Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise

Abstract

The present study was designed to determine whether consumption of an oral essential amino acid-carbohydrate supplement (EAC) before exercise results in a greater anabolic response than supplementation after resistance exercise. Six healthy human subjects participated in two trials in random order, PRE (EAC consumed immediately before exercise), and POST (EAC consumed immediately after exercise). A primed, continuous infusion of L-[ring-(2)H(5)]phenylalanine, femoral arteriovenous catheterization, and muscle biopsies from the vastus lateralis were used to determine phenylalanine concentrations, enrichments, and net uptake across the leg. Blood and muscle phenylalanine concentrations were increased by approximately 130% after drink consumption in both trials. Amino acid delivery to the leg was increased during exercise and remained elevated for the 2 h after exercise in both trials. Delivery of amino acids (amino acid concentration times blood flow) was significantly greater in PRE than in POST during the exercise bout and in the 1st h after exercise (P < 0.05). Total net phenylalanine uptake across the leg was greater (P = 0.0002) during PRE (209 +/- 42 mg) than during POST (81 +/- 19). Phenylalanine disappearance rate, an indicator of muscle protein synthesis from blood amino acids, increased after EAC consumption in both trials. These results indicate that the response of net muscle protein synthesis to consumption of an EAC solution immediately before resistance exercise is greater than that when the solution is consumed after exercise, primarily because of an increase in muscle protein synthesis as a result of increased delivery of amino acids to the leg.

Full text

Timing of amino acid-carbohydrate ingestion alters
anabolic response of muscle to resistance exercise
KEVIN D. TIPTON,
1,2
BLAKE B. RASMUSSEN,
1,2
SHARON L. MILLER,
1,2
STEVEN E. WOLF,
1
SHARLA K. OWENS-STOVALL,
1
BART E. PETRINI,
1
AND ROBERT R. WOLFE
1,2
1
Department of Surgery, University of Texas Medical Branch, and
2
Metabolism Unit,
Shriners Hospitals for Children, Galveston, Texas 77550
Received 5 September 2000; accepted in final form 6 March 2001
Tipton, Kevin D., Blake B. Rasmussen, Sharon L.
Miller, Steven E. Wolf, Sharla K. Owens-Stovall, Bart E.
Petrini, and Robert R. Wolfe. Timing of amino acid-car-
bohydrate ingestion alters anabolic response of muscle to
resistance exercise. Am J Physiol Endocrinol Metab 281:
E197–E206, 2001.—The present study was designed to de-
termine whether consumption of an oral essential amino
acid-carbohydrate supplement (EAC) before exercise results
in a greater anabolic response than supplementation after
resistance exercise. Six healthy human subjects participated
in two trials in random order, PRE (EAC consumed immedi-
ately before exercise), and POST (EAC consumed immedi-
ately after exercise). A primed, continuous infusion of
L-[ring-
2
H
5
]phenylalanine, femoral arteriovenous catheterization,
and muscle biopsies from the vastus lateralis were used to
determine phenylalanine concentrations, enrichments, and
net uptake across the leg. Blood and muscle phenylalanine
concentrations were increased by ⬃130% after drink con-
sumption in both trials. Amino acid delivery to the leg was
increased during exercise and remained elevated for the 2 h
after exercise in both trials. Delivery of amino acids (amino
acid concentration times blood flow) was significantly greater
in PRE than in POST during the exercise bout and in the 1st
h after exercise (P ⬍ 0.05). Total net phenylalanine uptake
across the leg was greater (P ⫽ 0.0002) during PRE (209 ⫾ 42
mg) than during POST (81 ⫾ 19). Phenylalanine disappear-
ance rate, an indicator of muscle protein synthesis from blood
amino acids, increased after EAC consumption in both trials.
These results indicate that the response of net muscle protein
synthesis to consumption of an EAC solution immediately
before resistance exercise is greater than that when the
solution is consumed after exercise, primarily because of an
increase in muscle protein synthesis as a result of increased
delivery of amino acids to the leg.
muscle protein synthesis; muscle protein breakdown; stable
isotopes; supplementation
BOTH EXERCISE AND NUTRITIONAL SUBSTRATES play impor-
tant roles in muscle protein metabolism. An acute bout
of resistance exercise increases muscle protein synthe-
sis more than breakdown, so that net muscle protein
balance (synthesis minus breakdown) is increased (5,
19, 20). Hyperaminoacidemia at rest has similarly
been demonstrated to increase net synthesis of muscle
protein, primarily by stimulating muscle protein syn-
thesis (1, 6). After intense resistance exercise, in-
creased amino acid availability via intravenous infu-
sion was shown to increase the rate of muscle protein
synthesis above levels observed with amino acid infu-
sion at rest (6). Thus exercise and amino acids seem to
have complementary effects on muscle protein synthe-
sis. Furthermore, the normal postexercise increase in
muscle protein breakdown was attenuated when
amino acids were infused after an exercise bout. Syn-
thesis, in this case, exceeded breakdown, resulting in
net muscle protein synthesis. Subsequently, we dem-
onstrated that a solution of amino acids given orally
was just as effective as intravenous amino acid infu-
sion for developing net muscle protein synthesis after
resistance exercise (27).
A combination of amino acids, to increase amino acid
availability, and carbohydrates, to stimulate insulin
release, should be a potent stimulator of net muscle
protein synthesis. We recently demonstrated that
ingestion of a bolus of6gofaminoacids combined
with 35 g of carbohydrates at both 1 and 3 h postex-
ercise resulted in muscle protein anabolism (21).
During an exercise bout, there may be a net loss of
muscle protein, because protein synthesis is either
decreased (8) or unchanged (9), whereas protein
breakdown is generally considered to be elevated (22).
Although muscle protein synthesis is increased after
exercise, it appears that this response is not stimulated
until some time after the exercise bout (17). Hyper-
aminoacidemia from ingestion of amino acids during
the exercise bout, as opposed to after exercise, may
counter the net loss of muscle protein, thereby creating
a more favorable situation for muscle growth. The
purpose of the present study was to determine whether
ingesting a combination of amino acid and carbohy-
drate before exercise is more effective in stimulating
net muscle protein synthesis than ingesting the mix-
ture after exercise.
Address for reprint requests and other correspondence: K. D.
Tipton, Metabolism Unit, Shriners Hospital for Children, 815 Mar-
ket St., Galveston, TX 77550 (E-mail: ktipton@utmb.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Am J Physiol Endocrinol Metab
281: E197–E206, 2001.
0193-1849/01 $5.00 Copyright
©
2001 the American Physiological Societyhttp://www.ajpendo.org E197

METHODS
Subjects
Six healthy volunteers (3 females, 3 males) were studied in
the postabsorptive state. The study design, purpose, and
possible risks were explained to each subject before written
consent was obtained. The Institutional Review Board and
the General Clinical Research Center (GCRC) of the Univer-
sity of Texas Medical Branch at Galveston approved the
study protocol. All subjects were healthy, nondiabetic, and
normotensive. They had a normal cardiac rhythm with no
abnormalities, as judged by medical history, physical exam-
ination, resting electrocardiogram, and laboratory blood and
urine tests. Subjects were recreationally active and were
instructed to refrain from physical exercise for 24 h before
being studied. Mean (⫾SE) age was 30.2 ⫾ 3.1 yr, height was
1.71 ⫾ 0.03 m, weight was 66 ⫾ 6 kg, body mass index was
22 ⫾ 1 kg/m
2
, and leg volume was 9.78 ⫾ 0.61 liters. At least
1 wk before the initial infusion study, each subject was
familiarized with the leg press and leg extension machine,
and their one-repetition maximum (1RM, the maximum
weight that can be lifted for one repetition) was determined
on each. Mean 1RM for the leg press was 122.9 ⫾ 12.8 kg and
for the leg extension was 92.3 ⫾ 13.7 kg.
Experimental Protocol
The protocol was designed to determine whether an oral
amino acid-carbohydrate solution (EAC) would be a more
effective stimulator of muscle protein anabolism if given
immediately before or immediately after a resistance exer-
cise bout. Each subject participated in two trials in random
order. The response of muscle protein metabolism was deter-
mined during and after an intense resistance exercise bout
while each subject consumed, on separate occasions, a bolus
of EAC immediately before exercise (PRE) or immediately
after exercise (POST). Study days were separated by 2 mo.
Subjects were instructed to maintain a consistent dietary
intake pattern throughout the duration of the study. One
female subject completed only the PRE trial; thus all data
reflect means of six subjects for PRE and five subjects for
POST. A schematic representation of the study protocol is
shown in Fig. 1.
Subjects reported to the GCRC on the evening before each
study day and began fasting at 2200. After the overnight fast,
at ⬃0600, an 18-gauge polyethylene catheter was inserted
into a large peripheral arm vein for the infusion of stable
isotopic tracers of amino acids. Catheters were inserted in
positions to prevent occlusion by bending of the arms. Sub-
jects were subsequently transported to the Exercise Metab-
olism Laboratory in the Shriners Hospital for Children,
Galveston. After background blood samples were taken, a
primed, continuous infusion of
L-[ring-
2
H
5
]phenylalanine
was started at ⬃0630 and continued throughout the protocol.
The priming dose was 2 ␮mol/kg, and the infusion rate was
0.05 ␮mol䡠 min
⫺1
䡠 kg
⫺1
. Catheters were then placed in the
femoral artery and vein, as well as a second peripheral arm
vein contralateral to the infusion site. The femoral arterial
catheter was also used for the continuous infusion of indo-
cyanine green (ICG).
After2hofinfusion to establish an isotopic steady state,
resting measurements were made of amino acid concentra-
tions and enrichments in the femoral artery and vein, as well
as muscle. Three blood samples, separated by ⬃10 min, were
taken from the femoral artery and vein for the measurement
of plasma arterial and venous amino acid enrichments and
concentrations. Blood samples were immediately placed into
preweighed tubes containing 1 ml of sulfosalicylic acid per
milliliter of blood and tubes containing lithium heparin. Leg
blood flow was measured by the dye-dilution technique dur-
ing this time (4). Briefly, ICG (0.5 mg/ml) was infused (60
ml/h) into the femoral artery. Blood samples were simulta-
neously taken from the femoral vein and a peripheral vein to
measure ICG concentration. The ICG infusion was briefly
halted and then quickly resumed to allow sampling from the
femoral artery for isotopic measurements. Immediately after
the blood sampling, a percutaneous muscle biopsy was taken
from the vastus lateralis. Muscle biopsies were taken from
the lateral portion of the vastus lateralis with sterile tech-
nique. The skin and subcutaneous tissue were anesthetized,
and an ⬃6-mm incision was made in the skin and muscle
fascia. A 5-mm Bergstro¨m biopsy needle (Depuy, Warsaw,
IN), with the cutting window closed, was advanced 3–5 cm
through the fascia deep into the muscle. With suction ap-
plied, the cutting cylinder was opened and then closed 2–3
times. A sample of ⬃50 mg of mixed muscle tissue was
obtained with each biopsy. Each sample was quickly (within
1 min) rinsed with ice-cold saline, blotted dry, and frozen in
liquid N
2
.
Immediately after the first muscle biopsy, subjects per-
formed an intense leg resistance exercise bout. Before initi-
ation of the resistance exercise routine, subjects consumed
either a 500-ml bolus of the EAC solution (PRE) or a placebo
solution (POST). The exercise bout consisted of 10 sets of 8
repetitions of leg press at 80% of 1RM and 8 sets of 8
repetitions of leg extension at 80% of 1RM. The rest interval
between sets was ⬃2 min, and the entire exercise bout was
completed in ⬃45–50 min. Blood samples were taken from
the femoral artery and vein after the 4th and 8th sets of leg
press (⬃10 and 20 min from the beginning of the exercise)
and the 2nd and 8th, or final, sets of leg extension (⬃30 and
45 min from the beginning of the exercise). A second muscle
biopsy was taken in the rest interval between the 7th and 8th
sets of leg extension. A second bolus drink, placebo for the
PRE trial and EAC for the POST trial, was consumed imme-
diately after exercise and the final blood draw. A series of
arterial and venous blood samples and two muscle biopsies
were taken in the 2 h after exercise. Blood samples were
drawn at 10, 20, 30, 45, 60, 90, and 120 min after exercise.
Fig. 1. Schematic representation of the study protocol. Time values
are in minutes from the end of exercise. AV, arteriovenous; EX,
exercise; EAC, essential amino acid-carbohydrate (supplement); ring
d
5
-Phe, L-[ring-
2
H
5
]phenylalanine.
E198 ANABOLIC RESPONSE OF MUSCLE TO SUPPLEMENT TIMING
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Muscle biopsies were taken at ⬃55 and 115 min after exer-
cise and the ingestion of the 2nd bolus drink.
EAC Solution
Each subject consumed two 500-ml bolus drinks during
each trial. The order of the trials was randomly selected.
During the PRE trial, the EAC drink was consumed imme-
diately before initiation of the exercise bout, and the placebo
was consumed immediately upon cessation of the exercise
bout. For the POST trial, the order was reversed. The EAC
consisted of6gofessential amino acids, in amounts designed
to increase muscle free intracellular amino acid levels in
proportion to their respective requirements for protein syn-
thesis, and 35 g of sucrose in 500 ml of deionized-distilled
water. The amounts of essential amino acids in a 500-ml
bolus EAC solution were (mg and ␮mol, respectively) histi-
dine 0.65, 4.2; isoleucine 0.60, 4.6; leucine 1.12, 8.5; lysine
0.93, 6.4; methionine 0.19, 1.3; phenylalanine 0.93, 5.6; thre-
onine 0.88, 7.4; and valine 0.7, 6.0. Additionally, 0.0605 g of
L-[ring-
2
H
5
]phenylalanine was added to the solution to main
-
tain isotopic steady state. A small amount of artificial sweet-
ener, containing aspartame, was added to the EAC to im-
prove palatability. The placebo solution was composed of
deionized-distilled water and an artificial sweetener contain-
ing aspartame. The placebo contained ⬍200 mg of phenylal-
anine.
Analysis of Samples
Blood. Amino acid enrichment and concentration of phe-
nylalanine in whole blood were measured by gas chromatog-
raphy-mass spectrometry (GC-MS; model 5989B, Hewlett-
Packard, Palo Alto, CA) (18). Upon thawing, 500 ␮lofthe
sulfosalicylic extract was passed over a cation exchange col-
umn (Dowex AG 50W-8X, 100–200 mesh H⫹ form; Bio-Rad
Laboratories, Richmond, CA) and dried under vacuum using
a Speed Vac (Savant Instruments, Farmingdale, NY). To
determine the enrichment of infused amino acids in whole
blood, the tert-butyldimethylsilyl (t-BDMS) derivative of each
amino acid was made according to previously described pro-
cedures (5, 18, 19). Isotopic enrichments were determined by
GC-MS (model 5989B, Hewlett-Packard) and expressed as a
tracer-to-tracee ratio (t/T) (16). Concentrations of free amino
acids were determined using an internal standard solution,
as previously described (4, 5, 18, 19). The internal standard
used was
L-[ring-
13
C
6
]phenylalanine (50 ␮mol/l) added in a
ratio of ⬃100 ␮l/ml of blood. Because the tube weight was
known, the amount of blood could also be determined, and
the blood amino acid concentration was determined from the
internal standard enrichments measured by GC-MS on the
basis of the amount of blood and internal standard added (4,
5, 18, 19). Appropriate corrections were made for overlapping
spectra that contributed to the t/T (23). Additionally, m⫹5
enrichments were corrected 6% for contributions from m⫹6.
Leg blood flow was determined by spectrophotometrically
measuring the ICG concentration in serum from the femoral
and the peripheral veins, as described previously (4, 5, 19).
Leg plasma flow was calculated from steady-state values of
dye concentration and converted to blood flow by use of the
hematocrit (4, 5, 18). Plasma insulin levels were determined
by radioimmunoassay (Diagnostic Products, Los Angeles,
CA). The intra-assay coefficient of variation (CV) was 1.45%.
Muscle. Muscle biopsy tissue samples were analyzed for
mixed protein-bound and free intracellular amino acid en-
richment and concentration, as previously described (4, 5, 18,
19). Tissue biopsies (⬃50 mg) of the vastus lateralis were
immediately blotted and frozen in liquid N
2
. Samples were
then stored at ⫺80°C until processed. Upon thawing, the
⬃25–30 mg of tissue were weighed and protein precipitated
with 0.5 ml of 10% perchloric acid. The tissue was then
homogenized and centrifuged, and the supernatant was col-
lected. This procedure was repeated two more times, and the
pooled supernatant (⬃1.3 ml) was processed, as were the
blood samples described above in Blood. To determine intra-
cellular enrichment of infused tracers, the t-BDMS deriva-
tive was prepared as previously described (4, 5, 19) and
analyzed by GC-MS. Intracellular enrichment was deter-
mined by correction for extracellular fluid on the basis of the
chloride method (2). Muscle free amino acid concentration
was measured with the internal standard method, with cor-
rections for the contribution of extracellular fluid and for
overlapping spectra, as described previously (4, 5, 18, 19).
The remaining pellet of muscle tissue was further washed,
twice with 0.9% saline and three times with absolute ethanol.
It was then placed in an oven and dried at 50°C overnight.
The dried pellet was then hydrolyzed at 110°C for 24 h with
6 N HCl. The protein hydrolysate was then passed over a
cation exchange column and dried by Speed Vac derivatized
with t-BDMS, as described in Blood. Enrichment of protein-
bound
L-[ring-
2
H
5
]phenylalanine was determined by GC-MS
(model 5973, Hewlett-Packard) with a splitless injection and
positive electron impact ionization. Mass-to-charge ratios
(m/z ) 338 and 341 were monitored. These ions are the m⫹3
and m⫹5 enrichments, respectively, where m⫹0 is the lowest
molecular weight of the ion. The ratio of m⫹5/m⫹3 was used
because it is more sensitive than the traditional m⫹5/m ⫹0
(used for blood samples). Enrichment from the protein-bound
samples was determined with a linear standard curve of
known m⫹5/m ⫹3 ratios and corrected back to the absolute
change in m⫹5 enrichment over the incorporation period.
Calculations
Chemical net amino acid balance (NB) across the leg was
calculated from the difference between the femoral arterial
and venous concentrations multiplied by the blood flow. Thus
NB  共C
a
 C
v
兲 䡠 BF
where C
a
is arterial amino acid concentration, C
v
is venous
amino acid concentration, and BF is leg blood flow.
Area under the curve was used to calculate total, as well as
essential and nonessential, amino acid uptake (mg) across
the leg for a given time period. The resting value was used as
baseline, so that all values reflected the uptake due to inges-
tion of EAC. The amount of phenylalanine that was taken up
by the leg and utilized for protein synthesis was calculated by
C
m4
 C
m1
 C
m4⫺m1
where C
m4
and C
m1
are the phenylalanine concentrations in
the intracellular pool of the final (4th) and initial (1st) muscle
biopsy. C
m4-m1
is the amount of phenylalanine remaining in
the muscle at the end of the study.
C
m4⫺m1
䡠 LV 䡠 0.6  total Phe
where total Phe is the total amount of phenylalanine remain-
ing in the leg at the end of the study, LV is leg volume, and
0.6 is the volume of leg that is muscle (10).
uptake  total Phe  Phe for MPS
where uptake is uptake of phenylalanine across the leg, and
Phe for MPS is the amount of phenylalanine taken up by the
leg and utilized for muscle protein synthesis.
Because phenylalanine is not metabolized in muscle, mus-
cle protein synthesis and breakdown can be estimated using
E199ANABOLIC RESPONSE OF MUSCLE TO SUPPLEMENT TIMING
AJP-Endocrinol Metab • VOL 281 • AUGUST 2001 • www.ajpendo.org

the NB across the leg and the arterial and venous enrich-
ments of
L-[ring-
2
H
5
]phenylalanine blood (26, 29). The rate of
appearance (R
a
) and rate of disappearance (R
d
)ofL-[ring-
2
H
5
]phenylalanine were calculated as estimations of muscle
protein breakdown and muscle protein synthesis, respec-
tively, from plasma amino acids in the blood (25, 29)
R
a
 共E
a
/E
v
 1兲 䡠 Ca 䡠 BF
where E
a
is arterial enrichment of L-[ring-
2
H
5
]phenyl-
alanine, E
v
is venous enrichment, and R
d
is NB ⫹ R
a
.
R
a
,R
d
, and NB were calculated for four time periods by
combining the individual measurements within each period
and using the mean values in the calculations.
Data Presentation and Statistical Analysis
Data are presented as means ⫾ SE. Results across time for
phenylalanine concentration were compared by one-way
ANOVA, with significance set at P ⬍ 0.05. When the overall
P ⬍ 0.05, Dunnett’s post hoc test was used to detect individ-
ual differences between rest and each time point. Differences
between PRE and POST for each time period and for total
phenylalanine uptake were detected with Student’s t-test
with unpooled variances, with significance set at P ⬍ 0.05.
Leg blood flow, phenylalanine enrichment, R
a
,R
d
, NB,
delivery to the leg, and muscle concentration are presented
as means of four periods: Rest, Exercise, Hour 1 Postexercise,
and Hour 2 Postexercise. The model used to determine sta-
tistical differences for each of these variables (except for
muscle concentration) is of the form
Y
s,Tr,t
 S
s

兺
j  1
3
A
Tr,t
t
j
 error
where s is 1,2,. . .,6 (S
s
is the effect of subject s), Tr is 0,1 (Tr
is the treatment, 0 is PRE, 1 is POST), t (the time period) is
1, 2, 3, or 4.
For muscle concentration, the model (which requires dele-
tion of the one subject who did not participate in the POST
part of the study) is
Y
s,Tr,t
 S
s,Tr

兺
j  1
3
A
Tr,t
t
j
 error
This change was made because of the apparently large
change in baseline between PRE and POST studies for some
of the subjects. The object of the analysis is to determine in
which, if any, time periods (t ⫽ 1, 2, 3, or 4) the conditions
兺
j  1
3
A
0,t
t
j

兺
j  1
3
A
1,t
t
j
are satisfied, and for
Tr  0, 1 and ␶
which of the following hold
兺
j  1
3
A
Tr,t
t
j

兺
j  1
3
A
Tr,␶
␶
j
and in those cases to obtain some idea of the magnitudes of
the change from the first to the second term. A general linear
model program was run with the measured data to address
these questions.
RESULTS
Blood Phenylalanine Concentrations
and Enrichments
Ingestion of EAC resulted in significant hyperami-
noacidemia in both the PRE and POST trials (Fig. 2).
Mean phenylalanine concentration increased by ⬃67%
in the first 10 min of exercise and was significantly
increased over resting levels by 10 min after exercise
during the PRE trial. Phenylalanine concentration in-
creased further after cessation of exercise and peaked
⬃30 min postexercise at levels ⬃135% above basal.
Phenylalanine concentration declined from 30 min post-
exercise until 120 min postexercise. During POST,
mean phenylalanine concentration was unchanged
during exercise, increased significantly at 20 min post-
exercise, peaked at ⬃130% of resting values 30 min
postexercise, and then declined steadily until 120 min
postexercise.
Mean enrichments of
L-[ring-
2
H
5
]phenylalanine are
presented as means of the four time periods in Table 1.
Arterial enrichment was decreased from rest during
exercise in both trials and in the 2nd h postexercise in
POST. Arteriovenous difference in enrichments was
decreased during exercise during both trials and dur-
ing the 1st h after exercise during PRE.
Fig. 2. Arterial and venous phenylalanine concentrations over time
for PRE trial (EAC before exercise, top) and POST trial (EAC after
exercise, bottom). *Significantly different from resting levels (time
⫺55), P ⬍ 0.05.
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Muscle Phenylalanine Concentrations
Muscle intracellular free phenylalanine concentra-
tions are summarized in Fig. 3. Phenylalanine concen-
trations in muscle were significantly greater at rest
during the PRE trial than during POST. During PRE,
muscle phenylalanine concentration was increased
46% by the end of exercise and was further increased to
86% above basal levels 1 h after exercise. Two hours
after exercise, and thus 3 h after ingestion of EAC,
muscle phenylalanine concentrations were 65% above
basal. During POST, muscle phenylalanine concentra-
tions were not increased during exercise but were sig-
nificantly elevated above rest and exercise levels at 2 h
postexercise, i.e., 2 h after ingestion of EAC, respec-
tively. When the differences in resting values are ac-
counted for, muscle phenylalanine concentration was
not significantly different between PRE and POST at
any time point.
Blood Flow and Phenylalanine Delivery to the Muscle
Blood flow to the leg at rest was not different be-
tween treatments (Table 2). Resistance exercise signif-
icantly increased leg blood flow by ⬃324% during PRE
and by ⬃201% during POST. In the 1st h after exer-
cise, leg blood flow was still significantly elevated
above rest during both trials, but there was no differ-
ence from rest during the 2nd h. During exercise and in
the 1st h after exercise, leg blood flow was significantly
greater for PRE than for POST.
Amino acid delivery to the leg (C
a
⫻ BF) at rest was
not significantly different between trials (Table 2).
During exercise, delivery was increased by ⬃650% in
the PRE trial and by almost 250% in the POST trial.
Delivery remained elevated above resting levels during
the 1st h after exercise for both trials but was not
increased in the 2nd h postexercise. Phenylalanine
delivery to the muscle was greater in PRE than POST
during exercise and the 1st h after exercise.
Plasma Insulin
Arterial insulin values for each time period are
shown in Table 3. Insulin levels significantly increased
after EAC consumption in each trial, i.e., during exer-
cise for PRE and immediately after exercise for POST.
Insulin remained elevated during the 1st h postexer-
cise in PRE and returned to resting levels by the 2nd h
postexercise in both trials.
Phenylalanine Uptake Across the Leg
Figure 4 shows the net phenylalanine uptake across
the leg measured over 3 h for the PRE and POST trials.
Net uptake of phenylalanine was ⬃160% greater in
PRE than in POST during the entire 3 h. The percent-
age of ingested phenylalanine that was taken up by the
leg was almost threefold greater (P ⫽ 0.01) during PRE
(21 ⫾ 4%) than during POST (8 ⫾ 2%), or 42 ⫾ 8 vs.
16 ⫾ 4% for PRE vs. POST, respectively, for both legs.
More phenylalanine remained in the muscle intracel-
lular pool of the leg at the end of the study in POST
than in PRE (P ⫽ 0.04; 24 ⫾ 3 and 42 ⫾ 8 for PRE and
POST, respectively). Thus, over the3hofthestudy,
180 ⫾ 50 mg of phenylalanine were taken up and
incorporated into protein during PRE and 39 ⫾ 18 mg
during POST (P ⫽ 0.02).
When these values are calculated for only the final
2 h of each trial, the differences narrow from the full
3 h and do not reach statistical significance, but the
trend for PRE values to be greater than POST remains.
Phenylalanine uptake for only the 2 h postexercise was
243 ⫾ 120 mg phenylalanine for PRE and 130 ⫾ 45 mg
phenylalanine for POST (P ⫽ 0.19). The mean percent-
age of ingested phenylalanine taken up by one leg in
the final 2 h postexercise only was 80% greater during
PRE (25 ⫾ 12%) than during POST (13 ⫾ 5%; P ⫽
Table 1. Mean arterial and venous phenylalanine enrichments and arteriovenous difference in enrichments
in PRE and POST trials
Rest Exercise 1 H Post-Ex 2 H Post-Ex
Artery PRE 0.0884⫾0.0134 0.0724⫾0.0084* 0.0691⫾0.0092 0.0759⫾0.0126
POST 0.0886⫾ 0.0165 0.0687⫾ 0.0108* 0.0664⫾ 0.0117 0.0691⫾ 0.121*
Vein PRE 0.0655⫾0.0087 0.0680⫾0.0078 0.0641⫾0.0079 0.0667⫾0.0092
POST 0.0656⫾ 0.0118 0.0643⫾ 0.0099 0.0598⫾ 0.0106 0.0601⫾ 0.0100
a-v Difference PRE 0.0151⫾0.0072 0.0044⫾0.0011* 0.0054⫾0.0018* 0.0092⫾0.0038
POST 0.0230⫾ 0.0049 0.0044⫾ 0.0010* 0.0067⫾ 0.0011 0.0090⫾ 0.0022
Values are enrichments ⫾ SE, expressed as tracer-to-tracee ratio (t/T). a-v, arteriovenous; PRE, value when essential amino acid-
carbohydrate (EAC) drink was consumed before exercise. POST, value when EAC was consumed after exercise. Rest, mean value for resting
time period. Exercise, mean value during exercise bout. 1 H Post-Ex, mean value for samples taken 0–60 min after exercise bout. 2 H Post-Ex,
mean value for samples taken 60–120 min after exercise bout. *Significantly different from Rest, P  0.05.
Fig. 3. Muscle free intracellular concentration (IC) of phenylalanine
for 4 muscle biopsies during PRE and POST trials. *Significantly
different from biopsy 1 (rest), P ⬍ 0.05.
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0.20). Similarly, the mean amount of phenylalanine
taken up during the final 2 h after EAC ingestion (i.e.,
during exercise and the 1st h after exercise for PRE
and the 2 h after exercise for POST) in each trial was
195 ⫾ 37 mg for PRE and 130 ⫾ 45 for POST, P ⫽ 0.14.
Phenylalanine Kinetics
Figure 5 summarizes phenylalanine R
a
,R
d
, and NB
for each time period during PRE and POST trials.
Phenylalanine R
a
did not change significantly from
resting levels during or after exercise in either PRE or
POST. PRE and POST R
a
values were not statistically
different at any time point. R
d
increased from Rest in
the hour immediately after EAC consumption by 216%
during PRE (exercise) and by 60% during POST (1st h
after exercise) trials. PRE R
d
was significantly greater
than POST R
d
during exercise and in the 1st h after
exercise. R
d
was not different for PRE and POST in the
2nd h after exercise.
During PRE, NB changed from negative at rest to
positive values during exercise and the 1st h postexer-
cise. During POST, NB was negative at rest and during
exercise but increased to positive values after exercise,
when the EAC drink was consumed. NB during POST
immediately returned to zero in the 2nd h after exer-
cise. NB was significantly greater during exercise and
in the 1st h after exercise in the PRE trial than in the
POST trial.
DISCUSSION
This study was designed to determine whether the
response of muscle protein metabolism to an EAC
solution was different if consumed immediately before
resistance exercise rather than immediately after re-
sistance exercise. Ingestion of EAC changed net muscle
protein balance from negative values, i.e., net release,
to positive net uptake, in both trials. However, the
total response to the consumption of EAC immediately
before exercise was greater than the response when
EAC was consumed immediately after exercise. Fur-
thermore, it appears that the change from a catabolic
state in the muscle to an anabolic state was primarily
due to an increase in muscle protein synthesis.
In the present study, the effectiveness of the drink
appeared to be greater when it was consumed imme-
diately before exercise (PRE) compared with immedi-
ately after exercise (POST). Approximately 209 ⫾ 42
mg of phenylalanine were taken up across the leg in
the PRE trial, whereas only 81 ⫾ 19 mg of phenylala-
nine were taken up during POST. Whereas the re-
sponse of muscle protein metabolism increased dra-
matically and then declined within1htobasal levels
after EAC consumption in the POST trial, the response
was sustained in the PRE trial. Net balance increased
during exercise, declined slightly, and then increased a
second time after exercise when the drink was con-
sumed before exercise. The length of the effect, plus
higher blood flow during exercise in the PRE trial,
resulted in significantly greater total uptake over the
entire study period.
In this study, the primary end point was to examine
the impact of the timing of EAC ingestion in relation to
resistance exercise on net muscle protein synthesis
and, as a result, the accretion of muscle. Thus the
response over the entire 3-h study period is the most
appropriate to compare between trials. On the other
hand, it could be argued that the results are biased
toward the PRE trial by calculating the data over the
entire 3-h study period. During PRE, the entire 3 h
follows the consumption of EAC, whereas during
POST, only 2 of the 3 h follow EAC ingestion. As a
result, we also calculated the uptake across the leg
over only the final 2 h after exercise of each trial, i.e.,
the 2nd and 3rd h after EAC ingestion during PRE and
the 1st and 2nd h after EAC ingestion during POST.
Calculated this way, the gap between the trials nar-
rowed, but the mean uptake across the leg was still
Fig. 4. Net phenylalanine uptake across the leg over 3 h for PRE and
POST trials. *Significantly different from POST (P ⫽ 0.013).
Table 2. Mean blood flow and delivery of phenylalanine to the leg for PRE and POST trials
Rest Exercise 1 H Post-Ex 2 H Post-Ex
Blood flow, ml䡠 min
⫺1
䡠 100 ml LV
⫺1
PRE 4.59⫾0.58 19.46⫾2.24* 7.64⫾1.73* 5.14⫾ 0.74
POST 3.67⫾0.46 11.05⫾1.28*† 4.72⫾0.36*† 3.35⫾0.32
Phe delivery, nmol䡠 min
⫺1
䡠 100 ml LV
⫺1
PRE 253⫾32 1,890⫾ 396* 828⫾129* 539⫾ 80
POST 191⫾28 654⫾80*† 506⫾ 97*† 341⫾59
Values are means ⫾ SE. Delivery of phenylalanine to the leg is calculated by blood flow ⫻ arterial concentration. LV, leg volume.
*Significantly different from Rest, P  0.05. †Significantly different from corresponding PRE value, P  0.05.
Table 3. Mean arterial insulin levels during 4 time
periods for PRE and POST trials
Rest Exercise 1 H Post-Ex 2 H Post-Ex
PRE 4.5⫾0.5 18.5⫾ 5.7 22.0⫾6.2 6.2⫾2.0
POST 4.1⫾0.8 8.5⫾2.4 27.0⫾ 5.8 6.6⫾ 1.2
Values are means ⫾ SE, expressed in IU/ml. Both PRE and POST
were significantly different across time, but individual significant
differences were not identifiable.
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80% greater for PRE than for POST (244 ⫾ 120 mg vs.
130 ⫾ 45 mg, respectively), although the difference did
not reach statistical significance (P ⫽ 0.09). If any-
thing, comparing only the final2hofeach trial biases
the results toward favoring the POST trial, because the
1st h after consumption of EAC during PRE is ignored.
Nonetheless, it is still evident that consuming EAC
before exercise is more effective than after exercise.
Fig. 5. Muscle phenylalanine rate of appearance
from muscle (R
a
), phenylalanine uptake from blood
(R
d
), and net phenylalanine balance across leg
(NB) for 4 time periods during PRE (open bars) and
POST (solid bars). Rest, mean of 3 resting values.
Ex, mean of 4 samples taken during resistance
exercise. Hr 1 PE, mean of 4 samples taken during
the 1st h after exercise. Hr 2 PE, mean of 3 sam-
ples taken during the 2nd h after exercise. *PRE
significantly different from POST, P ⬍ 0.05.
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Effectiveness of the timing of EAC ingestion is sup-
ported by comparing the amount of phenylalanine
taken up by the leg to the amount ingested in each
trial. During PRE, ⬃21% of ingested phenylalanine
was taken up by the leg, thus ⬃42% by both legs. The
proportion was much lower during POST, ⬃8% across
one leg or 16% for both legs. When EAC was consumed
1 h after exercise, ⬃125 mg of phenylalanine were
taken up across the leg (21), or about one-half of the
value found when EAC was consumed before exercise.
This represented ⬃11% of the ingested phenylalanine
for one leg, or 22% for both legs. When amino acids
were infused over a 3-h period after exercise, ⬃34% of
the infused amino acids were taken up across both legs
(6). Clearly, EAC consumption before exercise is more
effective than after exercise.
These data do not allow us to determine definitively
the reasons for the greater response of net muscle
protein synthesis to consuming essential amino acids
plus carbohydrates immediately before exercise rather
than after exercise. However, it is likely that the
greater delivery of amino acids to the muscle during
PRE accounts for the greater net uptake than during
POST. During exercise in the POST trial, net muscle
protein balance, as well as phenylalanine R
d
, an index
of muscle protein synthesis, was unchanged, whereas
in the PRE trial, phenylalanine R
d
and NB were in
-
creased. Consuming a source of amino acids before
exercise increases amino acid availability. Providing
amino acids at a time when blood flow is elevated, such
as during the exercise bout, maximizes delivery to the
muscle. Previous studies have demonstrated that mus-
cle protein synthesis is related to amino acid delivery
to the leg (5, 6, 27). Phenylalanine delivery during
exercise in the PRE trial was increased 6.5-fold over
resting levels and was more than twice that of POST.
Furthermore, delivery remained elevated after exer-
cise during PRE to a significantly greater extent above
that during POST. Similarly, in our previous study,
amino acid delivery was increased by EAC ingestion at
both 1 and 3 h postexercise (21) to levels comparable to
those obtained when EAC was consumed immediately
after exercise. Thus consumption of amino acids before
exercise results in greater amino acid delivery than
when they are consumed at various time points after
exercise, likely accounting for the greater response of
net muscle protein synthesis demonstrated during the
PRE trial.
Previously, we showed that hyperaminoacidemia
elicited by intravenous infusion of mixed amino acids
(6) and oral ingestion of both mixed and essential
amino acids (27) resulted in net muscle protein synthe-
sis after resistance exercise. In these studies, ⬃40gof
amino acids were provided steadily over a 3-h period.
We also demonstrated that nonessential amino acids
are unnecessary to stimulate net muscle protein syn-
thesis at rest (28) or after exercise (27). Subsequently,
we examined the response of muscle protein metabo-
lism to ingestion of a smaller amount of essential
amino acids plus carbohydrates (21) identical to the
one used in the present study. Similar levels of net
muscle protein synthesis resulted when subjects con-
sumed the bolus amino acid-carbohydrate solution at
both 1 and 3 h after exercise (21). Taken together with
the present results, it is clear that a relatively small
amount of essential amino acids, combined with carbo-
hydrates, is a potent stimulator of net muscle protein
synthesis when given either before or at various times
after resistance exercise.
It is not possible to delineate the effectiveness of the
separate components of the drink from this study. We
have previously demonstrated that muscle protein syn-
thesis is stimulated by essential amino acids alone (27,
28). Even single essential amino acids in a flooding
dose may stimulate muscle protein synthesis (24). It is
more difficult to assign a role to insulin in the change
from net negative protein balance to positive protein
balance. After exercise, insulin seems to be necessary
for protein synthesis to occur (11, 12, 14), yet increased
insulin does not stimulate muscle protein synthesis (7).
However, elevated insulin after resistance exercise
does diminish the increase of muscle protein break-
down in response to exercise (7). Consistent with this
notion, during the present study, phenylalanine R
a
,an
index of muscle protein breakdown, did not increase
after exercise in either trial. Thus stimulation of mus-
cle protein synthesis by essential amino acids, in addi-
tion to inhibition of the normal postexercise rise in
breakdown, likely accounts for the effectiveness of the
EAC drink for stimulating net muscle protein synthe-
sis after resistance exercise.
Determination of the response of the muscle in the
present study is based primarily on uptake of phenyl-
alanine across the leg. It is assumed that phenylala-
nine uptake corresponds to accretion of muscle protein.
However, it is possible that all of the amino acids taken
up by the muscle are not incorporated into protein, but
instead some fraction of the uptake simply expands the
muscle free intracellular pool. The amino acids could
then be released at some time after the conclusion of
the measurements, without ever being utilized for
muscle protein synthesis. Thus it is possible that net
uptake overestimated the extent of net muscle protein
synthesis. However, even if we assume the unlikely
circumstance that all of the phenylalanine remaining
in the muscle intracellular pool at the conclusion of the
study would be subsequently released, the amount
does not appear to be a substantial proportion of that
taken up by muscle, especially in the PRE trial. During
PRE, 24 ⫾ 3 mg of phenylalanine were taken up by
muscle but not utilized for protein synthesis, in con-
trast to 42 ⫾ 8 mg during POST. Thus the total amount
of phenylalanine taken up by the leg and utilized for
protein synthesis was ⬃180 mg (⬃86% of total uptake)
during PRE and ⬃39 mg (⬃48% of total uptake) during
POST. Clearly, even with this conservative estimate, a
large proportion of the phenylalanine taken up by
muscle was, in fact, utilized for muscle protein synthe-
sis during the study, further supporting the notion that
the EAC solution is an effective stimulator of muscle
protein anabolism.
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In the fasted state, muscle protein breakdown ex-
ceeds muscle protein synthesis, resulting in a net neg-
ative muscle protein balance. Net positive muscle pro-
tein balance can result only from an increase in muscle
protein synthesis and/or a decrease in muscle protein
breakdown. Resistance exercise alone has been shown
to increase muscle protein synthesis, but breakdown is
also increased, such that net muscle protein balance
remains negative (5). Additionally, net muscle protein
synthesis as a consequence of hyperaminoacidemia af-
ter resistance exercise is primarily due to increased
muscle protein synthesis (6, 27). In our previous study,
increased muscle protein synthesis was responsible for
the change from a catabolic to an anabolic state after
ingestion of EAC at both 1 and 3 h postexercise (21).
Similarly, in the present study, it is likely that the
increase in NB from negative to positive after EAC
consumption in both trials was also primarily due to an
increase in muscle protein synthesis. Mean R
d
, i.e.,
uptake of amino acids from the plasma pool, increased
dramatically (216 and 200% for PRE and POST, re-
spectively) after ingestion of EAC. The fact that phe-
nylalanine R
a
, an indicator of muscle protein break
-
down, did not change in response to EAC ingestion
further supports the notion that the change of net
muscle protein balance from positive to negative is
primarily due to an increase in protein synthesis.
In the present study, our arteriovenous tracer meth-
odology has quantified only the fate of blood-borne
amino acids (25, 29). Because the incorporation of
amino acids from the EAC solution into muscle protein
was of primary interest, R
d
and R
a
calculated using
blood-borne amino acids seemed the most appropriate
measures. In past studies we have utilized a three-
compartment model of muscle protein metabolism to
describe the effects of nutrition and exercise on muscle
protein synthesis and breakdown (3, 5, 6, 14, 15, 27).
However, in the present study, the combination of
sampling in close proximity to exercise and a bolus
ingestion of amino acids has made the use of that
model problematic. That model requires an isotopic
and physiological steady state, as well as a measurable
gradient between blood and intracellular phenylala-
nine enrichment. Instead, we calculated R
a
and R
d
by
use of data only from blood (25, 29). Whereas care must
be taken in interpreting R
a
and R
d
values from this
model (3, 30), it is the appropriate model to use in the
present study. The importance of the plasma amino
acids as a source for muscle protein synthesis is em-
phasized in this study. Therefore utilization of R
d
was
the appropriate parameter with which to compare the
effects of the timing of ingestion of the EAC drink.
Moreover, utilization of the blood-borne precursor for
measurement of R
d
allows us to relate these values to
net muscle protein synthesis determined by phenylal-
anine uptake.
The ingestion of a relatively small amount of essen-
tial amino acids, combined with carbohydrates, is an
effective stimulator of net muscle protein synthesis.
The stimulation of net muscle protein synthesis when
EAC is consumed before exercise is superior to that
when EAC is consumed after exercise. The combina-
tion of increased amino acid levels at a time when blood
flow is increased appears to offer the maximum stim-
ulation of muscle protein synthesis by increasing
amino acid delivery to the muscle and thus amino acid
availability.
We thank the nurses and staff of the General Clinical Research
Center (GCRC) at the University of Texas Medical Branch in
Galveston, TX. We also thank Dr. J. Rosenblatt for statistical assis-
tance, and the volunteers who participated in the studies for their
time and hard work.
This work was supported in part by Grants 8940 and 15489 from
the Shriners Hospitals for Children and National Institutes of
Health (NIH) Grant R01–38010. Studies were conducted at the
GCRC at the University of Texas Medical Branch at Galveston,
which is funded by a grant (M01 RR-00073) from the National Center
for Research Resources, NIH.
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