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Mixed muscle protein synthesis and breakdown after resistive exercise in humans

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Abstract

Mixed muscle protein fractional synthesis rate (FSR) and fractional breakdown rate (FBR) were examined after an isolated bout of either concentric or eccentric resistance exercise. Subjects were eight untrained volunteers (4 males, 4 females). Mixed muscle protein FSR and FBR were determined using primed constant infusions of [2H5]phenylalanine and 15N-phenylalanine, respectively. Subjects were studied in the fasted state on four occasions: at rest and 3, 24, and 48 h after a resistance exercise bout. Exercise was eight sets of eight concentric or eccentric repetitions at 80% of each subject's concentric 1 repetition maximum. There was no significant difference between contraction types for either FSR, FBR, or net balance (FSR minus FBR). Exercise resulted in significant increases above rest in muscle FSR at all times: 3 h = 112%, 24 h = 65%, 48 h = 34% (P < 0.01). Muscle FBR was also increased by exercise at 3 h (31%; P < 0.05) and 24 h (18%; P < 0.05) postexercise but returned to resting levels by 48 h. Muscle net balance was significantly increased after exercise at all time points [(in %/h) rest = -0.0573 +/- 0.003 (SE), 3 h = -0.0298 +/- 0.003, 24 h = -0.0413 +/- 0.004, and 48 h = -0.0440 +/- 0.005], and was significantly different from zero at all time points (P < 0.05). There was also a significant correlation between FSR and FBR (r = 0.88, P < 0.001). We conclude that exercise resulted in an increase in muscle net protein balance that persisted for up to 48 h after the exercise bout and was unrelated to the type of muscle contraction performed.
Mixed muscle protein synthesis and breakdown
after resistance exercise in humans
STUART M. PHILLIPS, KEVIN D. TIPTON, ASLE AARSLAND,
STEVEN E. WOLF, AND ROBERT R. WOLFE
Metabolism Unit, Shriners Burns Institute, and Departments of Surgery and Anesthesiology,
University of Texas Medical Branch, Galveston, Texas 77550
Phillips, Stuart M., Kevin D. Tipton, Asle Aarsland,
Steven E. Wolf, and Robert R. Wolfe. Mixed muscle
protein synthesis and breakdown after resistance exercise in
humans. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E99-
El07, 1997. -Mixed muscle protein fractional synthesis rate
(FSR) and fractional breakdown rate (FBR) were examined
after an isolated bout of either concentric or eccentric resis-
tance exercise. Subjects were eight untrained volunteers (4
males, 4 females). Mixed muscle protein FSR and FBR were
determined using primed constant infusions of L2HS]phenyl-
alanine and 15N-phenylalanine, respectively. Subjects were
studied in the fasted state on four occasions: at rest and 3,24,
and 48 h after a resistance exercise bout. Exercise was eight
sets of eight concentric or eccentric repetitions at 80% of each
subject’s concentric 1 repetition maximum. There was no
significant difference between contraction types for either
FSR, FBR, or net balance (FSR minus FBR). Exercise re-
sulted in significant increases above rest in muscle FSR at all
times: 3 h = 112%, 24 h = 65%, 48 h = 34% (P < 0.01). Muscle
FBR was also increased by exercise at 3 h (31%; P < 0.05) and
24 h (18%; P < 0.05) postexercise but returned to resting
levels by 48 h. Muscle net balance was significantly increased
after exercise at all time points [(in %A$ rest = -0.0573 ?
0.003 (SE), 3 h = -0.0298 ? 0.003, 24 h = -0.0413 t 0.004,
and 48 h = -0.0440 ? 0.0051, and was significantly different
from zero at all time points (P < 0.05). There was also a
significant correlation between FSR and FBR (r = 0.88, P <
0.001). We conclude that exercise resulted in an increase in
muscle net protein balance that persisted for up to 48 h after
the exercise bout and was unrelated to the type of muscle
contraction performed.
hypertrophy; muscle damage; fractional synthetic rate; frac-
tional breakdown rate
THE PROCESS OF MUSCLE HYPERTROPHY
after resistance
exercise is a fundamental adaptation to an increased
resistive workload. Muscle growth can only occur,
however, if there is net anabolism within the muscle.
That is, muscle protein net balance (synthesis minus
breakdown) is positive during the period in which
hypertrophy occurs. A variety of investigations have
shown that in the period after resistance and long-term
endurance exercise there is a stimulation of mixed
muscle protein synthesis (4, 7,s) that persists for up to
24 h (33). On the other hand several studies, using
indirect measures, have demonstrated that exercise
stimulates (10,21) or does not affect (9,16) myofibrillar
protein breakdown. We have recently confirmed that an
isolated bout of high-intensity resistance exercise stimu-
lated muscle protein breakdown over the first 4 h
postexercise (4). Nevertheless, because there was a
greater relative stimulation of synthesis, the overall
effect was an increase in muscle net protein balance
immediately after the exercise (4). However, the time
course of the responses of muscle protein breakdown
and, more importantly, muscle protein net balance
after resistance exercise has not been examined.
Activities that are comprised of repeated eccentric
contractions have been shown to result in disruption of
the ultrastructure of skeletal muscle (12, 14, 15).
Recently, Gibala et al. (15) showed that, immediately
after a single bout of either concentric or eccentric
resistance exercise in untrained subjects, there was
significant myofibrillar disruption that persisted for 48
h. There was, however, a far greater degree of myofibril-
lar damage observed after the eccentric phase of the
protocol (15). The degree of myofibrillar disruption and
myocellular enzyme release (an indicator of myofibril-
lar disruption) has consistenly been shown to be greater
after eccentric vs. concentric activities (12, 14, 15).
Whereas eccentric contractions result in greater myofi-
brillar disruption, as seen by electron microscopy, it is
not known whether there is an association between the
processes of myofibrillar disruption and the breakdown
of muscle proteins.
The purpose of this investigation was to examine the
time course of muscle protein synthesis (fractional
synthesis rate, FSR) and breakdown (fractional break-
down rate, FBR) after an isolated bout of resistance
exercise. In addition, we wished to determine whether
there was a difference in either FSR or FBR after the
performance of either concentric or eccentric exercise.
METHODS
Participants
Subjects were eight (4 male, 4 female) volunteers who were
advised of the purposes of the study and associated risks, and
all subjects gave written informed consent. The project was
approved by the Institutional Review Board and the Clinical
Research Centre (CRC) of The University of Texas Medical
Branch. The subjects’ descriptive characteristics are shown in
Table 1. Subjects were moderately active (recreational cycling
and running) and did not engage in any forms of resistance
training for 25 mo before or during the study. Subjects had
their concentric bilateral 1 repetition maximum (RM) deter-
mined (Table I) in the seated position in a Nautilus knee
extension machine. A subject’s 1 RM was defined as the
maximum weight he or she could lift to full extension and
hold for a l-s count. In the seated resting position in this
machine, the subject’s knee was flexed at -100”. Full exten-
sion required the movement of the subject’s knee through this
arc to the point at which the subject’s knee was fully
extended. During the I RM testing and the testing protocol no
assistance (“spotting”) was given, but subjects were verbally
encouraged to maintain their effort. After the initial testing
session, subjects were assigned in a counterbalanced manner
019%1849/97 $5.00 Copyright o 1997 the American Physiological Society E99
El00
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
Table 1. Descriptive characteristics of subjects
Age,
Yr 22.620.6
Ht, cm 171.0? 3.3
wt, kg 65.3 54.4
1 RM, kg 73.556.4
1 RM/Wt 1.43 + 0.04
Volume, kg 4,365?381
Values are means 5 SE (n. = 8). RM, repetition maximum; 1
RMNVt, repetition maximum per kg body weight; volume, total
weight lifted during protocol, which was calculated as the total no. of
repetitions completed multiplied by the mass lifted (in kg).
to either an eccentric or a concentric group and were matched
for 1 RM (per kg body weight) and gender. All female subjects
participating in this study were taking oral contraceptives
and were tested in the early (follicular) phase of their
menstrual cycle.
Experimental Protocol
The protocol was designed to examine the time course of
mixed muscle protein FSR and mixed muscle protein FBR after
an isolated resistance exercise bout. The intial response was
examined at -3 h postexercise and at two subsequent time points
(-24 h and -48 h) postexercise on consecutive days after the
initial exercise bout. The effect of contraction type was also
examined by looking at the effect of either eccentric or
concentric contractions on FSR and FBR. A schematic repre-
sentation of the infusion protocol is shown in Fig. 1.
Subjects reported to the CRC on the evening before the
beginning of testing and did the same for the remaining 4
days of the testing procedure. Subjects were instructed to
maintain a meat-free diet before and during the study
protocol. During the protocol, subjects consumed one meal at
the CRC after the infusion protocol; all other meals were
consumed away from the CRC at the subjects’ discretion.
Subjects were instructed to maintain a consistent dietary
pattern throughout the duration of the study. After an
overnight fast, at 0500 on the morning of day 1 (rest) of the
protocol, subjects had an l&gauge catheter inserted into a
dorsal hand vein, which was kept patent with a 0.9% saline
drip. This hand was also warmed with a heating pad to
“arterialize” the blood sample. Another Wgauge catheter was
inserted in a contralateral forearm vein for a primed constant
infusion of isotopically labeled amino acids. The catheters
were inserted in positions that were not occluded by arm
bending. Infusion of C2H5]phenylalanine was begun at -0600
(t
= 0). The initial (baseline) percutaneous muscle biopsy was
taken from the lateral vastus at -0800
(t
= 120). The muscle
biopsy was taken under suction after subcutaneous adminis-
* blood sample
muscle biopsy
+lSN-Phe ,-b
)EXERCISEI [*H,lPhe
: : : :
0 60 180 240 260 2u80 $0
Fig. 1. Schematic representation of infusion protocol on each day.
Note that exercise was performed on day 2 of study only. All values for
time are given as time postexercise (-3 h, -24 h, and -48 h), except
for day 1, which was before exercise protocol (Rest).
tration of 1% lidocaine. Baseline and all other muscle samples
were blotted dry, frozen in liquid nitrogen, and stored at
-80°C before analysis. Immediately after the biopsy a primed
constant infusion of 15N-labeled phenylalanine was initiated.
Blood samples were drawn before and throughout the infu-
sion protocol at
t
= 0, 120, 180, 2’10, 240, 260, 280, and 300
min (Fig. 1) for determination of the tracer-to-tracee ratio. In
addition, serum samples were drawn at
t
= 300 min for
determination of serum creatine kinase activity. After the
blood sample at
t
= 240 min, the 15N-phenylalanine infusion
was terminated for the determination of muscle protein FBR
according to the method of Zhang et al. (34). After termination
of the 15N-phenylalanine infusion, muscle biopsies were taken
from the same incision made previously
(t
= 120), at
t
= 280
(40 min after termination of the infusion; Fig. 1) and
t
= 300
min (60 min after termination of the infusion; Fig. 1). On
subsequent days, muscle biopsies were taken from the oppo-
site leg. Moreover, when biopsies were taken from the same
leg, the incision was made 25 cm proximal to the initial
incision. Care was also taken to ensure that the positioning of
the biopsy needle would not sample tissue from a previous
biopsy site by ensuring that the biopsy needle was proximal
or distal from the incision.
After the last biopsy and blood sample, subjects consumed
a meal at the CRC before being discharged. Subjects collected
24-h urine samples beginning at 0500 on day 1 (rest) for the
duration of the study. Urine samples were analyzed for
creatinine, urea, and 3-methylhistidine (3-MH) concentra-
tion. The same infusion protocol was repeated on days 1
(rest), 3 (24 h postexercise), and 4 (48 h postexercise), with the
exception of the 2nd day (-3 h postexercise), when subjects
performed the exercise test between
t
= 0 and
t
= 120 min of
the infusion of the protocol. The exercise test took place at the
University of Texas Medical Branch Alumni Field House. An
infusion of [2H5]phenylalanine was maintained throughout
the exercise protocol, which lasted for -45 min, by a battery-
operated calibrated infusion pump (Travenol, Hooksett, NH).
Subjects warmed up by cycling on a stationary cycle ergom-
eter with no load for 10 min. Subjects then performed eight
sets of eight repetitions at 80% of their predetermined
concentric 1 RM. Each set was followed by 2 min of rest.
Subjects who were randomized to the eccentric protocol had
the weight lifted for them by the investigators. The subjects
then extended their legs to full extension and lowered the
weight to a resting position. The opposite was true of the
concentric lifting protocol; that is, investigators lowered the
weight to a resting position for the subjects while they raised
the weight to full extension. All subjects, except for one in the
concentric group for whom the weight was lowered to -73% of
his 1 RM after two sets, were able to complete their predeter-
mined workload.
Is0 topes
All isotopes were dissolved in 0.9% saline before infusion.
Both C2H5]phenylalanine and 15N-phenylalanine were pur-
chased from Cambridge Isotopes (Andover, MA). All isotopes
were infused using a calibrated Harvard syringe pump
(Natick, MA), and the exact infusion rate was determined by
multiplying the infusate concentration, determined by gas
chromatography-mass spectrometry (GC-MS), by the mea-
sured infusion rate. The infusion rates of L2HJphenylalanine
and 15N-phenylalanine were 0.05 pmol
l
kg-l
l
minl (priming
dose 2.0 pmolkg). All isotopes were filtered through a 0.2~pm
filter before infusion. The infusion protocols were designed so
that steady state was achieved in both muscle and plasma
pools. This has been confirmed in previous studies (4) and is
shown in Figs. 2 and 3.
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
El01
Analyses
Urine. Urine collections were pooled, and the total volume
for each day was recorded. Analysis of urinary creatinine and
urea concentration was performed using commercially avail-
able kits (procedures 555 and 640, respectively; Sigma Chemi-
cal, St. Louis, MO). The concentration of 3-MH in urine was
determined by making the tert-butyl dimethylsilyl (t-BMDS)
derivative of 3-MH, using a GC-MS (Hewlett-Packard 5890/
5989B, Palo Alto, CA), and using 3-[methyl-13C]MH as an
internal standard (2.7 mM). The procedure for preparation of
urine samples for urinary 3-MH concentration by isotope
dilution was according to the procedures outlined by Rathma-
cher et al. (23). Briefly, -500 ~1 of urine (weighed) and -100
~1 of internal standard (weighed) were mixed. The sample
was then acidified by adding 20 ~1 of 1 N HCl. The urine
sample was then passed over an acid-washed cation exchange
column (Dowex AG 5OW-8X, 100-200 mesh, H+ form; Bio-
Rad Labs, Richmond, CA). The 3-MH and other amino acids
were then eluted from the column with two 1.5-ml washes of 2
N ammonium hydroxide. These aliquots were then dried
under vacuum using a Speed-Vat rotary drying apparatus
(Savant Instruments, Farmingdale, NY). The sample was
then incubated with urease (-25 U of Jack bean type IX
urease; Sigma Chemical) in a phosphate buffer (30 mM, pH
7.5) for 3 h at 37°C. The sample was again lyophilized
to dryness. To the dried sample, 50 ~1 of acetonitrile and
50 ~1 N-methyl-N-(t-butyldimethylsilyl) trifluoro-acetamide
(MTBSTFA; Pierce Chemical, Rockford, IL) were added, and
the sample was heated at 90°C for 1 h. The sample was then
analyzed using GC-MS (Hewlett-Packard 5890, series II;
Fullerton, CA) by injecting 1 ~1 and monitoring ions of
mass-to-charge ratio (m lx) 238 (m+O) and 239 (m+ 1) to
determine the concentration of 3-MH by use of the internal
standard method (3).
Blood. Blood samples for determination of amino acid
enrichment and concentration were immediately precipitated
in preweighed tubes containing 15% sulfosalicylic acid, which
contained a weighed amount of internal standard. The inter-
nal standard used was C2Hs]phenylalanine (85.5 PM), added
in a ratio of 100 $./ml of blood. To determine the enrichment of
infused phenylalanine and internal standards in whole blood,
the t-BDMS derivative of phenylalanine was made according
to previously described procedures (3). Analysis of t-BDMS
phenylalanine by GC-MS (Hewlett-Packard 5890, series II)
was performed using electron impact ionization and selected
ion monitoring of m/z 234, 235, 239, and 240, for the m+O,
m + 1, m + 5, and m +6 ions, respectively. Appropriate correc-
tions were made for any spectra that overlapped and contrib-
uted to the tracer (t)-to-tracee (T) ratio (t/T) (31). Concentra-
tion of phenylalanine in both blood and the muscle free pool
was calculated as described previously (3) and with the
assumption that interstitial water accounts for 13% of the
water content in muscle (3).
Serum samples were analyzed for creatine phosphokinase
(CPM) activity with a commercially available kit (no. 661;
Sigma Chemical, St. Louis, MO). CPK activity is expressed in
units (U) per milliliter, where one unit of CPK will phosphory-
late one nanomole of creatine per minute at 25°C (Sigma
Chemical definition). The intra-assay coefficient of variation
(CV) for this assay did not exceed 11% for any given day, and
the interassay CV was no greater than 13%.
Muscle. Muscle biopsy tissue samples were analyzed for
protein-bound and free intracellular enrichment, as well as
intracellular concentration, as described previously (3, 4).
Briefly, each sample was weighed and muscle protein was
precipitated with 800 ~1 of 14% perchloric acid (PCA). An
internal standard (2 pl/mg tissue) containing 3.3 PM L-[ring-
13C6]phenylalanine was added to the precipitate. The tissue
was then homogenized and centrifuged. The supernatant was
collected, and this procedure was repeated twice more with
additional 500+1 washes of 14% PCA. The remaining pellet of
muscle tissue was washed in distilled deionized H20, washed
three times in absolute ethanol, and then placed in a 50°C
oven to dry overnight. The dried pellet was placed in 6N HCl
and hydrolyzed for 24 h at 110°C. The protein hydrolysate
was then deionized using ion exchange columns as described
for blood analysis (3, 4). Briefly, an aliquot of the acid
hydrolysate (-4 mg wet weight of muscle) was passed over an
acid-washed cation exchange column (Dowex AG 5OW-8X,
100-200 mesh, H+ form; Bio-Rad Labs). The pooled PCA
washes (-1.3 ml) were prepared in the same manner as the
protein-bound acid hydrolysates for determination of the
intracellular phenylalanine enrichment. Amino acids were
eluted from the column with 3 ml of 3M NHdOH, and the
resulting eluate was collected and dried under vacuum with a
Speed-Vat rotary drying apparatus (Savant Instruments,
Farmingdale, NY). To make the heptaflurobutyric (HFB)
derivative of phenylalanine, 500 ~1 of 3.5 N HBr propanol
reagent were added to the dry residue, which was vortexed
and heated at 110°C for 1 h. The sample was then dried down
under a stream of dry N2 gas, and 100 ~1 of HFB anhydride
was added to the dry residue. The sample was then heated at
60°C for 20 min and was then dried down under N2. Ethyl
acetate (100 ~1) was added to resuspend the HFB-phenylala-
nine derivative for injection into the GC-MS. All phenylala-
nine enrichments were determined using chemical impact
ionization with methane gas and selected ion monitoring at a
variety of ions, depending on what was to be determined.
Protein bound [2Hs]phenylalanine enrichment was deter-
mined by monitoring m lx 407 and 409, which 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 to m +3
(m +5/m +3) was used, since it is much more sensitive than
the traditional m +5/m + 0 (used for plasma samples). Enrich-
ment from the protein-bound samples was determined using
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. Precursor enrichment for calcu-
lation of FSR was determined from intracellular 12H5]phenyl-
alanine enrichment by monitoring the m + 5/m +0 (mass 409
and 404 amu) enrichments of the HFB-phenylalanine. For
calculation of FBR, the decay in intracellular enrichment of
the infused 15N-phenylalanine was measured by monitoring
the ions of m/z 404 and 405 and was calculated according to
the calculations and assumptions outlined by Zhang et al.
(34). Muscle free phenylalanine concentration was deter-
mined using a [ring- 13C6]phenylalanine internal standard
(3.3 PM) and monitoring ion of m/z 410 and 404 and assuming
that interstitial water accounts for 13% of the water content
in muscle (3).
Cakulations
FSR of muscle protein was calculated from the determina-
tion of the rate of tracer incorporation into muscle protein and
with use of the muscle intracellular free phenylalanine
enrichment as the precursor, according to the equation
FSR (%/h) = ((Et, - Et,)I[E; (tl - to>l). 100 (1)
where Et0 is the enrichment in the protein-bound phenylala-
nine tracer from the first biopsy at t = 120 min, Et, is the
enrichment of the protein-bound phenylalanine tracer from
the second or third biopsy at t = 280 and t = 300 min, (ti - to)
is the incorporation time (-3 h); and E, is the mean intracellu-
El02
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
Table 2. Urinary indexes ofprotein metabolism
Day 1 (Rest) Day 2 (3 h) Day 3 (24 h) Day 4 (48 h)
Urea, mol/day 0.46 + 0.04 0.512 0.03 0.50 It 0.03 0.49 k 0.05
Creatinine, mmol/day 12.7 t 1.6 13.0 t 2.0 12.3 -+ 1.3 13.5 + 2.0
3-MH, pmol/day 173 t 11 180 t 13 189 t 14 205 Ifr 15
3-MWcreatinine, pmol * mmol-i * day-l 14.4 -t 1.0 15.6 + 1.9 15.9 2 1.0 17.12 2.2
Values are means + SE; n = 8 subjects. 3-MH, 3-methylhistidine. Day 1 (rest), day 2 (3 h), 3 h postexercise; day 3 (24 h), 24 h postexercise;
day 4 (48 h), 48 h postexercise.
lar (t = 120, 280, and 300) [2H5]phenylalanine enrichment using a Pearson product correlation and analyzed according
during the time period for determination of protein incorpora- to the appropriate degrees of freedom. AP value of co.05 was
tion. considered significant. Data are expressed as means t SE.
Phenylalanine was chosen as the tracer because it is not
oxidized in muscle or synthesized in the body. Thus appear-
ance of phenylalanine results entirely from protein break-
down. Whole body phenylalanine appearance was calculated
according to the equation
RESULTS
Urine
R, = F/(E,)
(2)
where R, is rate of appearance (pmol kg-l .mir+), F is
infusion rate (pmol kg-l. min+), and E, is whole blood
[2HS]phenylalanine enrichment (t/T).
By use of the tracee release method described previously
(34), the intracellular dilution of both plasma and muscle
amino acid enrichment after the cessation of infusion can be
measured and fit to equations to measure muscle FBR. This
was calculated in the current protocol by using the decay in
enrichment of 15N-phenylalanine and the equations outlined
previously (34). In calculating FBR in this manner, we have
assumed that our arterialized blood samples were representa-
tive of arterial enrichments (6). We have also assumed that
the arterial blood is the only source of tracer entering the
muscle intracellular free pool, such that there is no tracer
recycling (34), which during such a short infusion is a valid
assumption. Because we did not take a biopsy immediately
before stopping the 15N-phenylalanine infusion, we calcu-
lated the intramuscular 15N-phenylalanine at this time point
(240 min) by using the ratio of the mean intracellular
[12H5]phenylalanine (calculated as the mean of 120, 280 and
300 min) to arterial [2H5]phenylalanine enrichment and
multiplying by the arterial 15N-phenylalanine enrichment
(see Figs. 2 and 3). The validity of this approach has been
tested in animals (X.-J. Zhang, unpublished observations).
We will not discuss the mathematical assumptions or deriva-
tions of the equations necessary to calculate FBR, since this
has been previously presented (34). Given the assumptions
just presented, however, we were able to calculate a muscle
net balance by using FSR from _Eq. 1 and FBR, as
There was no difference in the excretion of urinary
urea throughout the study (Table 2). Urinary excretion
of creatinine was also unaffected during the protocol
(Table 2). The absolute excretion of 3-MH @mol./day)
was unchanged throughout the protocol, although the
time effect (P = 0.084) did show a trend toward an
increase (Table 2). The ratio of 3-MH excretion to
creatinine excretion also remained unchanged through-
out the protocol (P =
0.18;
Table 2).
Blood
Serum CPK activity remained unchanged over the
course of the study (day 1 = 6.9 t 0.3; day 2 = 6.8 t 0.4;
day 3 = 8 9 + 0.7; day 4 =
11.2 t 1.9;
P > 0.4).
-
Subjects’ blood 15N-phenylalanine and [2H5]phenyl-
alanine enrichment throughout the protocol (for day
I/rest) is shown in Fig. 2, A and B, respectively. As Fig.
2 shows, all subjects achieved a steady state in enrich-
net balance (%/h) = FSR (%/h) - FBR (%A$
Statistics
(3)
A 0.10.
-
I
y 0.05
CI
0.00
B
0 s1
v s2
+ s3
OS4
x s5
0 S6
A S7
+ S8
I I I I I
180 210 240 270 300
Data were analyzed using a two-way repeated-measures
analysis of variance (ANOVA), with time (days) and group
(eccentric or concentric) as the within and between factors,
respectively. Statistical analysis revealed that there were no
between-group differences in the measures of FSR, FBR, net
balance, 3-MH excretion, or any relevant variables. Sample
size analysis revealed that at least 22 subjects per group
would have to be studied to detect a difference between
groups. Hence, the data presented here are shown as one
group, with only the time effects analyzed and with use of a
one-way repeated-measures ANOVA. Wherever significant
differences were found, a Tukey post hoc test was used to
locate the pairwise difference. Correlations were performed
0.10
7
L-
l 0.05
w
0.00
0
ia Q
8 i 8
m A
0 sa
v s2
+ s3
OS4
x s5
•I S6
A S7
* S8
1
180
I
240
Time (min)
I
300
Fig. 2. Blood phenylalanine enrichment of individuals during infu-
sion (day 1). S, subject; t, tracer; T, tracee. A: blood i5N-phenylalanine
enrichment during infusion for each subject; B: blood [2H5]phenyl-
alanine enrichment during infusion for each subject.
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
El03
ment for both tracers during the course of the study,
and, more importantly, over the time course when
muscle biopsies were taken. Similar patterns in enrich-
ment were seen on all other study days (data not
shown).
Whole body phenylalanine turnover was calculated
from steady-state blood enrichment of [2Hs]phenyl-
alanine at 180, 240, and 300 min (Fig.
2B).
There was
no significant difference in whole body turnover (R,) of
phenylalanine on any study day (rest = 0.77 t 0.04;
3
h = 0.77 t 0.04; 24 h = 0.76 t 0.05; 48 h = 0.78 t
0.04, all in pmol
l
kg-l
l
minl).
Muscle
Muscle intracellular 15N-phenylalanine and [2Hs]phen-
ylalanine enrichment is shown in Fig. 3,
A
and
B,
respectively. The intracellular enrichments were consis-
tently lower than arterialized enrichments, presum-
ably because of dilution by protein breakdown (3,4). All
subjects achieved steady state in the intramuscular
pool for [2H5]phenylalanine enrichment; hence it ap-
pears that the 2-h infusion of 15N-phenylalanine was
also sufficient to achieve an intracellular steady state.
Intracellular enrichment of 15N-phenylalanine de-
creased in the time period between 280 and 300 min
(Fig.
3A)
for all subjects, allowing the calculation of
FBR. Muscle intracellular phenylalanine concentration
was not significantly different throughout the infusion
protocol (mean of biopsies at 120,280 and 300 min) and
was not different between days (rest= 86 t
16; 3
h =
93 t 17; 24 h = 82 ? 10; 48 h = 76 t 23 nmolfml
intracellular water;
P > 0.6).
Muscle protein FSR was calculated according to the
steady-state precursor-product equation outlined in
a
0.025
- I
0.000
B
240 260 280 300
0.075-
F 0.050- 8
II- 5!!
l 0
CI
o-025-
X X
0 0
8 0
V
0.000-1
I I I I I I I
120 150 180 210 240 270 300
Time (min)
0 Sl
v s2
+ s3
0 s4
x s5
0 S6
A s7
+ S8
0 Sl
v s2
+ s3
0 s4
x s5
0 S6
A S7
* S8
Fig. 3. Intramuscular phenylalanine enrichment of individual sub-
jects during infusion (day 1). A: muscle i5N-phenylalanine enrich-
ment during infusion for each subject (values at 240 min were
calculated according to assumptions outlined in METHODS). B: muscle
[2Hs]phenylalanine enrichment during infusion for each subject.
A
0.15
n
v
Ir
0.10
l
P
0
w
w
c
0.05
0.00
B
n
7
c
l
s
ii
LL
0.20
0.15
0.10
0.05
0.00 2
a
Rest 3h
b
b
-
a
Rest 3h
-
24h
C
1 48h
a
-
I
24h 48h
Fig. 4. Mixed muscle protein fractional synthesis rate (FSR, A) and
fractional breakdown rate (FBR, B) at rest and after exercise bout.
Means with different letters are statistically different (P < 0.05). A
main effect for time was found for FBR (P <
0.01).
Values are
means + SE (n = 8). Rest, day 1; 3h, 3 h postexercise; 24h, 24 h
postexercise; 48h, 48 h postexercise.
METHODS
(see Eq. 1). Muscle FSR increased from rest-
ing levels after exercise by 112% at 3 h postexercise
(P
< 0.01; Fig. 4A). In addition, FSR was significantly
elevated above rest at 24 and 48 h postexercise by 65
and 34%
(P
<
0.01;
Fig. 4A), respectively. However,
there was no significant difference in FSR between 24
and 48 h postexercise (Fig. 4A).
The FBR of mixed muscle proteins was also increased
after the exercise bout, at 3 h postexercise, by
31%
above resting values
(P
< 0.05; Fig.
4B).
FBR 24 h after
the exercise bout was still 18% above resting values
(P
c 0.05; Fig.
4B).
However, 48 h after the exercise
bout, FBR had returned to resting (day 1) values and
was not significantly different from rest (Fig.
4B).
There was a highly significant correlation (r = 0.88;
P
< 0.001) between the measured FSR and FBR (Fig.
5). Moreover, there was a significant correlation in the
change in FSR and FBR between days (r = 0.73;
P <
0.01; data not shown).
Ad’
0
2
pf’0
0 0
o B”&og
/‘“,”
0
0.0 1 I I I I I 1
0.0
0.1 0.2
FSR(%*h-')
Fig. 5. Correlation between mixed muscle protein FSR and FBR (y =
1.22x + 0.071;
r = 0.88,P <
0.01).
El04
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
Muscle protein net balance was calculated as the
difference between FSR and FBR and is shown for each
day during the protocol in Fig. 5. Net balance was
significantly negative on all study days despite the fact
that it was significantly increased at 3, 24, and 48 h
postexercise vs. rest (Fig. 6). Muscle protein net bal-
ances at 3, 24, and 48 h were 48, 28, and 23% higher
than at rest (Fig. 6). However, the net balance between
24 and 48 h was not significantly different (Fig. 6).
DISCUSSION
The findings from this study have shown that a
single isolated bout of concentric or eccentric resistance
exercise, in untrained subjects, results in elevations in
muscle FSR, FBR, and net protein balance. In addition,
there was a highly significant correlation between
muscle protein FSR and FBR, suggesting a tight rela-
tionship between these two processes in the fasted
state. To our knowledge, this is the first study to have
followed the time course of both synthesis and break-
down after a resistance exercise bout. After the exercise
bout, the FBR of mixed muscle proteins was elevated at
3 and 24 h. However, FBR returned to resting levels by
48 h postexercise. In contrast, the increase in FSR
persisted for 22 days after the exercise session. The
result of the elevation in FSR was that the net protein
balance within the exercised muscle increased and was
significantly higher postexercise at all time points
studied. It should be emphasized that all studies in the
present protocol were performed in the fasted state, so
it was expected that muscle net balance would be
negative (4).
Previous studies have demonstrated that an isolated
bout of resistance exercise resulted in an increase in
the mixed muscle protein synthetic rate (4, 8, 33).
Moreover, Biolo et al. (4) recently confirmed these
findings by use of a different method to calculate
muscle protein synthesis. The rate of biceps brachii
muscle protein synthesis after an isolated bout of
resistance exercise, in trained subjects, was reported to
be increased 50% above resting at 4 h and 109% at 24 h
postexercise (8). Subsequently, results from the same
group showed that, at 36 h postexercise, muscle mixed
FSR had returned to within 14% of the FSR in nonexer-
cised muscle (19), leading the authors to postulate that
FSR was initially increased but then abruptly de-
s
0.02
6
3 /I 0.00 c
rn’;
-c
-0.02-
2*
a, 5 -0.04-
0
5 -0.06-
> -0.08-
Rest 3h 24h 48h
Fig. 6. Muscle protein net balance (FSR minus FBR) at rest and after
exercise bout. Means with different letters are statistically different
(P
< 0.05). Values are means 2 SE (n = 8). See Fig. 4 for definitions of
legend.
creased at 36 h postexercise (19). These findings are
different from those of the current study, in which FSR
peaked at 3 h postexercise and was still elevated at 48 h
postexercise (Fig. 3A). The discrepancy may be due to
the training status of the subjects, since the time course
of muscle FSR after exercise may be different in trained
vs. untrained subjects. In addition, the time course
reported previously (8, 19) was constructed from three
independent groups of subjects, whereas the current
study used a repeated-measures design. It should also
be noted that the subjects studied at 4 and 24 h
postexercise by Chesley et al. (8) were studied in the fed
state, whereas the subjects at 36 h postexercise ate and
then slept (19). Relevant to this point, we have found
that an infusion of mixed amino acids after exercise, to
elevate blood amino acid concentrations to postpran-
dial levels, stimulated synthesis more than exercise
alone so that, in contrast to exercise alone, net balance
became positive (5). Moreover, we have shown that oral
amino supplementation, with 40 g of mixed amino acids
after resistance exercise, results in a positive net
balance and increases in amino acid uptake by muscle
(26).
The specific nature of the muscle proteins being
synthesized is not distinguishable when the present
technique is used to measure FSR. An elevation in
mixed muscle protein FSR, which represents an aver-
age synthetic rate of all myocellular proteins, has been
observed after resistance exercise (4, 8, 33; present
results). However, by weight myofibrillar proteins com-
prise -60% of all muscle proteins (28). Hence, given the
magnitude of the increase in mixed protein FSR that
we (4; present results) and others (8,33) have observed,
it seems likely that an increase in myofibrillar protein
synthetic rate must have occurred. In contrast to this
conclusion, a report in which myofibrillar protein syn-
thesis was examined (primarily actin and myosin, but
also including troponin, tropomyosin, C protein, and
titin) reported no change in myofibrillar protein synthe-
sis at 24 h postexercise in subjects who had completed a
3-mo resistance training program (29). However, we
recently reported that there was no change in mixed
muscle FSR after a bout of resistance exercise in
trained swimmers, all of whom were regularly engag-
ing in both resistance and endurance exercise (27).
Hence, the fact that there was no increase in mixed
muscle FSR (27) and myofibrillar protein FSR (29) in
trained subjects may be due to the training status of the
subjects. Given the results of the present study, it may
be that the response of muscle protein FBR, which
would be the predominant source of amino acids for
synthesis (FSR) in the fasted state, may be reduced in
trained individuals.
Muscle myofibrillar protein breakdown after resis-
tance exercise has been estimated using indirect mea-
sures such as 3-MH excretion. Because 3-MH is found
exclusively in actin and myosin and cannot be reuti-
lized once the protein is broken down, its appearance in
urine serves as a marker of myofibrillar protein degra-
dation (22, 23). Although the use of 3-MH has been
criticized, because of the unknown contribution of gut
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
El05
myofibrillar protein to 3-MH turnover, recent evidence
suggests that it is a reliable index of muscle myofibril-
lar breakdown (2224). However, results from previous
studies have been inconsistent, with some showing that
exercise results in an increase in (11, 16) and some
showing that it does not change (32, 33) 3-MH excre-
tion. In the present study we were unable to detect a
significant change in either total 3-MH excretion or
3-MH excretion expressed relative to creatinine excre-
tion (Table 2). This is somewhat surprising, given the
increase in muscle FBR that occurred at 3 and 24 h
postexercise. However, it may be that a 24-h urine
collection to measure 3-MH excretion is a relatively
insensitive marker of myofibrillar degradation. This
notion is supported by data showing that a single
isolated bout of weight lifting did not increase 3-M-H
excretion (32), whereas daily performance of resistance
exercise resulted in increased 3-MH excretion only
after the third bout (16). In addition, Fielding et al. (13)
did not report an increase in 3-MH excretion after an
intense bout of eccentric cycling until 10 days postexer-
cise, despite a significant elevation in leucine appear-
ance (protein breakdown).
We recently described an isotopic technique to mea-
sure mixed muscle protein FBR (34). The results from
this study show that muscle protein breakdown rate is
accelerated after resistance exercise, supporting our
earlier findings using a different technique (4). Muscle
protein FBR was not different between eccentric and
concentric contractions. This may be surprising given
the myofibrillar disruption that is generally observed
after eccentric work (12,X), which one might expect to
be associated with greater rate of protein breakdown.
However, if we assume that there is a relationship
between muscle protein breakdown and eccentric exer-
cise, the eccentric exercise in the present study may not
have been severe enough to cause damage that would
result in a greater increase in FBR after the eccentric,
vs. concentric, contractions. Evidence in support of this
can be seen in the comparatively moderate, and hetero-
geneous, responses of serum CPK. In addition, electron
micrographs of longitudinal muscle sections (two sub-
jects per condition) showed no significant difference in
the degree of myofibrillar damage between groups, and
in fact showed very little damage at all (data not
shown). Therefore, it is likely that the exercise was
simply not severe enough, for these subjects, to induce
different degrees of muscle damage between the two
groups. This is an indication that the two groups of
subjects were simply habitually performing enough
eccentric leg work in other activities (cycling, running,
stairmaster). It is well documented that the “protec-
tive” effect of eccentric exercise is long lasting (12, 13).
Despite there being no differences between the groups,
however, these data show that both FSR and FBR are
increased regardless of the type of contraction per-
formed.
A recent report indicated that muscle protein break-
down, measured as tyrosine release, after eccentric
contraction-induced muscle injury did not increase
significantly until 48 h postexercise and remained
elevated for 15 days (18). The difference in time course
of muscle protein breakdown in the previous study (18),
compared with the current study, likely relates to the
methodology for inducing and measuring protein break-
down. In support of a more rapid response of protein
breakdown after exercise, Balon et al. (1) found that
tyrosine and 3-MH release were increased only 30 min
after exercise. In the present study, the response of
breakdown was found to be elevated by 30% at -3 h
postexercise, and this response was attenuated at 24 h
to 18% above resting levels (Fig. 3B). These results
suggest a rapid postexercise activation of whatever
mechanism is responsible for the increase in muscle
protein degradation.
Eccentric activity has been shown to result in activa-
tion of neutral proteases such as calpain (2). It is
unknown, however, whether the same is true of concen-
tric activity. It is conceivable that these exercise-
activated proteases are responsible for the muscle
protein degradative response observed after exercise
(4, 11, 21; present results). Eccentric contractions also
result in the loss (breakdown) of muscle cytoskeletal
proteins (17). If muscular contractions result in an
activation of neutral proteases (2), then a sustained
increase in intracellular calcium, induced by the exer-
cise bout, could play a role in this process (12).
Despite the large increase in muscle protein FBR,
there was no corresponding change in whole body
protein turnover, as reflected by phenylalanine R,.
Similar disparities in muscle FSR and rates of whole
body protein turnover have been documented in the
postexercise period by others (4, 7, 8, 25, 27). These
observations have been interpreted as indicating that
whole body protein synthesis must have changed in the
opposite direction from muscle protein synthesis (8,
25). This is based on the assumption that muscle
protein synthesis accounts for -25-300/o of whole body
protein synthesis (20). However, we have shown that a
50% increase in muscle protein breakdown was accom-
panied by only a 5% increase in whole body protein
breakdown (4). This likely reflects the insensitive na-
ture of the measurement of whole body protein turn-
over, rather than a change occurring in the opposite
direction somewhere else in the body (splanchnic re-
gion). This interpretation is supported by our finding
that exercise stimulated splanchnic protein breakdown
in the dog (30). A possibility is that the lack of sensitiv-
ity of the whole body methodology, over a relatively
short time interval, may be due to transient changes in
tissue pool size. These changes would obscure any
direct relation between protein breakdown and the R,
into plasma of essential amino acids.
Given the rapid nature of the increase in muscle
protein FSR seen in the present study, it is likely a
posttranscriptionally regulated response to the exer-
cise stimulus. This is supported by the finding that an
increase in the synthetic response after exercise is
unrelated to the concentration of muscle RNA, which
remains unchanged even up to 24 h postexercise (8). It
should be noted, however, that large increases in
specific mRNAs could occur quite rapidly after exercise
El06
MUSCLE PROTEIN TURNOVER AFTER WEIGHT LIFTING
without a significant change in total RNA concentra-
tion. Large increases in mRNA are not always accompa-
nied, however, by equivalent, or even proportional,
increases in portein synthesis. The results of the pres-
ent study are consistent with our previous postulation,
that at least some of the control of the protein synthetic
response after exercise is related to increased intracel-
lular amino acid availability (4).
5.
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