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This study tests the hypothesis that a dose of 6 g of orally administered essential amino acids (EAAs) stimulates net muscle protein balance in healthy volunteers when consumed 1 and 2 h after resistance exercise. Subjects received a primed constant infusion of L-[(2)H(5)]phenylalanine and L-[1-(13)C]leucine. Samples from femoral artery and vein and biopsies from vastus lateralis were obtained. Arterial EAA concentrations increased severalfold after drinks. Net muscle protein balance (NB) increased proportionally more than arterial AA concentrations in response to drinks, and it returned rapidly to basal values when AA concentrations decreased. Area under the curve for net phenylalanine uptake above basal value was similar for the first hour after each drink (67 +/- 17 vs. 77 +/- 20 mg/leg, respectively). Because the NB response was double the response to two doses of a mixture of 3 g of EAA + 3 g of nonessential AA (NEAA) (14), we conclude that NEAA are not necessary for stimulation of NB and that there is a dose-dependent effect of EAA ingestion on muscle protein synthesis.
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Essential amino acids and muscle protein recovery
from resistance exercise
ELISABET BØRSHEIM, KEVIN D. TIPTON, STEVEN E. WOLF, AND ROBERT R. WOLFE
Metabolism Unit, Department of Surgery, Shriners Hospital for Children/Galveston,
University of Texas Medical Branch, Galveston, Texas 77550
Received 15 October 2001; accepted in final form 26 May 2002
Børsheim, Elisabet, Kevin D. Tipton, Steven E. Wolf,
and Robert R. Wolfe. Essential amino acids and muscle
protein recovery from resistance exercise. Am J Physiol En-
docrinol Metab 283: E648E657, 2002; 10.1152/ajpendo.
00466.2001.—This study tests the hypothesis that a dose of
6 g of orally administered essential amino acids (EAAs)
stimulates net muscle protein balance in healthy volunteers
when consumed 1 and 2 h after resistance exercise. Subjects
received a primed constant infusion of
L-[
2
H
5
]phenylalanine
and
L-[1-
13
C]leucine. Samples from femoral artery and vein
and biopsies from vastus lateralis were obtained. Arterial
EAA concentrations increased severalfold after drinks. Net
muscle protein balance (NB) increased proportionally more
than arterial AA concentrations in response to drinks, and it
returned rapidly to basal values when AA concentrations
decreased. Area under the curve for net phenylalanine up-
take above basal value was similar for the first hour after
each drink (67 17 vs. 77 20 mg/leg, respectively). Be-
cause the NB response was double the response to two doses
of a mixture of3gofEAA 3 g of nonessential AA (NEAA)
(14), we conclude that NEAA are not necessary for stimula-
tion of NB and that there is a dose-dependent effect of EAA
ingestion on muscle protein synthesis.
muscle protein metabolism; essential amino acids; stable
isotopes
MUSCLE PROTEIN SYNTHESIS is stimulated in the recovery
period after resistance exercise (4, 8). However, the
rate of muscle protein breakdown is also increased,
thereby blunting the change in the net balance be-
tween synthesis and breakdown. Although net muscle
protein balance is generally improved after resistance
exercise, it remains negative. Therefore, nutrient in-
take is necessary to achieve positive net muscle protein
balance.
The optimal composition and amount of nutrient
ingestion to maximally stimulate muscle protein syn-
thesis after resistance exercise are not known. It is
clear that amino acids or protein should be a compo-
nent, as we have previously shown that either the
infusion (5) or ingestion of a large amount (17) (3040
g) of amino acids after exercise stimulates muscle pro-
tein synthesis. Furthermore, muscle protein synthesis
was increased 3.5-fold when only a small amount (6 g)
of a mixture of essential amino acids (EAAs) was given
along with 35 g of carbohydrate after resistance exer-
cise (16). The latter results (16) suggest that a rela-
tively small amount (i.e., 6 g) of EAAs can effectively
stimulate muscle protein synthesis, but the indepen-
dent effects of amino acids and carbohydrate were not
assessed. Thus a principal goal of this study was to
determine the independent effect of ingestion of a bolus
of6gofEAAs on net muscle protein synthesis after
resistance exercise.
The proportional contributions of individual amino ac-
ids to a mixture ingested after resistance exercise can
potentially affect the response. In an earlier study (17),
we concluded that the ingestion of only EAAs was neces-
sary for stimulation of muscle protein synthesis, because
the effect on net muscle protein synthesis was similar
when subjects were given either 40 g of a balanced mix-
ture [21.4 g EAAs and 18.6 g nonessential amino acids
(NEAAs), roughly in proportion to their relative contri-
butions to muscle protein], or 40 g of only EAAs. How-
ever, if the NEAAs are not necessary for stimulation of
synthesis, it is unclear why ingestion of the EAAs alone
did not stimulate muscle protein synthesis to a greater
extent than did the balanced mixture. It could be that, in
fact, the NEAAs served no function, but the amount of
EAAs in the balanced mixture (21 g) exceeded the max-
imal effective dose. In this case, intake of more than 21 g
of EAAs (i.e., 40 g) would have no further effect than that
already elicited by 21 g. If true, then comparison of less
than the maximally effective dose of EAAs with a com-
parable amount of a balanced mixture of EAAs NEAAs
should reveal a significantly greater effect of the EAAs.
Thus a secondary goal of this project was to compare the
response to6gofanEAAmixture with the response to
6 g of a balanced mixture of amino acids, which included
3 g of EAAs and3gofNEAAs. We speculated that 6 g
of EAA would be less than the maximally effective dose of
EAA, and thus there would be a greater response to the
EAAs than to the mixture of EAAs and NEAAs. The
results of the response to6gofthebalanced mixture
have been published previously (14).
The composition of the mixture of EAAs we have
tested in this and previous studies (16) was originally
based on the composition of muscle protein, with the
Address for reprint requests and other correspondence: R. R. Wolfe,
815 Market St., Galveston, TX 77550 (E-mail: rwolfe@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 283: E648–E657, 2002;
10.1152/ajpendo.00466.2001.
0193-1849/02 $5.00 Copyright
©
2002 the American Physiological Society http://www.ajpendo.orgE648
idea being to increase the availability of each EAA in
proportion to its requirement for the synthesis of mus-
cle protein. However, different clearance rates of indi-
vidual amino acids could result in rates of uptake that
do not directly correspond to the composition of the
ingested mixture. This would be reected by dispropor-
tionate changes in concentrations of blood amino acids
compared with the composition of the ingested mix-
ture. Therefore, another goal of the current study was
to measure blood and intramuscular concentrations of
all amino acids before and after ingestion of the mix-
ture to determine whether the mixture achieved the
goal of causing proportional increases in all EAAs, and
if any NEAA dropped sufciently to become potentially
rate limiting for protein synthesis. This information
could help to formulate a new mixture that might be
more effective than the EAA mixture we have used.
Not only is the composition of nutrient ingestion
after exercise potentially important, but also the pat-
tern of ingestion may affect the response of muscle
protein synthesis. We have previously shown that
amino acid concentration, per se, is not a direct deter-
minant of muscle protein synthesis. Thus, when blood
amino acid concentrations were elevated to a steady-
state level about twice the basal values for 6 h, muscle
protein synthesis was stimulated over the rst 2 h but
thereafter returned to the resting level despite persis-
tent elevations in blood amino acid concentrations (6).
Similarly, in our earlier study after exercise (16), the
rate of net muscle protein synthesis returned to the
basal level 60 min after the ingestion of a bolus of
EAAs carbohydrate, even though the blood amino
acid concentrations were still approximately double
the basal level. These observations are best explained
by the muscle becoming refractory to a persistent ele-
vation in amino acid concentrations. If true, it follows
that the synthetic response to ingestion of a bolus of
amino acids would be refractory to a second dose until
the concentrations from the rst dose returned to the
basal level. It was therefore a further goal of this study
to determine whether the response of net muscle pro-
tein synthesis to a second dose of amino acids would be
affected by the ingestion of an initial dose 1 h earlier.
MATERIALS AND METHODS
Subjects. Six healthy subjects (3 men and 3 women), 1925
yr of age, participated in the study (Table 1). Subjects were
recreationally active but were not involved in a consistent
resistance or endurance-training program. They were fully
informed about the purpose and procedures of the study
before written consent was obtained. Before participation in
the experiments, each subject had a complete medical screen-
ing, including vital signs, blood tests, urine tests, and a
12-lead electrocardiogram, for determination of health status
at the General Clinical Research Center (GCRC) of the Uni-
versity of Texas Medical Branch (UTMB) at Galveston, TX.
The protocol was approved by the Institutional Review Board
of the UTMB.
Preexperimental procedures. At least 1 wk before an exper-
iment, subjects were familiarized with the exercise protocol,
and their one repetition maximum (1RM, the maximum
weight that can be lifted for one repetition) was determined
by the procedure described by Mayhew et al. (13) (Table 1).
The leg volume of each subject was estimated from anthro-
pometric measures of leg circumference and height at multi-
ple points down the length of the leg (Table 1).
Experimental protocol. Each subject was studied once. The
subjects were instructed not to exercise for 2 days before an
experiment, not to make any changes in their dietary habits,
and not to use tobacco or alcohol during the last 24 h before
an experiment. The subjects reported to the GCRC in the
evening before an experiment for an overnight stay and were
fasted from 10:00 PM.
The experimental protocol is shown schematically in Fig.
1. At 6:00 AM, an 18-gauge polyethylene catheter (Cook,
Bloomington, IN) was inserted into an antecubital arm vein
for the primed continuous infusion of stable isotopes of amino
acids. After obtaining a blood sample for measurement of
background amino acid enrichment, a primed, constant infu-
sion of [
15
N
2
]urea was started at 180 min (6:30 AM). At
120 min (7:30 AM), a primed, constant infusion of L-[ring-
2
H
5
]phenylalanine and L-[1-
13
C]leucine was started. The fol
-
lowing infusion rates (IR) and priming doses (PD) were used
L-[
2
H
5
]phenylalanine: IR0.10 mol kg
1
min
1
,
PD2 mol/kg
L-[1-
13
C]leucine: IR0.12 mol kg
1
min
1
,
PD4.8 mol/kg
15
N
2
]urea: IR0.2267 mol kg
1
min
1
,
PD88 mol/kg
Isotopes were purchased from Cambridge Isotopes (An-
dover, MA). They were dissolved in 0.9% saline and were
ltered through a 2-m lter before infusion. The infusion
protocol was designed to allow the quantication of the effect
of the drink on muscle protein synthesis and breakdown. The
net balance between protein synthesis and breakdown (net
muscle protein synthesis) was considered to be the primary
end point of the study, and urea production was measured
Table 1. Physical characteristics and exercise data for all subjects
Subject No. Gender, F/M Age, yr Height, m Weight, kg Leg Volume, l 1RM Leg Press, kg 1RM Leg Extension, kg
1 F 25 1.72 57.2 7.48 130.5 113.5
2 F 24 1.76 71.4 11.87 85.0 79.5
3 M 24 1.90 90.7 11.65 146.5 130.5
4 M 25 1.83 82.0 10.51 141.5 113.5
5 F 23 1.63 56.0 7.54 79.5 60.0
6 M 19 1.63 56.8 6.91 136.0 136.0
Means SD 23 2 1.74 0.11 69.0 14.9 9.332.27 119.8 29.6 105.5 29.7
1RM, one repetition maximum.
E649ESSENTIAL AMINO ACIDS AND EXERCISE
AJP-Endocrinol Metab VOL 283 OCTOBER 2002 www.ajpendo.org
isotopically to assess short-term changes in total amino acid
oxidation.
At 7:30 AM, 3-Fr 8-cm polyethylene catheters (Cook)
were inserted into the femoral vein and the femoral artery
with the subject under local anesthesia. Both femoral cathe-
ters were used for blood sampling, and the femoral arterial
catheter was also used for indocyanine green dye (ICG)
infusion for determination of leg blood ow (3). A constant
infusion of ICG (0.5 mg/min) was given at intervals during
the experiment (Fig. 1). The infusion ran for 10 min before
peripheral and femoral venous blood samples were drawn for
measurement of blood ow. The peripheral venous blood
samples were drawn from an 18-gauge polyethylene catheter
inserted into an antecubital vein of the arm opposite that into
which the amino acids were infused. Patency of catheters was
maintained by saline infusion.
Subjects rested in bed until the exercise started at 45
min (8:45 AM). Subjects performed 10 sets of 10 repetitions of
leg presses and 8 sets of 8 repetitions of leg extensions at 80%
of the 1RM. Each set was completed in 30 s with a 2-min
rest between sets, and the entire bout was completed in 40
min. This exercise bout was difcult for all subjects to com-
plete. The exercise ended 3 h after the start of the urea
infusion and 2 h after the start of the amino acid infusion.
Subjects then returned to bed, and the rst samples were
taken 30 min after the end of exercise.
At 1 and 2 h postexercise, the subjects were given an oral
supplement of 0.087 g of essential amino acids (EAA)/kg body
weight. The nutritional composition was designed to increase
intramuscular availability of EAA in proportion to the com-
position of muscle protein (Table 2). Each supplement solu-
tion was composed of 425 ml of double-distilled water, the
appropriate mixture of EAA, and an articial sweetener.
L-[ring-
2
H
5
]phenylalanine and L-[
13
C]leucine were added to
the drink in amounts to equal 8% enrichment, to allow
maintenance of isotopic equilibrium during ingestion of the
AA drinks.
To measure the isotopic enrichment of free amino acid
tracers in the muscle, muscle biopsies were sampled at 30,
90, 150, and 240 min after exercise (Fig. 1). With subjects
under local anesthesia, the biopsies were taken from the
lateral portion of the vastus lateralis 1015 cm above the
knee. A 5-mm Bergstrom biopsy needle (Depuy, Warsaw, IN)
was used to sample 3050 mg of mixed muscle tissue. The
sample was quickly rinsed, blotted, immediately frozen in
liquid nitrogen, and stored at 80°C for later analysis.
Blood samples were drawn for determination of net muscle
protein balance from the femoral artery and venous catheters
at 30 min after exercise, at 70, 80, 90, 105, 130, 140, 150, and
165 min after exercise (corresponding to 10, 20, 30, and 45
min after each drink), and at 180, 210, 220, and 240 min after
exercise (Fig. 1). The samples were analyzed for phenylala-
nine and leucine enrichments and concentrations. To allow
sampling from the femoral artery, the dye infusion was
stopped for 10 s and then quickly resumed.
Sample analyses. Blood samples for determination of
amino acid enrichment and concentrations were immediately
precipitated in preweighed tubes containing 15% sulfosali-
cylic acid (SSA), and a weighed amount of an appropriate
internal standard consisting of amino acids labeled differ-
ently from the infused amino acids was added (3, 4, 15). The
supernatant was passed over a cation exchange column
(Dovex AG 50W-8X, 100200 mesh H
form; Bio-Rad Labo
-
ratories, Richmond, CA) and dried under vacuum with a
Speed Vac (Savant Instruments, Farmingdale, NY). Enrich-
ments of intracellular free amino acids were then determined
on the tertiary-butyldimethylsilyl (t-BDMS) derivatives us-
ing GC-MS (Hewlett-Packard 5973, Palo Alto, CA) and se-
lected ion monitoring (21). Enrichments were expressed as
tracer-to-tracee ratios. Appropriate corrections were made
for overlapping spectra (21).
To determine muscle intracellular enrichment of infused
tracers, muscle tissue was weighed and the protein precipi-
tated with perchloroacetic acid. The tissue was then homog-
enized and centrifuged, and the supernatant was collected.
The procedure was then repeated, and the pooled superna-
tant was processed in the same way as the supernatant from
the blood samples.
Urea production was calculated from enrichment and
tracer infusion rates, as described previously (20). ICG con-
centration in serum for the determination of leg blood ow
was measured spectrophotometrically at ␭⫽805 nm (10, 19).
Plasma samples and muscle intracellular uid were also
analyzed for amino acid concentrations by high-performance
liquid chromatography (Waters Alliance HPLC System 2690,
Milford, MA). Plasma glucose concentration was determined
enzymatically by an automated system (YSI 1500, Yellow
Spring Instruments, Yellow Springs, OH). Plasma insulin
concentration was determined by a radioimmunoassay
method (Diagnostic Products, Los Angeles, CA).
Calculations. Net muscle phenylalanine balance, which
was considered as the primary end point, was calculated as
follows: (phenylalanine arterial concentration venous
concentration) blood ow. Because phenylalanine is nei-
ther produced nor metabolized in muscle, net phenylala-
nine balance reects net muscle protein synthesis, pro-
vided there are no signicant changes in the free
intracellular pool of phenylalanine. Area under the curve
(AUC) of net phenylalanine uptake was determined for
each individual hour after drink ingestion, with net uptake
at t 30 min postexercise used as the zero point for each
hour of the recovery period. This approach assumes a
Fig. 1. Infusion protocol. ICG, indocyanine green; D, drink; *, blood
sample; B, biopsy.
Table 2. Amino acid composition of drink
Amino Acid % of Total AAs Grams in EAA Drink
Histidine 10.9 0.6540
Isoleucine 10.1 0.6060
Leucine 18.6 1.1160
Lysine 15.5 0.9300
Methionine 3.1 0.1860
Phenylalanine 15.5 0.9300
Threonine 14.7 0.8820
Valine 11.5 0.6900
Total 99.9 5.994
AA, amino acid; EAA, essential amino acids. Composition is based
on a 70-kg person. Drink was given at 1 and 2 h after completion of
exercise.
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constant net balance after the basal sample if amino acids
are not given. The basis for this assumption is the rela-
tively constant net balance for 3 h after exercise in the
absence of nutrient intake that we have previously ob-
served when using the same exercise protocol (16).
Leg amino acid kinetics were calculated according to a
three-pool compartment model previously presented (2, 3).
Hourly averages for blood ow and blood and muscle amino
acid concentrations and enrichments were used in the
calculation of leg amino acid kinetics. Kinetic parameters
calculated for both amino acid tracers included intracellu-
lar de novo appearance, irreversible disappearance from
the intracellular compartment, and the rate of transport
from blood into muscle. In the case of phenylalanine,
irreversible loss from the intracellular pool can only be to
protein synthesis, because it is not oxidized in the muscle.
Because leucine can be oxidized in muscle, the irreversible
loss is due to synthesis oxidation. Neither leucine nor
phenylalanine can be synthesized in muscle, so de novo
appearance of both amino acids is due entirely to break-
down. We also calculated the rate of release of phenylala-
nine and leucine from protein breakdown into blood (R
a
)
and incorporation of phenylalanine from blood into muscle
protein (R
d
). Calculation of R
a
and R
d
, as well as the
three-pool kinetic factors, requires an isotopic, but not
physiological, steady state. By adding an appropriate
amount of tracer to the ingested amino acids, we were able
to maintain a relatively stable isotopic steady state, de-
spite changing concentrations of plasma amino acids (see
below). Thus the difference between total protein synthesis
and R
d
is the amount of recycling of phenylalanine that
was released from breakdown and directly incorporated
into protein without entering the blood. Similarly, the
difference between total protein breakdown and R
a
is the
amount of phenylalanine from breakdown that was di-
rectly reincorporated into protein, rather than being re-
leased into blood.
Statistical methods. Overall signicance of differences in
response with time was tested by repeated-measures analy-
sis of variance followed by Dunnetts test (SigmaStat 2.03,
SPSS, Chicago, IL). The response to the second drink was
compared with the response to the rst drink by Tukeys test.
Results were considered signicant if P 0.05. The results
are presented as means SE unless otherwise noted.
RESULTS
Phenylalanine concentration and balance. At 30 min
after exercise, the arterial blood phenylalanine concen-
tration was 59 4 nmol/ml. The concentration rose
signicantly within 10 min of ingestion of the EAA
drink (Fig. 2). Phenylalanine concentration declined
before the second drink but stayed signicantly above
predrink values until 240 min after exercise. The re-
sponse to the second drink was comparable to that of
the rst.
Phenylalanine net balance followed the same time
pattern as the blood concentration changes, with rapid
changes in response to arterial blood phenylalanine
changes (Fig. 2). However, the early increase of net
uptake was proportionately greater than the change in
arterial concentration, and the rate of net balance
returned to the basal value 40 min after ingestion of
the rst drink, despite persistent elevation of arterial
phenylalanine concentration. Net balance also in-
creased signicantly in response to the second drink
(Fig. 2) and returned to the basal value during the 3rd
h. The AUC for net uptake of phenylalanine above the
basal value was similar for the 1st h after the rst
drink (67 17 mg/leg) and the 1st h after the second
drink (77 20 mg/leg). However, the AUC decreased
signicantly by the 3rd h (5 20 mg/leg), despite the
fact that the arterial concentration was signicantly
elevated until 240 min after the rst drink.
Muscle intracellular phenylalanine concentration
was 57 3 nmol/ml before intake of the drinks. After
intake of drinks, the concentration increased, and the
value at 150 min (115 24 nmol/ml) was signicantly
higher than the baseline value. At 240 min, the con-
centration (88 14 nmol/ml) was not statistically
different from the baseline value.
Phenylalanine kinetics. Enrichment of phenylala-
nine in blood was relatively constant throughout the
experiment despite the large changes in concentration
(Fig. 3). No statistical changes in enrichment were
observed during the different calculation periods. This
was accomplished by adding tracer to the ingested
amino acids.
The rate of appearance of phenylalanine into the
blood from the muscle (R
a
) did not change during the
rst 2 h after the drink but increased signicantly
during the 3rd h (Fig. 4). The average rate of disap-
pearance (R
d
) of phenylalanine from the blood into the
muscle (i.e., protein synthesis from plasma phenylala-
nine) increased signicantly from the basal value dur-
ing the rst 2 h after intake of drink but was not
different from the basal value during the 3rd h.
Ingestion of the EAA drink caused inward transport
of phenylalanine from the artery to the muscle to
increase (Table 3, P 0.055). At 30 min after exercise,
the total rate of intracellular phenylalanine release
from protein breakdown was 43 9nmol min
1
100
ml leg
1
(Table 3). There was no change in R
a
during
the rst 2 h after the rst drink, but it increased signif-
icantly during the 3rd h to 75 13 nmol min
1
100 ml
leg
1
. The rate of utilization of phenylalanine for protein
synthesis was 30 9nmol min
1
100 ml leg
1
at 30 min
Fig. 2. Changes in arterial phenylalanine concentration and net
phenylalanine balance during the recovery period after a resistance
exercise bout (means SE; n 6). Essential amino acid (EAA) drink
was consumed at 60 and 120 min after exercise.
E651ESSENTIAL AMINO ACIDS AND EXERCISE
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after exercise, and this value increased signicantly dur-
ing the 1st and 2nd h after drink to 121 16 and 139
21 nmol min
1
100 ml leg
1
, respectively (Table 3). Dur
-
ing the 3rd h after drink, the value returned to the
basal level, despite the fact that the total intracellular
appearance of phenylalanine (inward transport
breakdown) was elevated approximately threefold
above the basal value.
Leucine concentration and kinetics. The pattern of
response of blood concentration and kinetics of leucine
was similar to that of phenylalanine. As was the case
for phenylalanine, leucine tracer was added to the
ingested amino acids. Therefore, enrichment of leucine
in blood was essentially constant throughout the ex-
periment despite changes in concentrations (Fig. 3).
Leucine concentration increased signicantly within
10 min of ingestion of the rst EAA drink, from a value
of 118 4 nmol/ml at 30 min after exercise to a peak of
407 24 nmol/ml at 20 min after ingestion of the
drink. The concentration declined before the second
drink, but not to the basal level. Leucine concentration
increased again after ingestion of the second drink, and
although the value again began falling 30 min after
ingestion of the second drink, it was still elevated
above basal at 210 min after ingestion of the rst
drink. Muscle intracellular leucine concentration in-
creased signicantly from the basal value of 144 7to
307 52 nmol/ml at 150 min. At 240 min, the concen-
tration was not different from basal value at 30 min
after exercise.
Leucine net uptake increased as a result of drink. No
signicant difference was found between the AUC
value for net uptake during the 1st h after the rst
drink (159 36 mg/leg) and that during the 1st h after
the second drink (165 32 mg/leg), but the area was
signicantly smaller by the 3rd h (20 22 mg/leg).
The average R
a
of leucine into the blood over each of
the hours after intake of drink did not change, whereas
the average rate of muscle protein synthesis and oxi-
dation from plasma leucine increased signicantly
from basal values during the rst 2 h after intake of
drink but was not different from basal values during
the 3rd h (Fig. 5). As a result, leucine net balance
increased from a slightly negative basal value to
250260 nmol min
1
100 ml leg
1
during the 2 h
after the rst drink (not signicant between hours; Fig.
5). During the 3rd h, net balance decreased again.
Table 4 shows leucine kinetics. The delivery of
leucine to the leg muscle increased as a result of intake
of the drink. Also, the movement of leucine into the
muscle increased and stayed elevated above the
predrink value throughout the following 3 h. The rate
of intracellular R
a
of leucine increased slightly after
the drink, and during the 1st h the value was signi-
cantly different from the predrink value. The rate of
utilization of intracellular leucine, which includes uti-
lization for both protein synthesis and oxidation, in-
creased signicantly during the 1st and 2nd h after
drink, whereas it returned to basal value during the
3rd h.
The rate of incorporation of leucine into protein can
be calculated from the rate of protein synthesis as
calculated with the phenylalanine tracer and the ratio
between leucine and phenylalanine in mixed muscle
protein. The ratio between leucine and phenylalanine
in mixed muscle protein was calculated from the ratio
between the rates of leucine and phenylalanine release
from muscle protein breakdown at the predrink time
point (30 min). The mean ratio for the group was 2.6
0.2. When calculated accordingly, the rate of leucine
incorporated into protein increased signicantly dur-
ing the 1st h after each drink, but during the 3rd h it
decreased to a value not different from the predrink
rate (Table 4). The rate of leucine oxidation was calcu-
lated from the difference between the rate of utilization
of intracellular leucine and the rate at which leucine
was used for protein synthesis. This calculation
showed that leucine oxidation increased signicantly
during the 1st h after the rst drink but that, during
Fig. 3. Arterial L-[
2
H
5
]phenylalanine and L-[1-
13
C]leucine enrich
-
ments during the recovery period after a resistance exercise bout
(means SE; n 6). EAA drink was consumed at 60 and 120 min
after exercise.
Fig. 4. Phenylalanine kinetics across leg before intake of drink and
during the 1st, 2nd, and 3rd h after the rst drink (means SE; n
6). R
a
, rate of appearance of phenylalanine into the blood; R
d
, rate of
disappearance of phenylalanine out of the blood. *P 0.05, value vs.
predrink value.
E652 ESSENTIAL AMINO ACIDS AND EXERCISE
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the 2nd and 3rd h, the values were not different from
the predrink value.
Plasma and muscle intracellular amino acid concen-
trations. Intake of drinks increased the arterial plasma
concentrations of total EAAs signicantly. At 150 min
after exercise (90 min after the rst drink and 30 min
after the second drink), the concentrations for most of
the EAAs given in the drinks were increased by 75
150%, except for isoleucine and leucine, which showed
greater responses (317 69 and 212 29% increase,
respectively; Fig. 6A). Arterial plasma concentrations
of the amino acids not given in the drink generally
showed smaller changes (Fig. 6B). Tyrosine increased
signicantly, whereas glycine, alanine, and tryptophan
decreased during the study.
Whereas there was no change in intracellular con-
centration of total EAAs or in total NEAAs during the
recovery period, some individual changes were ob-
served. Threonine was not different between rst and
second biopsies but increased thereafter to a concen-
tration at 150 min signicantly higher than predrink
concentration (741 99 vs. 561 59 nmol/ml H
2
O).
Similarly, valine, isoleucine, and leucine concentra-
tions all increased after the rst drink. The concentra-
tions increased further at 150 min but decreased again
at the end of the study at 240 min. For valine, the
values at 150 and 240 min were signicantly higher
than the predrink value (228 17 and 212 16 vs.
168 9 nmol/ml, respectively). For isoleucine, 90- and
150-min values were higher than the predrink value
(86 5 and 98 6 vs. 61 2 nmol/ml, respectively).
For leucine, 90-, 150-, and 240-min values were higher
than the predrink value (249 5, 297 22, and 240
15 vs. 186 7 nmol/ml, respectively). For the amino
acids not given in the drink, a signicant fall during
the day was found for asparagine, serine, arginine, and
alanine. The relative changes in intracellular concen-
trations of EAAs were small compared with the
changes in arterial plasma (Fig. 6).
Urea production. Plasma urea enrichment was sta-
ble at 4% throughout the study. Thus no change in
urea production was seen over time. The value was
stable at 330 mol kg
1
h
1
.
Plasma glucose and insulin concentrations. Arterial
glucose concentration decreased slightly during the
experiment from a value of 91 3 mg/dl at 30 min after
exercise to 87 3 mg/dl at the end of the study (P
0.05). No change was found in net uptake of glucose
during the study. Similarly, insulin concentrations did
not change over time.
DISCUSSION
The principal nding of this study was that ingestion
of a relatively small amount (6 g) of EAA effectively
stimulated net muscle protein balance after resistance
exercise. The response of net muscle protein balance
could be explained largely by a change in synthesis, as
the rate of breakdown was not signicantly affected.
The response of net balance was about twice the pre-
vious published response to6gofmixed amino acids
(14), leading to the conclusions that there is a dose
response to the amount of EAAs given and that inges-
tion of NEAAs is not necessary to stimulate net protein
synthesis. The latter conclusion is supported by
changes in NEAA concentrations after ingestion of the
EAA drink. Although plasma concentrations of some
individual NEAAs fell after ingestion of EAAs, intra-
cellular concentrations of NEAAs were generally main-
tained, indicating that availability of NEAAs did not
limit the response of muscle protein synthesis. Finally,
the response of net muscle protein synthesis to the
drink ingested 2 h after exercise was comparable to
that of the drink ingested 1 h after exercise.
Independent effect of EAA on net protein balance. In
previous studies, both intravenous infusion (5) and oral
intake of amino acids (17) after resistance exercise
have been shown to stimulate muscle protein synthe-
sis. However, optimal proportions and amounts of in-
dividual amino acids to stimulate muscle protein syn-
thesis are not known. Intake of only a small amount (6
g) of a mixture of EAA given along with 35 g of carbo-
hydrate after resistance exercise transiently increased
muscle protein synthesis 3.5-fold (14), but the indepen-
dent effects of amino acids and carbohydrate were not
assessed. The results of the present study show that
ingestion of6gofEAAalone without addition of
Fig. 5. Leucine kinetics across leg before intake of drink and during
the 1st, 2nd, and 3rd h after the rst drink (means SE; n 6). *P
0.05, value vs. predrink value.
Table 3. Calculated kinetics for phenylalanine before
drink and during 1st, 2nd, and 3rd h after intake of
first drink
Predrink 1st h 2nd h 3rd h
Inow 219 27 514 64* 59887* 54792*
Inward transport 201 31 590 74 659 193 454 105
Protein synthesis 30 9 121 16* 139 21* 75 31
Protein breakdown 43 940655575 13
Inow, rate at which phenylalanine enters leg via artery; inward
transport, rate of net phenylalanine movement from artery to mus-
cle; protein synthesis, rate of phenylalanine incorporation into pro-
tein; protein breakdown, rate of intracellular release of phenylala-
nine from protein breakdown. Values are nmol min
1
100 ml leg
1
(means SE; n 6). Value vs. predrink value: *P 0.05.
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carbohydrate effectively stimulated muscle protein
synthesis after resistance exercise (Figs. 4 and 5, Ta-
bles 3 and 4).
In a recent study, Miller et al. (14) compared the
independent and combined effects of a balanced mix-
ture of amino acids (i.e., EAAs NEAAs) and carbo-
hydrate on muscle protein synthesis after resistance
exercise. Addition of 35 g of carbohydrate to6gof
mixed AA did not cause a greater stimulation of net
muscle protein synthesis than the AAs alone. The
effect of adding carbohydrate to 6 g of EAA can be seen
in Fig. 7, which compares the AUC for net phenylala-
nine uptake for the 1st h after intake of drink (i.e.,
60120 min) in the present study with the previously
published response to6gofEAAs plus 35 g of carbo-
hydrate (16). The additional carbohydrate provided no
advantage to EAAs alone. From these results, it is
clear that the stimulation of protein synthesis by EAAs
is not a caloric effect, because ingestion of an additional
3 g of EAA (difference in EAA content between mixed
AA and EAA groups) caused a much larger effect than
addition of 35 g of carbohydrate to the amino acid
mixture (Fig. 7), and 35 g of carbohydrate alone had a
minimal effect (14). Although direct comparison with
historical data may be problematic, the cited studies
(14, 16) were performed in the same laboratory, ap-
proximately contemporaneously, and by use of the
same general experimental protocol and techniques.
The results of the current study indicate that 3.5
1.1 g of muscle protein were synthesized during the 3 h
after the rst drink in one leg, or 7.0 2.0 g in both
legs, on the basis of irreversible loss of phenylalanine
and the composition of muscle protein. This represents
27% of ingested EAAs, because each gram of muscle
protein synthesized includes both EAAs and NEAAs.
When account of the water content of muscle (73%) is
taken, this would represent a net gain of 26gof
muscle tissue synthesized in response to the drinks.
This magnitude of gain in muscle mass would therefore
require many weeks of comparable response to become
Fig. 6. Percentage changes in arterial
plasma concentrations of EAAs (A),
arterial plasma concentrations of non-
essential amino acids (NEAAs) and
tryptophan (B), intracellular concentra-
tions of EAAs (C), and intracellular
concentrations of NEAAs and trypto-
phan (D) from 30 min to 150 min after
exercise (means SE; n 6). EAA
drink was consumed at 60 and 120 min
after exercise.
Table 4. Calculated kinetics for leucine before drink and during 1st, 2nd, and 3rd h after intake of rst drink
Predrink 1st h 2nd h 3rd h
Inow 433 42 1228 128* 1366 143* 1318 167*
Inward transport 302 64 1008 210* 990 104* 718 91*
Utilization 100 16 458 58* 416 54* 17643
Protein synthesis 73 17 306 25* 360 60* 15841
Oxidation 27 7 15145* 56 34 18 10
Protein breakdown 10614 195 30* 16426 147 30
Utilization, rate of utilization of intracellular leucine (i.e., protein synthesis and oxidation); oxidation, rate of leucine oxidation. Values are
nmol min
1
100 ml leg
1
(means SE; n 6). *P 0.05, value vs. predrink value. P 0.05, 2nd h vs. 1st h value.
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detectable by available means to quantify changes in
muscle mass, during which time all variables such as
activity and other nutritional intake would have to be
absolutely controlled. Thus, although the stimulation
in net muscle synthesis resulting from the total of 12 g
of EAA reported in this study would eventually be
expected to enhance the rate of muscle gain during a
resistance training program, an outcome study based
on measured differences in muscle mass would have to
be carefully designed and executed to demonstrate an
effect of an EAA supplement.
EAA vs. NEAA. Because ingestion of 6 g of EAAs
alone stimulates muscle protein synthesis after resis-
tance exercise, NEAAs are apparently not required to
stimulate protein synthesis. In a previous study, Tip-
ton et al. (17) found no difference in net muscle protein
balance response to the ingestion of 40 g of mixed AAs
(roughly in proportion to their relative contributions to
muscle protein) or 40 g of EAAs. These results could
also be interpreted to indicate that NEAAs are not
needed for stimulation of muscle protein synthesis.
However, if the NEAAs were not necessary for stimu-
lation of synthesis, it was unclear why ingestion of the
EAAs alone did not stimulate muscle protein synthesis
to a greater extent than did the balanced mixture. The
response in the present study was about twice that
when6gofmixed AAs were given after a similar
exercise bout (Fig. 7) (14). Thus, it seems likely that
there is a dose response of muscle protein synthesis to
the intake of EAAs and that the amount of EAAs in the
balanced mixture (21 g) in the study by Tipton et al.
was equal to or exceeded the maximal effective dose.
However, it should be noted that, in this study by
Tipton et al., the amino acids were ingested as small
boluses over 3 h, and the different pattern of intake
may also have contributed to the lack of difference
between the two mixtures of amino acids.
Pattern of ingestion. No differences were found in the
protein synthesis response between the rst and the
second dose (Tables 3 and 4, Figs. 4 and 5), and the
AUC for net phenylalanine uptake was similar after
the rst and the second drinks. Net balance increased
rapidly after intake of the drink, and the relative
increase in net balance was greater than the change in
arterial phenylalanine concentrations (Fig. 2). How-
ever, when arterial AA concentrations started to drop,
net balance rapidly decreased to the basal level, even
though the arterial AA concentrations were still ele-
vated (Fig. 2). In fact, at 60 min after ingestion of the
rst drink, the phenylalanine concentration was still
higher than the maximal concentration previously ob-
served to coincide with stimulation of protein synthesis
when6gofmixed amino acids were given (14). Thus
muscle protein synthesis is apparently stimulated
when there is an increase in arterial, and presumably
interstitial, AA concentrations, rather than by the ab-
solute AA concentration. This observation is consistent
with our previous ndings (6) that, when blood amino
acid concentrations were elevated to a steady-state
level about twice the basal values for 6 h, muscle
protein synthesis was stimulated over the rst 2 h but
thereafter returned to the resting level despite the
persistent elevation in blood amino acid concentra-
tions.
The similarity of response to both boluses suggests
that there is little effect of the exact time of ingestion
after exercise. This is consistent with our previous
work, in which we observed similar responses to single
doses of amino acids and glucose given either immedi-
ately, 1 h, or 3 h after resistance exercise (16, 18). In
contrast, Esmarck et al. (9) recently reported that a
protein-carbohydrate-fat supplement was effective in
stimulating muscle protein gain over a period of resis-
tance training in elderly men only when ingested im-
mediately after, as opposed to 2 h after, exercise. Dif-
ferences between that study and our studies include
age of subjects, ingestion of protein rather than free
amino acids, and end point. Whereas we measured the
acute response of muscle protein, they measured net
muscle gain, which includes the response to all food
intake over a period of time. Thus it is possible that
ingestion of a protein-carbohydrate-fat supplement 2 h
after exercise might have interfered with either the
amount eaten at the next meal or the response to the
next ingested meal. In another study that addressed
the timing of intake on response, Levenhagen et al.
(11) found a greater stimulation of net muscle protein
synthesis when a protein-carbohydrate-fat supplement
was given immediately after aerobic exercise than
when it was given 2 h later. There likely is a difference
between timing after aerobic and timing after resis-
tance exercise, as performed in the current study. The
stimulation of muscle protein synthesis is modest after
aerobic exercise (7). Rather, the interaction effect of
exercise and supplement ingestion may be due to in-
creased muscle blood ow, and thus substrate delivery,
immediately after exercise. In contrast, fractional syn-
thetic rate remains elevated for 48 h after resistance
exercise (15), so an effect on timing of supplement
ingestion after resistance exercise is less likely.
Fig. 7. Area under curve for net uptake of phenylalanine over 1 h
after ingestion of6gofdifferent amino acid drinks. MAA, mixed
amino acids (n 7; values from Ref. 14); MAA CHO, MAA 35 g
carbohydrate (n 7; values from Ref. 14); EAA, present study;
EAA CHO, EAA 35 g carbohydrate (n 6; values from Ref. 16).
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Composition of drink. The composition of the mix-
ture of EAA we have tested in this and previous studies
(16, 18) was originally based on the composition of
muscle protein, with the notion of increasing the avail-
ability of each EAA in proportion to its requirement for
synthesis of muscle protein. The results of the current
study show that isoleucine and leucine increased more
than the others in the blood (Fig. 6), meaning that the
goal of causing proportional increases in all EAA was
not fully achieved. Different changes in concentrations
of blood amino acids after ingestion of the drink may be
caused by different clearance rates of individual amino
acids, thereby resulting in rates of uptake that do not
directly correspond to the composition of the ingested
mixture. Furthermore, it is possible that the effect is
largely, or even entirely, mediated by leucine alone (1).
Therefore, it is possible that adjustments in the com-
position of the drink could further improve the re-
sponse of net muscle balance.
Whereas NEAAs can be synthesized within the body
at a rate generally sufcient to meet requirements,
certain amino acids may be limited in the rapidity with
which changes in production can occur. Thus glycine is
known to be slowly transaminated (12), and this likely
explains the fall in plasma glycine concentration as its
utilization is increased because of the stimulated rate
of protein synthesis after EAA, not only in muscle but
also throughout the body. The fact that the EAA mix-
ture alone was twice as effective as the same amount of
the balanced mixture of AAs (14) in stimulating muscle
protein synthesis indicates that glycine was probably
not rate limiting, despite the decrease in plasma con-
centration, because the muscle concentration of free
glycine was maintained. Similarly, the fall in alanine
concentration in plasma probably reected increased
uptake elsewhere than in muscle for incorporation into
protein, as muscle is normally highly efcient in pro-
ducing alanine, and its rate of production is increased
during exercise (22).
Methodological considerations. Drinks normally are
ingested as boluses. However, quantifying the re-
sponse to a bolus ingestion of unlabeled amino acids
introduces potential methodological problems because
of rapid dilution of the tracer. The resulting isotopic
nonsteady state violates a fundamental assumption of
the three-pool compartment model (2, 3). We therefore
added tracer to the ingested amino acids so that a
relatively constant enrichment in the blood was main-
tained, even during absorption of the bolus. Thus the
calculated values for synthesis and breakdown should
theoretically be accurate. Nonetheless, we used net
protein balance, which is not dependent on the mea-
surement of isotopic enrichment, as our primary end
point, and the other variables were considered as sec-
ondary end points. This is not only because of method-
ological issues but also because, in terms of gain or loss
of muscle protein, the net balance (i.e., synthesis minus
breakdown) is the most relevant parameter. Further-
more, it can be determined in non-steady-state condi-
tions. In relation to net balance, the pertinent kinetic
parameters are the rate of synthesis from plasma
amino acids (phenylalanine and leucine, respectively)
and the rate of appearance of amino acids into plasma
from protein breakdown. This is because, although the
synthesis of protein from amino acids derived from
protein breakdown (i.e., intracellular recycling of
amino acids) is important from the standpoint of un-
derstanding the regulation of the process of synthesis,
it does not represent any net gain or loss of protein.
The protein synthesis from plasma amino acids and
appearance in plasma from protein breakdown are
calculated by the two-pool model. We also have calcu-
lated rates of synthesis and breakdown by use of the
three-pool model that we have described previously,
which includes synthesis from all sources and the total
rate of breakdown. However, even though we avoided
changes in enrichment by adding tracer to the exoge-
nous amino acids, the calculated values of synthesis
and breakdown were variable. This is because calcula-
tion of the parameters from the three-pool model re-
quires a gradient in enrichment from blood to the
intracellular phenylalanine pools, and the rapid inux
of amino acids from plasma caused the gradient in
enrichment between compartments to narrow to the
point where accurate measurement is difcult.
The primary potential problem in the interpretation
of net balance results in the nonsteady state is the
possibility that amino acids entered the intracellular
pool from the plasma that eventually reentered the
blood at some time after the nal blood sample was
drawn. To the extent that this occurred in this exper-
iment, we would have overestimated net uptake. How-
ever, the free intracellular phenylalanine concentra-
tion was not signicantly elevated at the time we drew
our last sample, so the magnitude of error due to this
potential problem was likely not large.
Because previous studies have shown that net mus-
cle protein balance remains slightly negative for sev-
eral hours after resistance exercise in the absence of
nutrient intake (4, 8), a control group was not included
in this study. Furthermore, in a previous study from
our laboratory, Rasmussen et al. (16) gave a placebo
drink at 60 min after a similar resistance exercise bout.
They found no signicant change in net balance over the
rst 3 h after exercise. Hence, the signicant positive net
balance observed after drinks in the present study can
be ascribed to the intake of the amino acids, rather
than to changes that would have occurred anyway.
We thank the nurses and the staff at the General Clinical Re-
search Center (GCRC) at the University of Texas Medical Branch
(UTMB) in Galveston, TX. We thank Julie M. Vargas for skillful
technical assistance. We also thank the volunteers who participated
in the study.
This work was supported by Shriners Hospital for Children Grant
8490 and the National Institutes of Health (NIH) Grants DK-38010
and AG-98006. Studies were conducted at the GCRC at the UTMB
in Galveston, funded by Grant M01 RR-00073 from the National
Center for Research Resources, NIH.
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... In addition, maximal carrying capacity was reduced by 16%, and endurance capacity was reduced by 26%. It is well known that a diet rich in EAAs induces gains in muscle net protein balance (i.e., gains in muscle mass) in normal [21,22] and pathological conditions [20]. Therefore, we performed the experiment to test whether ...
... In addition, maximal carrying capacity was reduced by 16%, and endurance capacity was reduced by 26%. It is well known that a diet rich in EAAs induces gains in muscle net protein balance (i.e., gains in muscle mass) in normal [21,22] and pathological conditions [20]. Therefore, we performed the experiment to test whether an EAA-enriched diet could protect the muscle against DEX-induced muscle atrophy. ...
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Dexamethasone (DEX) induces dysregulation of protein turnover, leading to muscle atrophy and impairment of glucose metabolism. Positive protein balance, i.e., rate of protein synthesis exceeding rate of protein degradation, can be induced by dietary essential amino acids (EAAs). In this study, we investigated the roles of an EAA-enriched diet in the regulation of muscle proteostasis and its impact on glucose metabolism in the DEX-induced muscle atrophy model. Mice were fed normal chow or EAA-enriched chow and were given daily injections of DEX over 10 days. We determined muscle mass and functions using treadmill running and ladder climbing exercises, protein kinetics using the D2O labeling method, molecular signaling using immunoblot analysis, and glucose metabolism using a U-13C6 glucose tracer during oral glucose tolerance test (OGTT). The EAA-enriched diet increased muscle mass, strength, and myofibrillar protein synthesis rate, concurrent with improved glucose metabolism (i.e., reduced plasma insulin concentrations and increased insulin sensitivity) during the OGTT. The U-13C6 glucose tracing revealed that the EAA-enriched diet increased glucose uptake and subsequent glycolytic flux. In sum, our results demonstrate a vital role for the EAA-enriched diet in alleviating the DEX-induced muscle atrophy through stimulation of myofibrillar proteins synthesis, which was associated with improved glucose metabolism.
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... On the other hand, nutritional balance based on adequate exercise is a useful strategy to combat the muscle atrophy derived from various clinical muscle wasting conditions [44]. Various studies suggested that amino acid supplementation, especially essential amino acids, contributes to muscle protein synthesis in humans [45][46][47]. In particular, BCAA leucine supplementation improves muscle protein synthesis in the elderly by stimulating anabolic processes [48,49]. ...
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Glucocorticoid excess is a critical factor contributing to muscle atrophy. Both endogenous and exogenous glucocorticoids negatively affect the preservation of muscle mass and function. To date, the most effective intervention to prevent muscle atrophy is to apply a mechanical load in the form of resistance exercise. However, glucocorticoid-induced skeletal muscle atrophy easily causes fatigue in daily physical activities, such as climbing stairs and walking at a brisk pace, and reduces body movements to cause a decreased ability to perform physical activity. Therefore, providing adequate nutrients in these circumstances is a key factor in limiting muscle wasting and improving muscle mass recovery. The present review will provide an up-to-date review of the effects of various nutrients, including amino acids such as branched-chain amino acids (BCAAs) and β–hydroxy β–methylbutyrate (HMB), fatty acids such as omega-3, and vitamins and their derivates on the prevention and improvement of glucocorticoid-induced muscle atrophy.
... In contrast, phenylalanine is oxidized in the liver [43] and would only be utilized for protein synthesis within skeletal muscle, which may explain our ability to delineate a correlation (albeit negative) between dietary phenylalanine incorporation and LAT1 expression. However, it should be noted that LAT1 is a bidirectional transporter and can coordinate both the influx and efflux of amino acids from skeletal muscle, both of which are enhanced in response to exogenous amino acids at rest and after exercise [1,44]. Therefore, it is possible that a higher basal LAT1 expression in skeletal muscle may promote a greater phenylalanine transmembrane flux, which could reduce the ability of exogenous phenylalanine to be utilized for de novo protein synthesis. ...
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The influx of essential amino acids into skeletal muscle is primarily mediated by the large neutral amino acid transporter 1 (LAT1), which is dependent on the glutamine gradient generated by the sodium-dependent neutral amino acid transporter 2 (SNAT2). The protein expression and membrane localization of LAT1 may be influenced by amino acid ingestion and/or resistance exercise, although its acute influence on dietary amino acid incorporation into skeletal muscle protein has not been investigated. In a group design, healthy males consumed a mixed carbohydrate (0.75 g·kg−1) crystalline amino acid (0.25 g·kg−1) beverage enriched to 25% and 30% with LAT1 substrates L-[1-13C]leucine (LEU) and L-[ring-2H5]phenylalanine (PHE), respectively, at rest (FED: n = 7, 23 ± 5 y, 77 ± 4 kg) or after a bout of resistance exercise (EXFED: n = 7, 22 ± 2 y, 78 ± 11 kg). Postprandial muscle biopsies were collected at 0, 120, and 300 min to measure transporter protein expression (immunoblot), LAT1 membrane localization (immunofluorescence), and dietary amino acid incorporation into myofibrillar protein (ΔLEU and ΔPHE). Basal LAT1 and SNAT2 protein contents were correlated with each other (r = 0.55, p = 0.04) but their expression did not change across time in FED or EXFED (all, p > 0.05). Membrane localization of LAT1 did not change across time in FED or EXFED whether measured as outer 1.5 µm intensity or membrane-to-fiber ratio (all, p > 0.05). Basal SNAT2 protein expression was not correlated with ΔLEU or ΔPHE (all, p ≥ 0.05) whereas basal LAT1 expression was negatively correlated with ΔPHE in FED (r = −0.76, p = 0.04) and EXFED (r = −0.81, p = 0.03) but not ΔLEU (p > 0.05). Basal LAT1 membrane localization was not correlated with ΔLEU or ΔPHE (all, p > 0.05). Our results suggest that LAT1/SNAT2 protein expression and LAT1 membrane localization are not influenced by acute anabolic stimuli and do not positively influence the incorporation of dietary amino acids for de novo myofibrillar protein synthesis in healthy young males.
Chapter
Protein is a critical part of the diet in individuals with inherited metabolic diseases (IMDs).Current Dietary Reference Intakes may underestimate protein needs for individuals with IMD.Distributing protein intake throughout the day facilitates anabolism.Additional protein and energy is required for catch-up growth in patients with growth failure.Proper use of protein substitutes (medical foods) is essential in assuring adequate balance of serum amino acids and promoting growth and development. Protein is a critical part of the diet in individuals with inherited metabolic diseases (IMDs). Current Dietary Reference Intakes may underestimate protein needs for individuals with IMD. Distributing protein intake throughout the day facilitates anabolism. Additional protein and energy is required for catch-up growth in patients with growth failure. Proper use of protein substitutes (medical foods) is essential in assuring adequate balance of serum amino acids and promoting growth and development.
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Background Chronic low back pain (LBP) is the most common musculoskeletal pain that affects a person’s daily activities. This present study aimed at evaluating the relationship between major dietary pattern and Chronic LBP. Methods This cross-sectional analysis was examined 7686 Kurdish adults. The RaNCD cohort study physician diagnosed chronic LBP. Dietary patterns were derived using principal component analysis. The three identified dietary patterns derived were named: 1) the vegetarian diet included vegetables, whole grain, legumes, nuts, olive, vegetable oil, fruits, and fruit juice; 2) high protein diet related to higher adherence to red and white meat, legumes, nuts, and egg; and 3) energy-dense diet characterized with higher intake of salt, sweet, dessert, hydrogenated fat, soft drink, refined grain, tea, and coffee. Dietary pattern scores were divided into tertiles. Binary logistic regression in crude, adjusted odds ratios (OR) and 95% confidence intervals (CI) were used to determine this association. Results Twenty-two per cent of participants had chronic LBP. Higher adherence to high protein dietary pattern was inversely associated with chronic LBP in crude (OR: 0.79, 95% CI: 0.69–0.9) and adjusted model (for age, sex, smoking, drinking, diabetes, physical activity, body mass index, and waist circumference) (OR: 0.84, 95% CI: 0.72–0.97). In addition, after controlling for the mentioned potential confounders, participants in the highest category of energy dense diet were positively associated with chronic LBP compared with those in the lowest category (OR: 1.13, 95% CI: 1.01–1.32). Conclusions Higher adherence to the high protein diet was inversely related to chronic LBP prevalence. In addition, we found that following energy dense diet was positively associated with chronic LBP.
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Background Sarcopenia plays a central role in the development of frailty syndrome. Nutrition and exercise are cornerstone strategies to mitigate the transition to frailty; however, there is a paucity of evidence for which dietary and exercise strategies are effective. Objective This large, multifactorial trial investigated the efficacy of different dietary strategies to enhance the adaptations to resistance training in pre-frail and frail elderly. Methods This was a single-site 16-week, double-blind, randomized, placebo-controlled trial conducted at the Clinical Hospital, School of Medicine - University of São Paulo, Sao Paulo, Brazil. Four integrated, sub-investigations were conducted to compare: 1) leucine vs. placebo; 2) whey vs. soy vs. placebo; 3) creatine vs. whey vs. creatine plus whey vs. placebo; 4) women vs. men in response to whey. Sub-investigations 1 to 3 were conducted in women, only. Two-hundred participants (154 women/46 men, mean age 72 ± 6 years) underwent a twice-a-week, resistance training program. The main outcomes were muscle function (assessed by dynamic and isometric strength and functional tests) and lean mass (assessed by DXA). Muscle cross-sectional area, health-related quality of life, bone and fat mass, and biochemical markers were also assessed. Results We observed that leucine supplementation was ineffective to improve muscle mass and function. Supplementation with whey and soy failed to enhance resistance-training effects. Similarly, supplementation with neither whey nor creatine potentiated the adaptations to resistance training. Finally, no sex-based differences were found in response to whey supplementation. Resistance exercise per se increased muscle mass and function in all sub-investigations. There were no adverse effects. Conclusion Neither protein (whey and soy), leucine, nor creatine supplementation enhanced resistance training-induced adaptations in pre-frail and frail elderly, regardless of sex. These findings do not support the notion that some widely used supplement-based interventions can add to the already potent effects of resistance exercise to counteract frailty-related muscle wasting and dynapenia. Clinical Trial Registry NCT01890382; https://clinicaltrials.gov/ct2/show/NCT01890382.
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• The aim of this study was to describe the time course of the response of human muscle protein synthesis (MPS) to a square wave increase in availability of amino acids (AAs) in plasma. We investigated the responses of quadriceps MPS to a ≈1.7-fold increase in plasma AA concentrations using an intravenous infusion of 162 mg (kg body weight)−1 h−1 of mixed AAs. MPS was estimated from D3-leucine labelling in protein after a primed, constant intravenous infusion of D3-ketoisocaproate, increased appropriately during AA infusion. • Muscle was separated into myofibrillar, sarcoplasmic and mitochondrial fractions. MPS, both of mixed muscle and of fractions, was estimated during a basal period (2.5 h) and at 0.5-4 h intervals for 6 h of AA infusion. • Rates of mixed MPS were not significantly different from basal (0.076 ± 0.008 % h−1) in the first 0.5 h of AA infusion but then rose rapidly to a peak after 2 h of ≈2.8 times the basal value. Thereafter, rates declined rapidly to the basal value. All muscle fractions showed a similar pattern. • The results suggest that MPS responds rapidly to increased availability of AAs but is then inhibited, despite continued AA availability. These results suggest that the fed state accretion of muscle protein may be limited by a metabolic mechanism whenever the requirement for substrate for protein synthesis is exceeded.
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The bidirectional transmembrane transport rates of leucine (Leu), valine (Val), phenylalanine (Phe), lysine (Lys), and alanine (Ala) were measured in vivo in the hindlimb muscle of five dogs and related to the rates of protein synthesis and degradation. The compartmental model was based on the systemic continuous infusion of stable isotopic tracers of the amino acids, and the measurement of the enrichment and concentration in the arterial and femoral vein plasma and the intracellular free water in muscle (obtained by biopsy). The transport rate from plasma to tissue (in micromoles per minute) was: Leu, 18.1 +/- 1.8; Val, 26.9 +/- 3.5; Phe, 10.5 +/- 1.6 Lys; 12.2 +/- 1.8; and Ala, 10.7 +/- 3.4. The transport rate from tissue to plasma (in micromoles per minute) was: Leu, 25.5 +/- 2.5; Val, 32.4 +/- 2.8; Phe, 17.0 +/- 2.8; Lys, 24.9 +/- 3.4; Ala, 34.4 +/- 9.0. When the transmembrane transport rate was normalized per unit of amino acid concentration in the source pool, we found that the transport of Leu, Val, and Phe was significantly faster (p less than .05) than the transport of Lys and Ala. The calculated rates of incorporation into hindlimb muscle protein of Phe and Lys (in micromoles per minute) were 4.2 +/- 1.3 and 19.4 +/- 5.3, respectively, and the rates of intracellular appearance from breakdown were 10.7 +/- 1.9 and 32.1 +/- 6.6, respectively. We concluded, therefore, that (1) the transmembrane amino acid transport rate can be measured in vivo in muscle with a relatively noninvasive technique, (2) in the dog hindlimb the equilibration between tissue and plasma free amino acid pool is different for each amino acid depending on the kinetics of the transmembrane transport systems, and (3) the transport rates of amino acids and their rate of appearance from protein breakdown are roughly comparable, suggesting that variations in transport rates could play a role in controlling the rate of protein synthesis.
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1. A dye-dilution method has been developed for the determination of leg blood flow in man. The method is based on the infusion of indicator into the distal part of the femoral vein with blood sampling from the same vein at the level of the inguinal ligament. Catheterization of the femoral artery is not required. Evidence of adequate mixing of dye and blood is presented, based on the finding of the same dye concentration in samples from two different levels in the femoral vein. 2. Leg blood flow measured by this technique at rest and during exercise in six healthy subjects was found to agree closely with simultaneous determinations with an intra-arterial indicator-dilution technique. The reproducibility of the blood-flow measurements, expressed as the coefficient of variation for a single determination, was 9·8%. 3. A routine procedure is suggested for leg blood-flow determination based on femoral venous dye infusion. Using this procedure, leg blood flow was measured in twelve healthy subjects at rest and during exercise at work loads of 100, 200, 400 and 600 kpm/min. A linear relationship was found between leg blood flow and pulmonary oxygen uptake. 4. The applicability of this method to the study of patients with occlusive arterial disease of the leg is illustrated by findings in two patients before and after vascular reconstruction.
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1. An indicator-dilution technique was used to determine human leg blood flow at rest and during exercise. The method is based on the infusion of Indocyanine Green into the femoral artery with blood sampling from the femoral vein at the level of the inguinal ligament. Evidence for mixing of dye and blood is presented, based on the finding of equal dye concentrations at two different sampling levels in the femoral vein. The minimum time of infusion required for equilibration at rest is 3 min and during exercise 1 min 20 s. 2. Leg blood flow was measured in eight healthy athletic subjects at rest and during upright exercise on a bicycle ergometer at 400, 800 and 1200 kpm/min. Linear relationships were found between blood flow on the one hand and work intensity and pulmonary oxygen uptake on the other. 3. Leg oxygen uptake was measured as the product of blood flow and femoral arterio-venous oxygen difference. Linear regressions were found for leg oxygen uptake in relation to both work intensity and pulmonary oxygen uptake. Leg mechanical efficiency during exercise averaged 34%. 4. A formula for the approximate calculation of leg blood flow is suggested, based on the pulmonary oxygen uptake and the femoral arterio-venous oxygen difference.
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We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)