of intracellular signaling proteins, especially those of
the mammalian target of rapamycin (mTOR) signaling cascade
(10). Stimulation of MPS in humans after feeding or resistance
exercise is accompanied by enhanced phosphorylation, and presumably
activity, of the mTOR signaling pathway, including the
70-kDa ribosomal protein S6 kinase (S6K1) and its target ribosomal
protein S6 (rpS6) (7, 11–16). In addition, global rates of
protein synthesis are tightly regulated by the activity of the
guanine nucleotide exchange factor eukaryotic initiation factor
2B (eIF2B), which is responsive to both amino acids and exercise
and is under the control of glycogen synthase kinase-3b in rats
(17, 18) and cultured myoblasts (19). Collectively, these observations
suggest that changes in MPS in response to feeding and
exercise may be regulated by specific downstream target proteins
of mTOR signaling such as S6K1, rpS6, and eIF2B.
Albumin is a major hepatic-derived plasma protein that is
unaffected by resistance exercise in young men (20). However,
albumin synthesis is stimulated by an increased availability of
amino acids (21–23). It has been suggested that dietary amino
acids may be incorporated into albumin protein in an effort
to minimize their irreversible oxidation (21). Thus, a feedinginduced
increase in albumin protein synthesis may serve as a
mechanism to ‘‘store’’ excess amino acids from the diet until
they are needed during periods of reduced supply (21).
The present study was designed to yield a dose-response relation
for ingested protein in the stimulation of muscle and albumin
protein synthesis after resistance exercise. We also wished
1 From the Exercise Metabolism Research Group, Department of Kinesiology
(DRM, MJR, JLF, JET, EIG, SBW, TP, and SMP), and Departments of
Neurology and Pediatrics (MAT), McMaster University, Hamilton, Canada.
2 Supported by a grant from the Natural Sciences and Engineering Research
Council (NSERC) of Canada. DRM and SBW were supported by
Canada Graduate Scholarships from the Canadian Institutes for Health Research
(CIHR). MJR was supported by an NSERC Undergraduate Student
Research Award. JLF was supported by a McMaster University Undergraduate
Student Research Award. JET and EIG were supported by CIHR Doctoral
Research Awards. SMP was the recipient of a CIHR New Investigator
career award.
3 Reprints not available. Address correspondence to SM Phillips, Department
of Kinesiology, 1280 Main Street West, Hamilton, ON, Canada, L8S
4K1. E-mail: phillis@mcmaster.ca.
Received May 9, 2008. Accepted for publication October 2, 2008.
doi: 10.3945/ajcn.2008.26401.
Am J Clin Nutr 2009;89:1–8. Printed in USA. 2009 American Society for Nutrition 1
AJCN. First published ahead of print December 3, 2008 as doi: 10.3945/ajcn.2008.26401.
Copyright (C) 2008 by the American Society for Nutrition
to characterize other fates of amino acids when the amount of
protein ingested might exceed the capacity for amino acids to be
used in MPS, namely irreversible oxidation.We hypothesized that
mixed MPS would demonstrate a dose response to dietary protein
after resistance exercise and that the maximal effective (ie,
maximally stimulatory for MPS) dose would be similar to what has
previously been reported to be maximal at rest (7). In addition, we
hypothesized that plasma albumin protein synthesis would display
a similar dose response to dietary protein as mixed muscle
protein. However, above an ingested dose of protein that maximally
stimulated muscle and albumin protein synthesis, we hypothesized
that amino acid oxidation would increase.
SUBJECT AND METHODS
Subjects
Six healthy active males (mean6SE: 2262 y; 86.167.6 kg;
1.82 6 0.1 m) who had 4 mo of previous recreational weightlifting
experience (range: 4 mo–8 y) volunteered to participate in
the study. Our repeated-measures design led us to recruit participants
with previous resistance training experience to minimize
the occurrence of exercise-induced muscle damage that can
occur with novel exercise in untrained individuals as well as to
minimize any learning effects during trials. Participants were
informed about the experimental procedure to be used as well as
the purpose of the study and all potential risks before giving
written consent. All participants were deemed healthy on the
basis of their response to a routine medical screening questionnaire.
The study conformed to all standards for the use of
human subjects in research as outlined in the Helsinki declaration
and was approved by the local Research Ethics Board of
McMaster University and Hamilton Health Sciences.
Study design
Participants reported to the laboratory on 5 separate occasions
separated by at least 1 wk. Each trial began with the participant
performing an acute bout of intense leg resistance exercise. After
exercise, participants consumed a drink containing 0, 5, 10, 20, or
40 g of egg protein in a randomized order. Whole-body leucine
oxidation as well as albumin protein synthesis (APS) and mixed
MPS were measured using a primed constant infusion of
[1-13C]leucine over 4 h. Leucine was chosen as a tracer, because
it is an essential amino acid that is primarily metabolized within
the lean tissues of the body. Furthermore, muscle tissue is both
enriched in branched-chain amino acids (BCAAs) such as leucine
and is also a major site for leucine oxidation.
Diet
Before each trial, participants were supplied with prepackaged
dietsfor2dthatprovidedamoderateproteinintake(1.4gkg21d21)
for resistance-trained athletes (24). Energy needs of the controlled
diets were estimated according to the Harris-Benedict equation and
were adjusted using a moderate activity factor (1.6) for all participants
to account for habitual activity. Body mass did not
change over the course of the controlled diet, suggesting
participants were in energy balance. Participants were also
required to complete diet records before the start of the study
to provide an estimate of habitual macronutrient consumption
as analyzed using a commercially available software program
(Nutritionist V; First Data Bank, San Bruno, CA). Reference
lists for portion size estimates were provided to participants
who were instructed to record all food or drink consumed in
a diet log during a 3-d period (ie, 2 weekdays and 1 weekend).
On the basis of the responses, the average habitual protein
intake was identical to the controlled diet (1.5 6 0.2 compared
with 1.4 6 0.1 g kg21 d21, respectively; P ¼ 0.8, paired t
test), whereas the energy intake was slightly less than the
controlled diet (130 6 10 compared with 170 6 3 kJ kg21 d21,
respectively; P , 0.05, paired t test). This apparent discrepancy in
energy intake between the controlled and the habitual diets is
likely related to the underreporting of true energy intake commonly
seen with self-reported dietary assessments (25). Nonetheless,
because body mass did not change throughout the
duration of the study, controlled diets were kept consistent to
ensure individuals had identical macronutrient consumption for
the 2 d before each trial.
Infusion protocol
Participants reported to the laboratory at 0700 after an overnight
fast, having refrained from all resistance exercise and any
strenuous physical activity for at least 3 d. Bilateral resistance
exercise was performed on guided-motion machines and involved
4 sets each of leg press, knee extension, and leg curl using a
predetermined load designed to elicit failure within 8–10 repetitions.
Each set was completed within’25 s with a rest period of
120 s between each set. After exercise, a baseline breath sample
was collected for determination of 13CO2 enrichment by isotope
ratio mass spectrometry (BreathMat Plus; Finnigan MAT GmbH,
Bremen, Germany). A polytetrafluoroethylene catheter was inserted
in the medial vein of each arm, one for tracer infusion and
the other for arterialized blood sampling. Arterialized blood samples
were obtained by wrapping the forearmin a heating blanket for
the duration of the infusion. Baseline blood samples were drawn,
and then participants received priming doses of NaH13CO2 (2.35
lmol/kg) and [1-13C]leucine (7.6 lmol/kg, 99 atom percent;
Cambridge Isotopes, Andover, MA) before beginning a constant
[1-13C]leucine infusion (7.6 lmol/kg/h) (Figure 1). Immediately
after the onset of the infusion, participants consumed a drink
containing 0, 5, 10, 20, or 40 g of whole-egg protein dissolved
in 400 mL of water. The amino acid content of the protein
was (in percent content, wt:wt): Ala, 6.3; Arg, 5.7; Asp, 8.7;
Cys, 2.4; Gln, 13.7; Gly, 3.6; His, 2.1; Ile, 5.9; Leu, 8.4; Lys,
5.9; Met, 3.8; Phe, 6; Pro, 3.6; Ser, 7.1; Thr, 4.3; Trp, 1.4; Tyr,
3.9; and Val, 7.2. On the basis of a leucine content of ’8% in
egg protein, drinks were enriched to 5% with [1-13C]leucine to
minimize disturbances in isotopic steady state. Arterialized
blood samples were collected every 0.5–1 h into evacuated
heparinized tubes and chilled on ice. Within 5 min of sampling,
100 lL of whole blood was deproteinized with 0.6 mol/L
perchloric acid (PCA) and the remaining sample was centrifuged
(4000 g) for 5 min to separate plasma. PCA extracts and
blood plasma were stored at 220C for further analysis. Biopsy
samples were taken from the vastus lateralis of a randomly
selected thigh using a 5-mm Bergstro¨m needle (modified for
manual suction) under 2% xylocaine local anesthesia. Muscle
biopsies were freed from any visible blood, fat, and connective
tissue and rapidly frozen in liquid nitrogen for further analysis.
Muscle biopsies for a given trial were taken from separate
2 MOORE ET AL
incisions (’4–5 cm apart) from the same leg at 1 and 4 h with
alternate legs being sampled for each subsequent trial.
Analysis
Blood amino acid concentrations were measured from the PCA
extract by HPLC as previously described (26). Plasma insulin
concentration was determined by a standard radioimmunoassay
kit (Coat-a-Count; Diagnostic Products, Los Angeles, CA). Blood
glucose concentration was measured spectrophometrically using
a standard glucose peroxidase enzymatic kit (Stanbio Laboratory,
Boerne, TX). Plasma urea concentration was measured using
a standard spectrophometric kit (Pointe Scientific Inc, Canton,
MI). Plasma enrichment of the t-BDMS derivative of a-[13C]-
ketoisocaproate acid (a-KIC) was measured by gas chromatography–
mass spectrometry (Hewlett-Packard 6890; MSD model
5973 Network; Agilent Technologies, Santa Clara, CA) as a surrogate
for intramuscular (27, 28) and hepatic (29) leucyl-transfer
RNA labeling.
Mixed muscle proteins were isolated from biopsy specimens
(’30 mg wet weight) by homogenizing with 10 lL/mg 100%
acetonitrile. The samples were mixed by vortex for 10 min and
then centrifuged (15,000 g) for 5 min. The resultant pellet was
washed again with 10 lL/mg of 100% acetonitrile, once with 1
mL of double-distilled H2O, and finally with 1 mL of 95%
ethanol. The pellet was lyophilized and amino acids were liberated
from mixed muscle tissue protein by 6N HCl (400 lL/
mg) acid hydrolysis at 110C for 24 h. Free amino acids were
purified using cation exchange chromatography (Dowex
50WX8-200 resin; Sigma-Aldrich Ltd, Oakville, Canada) and
converted to their N-acetyl-n-propyl ester derivatives for analysis
by gas chromatography (GC) combustion-isotope ratio mass
spectrometry (GC model; Hewlett-Packard 6890; Agilent Technologies;
IRMS model Delta Plus XP; Thermo Finnigan, Waltham,
MA). Derivitized amino acids were separated on a 30-m
DB-1701 column (temperature program: 110C for 2 min; 10C/
min ramp to 190C; hold for 2 min; 2C/min ramp to 210C;
20C/min ramp to 280C; hold for 5 min) before combustion.
To isolate intracellular proteins for Western blotting, a small
piece of wet muscle (’20 mg) was homogenized by hand on ice
in a tris-buffered lysis buffer (pH 7.6) containing 0.1% (wt:vol)
sodium dodecyl sulfate, 0.5% (wt:vol) deoxycholaic acid, 15
mmol/L tris-HCl, 167 mmol/L NaCl, and commercially available
phosphatase and protease inhibitors (Roche Diagnostics, Indianapolis,
IN). Protein content of the homogenates was determined
by bicinchoninic acid protein assay (Thermo Scientific,
Rockford, IL), and then samples (20 lg of protein) were loaded
on a 8–16% gradient sodium dodecyl sulfate polyacrylamide gel
(Pierce, Rockford, IL) before being transferred to a PVDF membrane
for blotting.Membraneswere blocked with5%(wt:vol) bovine
serum albumin (rpS6, eIF2Be, and actin) or 5%nonfat milk (S6K1)
in tris-buffered saline with 0.1% Tween (vol:vol) (TBST) and
then incubated overnight in primary antibody at 4C: S6K1
Thr389 (catalog no 11759, 1:1000; Santa Cruz Biotechnology,
Santa Cruz, CA); rpS6 Ser240/244 (2215, 1:4000; Cell Signaling,
Danvers, MA); and eIF2Be Ser539 (24775, 1:4000; Genetex,
San Antonio, TX). We measured rpS6 Ser240/244
phosphorylation, because this site is phosphorylated by S6K1
and is responsive to feeding after resistance exercise (16, 30).
As a loading control, protein phosphorylation was normalized
to total actin by incubating in primary antibody (A2066,
1:10,000; Sigma) for 1 h at room temperature. After washing
in TBST, membranes were incubated in horseradish peroxidase-
linked anti-rabbit immunoglobulin G secondary antibody
(NA934V, 1:50,000; Amersham Biosciences, Piscataway, NJ),
washed with TBST, and developed using Pierce’s Supersignal
West Dura HRP detection kit (Thermo Fisher Scientific, Nepean,
Canada). Images were captured on a Fluorochem SP
imaging system (Alpha Innotech, San Leandro, CA).
Fibrinogen was precipitated from plasma (’1.5 mL) by adding
4 IU of thrombin (Sigma-Aldrich Ltd) and 40 lL of 1 mol/L
CaCl2 and incubating at 4C for 12 h. Albumin was isolated
from plasma by its differential solubility in ethanol using an
assay adapted from a previously published method (31). Specifically,
plasma proteins were precipitated from 500 lL of
fibrinogen-free plasma by adding 1 mL of 10% trichloroacetic
acid (wt:vol). After centrifugation at 4500 g for 5 min, the supernatant
fluid was discarded and the resultant pellet was resuspended
in 500 lL of ddH2O. Albumin was solubilized by
adding 2.5 mL of 1% TCA (wt:vol) in 100% ethanol and
centrifuged at 4500 rpm for 5 min. The supernatant fluid was
collected, and albumin was precipitated by adding 1mL of 26.8%
ammonium sulfate (wt:vol). After centrifugation at 4500 rpm for 5
min, the supernatant fluid was removed and discarded and the resultant
pellet was washed once more with 1 mL of 26.8%
ammonium sulfate. To remove any residual free amino acids,
the albumin pellet was washed twice with 1 mL of 0.2 mol/L
PCA. Finally, the pellet was lyophilized and the bound amino
acids were liberated by acid hydrolysis and analyzed using
GC-combustion-isotope ratio mass spectrometry as described
above. Plasma albumin concentration was determined spectrophometrically
using a standard bromocresol green assay
procedure (Pointe Scientific Inc, Canton, MI).
Calculations
The rates of mixed muscle and albumin protein synthesis were
calculated using the standard precursor-product method:
Fractional protein synthesis(FSR, %/h)
¼ DEb=Ep 31=t 3100 ð1Þ
where DEb is the change in bound protein enrichment between 2
points, Ep is the mean enrichment over time of arterialized venous
plasma a-KIC, and t is the time between biopsies (for
mixed muscle FSR) or blood samples (for albumin FSR, t ¼ 60 and 240 min). Ep was calculated as the area under the venous
FIGURE 1. Schematic diagram of infusion protocol. Arrows correspond
to the time at which blood, breath, or muscle biopsy samples were taken. A
drink containing 0, 5, 10, 20, or 40 g of protein was ingested in a randomized
order immediately after a bout of resistance exercise.
PROTEIN INTAKE AFTER RESISTANCE EXERCISE 3
plasma a-KIC enrichment by time curve divided by time. Leucine
oxidation was calculated from the appearance of the 13C-label in
expiredCO2 using the reciprocal pool model with fractional bicarbonate
retention factors of 0.7 and 0.83 for fasted (0 g protein) and
fed (5–40 g protein) states, respectively (32, 33). The area under
the leucine oxidation by time curve was calculated using Prism 3.0
graphing software (GraphPad Software Inc, San Diego, CA) as an
estimate of total leucine oxidation. In addition, the area under the
plasma insulin concentration by time curve was also calculated
with the same graphing software.
Statistics
Because the present study used a within-subject design,
changes in MPS, APS, and plasma albumin concentrations were
analyzed using a one-factor (135; condition) repeated-measures
analysis of variance (ANOVA). A 2-factor ANOVA was used to
determine significant changes in plasma amino acid concentration
(5 3 7; condition, time), whole-body leucine oxidation (5 3 6;
condition, time), plasma urea concentration (2 3 5; time,
condition), and protein phosphorylation (2 3 5; time, condition).
Differences in means were determined using a Holm-Sidak post
hoc test. All data were analyzed using SigmaStat 3.1 statistical
software (Systat Software Inc, Chicago, IL) and statistical significance
was set at P 0.05. Values are expressed as mean 6 SEM.
RESULTS
Blood amino acid concentrations are summarized in Table 1.
There was no change in EAA, BCAA, or leucine concentrations
after exercise in the absence of protein ingestion (P.0.05). There
was a slight increase (P,0.01) in BCAA concentration 0.75–1 h
after ingestion of 5 g of protein that was not significantly different
from 0 g (main effect for condition, P . 0.05). EAA, BCAA, and
leucine concentrations were greater after ingestion of 10 g compared
with 0 g (main effect for condition, P,0.01) and peaked at
0.75 h after exercise (P,0.01). After ingestion of 20 g of protein,
concentrations ofEAAandBCAApeaked at 0.75 h (P,0.01) and
were .0 and 5 g (main effect for condition, P , 0.01), whereas
leucine was only .0 g (main effect for condition, P , 0.01). Ingestion
of 40 g of protein increased EAA, BCAA, and leucine
concentrations by 0.75 h that remained elevated for the duration of
the trial (P , 0.01) and were greater than all other conditions
(main effect for condition, P , 0.01).
Baseline plasma insulin concentrations (4.5 6 0.7, 5.8 6 0.7,
5.0 6 0.7, 5.6 6 0.5, and 6.1 6 0.9 lU/mL, conditions 0–40 g,
respectively; P . 0.05) were similar in all conditions. Although
insulin concentrations were not significantly altered over time
(time 3 condition interaction not significant, P . 0.05), the
insulin area under the curve over 4 h with 40 g (1648 6 202 lU/
mL) of protein was greater (P , 0.01) than after 0, 5, and 10 g
(1040 6 98, 1186 6 82, and 1258 6 201 lU/mL, respectively)
and tended to be greater than after 20 g (1340 6 118 lU/mL;
P ¼ 0.009, critical level ¼ 0.007) of protein ingestion. Baseline
blood glucose concentration (4.9 6 0.6, 4.5 6 0.4, 4.7 6 0.5,
5.060.5, and 4.560.5 mmol/L, conditions 0–40 g, respectively;
P . 0.05) was similar for all conditions and remained steady
throughout the entire study period (data not shown).
Plasma a-KIC enrichment was at plateau in all conditions
(7.8 6 0.2, 7.3 6 0.2, 7.6 6 0.1, 7.5 6 0.3, and 8.3 6 0.2 atom
percent excess for conditions 0–40 g, respectively). Mixed-muscle
TABLE 1
Blood amino acid concentrations after exercise1
Time (h)
Protein 0 0.75 1 1.5 2 3 4
lmol/L
EAAs
0 g*,2 628 (16) 609 (24) 637 (43) 618 (46) 566 (36) 524 (24) 537 (31)
5 gy 649 (37)a,b,3 706 (28)b,c 703 (23)b,c 697 (39)a,b 649 (19)a,b 581 (31)a,b 555 (35)a
10 gyz 670 (8)a,b 782 (25)c 764 (41)c 760 (50)c 690 (19)b,c 621 (23)b 568 (35)a
20 gz 656 (47)a 883 (78)b 864 (82)b,c 752 (47)c,d 731 (30)a,d 667 (15)a 625 (36)a
40 gd 660 (25)a 895 (40)b 892 (53)b 883 (71)b 878 (66)b 841 (45)b 788 (48)b
BCAAs
0 g* 274 (11) 256 (12) 266 (21) 255 (23) 241 (17) 232 (10) 244 (13)
5 g*y 288 (22) 313 (18) 309 (15) 308 (19) 290 (13) 262 (17) 251 (18)
10 gyz 298 (9)a,b 362 (19)c 346 (24)b,c 353 (32)a–c 323 (15)a–c 288 (12)a 272 (20)a
20 gz 296 (22)a 420 (40)c 412 (44)c 360 (34)b,c 353 (23)a–c 326 (14)a,b 311 (15)a,b
40 gd 303 (17)a 451 (31)b 454 (38)b 448 (46)b 456 (46)b 441 (34)b 413 (31)b
Leucine
0 g* 82 (3) 83 (3) 86 (6) 88 (6) 83 (7) 84 (4) 90 (5)
5 g*y 86 (5) 101 (4) 100 (3) 101 (5) 97 (2) 90 (5) 91 (6)
10 g*y 88 (2)a 120 (6)c 112 (7)b,c 115 (8)a–c 106 (4)a–c 99 (4)a–c 96 (6)a,b
20 gy 88 (6)a 146 (15)d 136 (13)c,d 118 (9)c 116 (5)b,c 108 (3)a–c 106 (4)a–c
40 gz 98 (10)a 167 (13)c 166 (12)c 155 (21)b,c 156 (18)b,c 148 (14)b 141 (13)b
1 Values are means (SEM), n ¼ 6. Blood amino acid concentration over 4 h after exercise. EAAs, essential amino acids
(sum of His, Ile, Leu, Lys, Met, Phe, Thr, Val; note Cys not measured); BCAAs, branched-chain amino acids (sum of Ile,
Leu, and Val. Data were analyzed using a 2-factor (protein 3 time) ANOVA. Differences in means were determined using
a Holm-Sidak post hoc test. Protein 3 time interactions for EAAs, BCAAs, and leucine were all significant, P , 0.01.
2 Protein doses with different symbols are significantly different from each other (main effect for protein, P , 0.01).
3 Means within each protein dose with different superscript letters are significantly different from each other, P , 0.01.
4 MOORE ET AL
FSR was increased (P , 0.01) above the fasted condition by ’37
and 56% after consumption of 5 and 10 g of protein, respectively
(Figure 2). At 20 g of protein, there was a ’93% increase (P ,
0.01) in mixed-muscle FSR above the fasted condition. There
was no difference in mixed-muscle FSR after consumption of
20 or 40 g of protein (P ¼ 0.29). Similarly, plasma albumin FSR
increased (P , 0.01) in a dose-dependent manner in response
to increasing amounts of dietary protein and reached a plateau
at 20 g (Figure 3). There was no difference in albumin FSR
after consumption of 20 or 40 g of dietary protein (P ¼ 0.65).
The average baseline plasma albumin concentration was 4.5 6
0.4 g/dL with no differences between conditions (data not
shown). A plot of the residuals for mixed muscle and plasma
albumin protein synthesis revealed no bias of trial order on the
measurement of FSR (data not shown).
Despite the marked changes in mixed-muscle FSR, there was
no statistically significant change (P . 0.05) in the phosphorylation
of S6K1 (Thr389), rps6 (Ser240/244), or eIF2Be (Ser539) 1
or 4 h after exercise with increasing protein intake (Figure 4).
Leucine oxidation was not different (P . 0.05) from the fasted
condition after ingestion of 5 and 10 g of protein (Table 2).
Leucine oxidation was stimulated at both 20 and 40 g of dietary
protein (main effect for condition; P . 0.05). There was a trend
toward a greater area under the leucine oxidation by time curve
(estimate of total leucine oxidized over 4 h) after ingestion of 40
g compared with 20 g of protein (153.1 6 12.3 compared with
129.3 6 9.0 lmol/kg; P ¼ 0.017, critical level ¼ 0.013). Plasma
urea was similar at baseline for all conditions (3.6 6 0.2, 3.5 6
0.5, 3.7 6 0.3, 3.7 6 0.3, and 4.0 6 0.1 mmol/L, conditions
0–40 g, respectively; P . 0.05). At 4 h, there were slight decreases
in plasma urea after ingestion of 0 and 5 g (’3 and
’10%, respectively) and slight increases after ingestion of 10,
20, and 40 g of protein (’7, ’5, and ’7%, respectively), although
none of these changes were significant (P . 0.1 for all
comparisons).
DISCUSSION
Our study is the first to our knowledge to describe the responses
of mixed-muscle and albumin protein synthesis as well as
whole-body leucine oxidation to increasing protein intake after
an acute bout of resistance exercise. We report that increasing
protein intake stimulates mixed-muscle and plasma albumin
protein synthesis in a dose-dependent manner up to 20 g of
dietary protein, after which there is a marked stimulation of
whole-body leucine oxidation and no further increase in protein
synthesis. The stimulation of muscle protein synthesis with increasing
protein intake was not associated with any consistent
change in the phosphorylation status of candidate signaling
proteins.
Borsheim et al (8) proposed the existence of a dose-response
relation between muscle protein synthesis and amino acid consumption
after resistance exercise on the basis of a comparison
of data from 2 studies (8, 9). They observed a postexercise
stimulation of muscle protein synthesis almost twice as great
after ingestion of 6 g compared with only 3 g of EAAs (8, 9).
Our present findings are in agreement with their conclusion in
that there is an apparent graded response of muscle protein
synthesis to low doses of dietary protein, as suggested by the
trend (P ¼ 0.059, critical level ¼ 0.025) toward a greater protein
synthetic response after ingestion of 10 g (’4.3 g EAAs)
compared with 5 g (’2.2 g EAAs) of whole protein. This is
similar to our previously published data demonstrating that 10 g
of whey protein is sufficient to enhance the postexercise stimulation
of muscle protein synthesis (4). The current data expand
on previous work that shows that muscle protein synthesis is
further stimulated with greater protein intakes but reaches a plateau
after ingestion of 20 g of high-quality protein (’8.6 g EAAs).
This is in agreement with a previous observation showing similar
postexercise net amino acid balance after 2 high doses of EAAs
(’21 compared with 40 g) (2). Thus, it appears that there is
a maximal effective dose of dietary amino acids for stimulating
muscle anabolism after resistance exercise. More important, our
data suggest that the dose of EAAs that maximally stimulates
muscle protein synthesis after resistance exercise (’8.6 g) is very
similar to that seen at rest (10 g) (7).
The phosphorylation of intracellular signaling proteins we
measured was unaffected by protein ingestion. This is in contrast
to previous work that has shown that amino acids robustly
FIGURE 2. Mean (6SEM) mixed-muscle fractional protein synthesis
(FSR) after resistance exercise in response to increasing amounts of
dietary protein. Data were analyzed using a one-factor (protein) repeatedmeasures
ANOVA to test for differences between conditions. Differences in
means were analyzed using a Holm-Sidak post hoc test. Means with different
letters are significantly different from each other (P , 0.01; n ¼ 6).
FIGURE 3. Mean (6SEM) plasma albumin fractional protein synthesis
(FSR) after resistance exercise in response to increasing amounts of dietary
protein. Data were analyzed using a one-factor (protein) repeated-measures
ANOVA to test for differences between conditions. Differences in means
were analyzed using a Holm-Sidak post hoc test. Means with different letters
are significantly different from each other (P , 0.01; n ¼ 6).
PROTEIN INTAKE AFTER RESISTANCE EXERCISE 5
enhance mTOR signaling and the activity of S6K1 and eIF2B (7,
13, 19). However, all of our measurements of the phosphorylation
state of these proteins were made against a background of
resistance exercise, which is a potent anabolic stimulus that has
also been shown to increase the activity of these signaling
pathways in the fasted state both in rats (18, 34) and humans (11,
12, 15, 16, 35). Thus, it is possible that resistance exercise already
stimulated the phosphorylation of proteins within the
mTOR-signaling pathway, thereby masking any amino acid–
induced changes with protein ingestion in our representative
downstream effectors. Although this would seem at odds with
work demonstrating the phosphorylation of S6K1 is enhanced
with feeding after exercise (12, 14, 16, 36), these studies
provided either additional carbohydrate, which would stimulate
insulin release that enhances the activation of the mTORsignaling
cascade in the presence of amino acids (37, 38), or
a source of crystalline EAA before, during, and after exercise
and are therefore difficult to compare with the present study in
which postexercise feeding of intact proteins was used. Alternatively,
training status has also been shown to influence signal
transduction after exercise, and it is possible that the resistancetrained
background of the individuals in the present study may
have resulted in a relatively blunted signaling response to exercise
and feeding (39). Moreover, although a reduction in
phosphorylation at residue Ser539 releases eIF2Be from the inhibitory
state mediated by GSK3b and likely contributes to an
increase in its guanine nucleotide exchange activity (40), Wang
and Proud (41) recently demonstrated that amino acids repress
the phosphorylation of eIF2Be on an as-yet-unstudied (in humans)
residue (Ser525), which may be the main mechanism by
which amino acids regulate eIF2B activity; this clearly warrants
further investigation. Nonetheless, our data demonstrate that
the phosphorylation status of certain signaling proteins, at least
at the time points we studied, shed little light on what is determining
the rate of muscle protein synthesis with protein ingestion
after exercise.
Because we saw no consistent change in the phosphorylation
of candidate signaling proteins, a reasonable question is what
is regulating muscle protein synthesis after resistance exercise
with increasing protein ingestion. We speculate that an increase
in delivery of amino acids might be the stimulus—that is, the
postexercise stimulation of muscle protein synthesis is primarily
related to substrate (ie, amino acid) availability (1). In short,
resistance exercise as an anabolic stimulus would facilitate the
transport of amino acids into the muscle (1, 42) and would also
prime the translational machinery of the muscle cell [see (10)
for review]. Amino acids taken up from the circulation are then
rapidly incorporated into new tissue proteins. Our data show that
there is, however, a maximal rate at which dietary amino acids
can be incorporated into muscle tissue and that with increasingly
higher concentrations of amino acids, there is no further stimulation
of muscle protein synthesis.
FIGURE 4. Mean (6SEM) phosphorylation of S6K1 at Thr389 (A), rpS6
at Ser240/244 (B), and eIF2B at Ser539 (C) 1 and 4 h after exercise in response
to increasing amounts of dietary protein. Data are expressed as a fold-change
from 0 g protein ingestion at 1 h. A separate gel was run for each participant
with 1- and 4-h samples loaded in adjacent lanes for all conditions (0–40 g
protein, respectively). Gel orientation was identical for all proteins. Data were
analyzed using a 2-factor (protein3time) repeated-measures ANOVA. There
were no significant main effects for time or protein (P . 0.05). There was no
significant time 3 protein interaction (P . 0.05; n ¼ 6).
TABLE 2
Whole-body leucine oxidation after exercise1
Time (h)
Protein 1a,2 1.5a,b 2b,c 2.5b,c 3c,d 4d
lmol kg21 h21
0 g*,
3 34 (4) 25 (2) 22 (2) 21 (2) 21 (3) 20 (3)
5 g* 37 (2) 32 (1) 31 (2) 27 (2) 27 (3) 23 (2)
10 g* 36 (3) 36 (5) 30 (3) 29 (3) 27 (3) 25 (2)
20 gy 48 (4) 49 (3) 45 (4) 44 (3) 40 (3) 36 (2)
40 gy 49 (4) 55 (4) 57 (4) 56 (7) 48 (6) 41 (4)
1 Values are means (SEM), n ¼ 6. Whole-body leucine oxidation over
4 h after exercise. Data were analyzed using a 2-factor (protein 3 time)
ANOVA. Differences in means were determined using a Holm-Sidak post
hoc test.
2 Times with different superscript letters are significantly different
(main effect for time, P , 0.01). There was no significant interaction (protein
3 time, P . 0.05).
3 Protein doses with different symbols are significantly different from
each other (main effect for protein, P , 0.01).
6 MOORE ET AL
We observed a dose-dependent stimulation of APS to increasing
amounts of dietary protein. Albumin synthesis appears
unaffected by resistance exercise (20), suggesting that the differences
in its fractional synthetic rate we observed is amino
acid mediated. This is consistent with the observation that albumin
protein synthesis is increased in response to the consumption of
a protein-containing meal (21–23) and can be influenced by the
level of protein in the diet (23). In fact, one thesis is that dietary
amino acids consumed in excess of their acute requirement to
synthesize lean tissue are directed toward albumin synthesis to
minimize their irreversible oxidative loss (21). In the present
study, rates of albumin synthesis (eg, mixed muscle proteins)
reached a plateau at 20 g of ingested protein, after which there was
a marked stimulation of whole-body leucine oxidation. Consequently,
whereas a fraction of dietary amino acids may be
sequestered in albumin protein as a conservatory mechanism,
we speculate that this is relatively minor in comparison with
the large storage capacity of skeletal muscle. It is more likely
that, similar to other lean tissues such as the splanchnic region
(43), feeding stimulates turnover (ie, synthesis and degradation)
of plasma albumin with little expansion of the protein
pool.
With graded protein intakes, the point at which amino acid
oxidation significantly increases has been suggested to reflect the
level at which protein intake becomes excessive (44). Suggestive
of a nutrient excess (45), leucine oxidation in the present study
was stimulated after ingestion of 20 and 40 g of protein. In
addition, muscle and plasma albumin protein synthesis were
maximally stimulated at 20 g of dietary protein, which suggests
an upper limit for incorporation of amino into these protein
pools had been reached.
Provided that adequate energy intake is met (46), our findings
have implications for protein recommendations for resistancetrained
athletes in terms of the quantity of dietary protein that
might maximize muscle growth. If we assume that a 20-g protein
dose maximally stimulates muscle protein synthesis after
exercise and we know that resistance exercise enhances the
synthesis of muscle protein for at least 24 h (26, 47, 48), one
could ask how many times in a day could someone consume
such a dose to stimulate muscle anabolism that would ultimately
translate into muscle growth? Because muscle protein synthesis
becomes refractory to persistent aminoacidemia (49) and excess
amino acids are lost to oxidation (44), we speculate that no more
than 5–6 times daily could one ingest this amount (’20 g) of
protein and expect muscle protein synthesis to be maximally
stimulated. Protein consumption in excess of this rate or dose
would ultimately lead to oxidative loss. In addition, given that
the capacity to oxidize amino acids adapts to the diet and can act
as a key regulator of protein stores (50), chronic protein consumption
in excess of this rate or dose could actually lead to
dampening of the protein synthetic response to suboptimal (ie,
,20 g) protein doses.
In summary, our data are the first, to our knowledge, to
demonstrate that muscle protein synthesis responds to increasing
protein intake in a dose-dependent manner after resistance exercise
and reaches a maximal stimulation after ingestion of 20 g
of high-quality protein. We observed little change in the phosphorylation
status of signaling proteins shown to activate the
translational machinery, which suggests that the main driver of
muscle anabolism with feeding after resistance exercise may
simply be amino acid availability. We also report that APS is
stimulated by increasing protein ingestion after resistance exercise
and reached a plateau at 20 g of ingested protein. The
stimulation of leucine oxidation after ingestion of 20 and 40 g of
protein suggests that these doses represent an excess of protein. In
conclusion, our data indicate that 20 g of intact high-quality
protein is sufficient to maximize the anabolic response to resistance
exercise.
We acknowledge the expert technical assistance of Drs. Kenneth Smith and
John Babraj in assisting with method development. We also thank Joe Hartman
for his help in data collection and the participants for their time and effort.
The authors’ responsibilities were as follows—DRM, JET,MAT, and SMP:
planned the study; DRM, MJR, JLF, TP, and SMP: collected the data; DRM,
MJR, JLF, JET, EIG, SBW, and TP: analyzed the data; and DRM, JET, EIG,
MAT, and SMP: wrote and edited the manuscript. None of the authors had any
financial or personal conflicts of interest to declare.
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