Availability of eIF4E regulates skeletal muscle
protein synthesis during recovery from exercise
T. A. GAUTSCH,1 J. C. ANTHONY,1 S. R. KIMBALL,2
G. L. PAUL,1 D. K. LAYMAN,1 AND L. S. JEFFERSON2
1Division of Nutritional Sciences and the Department of Food Science and Human Nutrition,
University of Illinois, Urbana, Illinois 61801; and 2Department of Cellular and Molecular
Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
eukaryotic initiation factor 4E; rats; isoleucine; translation
initiation; 4E-BP1
A DEPRESSION IN THE fractional rate of skeletal muscle
protein synthesis occurs with prolonged exercise (7, 8).
Changes in protein turnover after exercise suggest that
recovery from exercise occurs through stimulation of
protein synthesis (29); however, the mechanisms for
recovery of skeletal muscle protein synthesis after
exercise are currently unknown. It is generally accepted that short-term changes in protein synthesis are
achieved at the translation level, and, in many situations, it is thought that the rate of initiation limits
overall translation (24). Hence, it has been suggested
that recovery of muscle protein synthesis after exercise
occurs at the level of translation initiation (25), although there are no reports to confirm this hypothesis.
The initiation phase of protein synthesis is an important site of translational control, for it allows the cell to
alter protein synthesis rates rapidly and to reverse any
alteration equally as rapidly (15). Recent evidence from
several investigators has indicated that binding of the
mRNA to the 43S preinitiation complex is a ratecontrolling step in translation initiation (19, 32). This
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step requires the group four eukaryotic initiation factors (eIFs), eIF4B and eIF4F. eIF4F is a three subunit
complex consisting of 1) eIF4A, a RNA helicase that
functions in conjunction with eIF4B to unwind secondary structure in the 58-untranslated region (UTR) of the
mRNA; 2) eIF4E, a protein that binds the mRNA cap
structure and is important for selection and stability of
the mRNA to be translated; and 3) eIF4G, a 220-kDa
polypeptide that is suggested to function as a scaffold
for eIF4E, eIF4A, the mRNA, and the ribosome (via
association with eIF3) (32). The eIF4F complex collectively serves to recognize, unfold, and guide the mRNA
to the 43S preinitiation complex.
Alterations in either the phosphorylation state or in
the availability of eIF4E for binding eIF4G appear to
modulate the function of the eIF4F complex. Phosphorylation of eIF4E has been suggested to increase translation rates via increased association with eIF4G and
eIF4A (22) and/or increased mRNA cap-binding affinity
(21). Conversely, the availability of eIF4E for eIF4F
complex formation appears to be modulated by the
translational inhibitory phosphoprotein 4E-BP1 (27).
4E-BP1 competes with eIF4G for binding eIF4E and
has the ability to physically sequester eIF4E into an
inactive complex. Changes in the relative affinity of
eIF4E for eIF4G vs. 4E-BP1 due to phosphorylation of
one or more of these proteins is postulated to regulate
rates of translation.
Few studies have examined the effect of food intake
on recovery of muscle protein synthesis. Recent data
demonstrate that consumption of a complete meal after
acute food deprivation stimulates muscle protein synthesis via increases in initiation activity (36), whereas
other findings suggest that a combination of insulin
plus amino acids is required for stimulation of muscle
protein synthesis (28). However, to our knowledge, no
data exist examining the influence of postexercise meal
composition on recovery of skeletal muscle protein
synthesis.
The objectives of the present study were twofold: 1) to
examine the influence of postexercise feeding and meal
composition on the recovery rate of muscle protein
synthesis, and 2) to investigate if changes in muscle
protein synthesis with postexercise feeding and/or meal
composition can be attributed to changes in translation
initiation via alterations in eIF4E phosphorylation or
availability. This study demonstrates that inhibition of
muscle protein synthesis after prolonged exercise is
attributed to reduced availability of eIF4E. Feeding a
nutritionally complete meal, but not a carbohydrate
meal, resulted in a complete recovery of muscle protein
0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society
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Gautsch, T. A., J. C. Anthony, S. R. Kimball, G. L. Paul,
D. K. Layman, and L. S. Jefferson. Availability of eIF4E
regulates skeletal muscle protein synthesis during recovery
from exercise. Am. J. Physiol. 274 (Cell Physiol. 43): C406–
C414, 1998.—We examined the association of the mRNA cap
binding protein eIF4E with the translational inhibitor 4EBP1 in the acute modulation of skeletal muscle protein
synthesis during recovery from exercise. Fasting male rats
were run on a treadmill for 2 h at 26 m/min and were
realimented immediately after exercise with either saline, a
carbohydrate-only meal, or a nutritionally complete meal
(54.5% carbohydrate, 14% protein, and 31.5% fat). Exercised
animals and nonexercised controls were studied 1 h postexercise. Muscle protein synthesis decreased 26% after exercise
and was associated with a fourfold increase in the amount of
eIF4E present in the inactive eIF4E · 4E-BP1 complex and a
concomitant 71% decrease in the association of eIF4E with
eIF4G. Refeeding the complete meal, but not the carbohydrate meal, increased muscle protein synthesis equal to
controls, despite similar plasma concentrations of insulin.
Additionally, eIF4E · 4E-BP1 association was inversely related and eIF4E · eIF4G association was positively correlated
to muscle protein synthesis. This study demonstrates that
recovery of muscle protein synthesis after exercise is related
to the availability of eIF4E for 48S ribosomal complex formation, and postexercise meal composition influences recovery
via modulation of translation initiation.
EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
synthesis at 1 h postexercise and an increase in eIF4E
availability. The effect on eIF4E availability by postexercise nutrition is not ascribed to a differential insulin
response because plasma insulin concentrations in the
two meal groups were similar.
EXPERIMENTAL PROCEDURES
group) intubated with the nutritionally complete meal were
included in the study design to determine any parallel effects
of the food deprivation on muscle protein synthesis.
The experimental run was performed on the ninth day and
consisted of 2 h of treadmill running at 26 m/min (1.5%
grade). Previous reports on energy expenditure in this strain
of rat indicated this exercise intensity corresponds to ,75%
maximum oxygen consumption (31). Exactly 1 h after the
termination of the experimental run, animals were anesthetized by CO2 and killed by decapitation. The decision to
investigate muscle recovery at 1 h after exercise was based on
previous work in rodents which showed realimentation after
food deprivation to increase muscle protein synthesis within
1 h of refeeding (10, 20, 37). Trunk blood was collected and
centrifuged at 1,800 g for 10 min to obtain plasma.
Plasma measurements. Plasma glucose was analyzed by a
glucose oxidase-peroxidase automated method (YSI model
2300 analyzer, Yellow Springs Instruments, Yellow Springs,
OH). Plasma corticosterone and plasma insulin were analyzed using commercial RIA kits.
Administration of metabolic tracer. A bolus dose (0.7 ml/
100 g body wt) of L-[4,5-3H]isoleucine (200 mM containing 130
µCi/ml) was injected via the tail vein 50 min after the end of
exercise for the in vivo measurement of skeletal muscle
protein synthesis (11). The right gastrocnemius and plantaris
were excised as a unit 10 min after injection and quickly
frozen in liquid nitrogen before storage in a 280°C freezer.
The elapsed time from injection until freezing of muscle in
liquid nitrogen was recorded as the actual infusion time.
Muscle isoleucine specific radioactivity. Frozen muscle was
powdered under liquid nitrogen with a mortar and pestle. A
100-mg aliquot was combined with 1.25 ml extraction buffer
(40 mM sodium pyrophosphate, 20 mM sodium phosphate,
and 0.5 mM EDTA), then homogenized using a Polytron
(Kinematica, Kriens-Luzern, Switzerland). After homogenization, 150 µl of 100 g/l sodium dodecyl sulfate were added, and
the sample was boiled for 3 min and then centrifuged 10 min
at 1,000 g. Soluble protein was precipitated from a 100-µl
aliquot of the supernatant by adding 1.25 ml of cold 100 g/l
trichloroacetic acid and refrigerating overnight. The sample
was centrifuged for 3 min, and the resulting pellet was
washed three times with 1 ml of 50 g/l trichloroacetic acid and
once with 0.5 ml of 100% ethanol. The pellet was rinsed with
50 µl acetone and air dried. The dry pellet was transferred to
a hydrolyzing ampule combined with 1 ml of 6 M HCl, flame
sealed, and placed in a 110°C oven for 24 h. The solution was
transferred into 12 3 75-mm glass tubes and evaporated in a
speed vacuum (Savant Instruments, Farmingdale, NY). The
pellet was rinsed with 1 ml of distilled water and redried. The
resulting pellet was reconstituted in 750 µl of 1.5 mM
norleucine in distilled water. Ten milliliters of scintillation
cocktail were added to a 350-µl aliquot, and disintegrations
per minute were calculated from quenched standards (model
LS9000 Liquid Scintillation Counter, Beckman Instruments,
Palo Alto, CA). Sample isoleucine concentration (pmol/µl) was
determined by high-performance liquid chromatography
(HPLC) by derivatizing a 10-µl aliquot with phenylisothiocyanate (26).
Intracellular and plasma free isoleucine specific radioactivity. One milliliter of low-salt buffer (40 mM NaCl, 5 mM
NaPO4 at a pH of 7 by mixing di- and monobasic forms, 1 mM
MgCl2, 0.1 mM dithiothreitol, and 0.1 mM EDTA) was added
to 100 mg powdered muscle. The sample was mixed vigorously on a vortex for 45 s and then centrifuged for 3 min at
4°C. Amino acids were separated from proteins by filtering
400 µl of supernatant or 400 µl plasma through a 10,000
nominal molecular weight limit filter (Millipore, Milford, MA)
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Materials. Enhanced chemiluminescence (ECL) detection
reagents and horseradish peroxidase-conjugated sheep antimouse immunoglobulins were purchased from Amersham
Life Sciences. Polyvinylidene difluoride membranes were
obtained from Bio-Rad. The insulin radioimmunoassay (RIA)
kit was purchased from Linco Research (St. Louis, MO). The
corticosterone RIA kit was purchased from ICN Biomedicals
(Costa Mesa, CA). BudgetSolve scintillation cocktail was
purchased from Research Products International (Mount
Prospect, IL).
Animals and experimental design. The animal facilities
and protocol were reviewed and approved by the Institutional
Animal Care Review Board of the University of Illinois. Male
Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis,
IN), initially weighing 140–150 g, were individually housed
in wire-bottom cages in a room maintained at 23–25°C with a
12:12-h light-dark cycle. All animals were freely provided tap
water and a commercial pelleted diet (Harlan-Teklad Rodent
Chow, Madison, WI) until the experimental run. The day
after arrival, all animals began an 8-day treadmill acclimation schedule, which gradually increased either speed or
duration up to 26 m/min for 15 min. Animals that refused to
run during the acclimation period were eliminated from the
study. All exercise sessions began at the beginning of the light
cycle after recording of body weight and were performed on a
motor-driven treadmill.
The primary objective of the present study design was to
clearly define an exercise-induced decrease in skeletal muscle
protein synthesis and to determine the potential of carbohydrate alone or a complete macronutrient mixture to stimulate
protein synthesis during recovery. The study had five treatment groups (n 5 4 per group): CF, nonexercised controls
deprived of food for 10 h (serving as the baseline); CM,
nonexercised fed controls; EF, exercised and deprived of food
after the experimental run; EC, exercised and fed a 100%
carbohydrate meal after the experimental run; and EM,
exercised and fed a nutritionally complete meal after the
experimental run. The nutritionally complete meal was derived from a mixture of 55.5 g Ensure nutritional powder
(Ross Laboratories, Columbus, OH) reconstituted in 99 ml
distilled water. The caloric density was 8.8 kJ/ml (54.5%
energy from carbohydrate, 31.5% energy from fat, and 14%
energy from protein). The carbohydrate meal was isocaloric
with the complete meal (525 g/l consisting of 50% glucose,
50% sucrose by weight in distilled water). The test dose for
both experimental meals was 5 ml administered by gavage
immediately after the experimental run. Preliminary data
collected under conditions identical to the protocol used in the
present study indicated that this animal population consumed 293 6 9 kJ/day (n 5 20); therefore, the experimental
meals supplied ,15% of daily energy intake for this age and
strain of rats. This size meal was chosen to represent the
proportional amount of energy a human might realistically
consume after an exhaustive workout. Animals that were not
fed after exercise were intubated with 5 ml of 0.9% saline.
Food was removed from all animals 7 h before the experimental run. All animals were allowed free access to water after
the treadmill bout, but no food was available after exercise
beyond the defined postexercise meals. Fed controls (CM
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EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
performed to assess main effects with recovery group (exercise condition 1 meal) as the independent variable. When a
significant overall effect was detected, differences among
individual means were assessed with Duncan’s multiplerange post hoc test. The level of significance was set at P ,
0.05 for all statistical tests.
RESULTS
Preliminary experiments were performed with fooddeprived animals to validate use of isoleucine for the
flooding dose technique (11). Underlying assumptions
for an amino acid tracer are as follows: 1) the specific
activity of free isoleucine in plasma and muscle is
constant and equivalent to the infused dose during the
experimental period, and 2) the rate of incorporation of
labeled isoleucine into muscle protein increases over
time. Figure 1A demonstrates that the specific activity
of free isoleucine was constant over 15 min and accounts for .85% of the total infused specific activity. A
linear increase of labeled isoleucine (r 2 .0.9) incorporated into skeletal muscle protein is shown in Fig. 1B.
Fig. 1. Time course of specific radioactivities of free (A) and proteinbound (B) isoleucine in plasma and skeletal muscle (gastrocnemius
and plantaris) in rats food deprived for 10 h. A flooding dose of
isoleucine (200 µmol L-isoleucine with 4.77 MBq L-[4,5-3H]isoleucine)
was administered via tail vein. Values are means 6 SE; n 5 3 animals
per point. Infused dose is equal to 100% specific radioactivity.
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at 4°C. A 50-µl aliquot of the muscle-derived filtrate or a 20-µl
aliquot of the plasma-derived filtrate was filtered over a neutralized cation exchange column (50 WX4–400, Sigma Chemical, St. Louis, MO), rinsed three times with 1 ml distilled
water, then eluted with 2 ml of 25% (vol/vol) NaOH. The
solutions were evaporated in a speed vacuum as before. After
solutions were reconstituted in 110 µl distilled water, a 50-µl
aliquot of each was derivatized with phenyisothiocyanate and
reconstituted in 100 µl of diluent (Pico-Tag sample diluent for
free amino acid analysis, Waters Chromatography, Milford,
MA). A 30-µl aliquot of each sample was combined with 10 ml
scintillation cocktail for the determination of radioactivity.
The isoleucine concentration in 30-µl aliquots of remaining
diluent containing muscle free amino acids and 10-µl aliquots
of remaining diluent containing plasma free amino acids were
measured by HPLC. Specific activity was calculated from the
radioactivity divided by the amount of isoleucine. All specific
activity measurements were performed in duplicate.
Calculation of the fractional rate of protein synthesis.
Protein synthesis was calculated as a fractional rate of
synthesis (Ks, %/day) according to McNurlan et al. (18): Ks 5
(SI 3 100)/(SF 3 t), where SI represents isoleucine specific
activity incorporated into muscle protein, SF represents the
intracellular free pool isoleucine specific activity, and t represents the infusion time in days.
Quantitation of 4E-BP1·eIF4E and eIF4G·eIF4E complexes.
The left gastrocnemius and plantaris were excised, weighed,
and homogenized as a unit with a Polytron homogenizer in 7
vol of buffer A, consisting of (in mM) 20 N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid, 100 KCl, 0.2 EDTA, 2
ethylene glycol-bis(b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid, 50 NaF, 50 b-glycerophosphate, 1 dithiothreitol, 0.1
phenylmethylsulfonyl fluoride, 1 benzamidine, 0.5 sodium
vanadate, and 1 µM microcystin LR in 30-ml Oak Ridge
tubes. The homogenate was immediately centrifuged at
10,000 g for 10 min at 2°C, frozen in liquid nitrogen, and
stored at 280°C until analysis. eIF4E was isolated from
aliquots of the 10,000-g supernatant by immunoprecipitation,
using an anti-eIF4E monoclonal antibody as previously described (17). Immunoprecipitated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane,
and the membrane was incubated with either a rabbit antirat 4E-BP1 (kindly provided by Dr. J. C. Lawrence, University of Virginia School of Medicine, Charlottesville, VA) or a
polyclonal antipeptide antibody raised against amino acids
328–343 of human eIF4G (kindly provided by Drs. Virginia
M. Pain and Simon J. Morley, University of Sussex, Brighton,
UK) for 1 h at room temperature. The blots were then
developed using an ECL Western blotting kit as described
previously (16). The blots were routinely exposed to film for
various lengths of time, such that the signals of highest and
lowest intensities were within the linear range of analysis.
Quantitation of phosphorylated and unphosphorylated
eIF4E in skeletal muscle. The phosphorylated and unphosphorylated forms of eIF4E in skeletal muscle were separated
by isoelectric focusing of 10,000-g supernatants on a slab gel
and quantitated by protein immunoblot analysis as previously described (17).
Phosphorylation of p70S6K. Phosphorylation of p70S6K was
determined in 10,000-g supernatants by protein immunoblot
analysis as previously described (16). Membranes were incubated with a rabbit polyclonal p70S6K antibody (Santa Cruz
Biotechnology) for 1 h, and the bands were visualized using
an ECL Western blotting kit as previously described (16).
Statistical analysis. All data were analyzed by the STATISTICA statistical software package for the Macintosh, volume
II (StatSoft, Tulsa, OK). A two-way analysis of variance was
EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
Fig. 4. Plasma corticosterone in male rats 1 h after 2-h experimental
run. Values are means 6 SE; n 5 4 animals. Definitions are as in Fig.
2. Means not sharing same superscript(s) are different (P , 0.05).
Prolonged exercise did not depress plasma glucose
(Fig. 2) or insulin (Fig. 3) concentrations below the
nonfed controls. Feeding the carbohydrate meal immediately after exercise resulted in a significant increase
in plasma glucose at 1 h postexercise that was not seen
with feeding the complete meal. Conversely, insulin
concentrations increased similarly in response to feeding either the carbohydrate or the complete meal (P ,
0.06 for both EC vs. EF and EM vs. EF). Plasma
corticosterone was increased in the EF and EC groups
above meal-fed controls (Fig. 4) and tended to be
increased above food-deprived controls (P , 0.07).
Surprisingly, animals fed the complete meal after exercise had plasma corticosterone levels 54% below the
other exercise groups and were not different from
controls.
Skeletal muscle protein synthesis was depressed
26% in animals not fed after exercise compared with
baseline (CF group) (Fig. 5). This depression can be
attributed to the exercise protocol and not solely lack of
food, for muscle protein synthesis in the CF group was
not different from the nonexercised fed condition (CM
group). One hour after exercise, muscle protein synthesis in the carbohydrate-fed group was not significantly
stimulated above animals not fed after exercise. In
contrast, muscle protein synthesis rates in exercised
animals fed the complete meal were increased compared with the nonfed and carbohydrate-fed animals
and were recovered to rates that were not different
from baseline. The enhanced rate of recovery cannot be
attributed to a differential insulin response, for insulin
concentrations in the carbohydrate and complete meal
groups were similar. Interestingly, plasma corticoste-
Fig. 3. Plasma insulin in male rats 1 h after 2-h experimental run.
Values are means 6 SE; n 5 4 animals. Definitions are as in Fig. 2.
Means not sharing same superscript(s) are different (P , 0.05).
Fig. 5. Fractional rate of skeletal muscle protein synthesis (Ks,
%/day) in male rats 1 h after 2-h experimental run. Values are
means 6 SE; n 5 4 animals. Definitions are as in Fig. 2. Means not
sharing same superscript(s) are different (P , 0.05).
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Fig. 2. Plasma glucose in male rats 1 h after 2-h experimental run.
Values are means 6 SE; n 5 4 animals. CF, food-deprived controls;
CM, meal-fed controls; EF, exercised and food-deprived postexercise;
EC, exercised and fed a 100% carbohydrate meal postexercise; EM,
exercised and fed a complete meal postexercise. Means not sharing
same superscript(s) are different (P , 0.05).
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AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
Fig. 6. Amount of 4E-BP1 associated with eIF4E in skeletal muscle
of male rats 1 h after 2 h of treadmill exercise. A: a representative
immunoblot, with positions of a- and b-forms of 4E-BP1 noted to
right. B: values are means 6 SE; n 5 4 animals. Definitions are as in
Fig. 2. Means not sharing same superscript(s) are different (P ,
0.05).
Fig. 7. Amount of eIF4G associated with eIF4E in skeletal muscle of
male rats 1 h after 2 h of treadmill exercise. A: a representative
immunoblot. B: values are means 6 SE; n 5 3–5 animals. Definitions
are as in Fig. 2. Means not sharing same superscript(s) are different
(P , 0.05).
synthesis rate, consumption of the carbohydrate meal
did not significantly alter the association between
4E-BP1 and eIF4E (Fig. 6) or the amount of eIF4G
present in the eIF4E immunoprecipitate (Fig. 7). In
contrast, the exercise-induced increase in 4EBP1 · eIF4E association was completely abated in exercised animals fed the complete meal, and eIF4G · eIF4E
association was increased fivefold above the nonfed
exercised animals. The pattern of 4E-BP1 · eIF4E complex formation during recovery was inversely related to
the fractional rate of skeletal muscle protein synthesis
(r 5 20.65, P , 0.01). Additionally, the pattern of
eIF4G · eIF4E complex formation was positively related
(r 5 0.71, P , 0.01) to the fractional rate of skeletal
muscle protein synthesis. These results support a role
for eIF4E availability in the regulation of muscle
protein synthesis during exercise recovery.
To further investigate the mechanism through which
eIF4F function is altered, changes in the phosphorylation state of eIF4E were examined. The phosphorylation of eIF4E in vitro has been shown to be increased
under a variety of conditions where translation rates
are enhanced (32). Almost all of the eIF4E present in
muscle of exercised rats was in the phosphorylated
state (.90%), with the proportion not differing among
exercise groups (Fig. 8). In contrast, the phosphorylation of eIF4E in the meal-fed controls was 60% less
than nonfed controls (P , 0.018).
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rone concentrations were inversely related to muscle
protein synthesis (r 5 20.77, P , 0.01).
One mechanism through which muscle protein synthesis is depressed is via diminished availability of
eIF4E for eIF4F active complex formation, inhibiting
translation initiation. Decreased eIF4E availability
occurs via 4E-BP1 sequestering eIF4E into an inactive
complex, preventing eIF4G binding eIF4E (13). On an
immunoblot, this is visualized as an increase in the
amount of 4E-BP1 present in the eIF4E immunoprecipitate. Accompanying the depression in muscle protein
synthesis, 4E-BP1 · eIF4E complex formation was increased fourfold after prolonged exercise (Fig. 6). An
increase in eIF4E binding to 4E-BP1 implies a reduced
availability of eIF4E to associate with eIF4G, based on
in vivo data which suggest that eIF4E cannot bind both
eIF4G and 4E-BP1 concurrently (17). In accordance
with this report, the amount of eIF4G present in the
eIF4E immunoprecipitate was decreased 71% (Fig. 7).
These data suggest that after prolonged exercise, eIF4F
function was abated by a decrease in eIF4E availability,
which led to an inhibition of muscle protein synthesis.
In accordance with this mechanism, recovery of
muscle protein synthesis at the level of translation
initiation level would therefore occur via increased
availability of eIF4E for eIF4F active complex formation, stimulating translation initiation. Increased eIF4E
availability occurs via the dissociation of the eIF4E · 4EBP1 inactive complex, allowing the released eIF4E to
interact with eIF4G (13). Consistent with the fractional
EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
C411
meal-fed controls showed an increase in the phosphorylation of the kinase above the nonfed controls.
DISCUSSION
It has been suggested that regulation of translation
initiation might occur via the p70S6K signaling pathway.
The p70S6K signaling pathway is involved in the phosphorylation of the ribosomal protein S6 and has been
implicated in stimulating protein synthesis and increasing 4E-BP1 phosphorylation (32). The phosphorylation
of the p70S6K is associated with its activation. On a
polyacrylamide gel, this results in phosphoforms that
exhibit decreased electrophoretic mobility when subjected to electrophoresis. There were no differences in
the phosphorylation of p70S6K between the exercised
animals and the nonfed controls (Fig. 9). However,
Fig. 9. Phosphorylation of p70S6K in skeletal muscle of male rats 1 h
after 2-h experimental run. Arrows indicate multiple electrophoretic
forms of p70S6K. Data shown are representative of 4 animals.
Definitions are as in Fig. 2.
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Fig. 8. Amount of phosphorylated eIF4E as a percentage of total
eIF4E in skeletal muscle of male rats 1 h after 2-h experimental run.
A: results of a representative immunoblot, with phosphorylated and
unphosphorylated forms of eIF4E noted at right. B: values are
means 6 SE; n 5 4 animals. Definitions are as in Fig. 2. Means not
sharing same superscript(s) are different (P , 0.05).
It has been recognized for over a decade that the
primary change in muscle protein turnover during and
immediately after endurance exercise is a depression in
muscle protein synthesis (7), with the magnitude of
depression dependent on the intensity and duration of
exercise (2, 8). Additionally, prior exercise training has
no effect on increasing protein synthesis or reducing
degradation after prolonged exercise (7). Although the
acute effect of prolonged exercise on protein turnover is
catabolic, routine exercise programs do not lead to
muscle atrophy. Physical activity and exercise training
are associated with maintenance or enlargement of
skeletal muscles; therefore, at some point during recovery from endurance exercise, increases in protein synthesis and/or decreases in protein degradation must
occur. Carraro et al. (6) showed that protein synthesis
values in humans were elevated 22% 4 h after exercise
as compared with nonexercise conditions. Similarly,
Rennie et al. (29) demonstrated synthesis rates in
human subjects to increase 22% above the rested state
4.5 h after 3.75 h of treadmill exercise. Taken together,
protein turnover changes after exercise suggest that
the protein catabolic state induced by exercise continues beyond the end of exercise and that recovery from
endurance exercise is ultimately driven by increases in
protein synthesis.
Protein synthesis rates are mediated by extracellular
signals such as nutrients and hormones. One of the
signals that may mediate the depression in muscle
protein synthesis during and after endurance exercise
is insulin. Studies using perfused hindlimb preparations and incubated muscles have established an essential role for insulin in the maintenance of muscle
protein synthesis (15), while the absence of insulin in
the form of streptozotocin-induced diabetes causes extreme muscle catabolism in rats due to decreases in
protein synthesis (9). The ability of insulin to stimulate
glucose and amino acid uptake is enhanced after exercise (37); however, the role of insulin in the regulation
of muscle protein synthesis after exercise is unclear.
Balon et al. (2) found the ability of 200 µU/ml insulin to
stimulate protein synthesis in perfused rat muscles
was not enhanced after high- or moderate-intensity
exercise. Additionally, the results of the current study
suggest that low insulin concentrations cannot be
solely responsible for the depression in protein synthesis after exercise, for insulin concentrations between
the sedentary, food-deprived animals and the exercised,
food-deprived animals were similar.
Studies investigating the refeeding period after shortterm fasting have also identified insulin as an extracellular signal that mediates increases in muscle protein
synthesis. Injection of anti-insulin serum before food
intake suppresses the increase in muscle protein synthesis observed with refeeding (28). This finding is in
accordance with Yoshizawa et al. (36) and Svanberg et
al. (34), who found that administration of insulin
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EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
tions of insulin and reduced plasma concentrations of
corticosterone. In light of the present findings and the
available refeeding reports, it is attractive to suggest a
role for diminished corticosterone levels acting in concert with elevated insulin and amino acids in mediating
recovery of muscle protein synthesis.
Molecular mechanisms for the control of muscle
protein synthesis during and after exercise are uncharacterized. RNA concentrations are unchanged during
the relatively brief period of the exercise bout (25),
implying regulation occurring at the translation level.
The rate of initiation is thought to limit overall translation under most physiological conditions (24), and
studies investigating recovery of protein synthesis after acute food deprivation demonstrate increases in
polysome size and initiation activity (36). However, to
date, there are no studies examining translation initiation during recovery from endurance exercise.
One of the major sites of regulation in the initiation
process involves the recognition and unwinding of the
mRNA to allow binding to the 40S ribosome. This
tightly regulated step requires the participation of
eIF4B and eIF4F, a three-subunit complex consisting of
eIF4A, eIF4E, and eIF4G (19). One of the most studied
factors in this regard is eIF4E, a 24-kDa subunit of the
eIF4F complex which serves to select for the mRNA to
be translated by binding the m7GTP cap at the 58-end
of the mRNA. Binding of eIF4E to the mRNA cap
structure is succeeded by its associating with eIF4G, a
protein that facilitates the formation of the eIF4F
complex and the association of the mRNA with the 40S
ribosome (32). The association between eIF4E and
eIF4G is important for stabilizing the interaction between the mRNA and the 40S ribosome and is therefore
essential for successful 48S preinitiation ribosomal
complex formation.
There are currently two characterized mechanisms
in which changes in translation initiation occur as a
result of altered eIF4F function. The first mechanism
involves modulation of eIF4E availability via the binding of eIF4E to the translational repressor, 4E-BP1.
Studies in vivo show 4E-BP1 · eIF4E association to
increase in skeletal muscle of diabetic rats (16) and
overnight-fasted mice (33). Both these conditions demonstrate decreases in the phosphorylation state of
4E-BP1 and in eIF4G · eIF4E complex formation, supporting the suggestion that the binding of 4E-BP1 and
eIF4G to eIF4E is mutually exclusive and regulated by
4E-BP1 phosphorylation. In the current study, 4EBP1 · eIF4E complex formation was increased, and the
amount of eIF4G present in the eIF4E immunoprecipitate was decreased after prolonged exercise, similar to
the regulations seen during diabetes and starvation.
On the basis of these studies, the effect of exercise on
inhibiting translation initiation is most likely due to a
decrease in the phosphorylation of 4E-BP1, although
alternative explanations for the observed changes,
such as modulation of the activity of eIF2B, cannot be
ruled out at this time.
The extracellular signal(s) mediating the differential
effect of eIF4E availability by refeeding during recovery
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antibodies to fasting mice prevents the stimulatory
effect of refeeding a compete meal on muscle protein
synthesis. These studies establish the importance of
insulin in mediating the acute stimulation of muscle
protein synthesis during refeeding. However, Garlick et
al. (10) demonstrated that only supraphysiological concentrations of insulin could mimic the response in
muscle protein synthesis seen with refeeding when
infused in postabsorptive rats. Additionally, exogenous
insulin given to freely fed mice does not further enhance muscle protein synthesis (34). These findings
suggest that insulin is not solely responsible for the rise
in muscle protein synthesis by food intake. Thus,
instead of directly modulating the biosynthetic process
after exercise, insulin might play a permissive role in
the regulation of muscle protein synthesis.
Recent work in mice shows that refeeding a nutritionally complete meal after an overnight fast stimulates
muscle protein synthesis, but not when mice are refed a
protein-free meal (36). This effect is seen despite insulin concentrations not differing between meal conditions and plasma glucose being higher in animals refed
the protein-free meal. The authors conclude that amino
acids in addition to insulin are required for recovery of
muscle protein synthesis with refeeding. The results of
the current study concur with this proposal and support the hypothesis of Preedy and Garlick (28) that an
increase in muscle protein synthesis by food intake is
mediated by insulin acting in cooperation with amino
acids. These studies therefore suggest a functional role
for amino acids in enhancing the recovery of skeletal
muscle protein synthesis by feeding postexercise.
Another possible extracellular mediator in the acute
modulation of muscle protein synthesis during recovery
from exercise is corticosterone. Corticosterone is the
major glucocorticoid produced in the rat and is secreted
under conditions of low blood glucose and/or high stress
(4). Plasma levels of corticosterone are reported to be
elevated after acute exercise (35); however, there are no
reports describing the effects of corticosterone on protein synthesis during or after exercise. Administration
of corticosterone to streptozotocin-induced diabetic rats
for 5 days depressed muscle protein synthesis and RNA
activity (the rate of protein synthesis per unit of RNA),
suggesting impaired translation initiation (23). The
mechanism for the inhibitory effects of corticosterone is
unclear but appears to be dependent on low concentrations of insulin. The current data suggest that, under
the condition of low insulin, elevated corticosterone
levels may further contribute to the inhibition of protein synthesis seen after prolonged exercise.
Corticosterone concentrations are recognized to decline in food-deprived animals upon refeeding, decreasing significantly within an hour of initial food intake
(20, 28). Millward et al. (20) refed food-deprived rats
pretreated with anti-insulin serum or corticosterone
and determined that the acute increase in muscle
protein synthesis by refeeding requires insulin, a fall in
corticosterone, and at least one other independent
factor. In the current study, animals fed the complete
meal after exercise had increased plasma concentra-
EIF4E
AVAILABILITY REGULATES MUSCLE PROTEIN SYNTHESIS
,20-fold in the extracts of incubated muscle (1). However, activation of p70S6K did not occur during refeeding
after exercise. This result suggests that additional
intracellular signaling pathway(s) independent of p70S6K
have a role in the activation of muscle protein synthesis
during recovery from endurance exercise. Alternatively, observed differences in the phosphorylation of
the p70S6K may be affected by the timing of the measurements rather than the basic underlying mechanism.
Further study is required to characterize the pattern of
p70S6K phosphorylation during exercise recovery.
In summary, this investigation is the first to report
inhibition of translation initiation in skeletal muscle by
prolonged exercise. We are also the first to report
nutritional modulation of translation initiation during
recovery from endurance exercise. These data suggest
that feeding a nutritionally complete meal after exercise is more efficacious than feeding carbohydrate alone
for stimulating muscle protein synthesis after exercise.
Additionally, our data suggest that increasing plasma
insulin concentrations alone does not relieve inhibition
of skeletal muscle translation initiation after exercise.
We suggest that the protein component of the diet is
necessary for stimulating translation initiation in skeletal muscle during exercise recovery. The role of amino
acids in stimulating muscle protein synthesis requires
further study.
We are grateful to Lynne Hugendubler and Sharon Rannels for
technical assistance.
This work was supported in part by National Institutes of Health
Grants HD-27558 (to D. K. Layman) and DK-15658 (to L. S.
Jefferson).
Address for reprint requests: D. K. Layman, 439 Bevier Hall, 905
S. Goodwin Ave., Urbana, IL 61801.
Received 2 June 1997; accepted in final form 28 October 1997.
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