J Appl Physiol 106: 1730–1739, 2009.
First published June 5, 2008; doi:10.1152/japplphysiol.90395.2008.
HIGHLIGHTED TOPIC Regulation of Protein Metabolism in Exercise and Recovery
Essential amino acid and carbohydrate ingestion before resistance exercise
does not enhance postexercise muscle protein synthesis
Satoshi Fujita,1 Hans C. Dreyer,2,3 Micah J. Drummond,2,3 Erin L. Glynn,3 Elena Volpi,1
and Blake B. Rasmussen2,3
Departments of 1Internal Medicine and 2Physical Therapy, and 3Division of Rehabilitation Sciences, University of Texas
Medical Branch, Galveston, Texas
Submitted 12 March 2008; accepted in final form 2 June 2008
Fujita S, Dreyer HC, Drummond MJ, Glynn EL, Volpi E,
Rasmussen BB. Essential amino acid and carbohydrate ingestion
before resistance exercise does not enhance postexercise muscle
protein synthesis. J Appl Physiol 106: 1730 –1739, 2009. First pub-
lished June 5, 2008; doi:10.1152/japplphysiol.90395.2008.—Inges-
tion of an essential amino acid-carbohydrate (EAA ⫹ CHO) solution
following resistance exercise enhances muscle protein synthesis dur-
ing postexercise recovery. It is unclear whether EAA ⫹ CHO inges-
tion before resistance exercise can improve direct measures of pos-
texercise muscle protein synthesis (fractional synthetic rate; FSR). We
hypothesized that EAA ⫹ CHO ingestion before a bout of resistance
exercise would prevent the exercise-induced decrease in muscle FSR
and would result in an enhanced rate of muscle FSR during postex-
ercise recovery. We studied 22 young healthy subjects before, during,
and for 2 h following a bout of high-intensity leg resistance exercise.
The fasting control group (n ⫽ 11) did not ingest nutrients, and the
EAA ⫹ CHO group (n ⫽ 11) ingested a solution of EAA ⫹ CHO 1 h
before beginning the exercise bout. Stable isotopic methods were used
in combination with muscle biopsies to determine FSR. Immunoblot-
ting procedures were utilized to assess cell signaling proteins associ-
ated with the regulation of FSR. We found that muscle FSR increased
in the EAA ⫹ CHO group immediately following EAA ⫹ CHO
ingestion (P ⬍ 0.05), returned to basal values during exercise, and
remained unchanged at 1 h postexercise. Muscle FSR decreased in the
fasting group during exercise and increased at 1 h postexercise (P ⬍
0.05). However, the 2 h postexercise FSR increased by ⬃50% in both
groups with no differences between groups (P ⬎ 0.05). Eukaryotic
elongation factor 2 phosphorylation was reduced in both groups at 2 h
postexercise (EAA ⫹ CHO: 39 ⫾ 7%; fasting: 47 ⫾ 9%; P ⬍ 0.05).
We conclude that EAA ⫹ CHO ingestion before resistance exercise
does not enhance postexercise FSR compared with exercise without
nutrients.
essential amino acids; resistance exercise; human muscle protein
synthesis; protein metabolism; leucine
AN ACUTE BOUT of resistance exercise stimulates muscle protein
synthesis within 1– 4 h postexercise (3, 8, 9) that can remain
elevated for 24 – 48 h (8, 20, 22). Short-term resistance exercise
training of 1–12 wk also increases basal rates of muscle protein
synthesis (15, 17, 34, 35, 38).
Anabolic nutrients, primarily leucine and other essential
amino acids (EAAs), also stimulate muscle protein synthesis in
humans, whether given intravenously or ingested (24, 26, 27,
Address for reprint requests and other correspondence: B. B. Rasmussen,
Univ. of Texas Medical Branch, Dept. of Physical Therapy, Div. of Rehabil-
itation Sciences, Sealy Center on Aging, 301 Univ. Blvd., Galveston, TX
77555-1144 (e-mail: blrasmus@utmb.edu).
1730 8750-7587/09 $8.00 Copyright © 2009 the American Physiological Society
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33). The addition of carbohydrate (CHO) to an EAA solution
augments insulin secretion in humans (23, 32). Insulin stimu-
lation of human muscle protein synthesis is dependent on the
availability of amino acids (2, 12, 25) and is most likely
permissive in nature since the increase in muscle protein
synthesis is less than that seen with amino acid ingestion. We
have recently shown, in human subjects, that a leucine-en-
riched EAA and CHO ingestion potently and rapidly increases
muscle protein synthesis in association with a substantial
activation of the mammalian target of rapamycin (mTOR)
signaling pathway (13).
The provision of EAAs with or without CHO following
resistance exercise increases the rate of muscle protein synthe-
sis (4, 5, 10, 21, 23, 28). In addition, recent work has also
shown that the ingestion of whole proteins such as whey or
casein following resistance exercise augments muscle protein
synthesis following an acute bout of resistance exercise (11,
18, 19, 30, 31, 36) or when ingested chronically following 3
mo of resistance exercise training (14). On the other hand, it is
unclear whether the provision of anabolic nutrients before
resistance exercise can enhance postexercise muscle protein
synthesis. For example, it has been reported that the ingestion
of EAAs and CHO immediately before an acute bout of
resistance exercise produced a larger increase in indirect mea-
sures of muscle protein synthesis (i.e., leg net amino acid
balance and rate of disappearance) compared with when the
nutrients were ingested immediately postexercise (29). How-
ever, when 20 g of whey protein (rather than purified amino
acids) was ingested, there was no difference in leg amino acid
uptake between ingesting the protein immediately before ex-
ercise and ingestion 1 h postexercise (31). We have recently
shown that human muscle protein synthesis is decreased during
an acute bout of resistance exercise; however, during postex-
ercise recovery the rate of muscle protein synthesis is increased
in association with the activation of mTOR signaling (9). It is
not known whether EAA and CHO ingestion before resistance
exercise would overcome the reduction in direct measures of
muscle protein synthesis (i.e., mixed muscle fractional protein
synthetic rate; FSR) by activating signaling proteins linked to
the regulation of translation initiation and elongation such as
Akt/protein kinase B (PKB), tuberous sclerosis complex 2
(TSC2), mTOR, ribosomal S6 kinase 1 (S6K1), 4E-binding
protein 1 (4E-BP1), and eukaryotic elongation factor 2 (eEF2).
Exercising in the fed state may also prevent the resistance
exercise-induced increase in AMP-activated protein kinase
(AMPK␣2), a negative regulator of mTOR signaling. Although
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 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
signaling and muscle protein synthesis alterations during early
postexercise recovery may not be optimal markers of measur-
able muscle growth during training, it does provide necessary
data for determining how acute resistance exercise and/or
nutrient ingestion can activate cell signaling pathways that
promote muscle protein synthesis.
Therefore, we hypothesized that a leucine-enriched EAA
acid and CHO (EAA ⫹ CHO) ingestion before a bout of
high-intensity resistance exercise would prevent the exercise-
induced decrease in muscle protein synthesis and would stim-
ulate mTOR signaling and enhance muscle protein synthesis
during the early postexercise recovery period.
SUBJECTS AND METHODS
Subjects. We studied two groups of 22 young healthy subjects. In
the first group we recruited 11 young subjects (6 male and 5 female)
who consumed a solution of EAA ⫹ CHO 1 h before performing a
bout of heavy leg resistance exercise (EAA ⫹ CHO group). A second
group of 11 additional subjects (7 men and 4 women) performed the
exact same exercise bout but without consuming nutrients (fasting
control group). Subjects for both groups were recruited into the study
concurrently. Data from male and female subjects in each group were
combined because we did not detect significant sex differences in our
variables of interest. The postexercise signaling and muscle protein
synthesis data from the fasting control group have been previously
published and are not included in RESULTS (9). We have included
previously published fasting data in all tables and figures to provide
clear comparisons between groups (9). All subjects were healthy and
physically active but were not currently engaged in a resistance or
endurance exercise training program. All subjects gave informed
written consent before participating in the study, which was approved
by the Institutional Review Board of the University of Texas Medical
Branch. Screenings of subjects were performed with clinical history,
physical exam, and laboratory tests, including complete blood count
with differential, liver and kidney function tests, coagulation profile,
fasting blood glucose and oral glucose tolerance test (OGTT), hepa-
titis B and C screening, HIV test, pregnancy test, thyroid-stimulating
hormone (TSH), lipid profile, urinalysis, drug screening, and ECG.
All women were not taking oral contraceptives and were studied
during their follicular phase. There were no differences in subject
characteristics between groups (P ⬎ 0.05), and subject characteristics
are summarized in Table 1.
Study design. On two separate occasions (⬎5 days apart) and more
than 5 days before conducting the study, each subject was tested for
muscle strength by measuring their 1-repetition maximum (1RM) on
a leg extension machine (Cybex-VR2, Medway, MA) located in the
General Clinical Research Center (GCRC) Exercise Laboratory. The
higher of the two 1RM values obtained was used to determine the starting
weight (70% of 1RM) for the resistance exercise portion of our study.
Table 1. Subject characteristics
Fasting EAA ⫹ CHO
n 11 (M ⫽ 7, F ⫽ 4) 11 (M ⫽ 6, F ⫽ 5)
Age, yr 27⫾2 25⫾1
Height, m 1.68⫾0.03 1.70⫾0.01
Weight, kg 71.3⫾4.5 69.4⫾2
Body mass index, kg/m2 25.3⫾1.3 23.9⫾0.8
Lean body mass, kg 52.5⫾3.4 51.0⫾1.9
Body fat, % 23.4⫾2.4 23.3⫾2.4
Leg lean mass, kg 8.8⫾0.6 8.4⫾0.4
Values are mean ⫾ SE. Fasting data are from Ref. 9. EAA ⫹ CHO, essential
amino acids plus carbohydrates; M, men; F, women. There were no significant
differences between groups.
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1731
On the second visit a dual-energy X-ray absorptiometry (DXA) scan
(Hologic QDR 4500W, Bedford, MA) was also performed to measure
body composition and lean mass, and a pregnancy test was repeated
in the female subjects.
Each subject was admitted to the GCRC of the University of Texas
Medical Branch the day before the exercise study. The subjects were
then fed a standard dinner, and a snack was given at 2200. The
subjects were studied following an overnight fast under basal condi-
tions and refrained from exercise for 24 h before study participation.
The morning of the study, polyethylene catheters were inserted into a
forearm vein for tracer infusion, in the contralateral hand vein, which
was heated for arterialized blood sampling, and in the femoral artery
and vein (retrograde placement) of the leg for blood sampling. The
femoral lines were placed in the same leg from which muscle biopsies
were obtained. The arterial catheter was also used for the infusion of
indocyanine green (ICG, Akorn, Buffalo Grove, IL) to determine
blood flow.
After drawing a background blood sample, a primed continuous
infusion of L-[ring-2H5]phenylalanine (Cambridge Isotope Laborato-
ries, Andover, MA) was begun (time ⫽ 0 h at 0800) and maintained
at a constant rate until the end of the experiment (Fig. 1). The priming
dose for the labeled phenylalanine was 2 ␮mol/kg, and the infusion
rate was 0.05 ␮mol 䡠 kg⫺1 䡠 min⫺1. All subjects were studied at the
exact same time (i.e., between 0800 and 1400).
Subjects were studied during four time periods: preexercise (Pre-
Ex), exercise (Ex), first hour postexercise (1 h Post-Ex), and second
hour postexercise (2 h Post-Ex). During the Pre-Ex period, subjects
assigned to the EAA ⫹ CHO group ingested a nutrient solution,
whereas subjects in the fasting group did not receive nutrients al-
though water was provided ad libitum. Marking the beginning of the
Pre-Ex period, and 2 h following the initiation of the tracer infusions,
the first muscle biopsy was obtained from the lateral portion of the
vastus lateralis of the leg with the biopsy site between 15 and 25 cm
from the midpatella. The biopsy was performed using a 5-mm Berg-
ström biopsy needle, under sterile procedure and local anesthesia (1%
lidocaine). Once harvested the muscle tissue was immediately blotted
and frozen in liquid nitrogen (within seconds) and stored at ⫺80°C
until analysis. Immediately after the first biopsy, the EAA ⫹ CHO
group ingested the nutrient solution, and a continuous infusion of ICG
was started in the femoral artery (0.5 mg/min) and maintained for 50
min for both groups. Ten minutes after ICG infusion was started,
blood samples were drawn four times, at 10-min intervals, from the
femoral vein and the arterialized hand vein to measure ICG concen-
tration (Fig. 1). In addition to the blood obtained for ICG measure-
ment, blood samples were taken from the femoral artery and vein and
from the arterialized hand vein to measure blood glucose and amino
acid concentrations. At the end of baseline, a second biopsy was
obtained; however, the biopsy needle was inclined at a different angle
so that the second biopsy was taken ⬃2 in. (⬃5 cm) from the first.
Following the second biopsy, each subject was transported to the
exercise lab within the GCRC. The subjects performed 10 sets of 10
repetitions of leg extension exercises on a Cybex leg extension
machine (Cybex International, Medway, MA) set to 70% of their
1RM. All subjects started out at 70% of 1RM, but for a few subjects
the weight was slightly reduced (60 – 65% of 1 RM) to achieve 10
repetitions per set. The rest period between sets was 3 min, except
during blood collection, which required a few more minutes. As
during the basal period, ICG was continually infused during exercise
to accurately measure leg blood flow. Blood samples were again
drawn for blood pH, glucose, lactate, and amino acid concentrations
immediately after the 3rd, 6th, 8th, and 10th sets. Following the last
blood collection, subjects performed one final set of 10 repetitions,
and a third muscle biopsy was obtained within seconds of completing
the last muscle contraction. The subjects were then transported back
to the special procedures room of the GCRC for the duration of the
study.
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1732 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
Fig. 1. Study design. Blood and muscle sampling are indi-
cated by arrows. A detailed description of the study design is
provided in the text. Study design was identical for the
fasting control group and the leucine-enriched essential
amino acid and carbohydrate (EAA ⫹ CHO) group except
for the ingestion of nutrients (⫹/⫺).
During the 1 h Post-Ex period, ICG was again infused continuously
(as during the Pre-Ex and Ex periods) to measure leg blood flow, and
blood was drawn for the measurement of blood gas, electrolytes, and
glucose and lactate concentrations. Samples were obtained every 10
min (as during the Pre-Ex and Ex periods). At the end of the first hour
postexercise, a fourth muscle biopsy was obtained through a new
incision site ⬃5 cm from the first incision.
During the 2 h Post-Ex period (2nd hour postexercise), blood
samples were collected in the same manner as during the previous
periods. At the end of the second hour postexercise, a final muscle
biopsy was collected as described above from the second incision;
however, the biopsy needle was inclined at a different angle again so
that the biopsy was taken ⬃5 cm apart from the prior biopsy.
Composition of the EAA ⫹ CHO solution. The nutrient solution
contained leucine-enriched EAAs and CHO (sucrose) as described in
our previous study (13). The solution contained EAAs in the follow-
ing proportions: histidine (8%), isoleucine (8%), leucine (35%), lysine
(12%), methionine (3%), phenylalanine (14%), threonine (8%), and
valine (10%). To minimize the potential tracer dilution with the
addition of the amino acids, phenylalanine tracer was added to the
solution at 6.5% of phenylalanine content. Lean mass as determined
by DXA was used to calculate the proportion of each EAA [0.35 g/kg
of fat-free mass (FFM)] added to the nutrient solution. Similarly,
CHO (sucrose) was added at 0.5 g/kg FFM to each nutrient solution.
All ingredients (EAA and CHO) were dissolved in a noncaloric,
caffeine-free, flavored beverage to increase palatability. The rationale
for using this amount of nutrients was to provide a maximal stimulus
for muscle protein synthesis via large increases in amino acid avail-
ability in combination with an increase in circulating insulin concen-
trations.
Blood flow, insulin, and glucose uptake. Serum ICG concentration
for the determination of leg blood flow was measured spectrophoto-
metrically (Beckman Coulter, Fullerton, CA) at wavelength ␭ ⫽ 805
nm (36). Plasma glucose concentration was measured using an auto-
mated glucose and lactate analyzer (YSI, Yellow Springs, OH).
Plasma insulin concentrations were determined by ELISA (Linco
Research, St. Charles, MO). Leg glucose utilization was calculated as
net glucose uptake across the leg:
leg glucose uptake ⫽ 共CA ⫺ CV兲 䡠 BF
where CA and CV are the blood glucose concentrations in the femoral
artery and vein, respectively, and BF is leg blood flow. Data are
expressed as micromoles of glucose per minute per kilogram of FFM
of the leg (␮mol 䡠 min⫺1 䡠 kg leg FFM⫺1).
Phenylalanine net balance. Phenylalanine net balance across the
leg was calculated as:
net balance ⫽ 共CA ⫺ CV兲 䡠 BF
where CA and CV are the blood phenylalanine concentrations in the
femoral artery and vein, respectively, and BF is leg blood flow. Data
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are expressed as hourly averages for each period (nmol 䡠 min⫺1 䡠 100
ml leg volume⫺1).
Muscle FSR. Muscle tissue samples were ground, and intracellular
free amino acids and muscle proteins were extracted as previously
described (37). Blood and muscle intracellular free concentration of
phenylalanine and leucine and the intracellular free enrichment of
phenylalanine were determined by gas chromatography-mass spec-
trometry (GCMS, 6890 Plus GC, 5973N MSD, 7683 autosampler,
Agilent Technologies, Palo Alto, CA) using appropriate internal
standards (37). Mixed muscle protein-bound phenylalanine enrich-
ment was analyzed by GCMS after protein hydrolysis and amino acid
extraction (37), using the external standard curve approach (6). We
calculated the fractional synthetic rate of mixed muscle proteins
(FSR) by measuring the incorporation rate of the phenylalanine tracer
into the proteins (⌬Ep/t) and using the precursor-product model to
calculate the synthesis rate:
FSR ⫽ 共⌬Ep /t兲/兵关EM共1兲 ⫹ EM共2兲兴/2其 䡠 60 䡠 100
where ⌬Ep is the increment in protein-bound phenylalanine enrich-
ment between two sequential biopsies, t is the time between the two
sequential biopsies, and EM(1) ⫹ EM(2) are the phenylalanine enrich-
ments in the free intracellular pool in the two sequential biopsies. Data
are expressed as percent per hour.
Immunoblotting and enzyme activity. We have published the pro-
cedures for SDS-PAGE and immunoblotting in detail elsewhere (9).
In short, the amount of total protein loaded per lane was identical,
done in duplicate and separated by SDS-PAGE. For mTOR, Akt/PKB,
TSC2, S6K1, and eEF2 separation, 7.5% gels were used, whereas for
4E-BP1, separation was accomplished with 15% gels. Following
SDS-PAGE, proteins were transferred to polyvinylidene diflouride
membranes (PVDF) (Hybond-P, Amersham Biosciences, Piscataway,
NJ) at 50 V for 1 h. Once transferred, PVDF membranes were placed
in blocking buffer [5% nonfat dry milk (NFDM) in TBST (Tris-
buffered saline and 0.1% Tween-20)] for 1 h. Blots were then serially
washed two times in deionized water and two more times in TBST
and incubated with primary antibody in 5% NFDM in TBST (except
4E-BP1 and TSC2, which were incubated in 5% BSA) overnight at
4°C with constant agitation. The next morning, the blots were washed
in TBST twice and incubated with secondary antibody for 1 h in 5%
NFDM in TBST at room temperature with constant agitation. After
secondary incubation, the blots were washed for 15 min and then
serially washed (3 ⫻ 5 min) with TBST. Blots were then incubated for
5 min with enhanced chemiluminescence reagent (ECL plus Western
Blotting Detection System, Amersham Biosciences, Piscataway, NJ)
to detect horseradish peroxidase activity. Optical density measure-
ments were obtained with a CCD camera mounted in a ChemiDoc
XRS imaging system (Bio-Rad, Hercules, CA). Once the appropriate
image was captured, densitometric analysis was performed using
Quantity One 1-D analysis software (version 4.5.2) (Bio-Rad). We
found that protein abundance did not change over the short time frame
of the study; therefore, all data were expressed as the change in
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 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE 1733
Table 2. Leg blood flow, glucose uptake, and blood insulin
Pre-Ex Ex 1 h Post-Ex 2 h Post-Ex

Leg blood flow, ml 䡠 100 ml leg⫺1 䡠 min⫺1

EAA⫹CHO 5.2⫾0.7 13.4⫾2.0* 5.8⫾0.8 5.8⫾0.7
Fasting 3.5⫾0.4 13.1⫾1.5* 5.0⫾0.6 3.9⫾0.5
Insulin, ␮U/ml
EAA⫹CHO 20.5⫾2.8† 12.3⫾2.8* 7.4⫾2.0 4.6⫾1.0
Fasting 4.9⫾0.8 7.6⫾0.8 4.6⫾0.6 3.6⫾0.6
Glucose uptake, ␮mol 䡠 min⫺1 䡠 100 g LM⫺1
EAA⫹CHO 16.4⫾4.4† 31.7⫾7.2* 16.3⫾2.5† 13.6⫾2.4
Fasting 3.6⫾0.8 25.8⫾3.4* 7.3⫾1.9 8.9⫾2.0
Values are mean ⫾ SE. Measurements are hourly averages for each time period as shown in Fig. 1: the hour before beginning exercise (Pre-Ex), during exercise
(Ex), and during postexercise recovery (1 h Post-Ex and 2 h Post-Ex). LM, lean mass. *P ⬍ 0.05 vs. Pre-Ex. †P ⬍ 0.05 vs. fasting at respective time period.

phosphorylation in arbitrary units as we have previously described (9,
10, 13).
AMPK␣2 activity was measured as previously described (9). Ac-
tivity is expressed in picomoles of phosphate incorporated per milli-
gram of muscle protein subjected to immunoprecipitation per minute
(pmol 䡠 min⫺1 䡠 mg of protein⫺1).
Antibodies. The primary antibodies used were all purchased from
Cell Signaling (Beverly, MA): phospho-mTOR (Ser2448; 1:1,000);
phospho-p70 S6K1 (Thr389; 1:500); phospho-Akt/PKB (Ser473;
1:500); phospho-tuberin/TSC2 (Thr1462; 1:500); phospho-4E-BP1
(Thr37/46; 1:500); and phospho-eEF2 (Thr56; 1:1,000). Anti-rabbit
IgG horseradish peroxidase-conjugated secondary antibody was pur-
chased from Amersham Bioscience (1:2,000).
Statistical analysis. All values are expressed as means ⫾ SE.
Primary outcomes were muscle protein synthesis, phenylalanine net
balance, and blood metabolite and amino acid concentrations. Sec-
ondary outcomes include intracellular signaling proteins and enzyme
activity. Comparisons between EAA ⫹ CHO and fasting were per-
formed using ANOVA with repeated measures, the effects being
group and time (Pre-Ex, Ex, 1 h Post-Ex, and 2 h Post-Ex), using
JMP statistical software version 4.0.5 (SAS Institute). Post hoc
testing was performed using t-test as appropriate. Significance was
set at P ⬍ 0.05.
RESULTS
Blood flow, insulin, and glucose uptake. Leg blood flow,
insulin, and glucose uptake results are shown in Table 2. Leg
blood flow was significantly higher after nutritional intake in
the EAA ⫹ CHO group compared with the fasting group in the
Pre-Ex period (baseline; P ⬍ 0.05). Leg blood flow increased
significantly during exercise in both groups compared with the
basal value (P ⬍ 0.05) and returned to preexercise baseline
values at 1 h and 2 h postexercise (P ⬎ 0.05). Insulin concen-
tration significantly increased following EAA ⫹ CHO inges-
tion during the Pre-Ex period (P ⬍ 0.05) and remained higher
than basal values during the exercise period (P ⬍ 0.05). Insulin
concentration returned to basal values during recovery in the
EAA ⫹ CHO group, which was not different from the fasting
group (P ⬎ 0.05). Glucose uptake across the leg was signifi-
cantly higher after nutritional intake in the EAA ⫹ CHO group
compared with fasting group in the Pre-Ex period (P ⬍ 0.05).
Glucose uptake remained higher during exercise and 1 h
postexercise in the EAA ⫹ CHO group compared with the
fasting group preexercise baseline values (P ⬍ 0.05). In the
fasting group, leg glucose uptake increased only during the exer-
cise period (P ⬍ 0.05).
Muscle intracellular leucine and phenylalanine concentra-
tions. Muscle intracellular leucine and phenylalanine concen-
trations are shown in Table 3. There was no difference in
muscle leucine or phenylalanine concentration between groups
at baseline. Muscle leucine concentration increased signifi-
cantly in response to nutritional intake and remained signifi-
cantly higher immediately following exercise in the EAA ⫹
CHO group compared with the fasting group (P ⬍ 0.05).
Muscle leucine concentrations decreased slightly during pos-
texercise recovery in the fasting group (P ⬍ 0.05). Similarly,
muscle phenylalanine concentration increased significantly fol-
lowing EAA ⫹ CHO ingestion compared with the fasting
group (P ⬍ 0.05). Muscle phenylalanine concentration was
also higher immediately postexercise in the EAA ⫹ CHO
group (P ⬍ 0.05) and gradually declined to baseline levels by
2 h postexercise. Phenylalanine concentration was unchanged
throughout the study except for a slight decrease at 2 h
postexercise in the fasting group (P ⬍ 0.05).
Muscle protein synthesis and phenylalanine net balance
across the leg. The phenylalanine tracer was added to the
nutrient solution to minimize changes in enrichment following
nutrient ingestion. In fact, blood and intracellular phenylala-
nine enrichment was stable throughout the experiment (i.e.,
enrichments were not different between study periods) in the

Table 3. Muscle intracellular amino acid concentrations

1 h Pre-Ex Pre-Ex Post-Ex 1 h Post-Ex 2 h Post-Ex

Leucine, ␮mol/l
EAA⫹CHO 161⫾15 601⫾120*† 430⫾53*† 334⫾32*† 195⫾15*
Fasting 204⫾12 165⫾15 239⫾28 149⫾18* 132⫾11*
Phenylalanine, ␮mol/l
EAA⫹CHO 67⫾3 140⫾15*† 149⫾10*† 118⫾12* 77⫾2*
Fasting 77⫾6 70⫾4 86⫾5 72⫾5 64⫾3*
Values are means ⫾ SE. Muscle amino acid concentrations taken at baseline (1 h Pre-Ex), before exercise but following nutrient ingestion in the EAA⫹CHO
group (Pre-Ex), immediately following exercise (Post-Ex), and during postexercise recovery (1 h Post-Ex and 2 h Post-Ex). *P ⬍ 0.05 vs. 1 h Pre-Ex. †P ⬍
0.05 vs. fasting at respective study period.

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1734 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE

exercise to the 2 h postexercise biopsy). We found that there

was no difference (P ⫽ 0.36) in the entire postexercise muscle
FSR between the fasting group (exercise without nutrients) and
the EAA ⫹ CHO group (nutrient ingestion before exercise),
indicating no significant anabolic advantage for ingesting an
EAA ⫹ CHO solution before exercise on postexercise muscle
protein synthesis (Fig. 2B). In addition, we calculated the total
muscle FSR during both exercise and recovery [i.e., the rate of
tracer incorporation from the Pre-Ex biopsy to the 2 h postex-
ercise biopsy. We found that there was no difference (P ⫽
0.20) between groups (0.089 ⫾ 0.01 vs. 0.073 ⫾ 0.005%/h) for
EAA ⫹ CHO and fasting groups, respectively].
Blood phenylalanine concentration did not change through-
out the entire study in the fasting group (P ⬎ 0.05; Fig. 3A). In
the EAA ⫹ CHO group, blood phenylalanine concentration
was significantly higher than in the fasting group during the
preexercise, exercise, and 1 h postexercise periods (P ⬍ 0.05;

Fig. 2. A: muscle protein fractional synthetic rate (FSR) time course in both
the fasting control group and the EAA ⫹ CHO group. FSR was determined
during a preexercise period (Pre-Ex), during exercise (Ex), 1 h postexercise (1
h Post-Ex), and 2 h postexercise (2 h Post-Ex). Nutrients were ingested
immediately following the first muscle biopsy in the EAA ⫹ CHO group
during the Pre-Ex period. B: FSR in both the fasting control and EAA ⫹ CHO
groups over the entire 2 h postexercise recovery period (i.e., rate of incorpo-
ration calculated from the biopsy collected immediately postexercise to the
biopsy collected at 2 h postexercise). Fasting control group data have been
previously published (9). Values are means ⫾ SE. *P ⬍ 0.05 vs. fasting
Pre-Ex. # P ⬍ 0.05 vs. fasting at respective time point.

EAA ⫹ CHO group and the fasting group (P ⬎ 0.05; data not
shown). Mixed muscle protein FSR increased significantly in
response to the EAA ⫹ CHO ingestion compared with the
fasting group basal value (P ⬍ 0.05; Fig. 2A). The mixed
muscle FSR in the EAA ⫹ CHO group returned to basal values
during exercise (P ⬎ 0.05), remained unchanged during the 1 h
postexercise period (P ⬎ 0.05), and increased significantly at
2 h postexercise (P ⬍ 0.05) compared with basal FSR. The
FSR time course for the fasting group has been previously
published (9), and the data show that FSR decreased during Fig. 3. Time course of blood phenylalanine concentration (A) and net balance
exercise (P ⬍ 0.05); however, FSR increased (compared with (B) across the leg in the preexercise state (Pre-Ex), during exercise (Ex), 1 h
basal FSR) during the 1 h and 2 h postexercise periods (P ⬍ postexercise (1 h Post-Ex), and 2 h postexercise (2 h Post-Ex) for both the
0.05; Fig. 2A). fasting control and EAA ⫹ CHO groups. Data shown are presented as the
hourly average. Pre-Ex data for the fasting group are basal data without
We also determined the muscle FSR over the entire 2-h nutrients, whereas Pre-Ex data for the EAA ⫹ CHO are the hourly average
postexercise recovery period (i.e., the rate of phenylalanine following nutrient ingestion. Values are means ⫾ SE. # P ⬍ 0.05 vs. fasting
incorporation from the biopsy collected immediately after at respective time point.

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 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
Fig. 3A). During the first period following ingestion of EAA ⫹
CHO, the phenylalanine net balance across the leg significantly
increased and became positive in the EAA ⫹ CHO group (P ⬍
0.05; Fig. 3B). Postexercise phenylalanine net balance in the
EAA ⫹ CHO group gradually decreased and became negative
during 1–2 h of recovery. The fasting group phenylalanine net
balance tended to become less negative during postexercise
recovery, but this change did not reach significance (P ⬎ 0.05).
Upstream regulators of mTOR signaling. The phosphoryla-
tion of Akt/PKB at Ser473 increased significantly in response
to the EAA ⫹ CHO ingestion before exercise and remained
elevated during exercise and 1 h postexercise (P ⬍ 0.05; Fig. 4A).
Akt/PKB phosphorylation returned to basal levels at 2 h
postexercise. Akt/PKB phosphorylation increased only at the
end of the first hour postexercise in the fasting group (9).
Fig. 4. Upstream regulators of the mammalian target of rapamycin (mTOR)
signaling pathway in the EAA ⫹ CHO and fasting control group 1 h before
exercise (1 h Pre-Ex), immediately before beginning exercise (Pre-Ex), im-
mediately following exercise (Post-Ex), 1 h postexercise (1 h Post-Ex), and 2 h
postexercise (2 h Post-Ex) . Nutrients were ingested immediately following the
first muscle biopsy (1 h Pre-Ex) in the EAA ⫹ CHO group. A: Akt/protein
kinase B (PKB) phosphorylation at Ser473. B: AMP-activated protein kinase
(AMPK␣2) activity. The time course data for the fasting control group have
been previously published (9). Values are means ⫾ SE. *P ⬍ 0.05 vs. 1 h
Pre-Ex. # P ⬍ 0.05 vs. fasting at respective time point. †P ⬍ 0.05 for time
effect. There were no differences between groups for the AMPK data.
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1735
The phosphorylation of TSC2 at Thr1462 did not change
throughout the entire study period in either group (P ⬎ 0.05;
data not shown) although there was a slight decrease at the end
of the first hour postexercise in the fasting group (9).
Muscle AMPK␣2 activity tended to decrease following
nutrient ingestion in the EAA ⫹ CHO group (P ⬎ 0.05; Fig.
4B). There was a significant time effect for AMPK␣2 activity
to be increased postexercise in both groups (P ⬍ 0.05; Fig.
4B). There were no differences between groups at any time
point (P ⬎ 0.05). AMPK␣2 activity in the fasting group has
been previously published (9).
mTOR and downstream regulators of mTOR signaling and
translation elongation. The phosphorylation of mTOR at
Ser2448 increased significantly in response to the EAA ⫹
CHO ingestion before exercise (P ⬍ 0.05; Fig. 5A). mTOR
phosphorylation returned to 1 h Pre-Ex (basal) levels during
exercise (P ⬎ 0.05) and was not significantly increased at 1 h
and 2 h postexercise in the EAA ⫹ CHO group compared with
baseline or the fasting group (P ⬎ 0.05). In comparison, the
fasting group mTOR phosphorylation was significantly in-
creased at 1 and 2 h postexercise (P ⬍ 0.05) (9).
Phosphorylation of 4E-BP1 at Thr37/46 increased signifi-
cantly in response to the EAA ⫹ CHO ingestion before
exercise (P ⬍ 0.05; Fig. 5B) and returned to 1 h Pre-Ex basal
levels during exercise (P ⬎ 0.05). 4E-BP1 phosphorylation at
1 h and 2 h postexercise also did not differ from 1 h Pre-Ex
basal values; however, phosphorylation was higher than the
fasting group immediately and 1 h postexercise (P ⬍ 0.05).
The fasting group 4E-BP1 phosphorylation decreased during
exercise (P ⬍ 0.05) and was not different from baseline during
postexercise recovery (9).
Phosphorylation of S6K1 at Thr389 increased significantly
in response to the EAA ⫹ CHO ingestion before exercise (P ⬍
0.05; Fig. 5C) and remained higher immediately postexercise
and 1 h and 2 h postexercise (P ⬍ 0.05). In comparison, the
fasting group showed a significant increase in S6K1 phosphor-
ylation at the end of the 2 h postexercise period (9); however,
the phosphorylation of S6K1 was higher in the EAA ⫹ CHO
group compared with the fasting group at Pre-Ex and Post-Ex
(P ⬍ 0.05).
Phosphorylation of eEF2 at Thr56 decreased significantly in
response to EAA ⫹ CHO ingestion (P ⬍ 0.05; Fig. 5D) and
increased to baseline levels during exercise (P ⬎ 0.05). eEF2
phosphorylation was significantly reduced (compared with
baseline) at the end of the 1 and 2 h postexercise periods (P ⬍
0.05). eEF2 phosphorylation was significantly higher in the
fasting group compared with the EAA ⫹ CHO group Post-Ex;
however, phosphorylation was reduced to a similar extent
during postexercise recovery in the fasting group (9).
DISCUSSION
The primary finding from our study was that muscle protein
synthesis increased to a similar extent postexercise in both the
EAA ⫹ CHO (fed) group and control (fasted) group. Thus,
performing a bout of resistance exercise in the fed state did not
further increase muscle protein synthesis during postexercise
recovery compared with the fasted state. However, the postex-
ercise time course for FSR was different between the two
groups in that during exercise the FSR did not decrease below
basal values in the EAA ⫹ CHO group, and the postexercise
www.jap.org
1736 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
Fig. 5. Phosphorylation status of down-
stream components of mTOR signaling path-
way in the EAA ⫹ CHO and fasting control
group 1 h before exercise (1 h Pre-Ex), im-
mediately before beginning exercise (Pre-
Ex), immediately following exercise (Post-
Ex), 1 h postexercise (1 h Post-Ex), and 2 h
postexercise (2 h Post-Ex). mTOR phosphor-
ylation at Ser2448 (A), 4E-binding protein 1
(4E-BP1) phosphorylation at Thr37/46 (B),
S6 kinase 1 (S6K1) phosphorylation at
Thr389 (C), and eukaryotic elongation factor
2 (eEF2) phosphorylation at Thr56 (D) are
shown. The time course phosphorylation data
for the fasting group have been previously
published (9). Values are means ⫾ SE. *P ⬍
0.05 vs. 1 h Pre-Ex. #P ⬍ 0.05 vs. fasting at
respective time point.
increase in FSR was delayed compared with the fasting control
group. On the other hand, our recent work has shown that
providing EAA ⫹ CHO following a bout of resistance exercise
increases the rate of muscle protein synthesis to a greater extent
than when resistance exercise is performed without nutrient
ingestion (10). Therefore, our data support the hypothesis that
EAA ⫹ CHO ingestion following resistance exercise is more
effective at enhancing muscle protein synthesis during postex-
ercise recovery.
Our recent data (10) are in agreement with a large number of
studies in which nutrient ingestion (i.e., amino acids, protein,
or EAA ⫹ CHO) following a bout of resistance exercise has
been shown to increase muscle protein synthesis in human
subjects (4, 5, 10 –11, 18 –19, 21, 28, 30 –31, 36). In addition,
recent work has shown that when subjects ingested fat-free
milk (i.e., protein ⫹ CHO) following each resistance exercise
bout (during 3 mo of training), the increase in muscle mass was
greater than in either an isoenergetic soy protein group or an
isoenergetic CHO control group (14). In contrast to these
studies, one study has shown that EAA ⫹ CHO ingestion
immediately before an acute bout of resistance exercise pro-
duced a larger increase in indirect measures of muscle protein
synthesis (i.e., leg net amino acid balance and rate of disap-
pearance) compared with when the nutrients were ingested
immediately postexercise (29). We cannot completely explain
the discrepancies between that study and our present study, but
it should be noted that several differences in study design exist.
For example, in the prior study (29) the authors used a smaller
dose of EAA ⫹ CHO than we did, and they provided the
nutrients immediately before exercise, whereas we adminis-
tered the nutrients 1 h before beginning exercise. In addition, it
is difficult to determine if muscle protein synthesis was actu-
ally increased in the aforementioned study (29) because it was
not measured directly. In addition, the ingestion of whey
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protein immediately before exercise (31) also resulted in sim-
ilar increases in nondirect measures of muscle protein synthesis
and anabolism. Therefore, we cannot rule out the possibility
that intact protein ingestion before exercise may have produced
different results than what we have reported in the present
study. Recently, it has been shown that protein and CHO
ingestion “during” a 2-h whole body resistance exercise work-
out resulted in an increase in leg muscle FSR (although this
difference was not significant when the muscle intracellular
enrichment was used to calculate FSR) (1). There are a couple
of reasons for the discrepancy between that study and the
present study. First, the authors (1) studied subjects in the fed
state, whereas our subjects were fasted before they consumed
a bolus of EAA ⫹ CHO 1 h before exercise. Second, we
measured FSR during ⬃1 h of leg muscle contractions,
whereas the subjects in the Beelen et al. (1) study underwent an
intermittent exercise routine (i.e., upper body exercise was
performed during the first hour of exercise and lower body
exercises in the last hour). Thus it is possible that consuming
protein/amino acids during an intermittent resistance exercise
workout may result in a stimulation of FSR. Future work is still
required to determine if muscle protein synthesis is increased
during exercise or recovery when EAA ⫹ CHO are ingested
immediately before performing an exercise bout. In any event,
it is clear that net muscle anabolism during recovery is not
greater when exercise is performed in the fed state because
phenylalanine net balance across the leg is similar to subjects
who exercised in the fasted state (Fig. 3B).
Our data do not support the view that exercising in the fed
state can increase muscle protein synthesis during a bout of
resistance exercise. Specifically, our data show that muscle
protein synthesis returns to basal values during a bout of
resistance exercise performed while in the fed state (Fig. 2).
We have previously shown that muscle protein synthesis de-
106 • MAY 2009 • www.jap.org
 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
creases during a bout of resistance exercise performed while in
the fasting state (9). In the present study, muscle protein
synthesis decreased during exercise compared with the large
increase detected during the hour following nutrient ingestion.
However, muscle protein synthesis was not significantly lower
than the fasting group basal values, which may indicate that
performing a bout of resistance exercise while in the fed state
may prevent muscle protein synthesis from decreasing (i.e.,
below basal FSR values) during exercise as we originally
hypothesized. It remains to be determined whether the blunted
decrease in FSR during exercise is physiologically relevant
because of the relatively short exercise duration. In fact, we
were unable to detect a difference between groups in the FSR
calculated during both exercise and recovery periods. During
postexercise recovery, the reduction or attenuation of muscle
protein synthesis during resistance exercise is rapidly reversed
and the rate of protein synthesis is increased within 1 h
postexercise (9). In the present study the postexercise time
course for muscle protein synthesis in the EAA ⫹ CHO group
was slightly altered, in that protein synthesis did not increase
during the first hour of recovery but an increase was detected
at 2 h postexercise. Contrary to our original hypothesis, per-
forming a bout of resistance exercise while in the fed state
produces a similar increase in postexercise muscle protein
synthesis compared with when exercise is performed in the
fasted state (Fig. 2B).
The most likely explanation for no increase in muscle
protein synthesis “during” resistance exercise is that the prior-
ity of the muscle cell is to provide ATP to support muscle
contraction. Thus the anabolic and ATP-consuming process of
protein synthesis is given less priority in favor of stimulating
catabolic ATP-producing processes such as glycolysis. Our
data show that exercising in the fed state may prevent an actual
decrease in protein synthesis during exercise; however, it is
clear that muscle protein synthesis is not stimulated during
exercise. We have previously identified potential cellular
mechanisms responsible for reducing muscle protein synthesis
during exercise, which include the large increase in muscle
acidosis induced by the resistance exercise bout or an increase
in cellular energetic stress (9). AMPK, a cellular energy sensor,
is increased immediately and 1 h following a bout of resistance
exercise in the fasted state (9). In the present study we show
that AMPK increased to a similar extent immediately follow-
ing exercise and during recovery in both groups (Fig. 4B).
Therefore, feeding before exercise did not prevent AMPK
activation during exercise. It has also been shown that acidosis
can reduce the rate of skeletal muscle protein synthesis in both
animals and humans (7). In any event, our data clearly show
that the local environment within the muscle during exercise in
the fed state is not conducive to promoting muscle protein
synthesis.
There was an interesting difference in the FSR response
between our two groups. In the fasting group, muscle FSR
decreased during exercise; however, FSR rebounded quickly
and was significantly increased at both 1 and 2 h postexercise.
On the other hand, as previously mentioned, in the EAA ⫹
CHO group, muscle FSR did not decrease as much during the
resistance exercise bout, indicating that the nutrient ingestion
before exercise may have prevented the decrease in FSR during
exercise. The postexercise increase in FSR was also delayed in
the EAA ⫹ CHO such that FSR did not increase until 2 h
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1737
postexercise. The mechanism(s) responsible for the delay in
FSR activation in the EAA ⫹ CHO group cannot be directly
determined in the present study. However, it is possible that the
large activation of Akt/mTOR signaling and muscle protein
synthesis by the abundant supply of amino acids and insulin
immediately following the ingestion of EAA ⫹ CHO (before
exercise) may have resulted in a partial refractory period for
muscle protein synthesis after exercise. In any event, the
increase in muscle FSR during recovery was also linked to an
elevated muscle protein breakdown rate in both groups since
phenylalanine net balance across the leg was negative during
postexercise recovery. This would indicate that nutrient inges-
tion before exercise did not prevent the normal increase in
muscle protein breakdown following the resistance exercise
bout and provides more support for the hypothesis that EAA ⫹
CHO ingestion before exercise does not enhance muscle anab-
olism during postexercise recovery.
Muscle protein synthesis is stimulated in the recovery period
following a bout of resistance exercise (3, 9, 15, 22, 38). We
have recently shown that activation of the muscle Akt/mTOR
signaling pathway is associated with the increase in muscle
protein synthesis following an acute bout of resistance exercise
(9 –10). In particular, mTOR and S6K1 phosphorylation are
increased and eEF2 phosphorylation is decreased during the
first 1–2 h postexercise, indicating that both translation initia-
tion and elongation are activated. In the present study, we
detected a large activation of the Akt/mTOR-associated sig-
naling proteins 1 h following the ingestion of the EAA ⫹ CHO
as previously reported (13). Specifically, we found that Akt
phosphorylation was increased following the ingestion of the
EAA ⫹ CHO solution and remained elevated during exercise
and at 1 h postexercise. However, phosphorylation of mTOR,
S6K1, 4E-BP1, and eEF2 during postexercise recovery were
not different between the fasting and EAA ⫹ CHO groups
(Fig. 5). In fact, eEF2 phosphorylation was significantly re-
duced at 1 and 2 h postexercise in association with the
increased rate of protein synthesis in the fasting group; how-
ever, the reduced eEF2 phosphorylation at 1 h postexercise in
the EAA ⫹ CHO group did not correspond with an increase in
muscle protein synthesis. In our previous work we have clearly
shown a direct relationship between eEF2 dephosphorylation
and an increase in muscle protein synthesis following resis-
tance exercise and/or nutrient ingestion (9, 10, 13), indicating
an important role for elongation in the control of muscle FSR.
The disconnect between eEF2 phosphorylation and muscle
FSR at 1 h postexercise in the EAA ⫹ CHO group may be due
to the fact that we collected the biopsy at hourly intervals
whereas the FSR is a calculated hourly average. It is likely that
at 1 h postexercise elongation was activated in the EAA ⫹
CHO group, but this was not reflected in the hourly FSR
immediately following exercise. Finally, our study was focused
on the early postexercise recovery response in signaling and
FSR, and we acknowledge the possibility that long-term acti-
vation of mTOR signaling and FSR such as at 6, 24, or 48 h
postexercise may be better indicators of muscle growth over
time.
In summary, we show that performing a bout of resistance
exercise in the fed state prevented the normal decrease in FSR
during exercise and delayed the normal increase in FSR pos-
texercise. However, the eventual increase in FSR in the fed
group did not further increase muscle protein synthesis during
106 • MAY 2009 • www.jap.org
1738 NUTRIENT INGESTION BEFORE RESISTANCE EXERCISE
postexercise recovery compared with performing resistance
exercise in the fasted state. In contrast, our recent findings
indicated an enhanced stimulation of muscle protein synthesis
when EAA ⫹ CHO are ingested 1 h following a bout of
resistance exercise (10). We conclude that EAA ⫹ CHO
ingestion postexercise appears to be more effective (than in-
gestion preexercise) at inducing further increases in the rate of
human skeletal muscle protein synthesis during early postex-
ercise recovery.
ACKNOWLEDGMENTS
We thank the nurses and personnel of the General Clinical Research Center
of the University of Texas Medical Branch and Dr. Jerson Cadenas for help
with the conduct of the clinical portion of this study. We also thank Ming
Zheng, Jessica Lee, Shelley Medina, and Sarah Toombs Smith for technical
assistance.
GRANTS
This research was supported by Grant R01-AR-049877 from the National
Institute for Arthritis and Musculoskeletal and Skin Diseases, Grant S10-RR-
16650 from the Shared Instrumentation Grant Program, Grant-P30-AG024832
from the National Institute on Aging, and Grant M01-RR-00073 from the
General Clinical Research Branch, National Center for Research Resources.
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