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Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: A double-blind, randomized trial

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Leucine is a key amino acid involved in the regulation of skeletal muscle protein synthesis. We assessed the effect of the supplementation of a lower-protein mixed macronutrient beverage with varying doses of leucine or a mixture of branched chain amino acids (BCAAs) on myofibrillar protein synthesis (MPS) at rest and after exercise. In a parallel group design, 40 men (21 ± 1 y) completed unilateral knee-extensor resistance exercise before the ingestion of 25 g whey protein (W25) (3.0 g leucine), 6.25 g whey protein (W6) (0.75g leucine), 6.25 g whey protein supplemented with leucine to 3.0 g total leucine (W6+Low-Leu), 6.25 g whey protein supplemented with leucine to 5.0 g total leucine (W6+High-Leu), or 6.25 g whey protein supplemented with leucine, isoleucine, and valine to 5.0 g total leucine. A primed continuous infusion of l-[ring-(13)C6] phenylalanine with serial muscle biopsies was used to measure MPS under baseline fasted and postprandial conditions in both a rested (response to feeding) and exercised (response to combined feeding and resistance exercise) leg. The area under the blood leucine curve was greatest for the W6+High-Leu group compared with the W6 and W6+Low-Leu groups (P < 0.001). In the postprandial period, rates of MPS were increased above baseline over 0-1.5 h in all treatments. Over 1.5-4.5 h, MPS remained increased above baseline after all treatments but was greatest after W25 (∼267%) and W6+High-Leu (∼220%) treatments (P = 0.002). A low-protein (6.25 g) mixed macronutrient beverage can be as effective as a high-protein dose (25 g) at stimulating increased MPS rates when supplemented with a high (5.0 g total leucine) amount of leucine. These results have important implications for formulations of protein beverages designed to enhance muscle anabolism. This trial was registered at clinicaltrials.gov as NCT 1530646.
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Leucine supplementation of a low-protein mixed macronutrient
beverage enhances myofibrillar protein synthesis in young men:
a double-blind, randomized trial
1–3
Tyler A Churchward-Venne, Leigh Breen, Danielle M Di Donato, Amy J Hector, Cameron J Mitchell, Daniel R Moore,
Trent Stellingwerff, Denis Breuille, Elizabeth A Offord, Steven K Baker, and Stuart M Phillips
ABSTRACT
Background: Leucine is a key amino acid involved in the regula-
tion of skeletal muscle protein synthesis.
Objective: We assessed the effect of the supplementation of a lower-
protein mixed macronutrient beverage with varying doses of leucine
or a mixture of branched chain amino acids (BCAAs) on myofibril-
lar protein synthesis (MPS) at rest and after exercise.
Design: In a parallel group design, 40 men (21 6 1 y) completed
unilateral knee-extensor resistance exercise before the ingestion of
25 g whey protein (W25) (3.0 g leucine), 6.25 g whey protein (W6)
(0.75g leucine), 6.25 g whey protein supplemented with leucine to
3.0 g total leucine (W6+Low-Leu), 6.25 g whey protein supplemented
with leucine to 5.0 g total leucine (W6+High-Leu), or 6.25 g whey
protein supplemented with leucine, isoleucine, and valine to 5.0 g
total leucine. A primed continuous infusion of
L-[ring-
13
C
6
] phenyl-
alanine with serial muscle biopsies was used to measure MPS under
baseline fasted and postprandial conditions in both a rested (response
to feeding) and exercised (response to combined feeding and resis-
tance exercise) leg.
Results: The area under the blood leucine curve was greatest for the
W6+High-Leu group compared with the W6 and W6+Low-Leu
groups (P , 0.001). In the postprandial period, rates of MPS were
increased above baseline over 0–1.5 h in all treatments. Over 1.5–
4.5 h, MPS remained increased above baseline after all treatments
but was greatest after W25 (w267%) and W6+High-Leu (w220%)
treatments (P = 0.002).
Conclusions: A low-protein (6.25 g) mixed macronutrient beverage
can be as effective as a high-protein dose (25 g) at stimulating in-
creased MPS rates when supplemented with a high (5.0 g total
leucine) amount of leucine. These results have important implica-
tions for formulations of protein beverages designed to enhance
muscle anabolism. This trial was registered at clinicaltrials.gov as
NCT 1530646. Am J Clin Nutr 2014;99:276–86.
INTRODUCTION
The provision of a complete mixture of amino acids increases
myofibrillar protein synthesis (MPS)
4
rates (1) through the ac-
tivation of the target of rapamycin complex-1 (2). This effect on
MPS is primarily attributable essential amino acids (EAAs)
because nonessential amino acids do not stimulate MPS (3–5).
The relation between amino acid (6) and protein (7, 8) intakes and
MPS is dose dependent and saturable. Of EAAs, the branched-
chain amino acid (BCAA) leucine is a key determinant of the
postprandial stimulation of MPS after protein intake in rodents
(9). In vivo animal studies have shown that the independent ad-
ministration of leucine, but not isoleucine or valine, can stimulate
MPS rates (10, 11) to the same extent as complete mixtures of
EAA or complete protein (12, 13). Some research in humans has
focused on the efficacy of leucine supplementation to promote
increases in MPS (14–17) and augment skeletal muscle mass (18,
19). Although some studies have shown increased rates of MPS
with leucine administration (20–22), other studies have not (17,
23–25). However, the provision of leucine can result in reduced
circulating concentrations of isoleucine and valine (18, 26, 27),
which could lower MPS (28); thus, the inclusion of all BCAAs as
opposed to leucine alone may be efcacious. The addition of
BCAAs, and in particular leucine, to stimulate MPS in a less than
optimally effective dose of protein may represent an effective
strategy to increase MPS after feeding or under the influence of
the markedly anabolic stimulus of resistive exercise.
The aim of the current study was to assess the potential
to enhance the effect of a dose of protein that contained a quan-
tity of EAAs previously shown to be suboptimal in maximally
1
From the Exercise Metabolism Research Group, Departments of Kinesi-
ology (TAC-V, LB, DMDD, AJH, CJM, and SMP) and Neurology (SKB),
McMaster University, Hamilton, Canada, and the Nestle
´
Research Centre,
Nestec Ltd, Lausanne, Switzerland (DRM, TS, DB, and EAO).
2
Supported by Nestec Ltd and the Natural Sciences and Engineering Re-
search Council of Canada (postgraduate scholarship to TAC-V).
3
Address correspondence to SM Phillips, McMaster University, 1280
Main Street West, Hamilton, Ontario L8S 4K1, Canada. E-mail: phillis@
mcmaster.ca.
4
Abbreviations used: Akt, protein kinase B; AUC
neg
, AUC below base-
line; AUC
pos
, AUC above baseline; BCAA, branched-chain amino acid;
C
max
, maximum concentration; EAA, essential amino acid; eEF2, eukaryotic
elongation factor 2; EX-FED, response to combined feeding and resistance
exercise; FED, response to feeding; FSR, fractional synthetic rate; MPS,
myofibrillar protein synthesis; mTOR, mechanistic target of rapamycin;
T
max
, time of maximum concentration; W6, 6.25 g whey protein; W6
+BCAAs, 6.25 g whey protein supplemented with leucine, isoleucine, and
valine to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supple-
mented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey pro-
tein supplemented with leucine to 3.0 g total leucine; W25, 25 g whey
protein; 1-RM, single-repetition maximum strength; 4E-BP1, eukaryotic ini-
tiation factor 4E binding protein 1.
Received June 21, 2013. Accepted for publication November 8, 2013.
First published online November 27, 2013; doi: 10.3945/ajcn.113.068775.
276 Am J Clin Nutr 2014;99:276–86. Printed in USA. Ó 2014 American Society for Nutrition
at MCMASTER UNIVERSITY on January 21, 2014ajcn.nutrition.orgDownloaded from
68775.DCSupplemental.html
http://ajcn.nutrition.org/content/suppl/2014/01/11/ajcn.113.0
Supplemental Material can be found at:
stimulating MPS rates with feeding (6) and after exercise (7) on
MPS rates when ingested as part of a mixed macronutrient bev-
erage. Subjects were randomly assigned to a positive control [25 g
of whey protein (W25); 3.0 g leucine], a negative control [6.25 g
whey protein (W6); 0.75 g leucine], or treatments that consisted
of 6.25 g whey supplemented with a lower dose of leucine [6.25 g
whey protein supplemented with leucine to 3.0 g total leucine
(W6+Low-Leu)], a higher dose of leucine [6.25 g whey protein
supplemented with leucine to 5.0 g total leucine (W6+High-Leu],
or a higher dose of leucine plus isoleucine and valine [6.25 g
whey protein supplemented with leucine, isoleucine, and valine
to 5.0 g total leucine (W6+BCAAs)]. Rates of MPS and the
phosphorylation status of protein targets of the protein kinase B
(Akt)–mechanistic target of rapamycin (mTOR) C-1 pathway
were examined under postabsorptive conditions and in the post-
prandial state under rested and postexercise conditions. We examined
a temporally early (0–1.5 h) and late (1.5–4.5 h) postprandial
period during both resting and postexercise recovery conditions
because leucine has been suggested to direct the peak activation
but not the duration of MPS (9). We hypothesized that W6+Low-
Leu, W6+BCAAs, W25, and W6+High-Leu would stimulate
greater postprandial MPS rates than W6 would under resting
conditions with no differences between treatments. During post-
exercise recovery, we hypothesized that W6+BCAAs, W25, and
W6+High-Leu would elicit increases in MPS that were equiva-
lent but greater than with W6+Low-Leu and W6 because of
the maintenanc e of the MPS response over the later time pe-
riod examined.
SUBJECTS AND METHODS
Study participants
Forty young men between 18–35 y of age were recruited via
advertisements posted on the McMaster University campus to
participate in the study. Characteristics of study participants are
presented in Table 1. In a double-blind manner, participants
were randomly assigned to 1 of 5 parallel treatment groups (n =
8/group) in a block design balanced for body weight. The ran-
domization technique was performed by using the minimization
technique implemented with the Trialbalance computer program
(Nestle
´
). An individual at McMaster, who was not directly in-
volved with the study, was responsible for random assignment
and treatment preparation. Five codes were generated, and their
corresponding group assignments were stored in 5 separate
code-break envelopes that were held and sealed until the com-
pletion of all data analyses. After a participant’s body weight
was entered in the Trialbalance system, an individual subject
code was generated that corresponded to the treatment-group
allocation, which was known only to the individual who was
responsible for random assignment and treatment preparation.
Only the individual participant’s code was placed in the treat-
ment container. None of the study participants reported having
engaged in a structured program of resistance exercise within
the past year but reported being recreationally active w 2–3
times/wk. Participants were deemed healthy on the basis of re-
sponses to a routine health-screening questionnaire. Each par-
ticipant was informed of the purpose of the study, experimental
procedures, and potential risks before providing written consent.
The study was approved by the Hamilton Health Sciences Re-
search Ethics Board and conformed to the standards for the use
of human subjects in research as outlined in the most recent
update of the Declaration of Helsinki. The study also conformed
to standards established by the Canadian Tri-Council Policy on
the ethical use of human subjects (29). The study took place at
the Ivor Wynne Centre, Department of Kinesiology, McMaster
University, Ontario, Canada, from 2011 to 2012. The primary
outcome measure wa s a change in myofibrillar fractional
synthetic rate (FSR) (%/h) as assessed by the incorporation of
13
C
6
-labeled phenylalanine into myofibrillar proteins.
Pretesting
Approximately 1 wk before the experimental infusion trial,
study participants underwent unilateral strength testing of the
knee-extensor muscles. Participants performed a series of graded
knee-extensions to determine their single-repetition maximum
strength (1-RM) with their self-reported dominant leg by using
a seated knee-extension device (Atlantis Precision Series C-105).
In addition, each participant underwent a whole-body dual-
energy X-ray absorptiometry scan (QDR-4500A, software ver-
sion 12.31; Hologic) to measure body composition (Table 1).
Participants were provided with prepackaged standardized diets
that were consumed during the 2 d immediately preceding the
experimental infusion trial. Diets were designed to provide
sufficient energy to maintain an energy balance as determined by
the Harris-Benedict equation and were adjusted by using a
moderate activity factor (1.4–1.6) to account for participants self-
reported physical activity patterns. The macronutrient distribution
TABLE 1
Participant characteristics
1
W6 W6+Low-Leu W25 W6+BCAAs W6+High-Leu
Age (y) 20.5 6 1.1 20.4 6 0.6 20.9 6 0.6 20.8 6 0.8 19.5 6 0.1
Height (m) 1.80 6 0.03 1.76 6 0.02 1.76 6 0.02 1.83 6 0.03 1.76 6 0.03
Weight (kg) 79.4 6 3.5 77.7 6 3.3 78.1 6 2.8 81.3 6 3.8 79.4 6 3.4
BMI (kg/m
2
) 24.5 6 0.7 25.0 6 1.0 25.2 6 1.0 24.3 6 0.8 25.7 6 1.2
Fat-free mass (kg) 66.6 6 2.4 64.2 6 2.3 66.0 6 2.8 70.4 6 2.9 64.8 6 2.7
Fat mass (kg) 12.9 6 1.3 13.6 6 1.6 12.1 6 0.8 10.9 6 1.1 14.6 6 1.2
1-RM (kg) 64.5 6 1.7 71.3 6 4.2 65.1 6 3.2 67.3 6 3.6 59.4 6 3.5
1
All values are means 6 SEMs. n = 8/treatment group. W6, 6.25 g whey protein; W6+BCAAs, 6.25 g whey protein
supplemented with leucine, isoleucine, and valine to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supplemented
with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey protein supplemented with leucine to 3.0 g total leucine;
W25, 25 g whey protein; 1-RM, single-repetition maximum strength.
LEUCINE AND MYOFIBRILLAR PROTEIN SYNTHESIS 277
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of diets was 55% carbohydrate, 30% fat, and 15% protein. Par-
ticipants were instructed to consume all food and beverages
provided and avoid the consumption of food and beverages
(other than water) that were not provided as part of the stan-
dardized diet. Participants were instructed to abstain from
strenuous physical exercise for 72 h before the experimental
infusion trial and to consume their evening meal no later than
2000 the evening before the trial.
Experiment
Participants reported to the laboratory at w0600 on the morning
of the experimental infusion trial after an overnight fast. A
catheter was inserted into an antecubital vein, and a baseline
blood sample was taken before the initiation of a 0.9% saline
drip to keep the catheter patent to allow for repeated arterialized
blood sampling. Arterialized blood samples (30) were obtained
repeatedly during the infusion trial (see Online Supplemental
Material Figure 1 under “Supplemental data” in the online issue)
by wrapping a heating blanket around the forearm. Blood
samples were collected into 4 mL heparinized evacuated tubes
and chilled on ice. A second catheter was inserted into the an-
tecubital vein of the opposite arm before the initiation of a
primed continuous infusion (0.05 mmol $ kg
21
$ min
21
; 2.0
mmol $ kg
21
prime) of [ring-
13
C
6
] phenylalanine (Cambridge
Isotope Laboratories). The infusate was passed through a 0.2-
mm filter before entering the blood. The baseline (fasted) FSR
was calculated on the basis of the
13
C enrichment of mixed
plasma proteins obtained from the preinfusion blood sample and
skeletal muscle biopsy after w3 h tracer incorporation (31, 32).
Participants performed an acute bout of unilateral seated knee-
extension resistance exercise (Atlantis Precision Series C-105)
that consisted of 8 sets of 10–12 repetitions at w80% of their
previously determined 1-RM with an interset rest interval of
2 min. Immediately after completion of the resistance exercise,
participants underwent bilateral biopsies from both rested and
exercised legs and immediately ingested their designated nutri-
ent treatment (Table 2). Bilateral biopsy samples were obtained
at 1.5 and 4.5 h after treatment administration from a rested fed
[response to feeding (FED)] and exercise-fed [response to com-
bined feeding and resistance exercise (EX-FED)] leg. Muscle
biopsies were obtained from the vastus lateralis muscle by using
a 5 mm Bergstro
¨
m needle that was custom adapted for manual
suction under 2% xylocaine local anesthesia. Tissue samples
were freed from visible blood, fat, and connective tissue and
immediately frozen in liquid nitrogen for additional analysis as
previously described (33, 34). Each biopsy sample was obtained
from a separate incision w2–3 cm apart. Each participant un-
derwent a total of 6 skeletal muscle biopsies (3 biopsies from
each leg). See Online Supplemental Material Figure 1 under
“Supplemental data” in the online issue for an outline of details
of the infusion protocol.
Beverage composition
Study participants were administered nutrient treatments
orally in a double-blinded manner immediately after resistance
exercise. All treatments were provided in colored plastic con-
tainers. Treatments were similar in color, smell, and taste because
their main constituents were the same but provided in different
quantities depending on the treatment. The macronutrient and
amino acid composition of each of the 5 treatments is outlined in
Table 2. The W6+Low-Leu, W6+BCAAs, W6+High-Leu, and
W6 treatme nts were isonitrogenous, isoenerge tic, and macro-
nutrient-matched, whereas the positive control (W25) contained
a reduced amount of carbohydrate and more protein to be energy-
matched to the other treatments. The whey protein isolate (biPro;
Davisco Foods) was independently tested (Telmark) in triplicate
for content analysis. Free-form amino acids used were as follows:
L-leucine, L-isoleucine, L-valine, L-alanine, and L-glycine (Sigma
Life Science; Sigma-Aldrich). The carbohydrate source was
TABLE 2
AA, protein, carbohydrate, and fat contents of nutritional treatments
1
Nutritional treatment group
W6
W6+
Low-Leu W25
W6+
BCAAs
W6+
High-Leu
Endogenous AAs (g)
Alanine 0.29 0.29 1.15 0.29 0.29
Arginine 0.13 0.13 0.53 0.13 0.13
Aspartic acid 0.70 0.70 2.80 0.70 0.70
Cystine 0.19 0.19 0.78 0.19 0.19
Glutamic acid 1.03 1.03 4.10 1.03 1.03
Glycine 0.11 0.11 0.43 0.11 0.11
Histidine 0.14 0.14 0.55 0.14 0.14
Isoleucine 0.34 0.34 1.35 0.34 0.34
Leucine 0.75 0.75 3.00 0.75 0.75
Lysine 0.68 0.68 2.70 0.68 0.68
Methionine 0.14 0.14 0.58 0.14 0.14
Phenylalanine 0.22 0.22 0.88 0.22 0.22
Proline 0.26 0.26 1.05 0.26 0.26
Serine 0.16 0.16 0.63 0.16 0.16
Threonine 0.28 0.28 1.10 0.28 0.28
Tryptophan 0.17 0.17 0.68 0.17 0.17
Tyrosine 0.22 0.22 0.88 0.22 0.22
Valine 0.35 0.35 1.38 0.35 0.35
Added AAs (g)
Alanine 3.18 2.05 0.00 0.03 1.05
Glycine 3.17 2.05 0.00 0.03 1.05
Leucine 0.00 2.25 0.00 4.25 4.25
Isoleucine 0.00 0.00 0.00 1.01 0.00
Valine 0.00 0.00 0.00 1.03 0.00
Added carbohydrate (g) 35.0 35.0 22.60 35.0 35.0
Added fat (g) 5.68 5.68 5.68 5.68 5.68
Totals
Whey protein (g) 6.15 6.15 24.57 6.15 6.15
EAAs (g) 2.89 5.14 11.54 9.18 7.14
NEAAs (g) 9.61 7.36 13.03 3.32 5.36
Total protein (g) 12.5 12.5 24.57 12.5 12.5
Leucine (g) 0.75 3.00 3.00 5.00 5.00
Isoleucine (g) 0.34 0.34 1.35 1.35 0.34
Valine (g) 0.35 0.35 1.38 1.38 0.35
BCAAs (g) 1.43 3.68 5.73 7.73 5.68
Carbohydrate (g) 35.0 35.0 22.90 35.0 35.0
Fat (g) 5.68 5.68 5.68 5.68 5.68
kcal 241 241 241 241 241
1
AA, amino acid; BCAA, branched-chain amino acid; EAA, essential
amino acid; NEAA, nonessential amino acid; W6, 6.25 g whey protein; W6
+BCAAs, 6.25 g whey protein supplemented with leucine, isoleucine, and
valine to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supple-
mented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey
protein supplemented with leucine to 3.0 g total leucine; W25, 25 g whey
protein.
278 CHURCHWARD-VENNE ET AL
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sucrose, whereas the fat source was hydrogenated coconut oil
(Nestle
´
Coffee Mate; Nestec Ltd). All nutrient treatments were
prepared in 300 mL H
2
O (Table 2). To minimize disturbances in
the isotopic equilibrium after amino acid ingestion, beverages
were enriched to 4% with
L-[ring-
13
C
6
] phenylalanine on the
basis of a phenylalanine content of 3.5% in the whey protein.
Analytic methods
Blood glucose was measured by using a blood glucose meter
(OneTouch Ultra 2; Lifescan Inc). Blood amino acid concen-
trations were analyzed via HPLC as described previously (35).
Plasma
L-[ring-
13
C
6
] phenylalanine enrichment was determined
as previously described (36). Plasma insulin concentrations were
measured with the use of a commercially available immunoas-
say kit (ALPCO Diagnostics).
Muscle samples (w40–50 mg) were homogenized on ice in
buffer [10 mL/mg 25 mM Tris 0.5% vol:vol Triton X-100 and
protease/phosphatase-inhibitor cocktail tablets (Complete Pro-
tease Inhibitor Mini-Tabs, Roche; PhosSTOP, Roche Applied
Science)]. Samples were centrifuged at 15,000 3 g for 10 min at
48C. The supernatant fluid was removed, and protein concen-
trations were determined via a bicinchoninic acid protein assay
(Thermo Scientific). The pellet that contained myofibrillar pro-
teins was stored at 2808C for future processing. Working samples
of equal concentrations were prepared in Laemmli buffer
(37). Equal amounts (20 mg) of protein were loaded onto 10%
or gradient precast gels (BIO-RAD Mini-PROTEAN TGX
Gels; Bio-Rad Laboratories) for separation by electrophoresis.
Proteins were transferred to a polyvinylidene fluoride mem-
brane, blocked (5% skim milk), and incubated overnight at 48C
in primary antibody as follows: phospho-Akt
Ser473
(1:1000, no.
4058; Cell Signaling Technology), phospho-mTOR
Ser2448
(1:1000,
no. 2971; Cell Signaling Technology), phospho-70 kDa ribosomal
protein S6 kinase 1
Thr389
(1:1000, no. 9234; Cell Signaling
Technology), phospho-eukaryotic initiation factor 4E binding
protein 1 (4E-BP1)
Thr37/46
(1:1000, no. 2855; Cell Signaling T ech-
nology), phospho-eukaryotic elongation factor 2 (eEF2)
Thr56
(1:1000, no. 2331; Cell Signaling Technology), and phospho-S6
ribosomal protein (1:2000, no. 2215; Cell Signaling Technology).
Membranes were washed and incubated in secondary antibody
(1 h at room temperature) before detection with chemiluminescence
(SuperSignalWest Dura Extended Duration Substrate, no.
34075; ThermoScientific) on a FluorChem SP Imaging system
(Alpha Innotech). Phosphorylation status was expressed relative
to a-tubulin (1:2000, no. 2125; Cell Signaling Technology) and
is presented for each protein as the fold change from baseline
(fasted) conditions. Images were quantified by spot densitometry
with ImageJ software (version 1.45; NIH).
Muscle biopsy samples were processed as previously described
(38). To determine the intracellular
13
C
6
phenylalanine enrichment,
w20–25 mg muscle was homogenized in 0.6 M perchloric acid/L.
Free amino acids in the resulting supernatant fluid were passed
over an ion-exchange resin (Dowex 50WX8-200 resin; Sigma-
Aldrich Ltd) and converted to their heptafluorobutyric derivatives
for analysis via gas chromatography–mass spectrometry (models
6890 GC and 5973 MS; Hewlett-Packard) by monitoring ions
316 and 322 after electron ionization. To determine muscle free
intracellular amino acid concentrations, samples were processed
as previously described (35). Briefly , muscle samples were derivatized
and analyzed by using HPLC (HPLC: Waters model 2695; column:
Waters Nova-Pak C
18
,4mm; detector: Waters 474 scanning
fluorescence detector; Waters). To determine myofibrillar protein-
bound enrichments, a separate piece (w40–50 mg) of muscle
was homogenized in a standard buffer that contained protease
and phosphatase inhibitors as described previously. The super-
natant fluid was collected for Western blot analysis, and the pellet
was further processed to extract myofibrillar proteins as pre-
viously described (38). The resulting myobrillar enriched protein
pellet was hydrolyzed in 6 M HCL at 1108C overnight. Subse-
quently, the free amino acids were purified by using ion-exchange
chromatography and converted to their N-acetyl-n-propyl ester
deriv ati v es for analysis by using gas chromatography–combustion–
isotope ratio mass spectrometry (GC-C-IRMS: Hewlett-Packard
6890; IRMS model: Delta Plus XP; Thermo Finnagan).
Calculations
The FSR of myofibrillar protein was calculated by using the
standard precursor-product equation
FSR ¼½ðE
2b
2 E
1b
ÞOðE
IC
3 tÞ 3 100 ð1Þ
where E
b
is the enrichment of bound (myofibrillar) protein, E
IC
is the average enrichment of the intracellular free amino acid
precursor pool of 2 muscle biopsies, and t is the tracer incorpo-
ration time in hours. The use of tracer-naı
¨
ve subjects allowed us
to use a preinfusion blood sample (ie, a mixed plasma protein
fraction) as the baseline enrichment (E
1b
) for the calculation
of the baseline (fasted) FSR (31, 39, 40), which is an approach
that has been validated by our research group (33) and other
researchers (41).
Statistics
Strength tests and dietary run-in variables were compared by
using a 1-factor (treatment) ANOVA. Blood glucose and plasma
insulin were analyzed by using a 2-factor (treatment 3 time)
repeated-measures ANOVA. Data for the AUC above baseline
(AUC
pos
), maximum concentration (C
max
,), time of maximum
concentration (T
max
), and AUC below baseline (AUC
neg
) were
analyzed by using a 1-factor (treatment) ANOVA. Plasma en-
richments were analyzed by using a 2-factor (treatment 3 time)
repeated-measures ANOVA and linear regression. Intracellular
precursor pool enrichments were analyzed by using a 3-factor
(treatment 3 time 3 condition) mixed-model ANOVA and
linear regression. Intracellular amino acids (leucine, isoleucine,
valine, and the sum of EAAs), protein phosphorylation, and
myofibrillar FSR were analyzed by using a 3-factor (treatment
3 time 3 condition) mixed-model ANOVA. Protein phosphor-
ylation is expressed as the fold change from baseline (fasted).
Tukey’s post hoc analysis was performed whenever a significant
F ratio was shown to isolate specific differences. Statistical
analyses were performed with a software package (SPSS version
16; SPSS Inc). For data that did not pass normality, values were
transformed by using the square root, reciprocal, or ln of the
value. The statistical analysis was performed on transformed
data but means (6SEMs) of nontransformed data are presented
in graphic or tabular form for clarity. Means were considered to
be statistically significant for P values ,0.05. The study was
powered on the basis of previous work from our group (7) that
LEUCINE AND MYOFIBRILLAR PROTEIN SYNTHESIS 279
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showed that 20 g protein stimulates significantly greater MPS
rates than 5 g protein does during the initial 4 h postexercise
with no additional increase in MPS when 40 g protein was in-
gested. Therefore, W6+Low-Leu was chosen to show a signifi-
cantly greater average postexercise myofibrillar FSR than that of
W6. W6+Low-Leu was chosen over W6+BCAAs and W6
+High-Leu because this treatment was hypothesized to be less
likely to show efficacy. Therefore, the trial was designed to show
a relevant effect in the myofibrillar FSR of 0.033%/h. The SD
was assumed to be 0.022%/h on the basis of our previous ob-
servations (7). To show this effect as significant with a 2-sided
statistical test with an experiment-wise false-positive rate of 5%
and a power of 80%, a total of n = 8/group was needed.
RESULTS
Participant characteristics
Participant characteristics are shown in Table 1. Each treat-
ment group consisted of 8 randomly assigned participants, all of
whom received their intended treatment and whose results were
analyzed for primary and secondary outcomes.
Exercise variables
There were no significant differences between treatment
groups for 1-RM (Table 1) or the product of load (kg) 3 volume
(no. of repetitions) for exercise performed during the experiment
(data not shown; all P . 0.05).
Dietary run-in
Participants received w1.2gprotein$ kg body weight
21
$ d
21
during the standardized diet with no significant differences be-
tween treatment groups. There were no differences between
treatment groups for total energy, protein, carbohydrate, or fat
(data not shown) (all P . 0.05).
Blood glucose, plasma insulin, and blood amino acid
concentrations
Blood glucose concentration showed a rapid but transient
increase and was elevated above baseline at 20 and 40 min after
treatment administration (P-main effect for time , 0.001). In
addition, blood glucose concentrations were greater in the W6
group than in the W25 group (5.50 6 0.33 compared with 4.95 6
0.20 mmol/L, respectiv ely; P-main ef fect for treatment = 0.019).
Plasma insulin concentrations increased rapidly after treat-
ment administration showing a main effect for time (P , 0.001;
Figure 1). The area under the insulin curve (Figure 1, inset)
after treatment administration was not different between treat-
ment groups (P = 0.497).
Concentrations over time for blood leucine, isoleucine, valine,
and the sum of the EAAs are shown in Figure 2 (A–D, respectively).
No statistical analysis was performed on the concentration over
time data. The AUC
pos
,C
max
,T
max
,andAUC
neg
were analyzed for
blood leucine, isoleucine, valine, and SEAAs and are presented in
Table 3.BoththeAUC
pos
and C
max
for blood leucine were greatest
after the W6+High-Leu treatment and were significantly different
from that with W6+Lo w-Leu and W6, while the C
max
was also
dif ferent from W25. For both isoleucine and val ine, the AUC
neg
was
reduced after W6+BCAAs and W25 treatment s and were signifi-
cantly different from that after W6+High-Leu treatment. The T
max
for
leucine, isoleucine, val ine, and SEAAs tended to occur latest in the
W25 group and most rapidly for the W6+High-Leu group (T able 3).
Intracellular leucine, isoleucine, valine, and SEAAs
Intracellular concentrations of leucine, isoleucine, valine and
SEAAs are shown in Table 4. Intracellular leucine showed a time
3 treatment interaction (P = 0.031), whereby it increased at 1.5 h
posttreatment in all treatment groups except the W6 group but
returned to values not dif ferent from baseline by 4.5 h. Intracellul ar
isoleucine showed a time 3 condition interaction (P = 0.012) that
FIGURE 1. Mean (6SEM) plasma insulin concentrations (mmol/L) after treatment administration (n = 8/treatment group). Time-course data were analyzed
by using a 2-factor (treatment 3 time) repeated-measures ANOVA with Tukey’s post hoc test (P-main effect for time , 0.001; P-treatment 3 time interaction
= 0.26). Times with different lowercase letters were significantly different from each other. Inset: The AUC was analyzed by using a 1-factor (treatment)
ANOVA with Tukey’s post hoc test (P = 0.497). W6, 6.25 g whey protein; W6+BCAA, 6.25 g whey protein supplemented with leucine, isoleucine, and valine
to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey protein supplemented
with leucine to 3.0 g total leucine; W25, 25 g whey protein.
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increased abov e baseline at 1.5 h in the FED condition only . In-
tracellular valine showed a main effect for time (P = 0.006) that fell
below baseline fasted concentrations at 4.5 h. There were no time (P =
0.69), treatment (P = 0.66), or condition (P = 0.71) ef fects for SEAAs.
Plasma and intracellular free phenylalanine enrichments
Intracellular free phenylalanine enrichments were not different
across time (P = 0.337), between conditions (P = 0.128), or
between treatment groups (P = 0.746). In addition, there were no
FIGURE 2. Mean (6SEM) blood concentrations (mmol/L) of leucine (A), isoleucine (B), valine (C), and SEAAs (D) after treatment administration (n =8/
treatment group). No statistical analysis was performed on time-course data. EAA, essential amino acid; W6, 6.25 g whey protein; W6+BCAA, 6.25 g whey
protein supplemented with leucine, isoleucine, and valine to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total
leucine; W6+Low-Leu, 6.25 g whey protein supplemented with leucine to 3.0 g total leucine; W25, 25 g whey protein.
TABLE 3
Variables of blood leucine, isoleucine, valine, and SEAAs after treatment administration
1
W6 W6+Low-Leu W25 W6+BCAAs W6+High-Leu P
Leucine
AUC
pos
3223 6 1465 12,234 6 1629
z
19,252 6 3393
z
27,517 6 4493
z,
* 35,278 6 6016
z,
* ,0.001
C
max
(mmol/L) 145 6 18 295 6 40 309 6 51 459 6 75
z
554 6 74
z,
*
,#
,0.001
T
max
(min) 49 6 8436 5756 14 51 6 7416 9 0.084
AUC
neg
24654 6 1906
#
21510 6 903 2196 6 157 2991 6 469 2565 6 282 0.025
Isoleucine
AUC
pos
1145 6 764 432 6 142 6692 6 1766
z,
*
,+
4225 6 883
z,
*
,+
344 6 157 ,0.001
C
max
(mmol/L) 61 6 9556 10 131 6 29
z,
*
,+
122 6 22*
,+
55 6 8 ,0.001
T
max
(min) 57 6 9406 5706 8*
,+
45 6 6356 6 0.008
AUC
neg
22919 6 721 23503 6 581
#
2643 6 261 22073 6 445 25618 6 1039
y,#
,0.001
Valine
AUC
pos
2189 6 1741 489 6 165 7752 6 2516
z,
*
,+
5347 6 1373
z,
*
,+
665 6 337 ,0.001
C
max
(mmol/L) 178 6 21 150 6 14 229 6 44 246 6 40* 162 6 16 0.035
T
max
(min) 53 6 10 40 6 5746 10
+
58 6 10 33 6 5 0.015
AUC
neg
29690 6 2451 29457 6 1689 22174 6 486 25247 6 2163 213,988 6 2012
#,y
0.001
SEAAs
AUC
pos
11,739 6 6425 11,841 6 2310 62,722 6 18,780
z,
* 48,937 6 11,917
z,
* 31,047 6 7267 ,0.001
C
max
(mmol/L) 742 6 85 801 6 95 1175 6 188 1183 6 175
z
1088 6 90 0.008
T
max
(min) 53 6 10 40 6 5836 17 56 6 7396 9 0.054
AUC
neg
222,492 6 9420 218,734 6 4681 23028 6 1282* 26135 6 2122 216,198 6 5315 0.013
1
All values are means 6 SEMs. n = 8/treatment group. Blood concentrations (mmol/L) of leucine, isoleucine, valine, and SEAAs after treatment
administration are shown. The AUC
pos
,C
max
,T
max
, and AUC
neg
were each analyzed for blood leucine, isoleucine, valine, and SEAAs by using a 1-factor
(treatment) ANOVA with Tukey’s post hoc test.
z
Significantly different from W6; *significantly different from W6+Low-Leu;
#
significantly different from
W25;
y
significantly different from W6+BCAAs; + significantly different from W6+High-Leu. AUC
neg
, AUC below baseline; AUC
pos
, AUC above baseline;
C
max
, maximum concentration; EAA, essential amino acid; T
max
, time of maximum concentration; W6, 6.25 g whey protein; W6+BCAAs, 6.25 g whey
protein supplemented with leucine, isoleucine, and valine to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total
leucine; W6+Low-Leu, 6.25 g whey protein supplemented with leucine to 3.0 g total leucine; W25, 25 g whey protein.
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interaction effects for any of the factors (P-time 3 treatment=
0.941; P-condition 3 treatment = 0.992; P-time 3 condition =
0.117; P-time 3 condition 3 treatment = 0.975). The slope of
the intracellular free phenylalanine enrichment was not different
from zero for any of the treatment groups in either FED or EX-
FED conditions (see Online Supplemental Material Figure 2
under “Supplemental data” in the online issue).
Plasma free phenylalanine enrichments did not differ between
treatment groups (P = 0.917) or across time (P = 0.58). The
slope of the plasma free phenylalanine enrichment was not
different from zero for any treatment group (see Online Sup-
plemental Material Figure 3 under “Supplemental data” in the
online issue).
MPS
Myofibrillar FSR is shown in Figure 3 (A–D, respectively).
The myofibrillar FSR (%/h) during the early 0–1.5 h and late
1.5–4.5 h response is shown in Figure 3, A and B, whereas the
aggregate 0–4.5 h response under both FED and EX-FED con-
ditions is shown in Figure 3, C and D. Over the 0–1.5- and 1.5–
4.5-h periods, the myofibrillar FSR showed a treatment 3 time
interaction (P = 0.002), whereby the FSR was increased com-
pared with at baseline (fasted) in all treatment groups when
measured over 0–1.5 h. Over 1.5–4.5 h postexercise, the FSR
remained increased compared with at baseline in all treatment
groups; however, the FSR in W25 and W6+High-Leu groups
was greater than in W6+Low-Leu, W6+BCAAs, and W6
groups. Similarly, the aggregate myofibrillar FSR response over
0–4.5 h showed a treatment 3 time interaction (P = 0.005),
whereby the FSR was increased compared with at baseline in all
treatment groups during the 0–4.5-h postprandial period; how-
ever, the FSR was greater in W25 and W6+High-Leu than W6
+Low-Leu, W6+BCAAs, and W6 groups. There were no sig-
nificant differences between FED and EX-FED conditions when
examined over the 0–1.5- and 1.5–4.5-h periods (P = 0.483) or
over the aggregate 0–4.5-h period (P = 0.419).
Muscle anabolic signaling
Changes in the phosphorylation status of signaling proteins
involved in the regulation of messenger-RNA translation initi-
ation and elongation are shown in Table 5. Akt (phospho-
Akt
Ser473
) showed a treatment 3 time 3 condition interaction
(P = 0.025). Phospho-mTOR
Ser2448
showed a treatment 3 time
interaction (P = 0.041), whereby at 1.5 h, phospho-mTOR
Ser2448
was increased after W6+Low-Leu, W25, and W6+High-Leu
treatments. At 4.5 h, phospho-mTOR
Ser2448
remained increased
above baseline (fasted) only after W6+High-Leu. Phospho-
p70S6k
Thr389
showed no effect of time (P = 0.377), treatment
(P = 0.353), or condition (P = 0.062) at the times examined.
Phospho-4E-BP1
Thr37/46
showed a condition 3 time interaction
TABLE 4
Intracellular concentrations (mmol/L) of leucine, isoleucine, valine, and SEAAs after treatment administration
1
W6 W6+Low-Leu W25 W6+BCAAs W6+High-Leu P
Leucine
Baseline 214 6 11
a
209 6 15
a
212 6 16
a
229 6 17
a
222 6 18
a
Treatment 3 time = 0.031
1.5-h FED 215 6 18
a
248 6 16
b
274 6 32
b
313 6 24
z,b
344 6 16
z,
*
,#,b
4.5-h FED 210 6 8
a
221 6 14
a
248 6 8
z,a
232 6 12
a
263 6 27
z,
*
,a
1.5-h EX-FED 222 6 19
a
267 6 12
b
255 6 23
b
299 6 36
z,b
351 6 17
z,
*
,#,b
4.5-h EX-FED 188 6 14
a
205 6 20
a
224 6 17
z,a
225 6 15
a
249 6 31
z,
*
,a
Isoleucine
Baseline
a
216 6 27 254 6 33 322 6 41 296 6 38 290 6 37 Time 3 condition = 0.012
1.5-h FED
b
219 6 32 294 6 65 365 6 47 389 6 67 312 6 46
4.5-h FED
a
208 6 36 276 6 64 333 6 39 297 6 52 261 6 50
1.5-h EX-FED
a
218 6 44 299 6 64 340 6 38 321 6 44 284 6 56
4.5-h EX-FED
a
215 6 57 311 6 76 330 6 30 348 6 71 302 6 65
Valine
Baseline
a
232 6 23 280 6 26 259 6 24 261 6 16 288 6 25 Time = 0.006
1.5-h FED
a
252 6 27 224 6 9 280 6 29 262 6 15 308 6 22
4.5-h FED
b
222 6 16 216 6 14 295 6 6 240 6 17 195 6 16
1.5-h EX-FED
a
246 6 21 250 6 13 274 6 30 248 6 21 255 6 33
4.5-h EX-FED
b
208 6 27 212 6 16 264 6 26 246 6 25 232 6 33
SEAAs
Baseline 2966 6 185 2730 6 157 2718 6 217 3142 6 273 3016 6 229 Time = 0.69
1.5-h FED 3336 6 366 2837 6 201 3118 6 288 2946 6 134 3212 6 269 Treatment = 0.66
4.5-h FED 3301 6 256 2801 6 176 2925 6 136 2741 6 115 2818 6 350 Condition = 0.71
1.5-h EX-FED 3190 6 171 3110 6 251 2684 6 212 2732 6 277 2626 6 149
4.5-h EX-FED 3163 6 277 2657 6 273 3198 6 290 3066 6 181 3261 6 359
1
All values are means 6 SEMs. n = 8/treatment group. Intracellular concentrations (mmol/L) of leucine, isoleucine, valine, and SEAAs after treatment
administration. Data for leucine, isoleucine, valine, and SEAAs were each analyzed by using a 3-factor (treatment 3 time 3 condition) mixed-model ANOVA
with Tukey’s post hoc test. Times with different superscript letters within treatment columns were significantly different from each other within that treatment.
Times with different superscript letters within the time/condition column were significantly different from each other.
z
Significantly different from W6;
*significantly different from W6+Low-Leu;
#
significantly different from W25. EAA, essential amino acid; EX-FED, response to combined feeding and
resistance exercise; FED, response to feeding; W6, 6.25 g whey protein; W6+BCAAs, 6.25 g whey protein supplemented with leucine, isoleucine, and valine
to 5.0 g total leucine; W6+High-Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey protein supplemented
with leucine to 3.0 g total leucine; W25, 25 g whey protein.
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(P = 0.044), whereby both conditions (FED and EX-FED) were
increased above baseline (fasted) at 1.5 h. For phospho-ribosomal
protein S6
Ser240/244
, there was a condition 3 time interaction (P ,
0.001) whereby both conditions (FED and EX-FED) were in-
creased above baseline fasted at both 1.5 and 4.5 h; however, the
increase in the EX-FED condition at 1.5 h was greater than in
the FED condition. Phospho-eEF2
Thr56
showed no effect of time
(P = 0.197), treatment (P = 0.384), or condition (P = 0.091) at
the times examined. See Online Supplemental Material Figure 4
under “Supplemental data” in the online issue for representative
blot images.
DISCUSSION
Our results showed that the addition of a higher dose of leucine
to a smaller amount of protein (6.25 g) within a mixed macro-
nutrient beverage enhanced MPS to the same level as that seen
with 4 times as much whey protein (25 g). Muscle protein synthesis
is increased in response to exercise and protein feeding in healthy
individuals (42) and is the primary variable that determines di-
urnal changes in the net muscle-protein balance (43). It has been
shown that MPS is stimulated in a protein-EAA dose-dependent
manner up to w10 g EAAs at rest (6) and w20 g protein (w8.6
g EAAs) after resistance exercise (7). Whey protein was used as
the base protein source in this study because it is a high-quality
protein source that robustly stimulates postprandial MPS rates
(44). However, protein is typically co-ingested with carbohy-
drate and fat during meals, which may alter the kinetics of gut
amino acid absorption (45). Thus, in this trial, we tested the efficac y
of mixed macronutrient beverages with varying doses of whey
protein and amino acids to stimulate MPS.
Consistent with our previous results when we used protein
feeding alone (7), we showed that a low dose of protein (W6) was
suboptimal for the stimulation of maximal MPS rates compared
with 4 times as much whey protein (W25) even within a mixed
macronutrient beverage over the aggregate 0–4.5-h postprandial
period. The supplementation of this low-protein dose with a high
proportion of leucine (W6+High-Leu) stimulated MPS to an
equivalent magnitude and duration as that stimulated after in-
gestion of an energy-matched mixed macronutrient beverage
that contained W25. Previous work has shown that W25 is a
dose of protein and EAAs that is sufficient to induce a maximal
stimulation of MPS rates at rest (6) and after resistance exercise
(7). There were no differences in treatments in MPS during the
0–1.5-h postprandial period; however, W6+High-Leu and W25
stimulated greater MPS over the 1.5–4.5- and aggregate 0–4.5-h
periods than did each of the other treatments (Figure 3). The
somewhat surprising lack of difference in MPS rates in treat-
ments during the early postexercise and postfeeding period oc-
curred despite markedly divergent blood leucine, isoleucine,
valine, and EAA concentrations (Figure 2, Table 3). Presumably,
this lack of difference early after feeding (ie, #1.5 h) suggested
that amino acid availability or nutrient signals (leucine) that
served to trigger MPS were equivalent in all conditions. In contrast,
in the latter portion of the protocol, only with the W6+High-Leu
treatment was an MPS response shown that was equivalent to
that with the W25 treatment, despite containing only one-quarter
of the whey protein dose and w62% of the EAA content. That
FIGURE 3. Values are means 6 SEMs (n = 8/treatment group). Mean (6SEM) myofibrillar FSRs (%/h) calculated during baseline (fasted) conditions, over
both early (0–1.5 h) and late (1.5–4.5 h) time periods (A and B), and over the aggregate 0–4.5-h postexercise recovery period (C and D) in both FED and EX-
FED conditions after treatment administration. Data were analyzed by using a 3-factor (treatment 3 time 3 condition) mixed-model ANOVA with Tukey’s
post hoc test for analysis of the 0–1.5- and 1.5–4.5-h responses (P-treatment 3 time interaction = 0.002; P-treatment 3 time 3 condition = 0.799) and the
aggregate 0–4-h response (P-treatment 3 time interaction = 0.005; P-treatment 3 time 3 condition = 0.942). Times with different lowercase letters were
significantly different from each other within that treatment.
z
Significantly different from W6 within that time; *significantly different from W6+Low-Leu
within that time;
y
significantly different from W6+BCAA within that time. EX-FED, response to combined feeding and resistance exercise; FED, response to
feeding; FSR, fractional synthetic rate; W6, 6.25 g whey protein; W6+BCAA, 6.25 g whey protein supplemented with leucine, isoleucine, and valine to 5.0 g
total leucine; W6+High-Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey protein supplemented with
leucine to 3.0 g total leucine; W25, 25 g whey protein.
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the W6+High-Leu treatment was effective may relate to the fact
that this treatment elicited the greatest blood leucine AUC
pos
(Table 3), the greatest intracellular leucine concentration when
assessed at 1.5 h (Table 4), and led to a sustained increase in the
phosphorylation of mTOR
Ser2448
at 4.5 h (Table 5). We also
observed an increase in the phosphorylation of targets down-
stream of mTOR
Ser2448
including 4E-BP1
Thr37/46
and ribosomal
protein S6
Ser240/244
although there were no significant treatment
effects (Table 5). We powered our study to detect relevant dif-
ferences in the myofibrillar FSR (%/h), and thus, we may have
lacked the statistical power to detect important diff erences in in-
tracellular signaling molecule phosphorylation. In partial agree-
ment with our observation of the MPS response after the W6
+High-Leu treatment, previous studies have shown that a high
proportion of leucine (3.5 compared with 1.8 g) within a 10-g
EAA solution resulted in greater intramuscular cell signaling
and a more prolonged mixed MPS response (24).
Contrary to our hypothesis, the W6+BCAAs treatment resulted
in MPS rates that were less robust than with W6+High-Leu and
W25 treatments. These differences occurred despite the fact that
supplemental isoleucine and valine attenuated the decline in
concentrations of these amino acids in the blood compared with
that observed after the W6+High-Leu treatment (Table 3); how-
ever, intracellular concentrations of isoleucine and valine were
not different between these treatments (Table 4). In addition, the
W6+BCAAs treatment was associated with a lower intracellular
leucine concentration at 1.5 h, a lower mean leucine AUC
pos
,
alowerleucineC
max
, and a greater T
max
than with the W6+High-
Leu treatment (Table 3). We have shown that a rapid amino-
acidemia after protein feeding stimulates greater MPS rates after
resistance exercise than a slow protracted aminoacidemia (46).
Therefore, we speculate that the greater T
max
for leucine, iso-
leucine, valine and SEAAs after W6+BCAAs compared with
W6+High-Leu may partially explain the observed differences in
MPS rates. Because BCAAs share a common intestinal trans-
porter, differences in amino acid appearance profiles between
W6+BCAAs and W6+High-Leu treatments likely represents
antagonism between BCAAs for uptake from the gut, which is
TABLE 5
Western-blot analysis of protein synthesis–associated signaling proteins after treatment administration
1
W6 W6+Low-Leu W25 W6+BCAAs W6+High-Leu P
Phospho-Akt
Ser473
1.5-h FED 1.61 6 0.30 2.89 6 0.69
z,y,a
2.09 6 0.38
a
1.52 6 0.25 1.76 6 0.18 Treatment 3 time 3 condition = 0.025
4.5-h FED 0.70 6 0.17 1.67 6 0.40
z,y,+,$
1.01 6 0.24 0.71 6 0.16 0.74 6 0.09
1.5-h EX-FED 1.45 6 0.18 4.23 6 1.40
z,+,$,a
3.69 6 0.64
z,$,a
2.47 6 0.24 2.03 6 0.46
4.5-h EX-FED 1.38 6 0.41
$
1.07 6 0.10 1.50 6 0.30 1.20 6 0.16 1.17 6 0.21
Phospho-mTOR
Ser2448
1.5-h FED 1.05 6 0.15 1.59 6 0.49
a
1.52 6 0.29
a
1.35 6 0.15 1.89 6 0.31
z,y,a
Treatment 3 time = 0.041
4.5-h FED 0.62 6 0.15 0.86 6 0.19 1.25 6 0.22 0.89 6 0.15 1.63 6 0.26
z,y,a
1.5-h EX-FED 1.23 6 0.16 1.45 6 0.29
a
1.60 6 0.09
a
1.45 6 0.14 2.14 6 0.36
z,y,a
4.5-h EX-FED 0.87 6 0.11 1.41 6 0.39 1.10 6 0.23 1.11 6 0.14 1.71 6 0.25
z,y,a
Phospho-p70S6k1
Thr389
1.5-h FED 1.04 6 0.10 1.13 6 0.09 1.02 6 0.07 0.98 6 0.08 1.12 6 0.13 Time = 0.38
4.5-h FED 0.97 6 0.13 1.02 6 0.11 0.97 6 0.13 0.86 6 0.12 1.14 6 0.14 Treatment = 0.35
1.5-h EX-FED 1.15 6 0.11 1.13 6 0.09 1.06 6 0.06 0.92 6 0.10 1.07 6 0.11 Condition = 0.06
4.5-h EX-FED 1.01 6 0.12 1.06 6 0.08 1.02 6 0.10 0.93 6 0.09 1.33 6 0.09
Phospho-4E-BP1
Thr37/46
1.5-h FED 1.02 6 0.12 1.19 6 0.12 1.25 6 0.18 1.20 6 0.12 1.58 6 0.24 Condition 3 time = 0.044
4.5-h FED 0.73 6 0.07 0.95 6 0.15 0.86 6 0.17 1.04 6 0.19 0.85 6 0.14 (1.5 h . baseline)
1.5-h EX-FED 1.05 6 0.17 1.28 6 0.16 1.77 6 0.48 1.38 6 0.20 1.59 6 0.38 (1.5-h EX-FED . FED)
4.5-h EX-FED 1.02 6 0.18 1.05 6 0.18 1.20 6 0.29 1.12 6 0.10 1.45 6 0.22
Phospho-rpS6
Ser 240/244
1.5-h FED 1.72 6 0.41 2.46 6 0.93 1.93 6 0.32 2.66 6 0.65 2.45 6 0.56 Condition 3 time , 0.001
4.5-h FED 1.31 6 0.22 2.31 6 0.59 2.09 6 0.42 1.63 6 0.32 1.12 6 0.30 (All times . baseline)
1.5-h EX-FED 3.25 6 1.08 3.34 6 0.93 4.45 6 1.63 3.67 6 1.27 4.49 6 1.48 (1.5-h EX-FED . FED)
4.5-h EX-FED 1.47 6 0.42 2.86 6 1.26 1.38 6 0.15 2.41 6 0.65 2.44 6 0.59
Phospho-eEF2
Thr56
1.5-h FED 0.95 6 0.06 0.96 6 0.05 1.02 6 0.06 1.11 6 0.09 1.08 6 0.04 Time = 0.197
4.5-h FED 0.93 6 0.07 1.08 6 0.13 1.12 6 0.12 0.99 6 0.07 1.00 6 0.06 Treatment = 0.384
1.5-h EX-FED 0.92 6 0.03 1.15 6 0.08 1.12 6 0.10 1.09 6 0.05 1.13 6 0.04 Condition = 0.091
4.5-h EX-FED 0.88 6 0.06 1.11 6 0.10 1.03 6 0.07 1.07 6 0.11 1.05 6 0.04
1
All values are fold difference from baseline and are means 6 SEMs. n = 8/treatment group. The phosphorylation status of Akt
Ser473
,mTOR
Ser2448
,
p70S6k
Thr389
, 4E-BP1
Thr37/46
, rpS6
Ser240/244
, and eEF2
Thr56
is shown and expressed as the fold difference from baseline (fasted), at 1.5 h and 4.5 h
postexercise recovery in both FED and EX-FED conditions after treatment administration. Data for Akt
Ser473
,mTOR
Ser2448
, p70S6k
Thr389
, 4E-BP1
Thr37/46
,
rpS6
Ser240/244
, and eEF2
Thr56
were analyzed by using a 3-factor (treatment 3 time 3 condition) mixed-model ANOVA with Tukey’s post hoc test.
a
Difference
from baseline;
z
significantly different from W6;
y
significantly different from W6+BCAAs;
+
significantly different from W6+High-Leu;
$
significantly different
from the opposite condition at the same time point. Akt, protein kinase B; eEF2, eukaryotic elongation factor 2; EX-FED, response to combined feeding and
resistance exercise; FED, response to feeding; mTOR, mechanistic target of rapamycin; p70S6k, 70 kDa ribosomal protein S6 kinase 1; rpS6, ribosomal
protein S6; W6, 6.25 g whey protein; W6+BCAAs, 6.25 g whey protein supplemented with leucine, isoleucine, and valine to 5.0 g total leucine; W6+High-
Leu, 6.25 g whey protein supplemented with leucine to 5.0 g total leucine; W6+Low-Leu, 6.25 g whey protein supplemented with leucine to 3.0 g total
leucine; W25, 25 g whey protein; 4E-BP1, eukaryotic initiation factor 4E binding protein 1.
284 CHURCHWARD-VENNE ET AL
at MCMASTER UNIVERSITY on January 21, 2014ajcn.nutrition.orgDownloaded from
congruent with data showing that isoleucine and valine compete
with and can impede leucine absorption (47). The same effect
could be true for the transsarcolemmal BCAA transport because
BCAAs share the same transporter at that site (48).
We have previously shown that a suboptimal protein dose (6.25
g whey) supplemented with leucine (total leucine: 3.0 g) or
a complete mixture of EAAs devoid of leucine (total leucine:
0.75 g) can stimulate postprandial MPS rates equivalent to that
stimulated after the ingestion of 25 g whey protein (total leucine:
3.0 g) under resting but not postresistance exercise conditions
(14). Similarly, in this study, W6+Low-Leu (total leucine: 3.0 g)
and W6 (total leucine: 0.75 g) were as effective as W25 (total
leucine: 3.0 g) at stimulating MPS rates when assessed during the
early 0–1.5 h but not the later 1.5–4.5 h period. We showed no
difference between FED compared with EX-FED rates of MPS,
likely because of our choice of tissue sampling times. Our current
results extend those of our previous work (14) by showing that,
within the context of a mixed macronutrient beverage, a sub-
optimal protein dose (6.25 g) supplemented with a higher pro-
portion of leucine (5.0 g total) was as effective at stimulating
increased MPS rates as a dose of protein (25 g) able to induce
a maximal stimulation of MPS rates after resistance exercise (7)
and a dose of EAAs that maximally stimulates MPS at rest (6).
A novel aspect of our current study was that protein and amino
acids were co-ingested with carbohydrate and fat. In our previous
work (14), in which protein and free amino acids were ingested in
isolation, the supplementation of 6.25 g whey to contain 3.0 g
leucine induced peak blood amino acid concentrations w550.0
mM, whereas in the current study, the same protein dose sup-
plemented up to 5.0 g leucine was necessary to achieve similar
peak blood leucine concentrations when co-ingested with car-
bohydrate and fat as part of a mixed macronutrient beverage.
Thus, as has been reported previously (45, 49), the co-ingestion
of protein with additional macronutrients attenuated the post-
prandial rise in blood amino acid concentrations.
Although several studies have assessed the effects of protein-
carbohydrate co-ingestion on MPS rates (49, 50), few studies
have examined the MPS response after a physiologic (ie single
bolus) co-ingestion of protein, carbohydrate, and fat (51). Al-
though the addition of carbohydrate to protein does not further
stimulate increased MPS rates when adequate protein is provided
(49, 50), it is not clear whether insulin can enhance MPS rates
after the intake of a suboptimal protein dose in young individuals.
In the current study, we observed robust increases in MPS rates in
the W6 treatment that consisted of only 6.25 g whey protein but
was co-ingested with 35.0 g carbohydrate. Whether the MPS
response to W6 was enhanced by the addition of carbohydrate or
whether only 0.75 g leucine serves as a sufficient nutrient signal
to stimulate early increases in MPS rates in young, healthy in-
dividuals (14) requires additional investigation.
In conclusion, our results show that, when a suboptimal dose of
protein (6.25 g) is supplemented with a relatively high dose of
leucine (W6+High-Leu), rates of MPS are equivalent in both
magnitude and duration to those observed after ingestion of an
energy-matched beverage containing a saturating for MPS, 25-g
dose of protein (W25) (7). These findings show that, within the
context of mixed macronutrient intake, suboptimal protein doses
can be made more effective in stimulating MPS through the
addition of a high proportion of free leucine. This effect may be
of importance in the development of nutritional formulations
designed to promote skeletal muscle anabolism, which may be of
particular significance to individuals in whom total protein intake
is restricted or inadequate.
We thank Tracy Rerecich and Todd Prior for their technical assistance and
study participants for their time and effort
The authors’ responsibilities were as follows—SMP, TS, DRM, DB, and
EAO: designed the study; TAC-V, LB, DMDD, CJM, TS, SKB, and SMP:
conducted the research; TAC-V, AJH, and SMP: analyzed data; TAC-V and
SMP: wrote the manuscript and had primary responsibility for the final
content of the manuscript; and all authors: assisted in editing and providing
meaningful input into the manuscript and read and approved the final man-
uscript. DRM and TS were, and DB and EAO are, employees of Nestec SA,
which is a subsidiary of Nestle
´
Ltd and provides professional assistance,
research, and consulting services for food, dietary, dietetic, and pharmaceu-
tical products of interest to Nestle
´
Ltd. DRM, TS, DB, and EAO declared no
financial conflicts of interest. TAC-V, LB, DMDD, AJH, CJM, SKB, and
SMP had no conflicts of interest.
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... The BCAAs are Leucine, Isoleucine, and Valine, and benefits related to energy metabolism in the muscle are attributed to them [17]. Specifically, leucine has been shown to promote muscle protein synthesis, decrease central fatigue and improve performance [18][19][20][21][22]. ...
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... [35,36] Lysine and Threonine are deficient in grains, while methionine, tryptophan, and cystine are deficient in pulses/legumes/beans, and lysine is deficient in nuts and seeds. [37] Tryptophan is also deficient in maize. As a result, people who take a VD must eat a wide array of protein sources to meet their diverse requirements for amino acids. ...
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