J Appl Physiol 96: 679–687, 2004.
First published October 17, 2003; 10.1152/japplphysiol.00962.2003.
Alterations in the expression of mRNAs and proteins that code for species
relevant to eIF2B activity after an acute bout of resistance exercise
Neil Kubica,1,2 Scot R. Kimball,2 Leonard S. Jefferson,2 and Peter A. Farrell1
1
Noll Physiological Research Center, The Pennsylvania State University, University Park 16802; and 2The Pennsylvania State
University College of Medicine, Department of Cellular and Molecular Physiology, Hershey, Pennsylvania 17033
Submitted 8 September 2003; accepted in final form 13 October 2003
RESISTANCE EXERCISE HAS BECOME a crucial component in the
training of the modern athlete. However, at this time our
understanding of the cellular mechanisms leading to increases
in skeletal muscle protein synthesis as a result of muscular
contractions is limited. Elevations in protein synthesis in response to contractions can potentially be controlled at several
stages, including mRNA transcription from DNA and translation of mRNA into protein at the ribosome. The majority of
studies in the literature concerning the regulation of protein
synthesis have focused on translation efficiency, a process in
which translation initiation is the rate-limiting step.
Our laboratory has previously found that resistance exercise
elevates the activity of eukaryotic initiation factor 2B (eIF2B)
16 h postexercise, concomitant with elevations in skeletal
muscle protein synthesis (12, 27). eIF2B serves as a guanine
nucleotide exchange factor that substitutes GTP for GDP on
eukaryotic initiation factor 2 (eIF2), thereby allowing eIF2 to
complex with initiator methionyl-tRNA (met-tRNAi) and subsequently associate with the 40S ribosomal subunit to form the
43S preinitiation complex (36, 37). Binding of met-tRNAi to
the 40S ribosomal subunit is an essential step in the translation
of all mRNA species, and regulation of this step of the
initiation process is frequently associated with the control of
global protein synthesis. As eIF2 is released from the ribosome
after each round of initiation, the GTP is hydrolyzed and eIF2
is found in an inactive state bound to GTP. To bind another
met-tRNAi, and thus participate in a subsequent round of
initiation, eIF2B must exchange GTP for GDP. Therefore,
regulation of eIF2B activity and availability can be an important regulator of translation initiation and protein synthesis.
Several potential mechanisms have been proposed to regulate eIF2B activity, including 1) phosphorylation of the ⑀-subunit (2, 9, 39, 49) by kinases such as glycogen synthase
kinase-3 (GSK-3) (50), casein kinase I (CKI) (45), and casein
kinase II (CKII) (2, 44, 45); 2) phosphorylation of the ␣-subunit of eIF2 (19) by various kinases such as the doublestranded RNA-activated protein kinase (PKR) and the hemeregulated inhibitor (6, 34); and 3) phosphorylation of eIF2␤ by
protein kinase A (PKA) (24). Although the effect of eIF2B⑀
phosphorylation by CKI and CKII remains unclear, GSK-3␤
has been shown to phosphorylate eIF2B⑀ on Ser535, thereby
inhibiting the exchange activity of the factor (22, 50). Activated GSK-3␤ has been shown to be a negative regulator of
hypertrophy in cardiomyocytes (16), the heart (1), and skeletal
muscle myotubes (48). Second, phosphorylation of the ␣-subunit of eIF2 increases the binding affinity between eIF2 and
eIF2B, thereby reducing protein synthesis via competitive
inhibition (19). Therefore, phosphorylation of eIF2␣ on the
Ser51 residue converts eIF2 from an exchange substrate of
eIF2B to a competitive inhibitor of eIF2B activity. Finally, the
␤-subunit of eIF2 has been shown to bind eIF2B in vitro, and
phosphorylation of eIF2␤ by PKA has been shown to increase
eIF2B activity in vitro (24). The roles of eIF2␣ and eIF2␤
phosphorylation have yet to be determined in response to
exercise.
Virtually all of the present knowledge concerning the control
of translational events involves alterations in protein abundance and protein phosphorylation. Even at the protein level, it
remains unclear which of the aforementioned mechanisms are
relevant in regulating increases in translation associated with
muscular contractions. Information concerning alterations in
expression levels of the associated mRNA species in response
Address for reprint requests and other correspondence: L. S. Jefferson, Dept.
of Cellular and Molecular Physiology, The Pennsylvania State Univ. College
of Medicine, PO Box 850, Hershey, PA 17033 (E-mail: [email protected]).
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.
eukaryotic initiation factor 2␣; eukaryotic initiation factor 2␤; glycogen synthase kinase 3␤; casein kinase I; casein kinase II; doublestranded RNA-activated protein kinase
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8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society
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Kubica, Neil, Scot R. Kimball, Leonard S. Jefferson, and Peter
A. Farrell. Alterations in the expression of mRNAs and proteins that
code for species relevant to eIF2B activity after an acute bout of
resistance exercise. J Appl Physiol 96: 679–687, 2004. First published
October 17, 2003; 10.1152/japplphysiol.00962.2003.—The focus of
the study described herein was to examine the relative expression
levels of mRNAs and proteins relevant to the regulation of translational initation, and hence protein synthesis, in the time course after an
acute bout of resistance exercise in male Sprague-Dawley rats. Significant increases in the relative abundance of the mRNAs coding for
the epsilon (33%) and gamma (26%) subunits of eukaryotic initiation
factor (eIF) 2B were observed 48 h after the exercise bout. Furthermore, the mRNA coding for the delta subunit of eIF2B was also
significantly increased, both 24 h (46%) and 48 h (44%) postexercise.
There was a relative decrease in three eIF2B⑀ kinase mRNAs, namely
sequences coding for glycogen synthase kinase 3␤ (49%), casein
kinase I (48%), and casein kinase II (42%) 48 h into the recovery
period. Additionally, there was a significant decrease in expression of
the mRNAs coding for eIF2␣ (28% 24 h postexercise) and one of its
regulatory kinases, double-stranded RNA-activated protein kinase
(33% 48 h postexercise). Finally, an increase in eIF2B total protein
(124%) was observed within 3 h postexercise. These results suggest
that there may be rapid translational regulation of mRNAs coding for
species relevant to translational initiation after an acute bout of
resistance exercise. Furthermore, transcription of these mRNAs is
altered further into the recovery period, and this might play a role in
protein synthetic capacity on subsequent bouts of resistance exercise.
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RESISTANCE EXERCISE AND EIF2B REGULATION
MATERIALS AND METHODS
Animal care. All experimental procedures were approved by the
Institutional Animal Care and Use Committee of The Pennsylvania
State University College of Medicine. Male Sprague-Dawley rats
(⬃300–350 g) were housed in temperature- and humidity-controlled
holding facilities on a 12:12-h light-dark cycle. Rats were fed a
standard rodent diet (Harlan-Teklad Rodent Chow, Madison, WI), and
both food and water were provided ad libitum.
Acute resistance exercise protocol. Details of the exercise protocol
have been described previously (13). Briefly, male Sprague-Dawley
rats were operantly conditioned to stand on their hindlimbs and touch
an illuminated bar located high on the wall of a Plexiglas cage. This
movement was reinforced with the use of a mild foot shock (⬍2 mA,
60 Hz) over the course of four familiarization sessions. Once this
learning process was completed, weighted Velcro vests were strapped
over the scapulae and the animals were required to touch the overhead
bar 50 times during a given exercise session. The “acute” resistance
exercise protocol consisted of four separate sessions with 1 day of rest
between sessions. The rats performed 50 repetitions in each session
with 0.2 (day 1), 0.4 (days 2 and 3), and 0.6 (day 4) g weighted vest/g
body wt, respectively.
It is important to note that even the 60% of body weight intensity
level is insufficient to cause muscle hypertrophy with repeated exercise bouts. In fact, a progressive overload paradigm culminating in
several weeks of resistance exercise with a load in excess of 1 g
weighted vest/g body wt is necessary to induce muscle hypertrophy
(unpublished results). However, the 60% of body weight exercise
session is considered to be representative of resistance exercise
because such a load has been shown in numerous published studies to
cause an increase in muscle protein synthesis in untrained rats.
Conversely, the 20 and 40% of body weight sessions are included
simply as a training tool to teach the rats to maintain their balance
while performing the desired exercise motion. These exercise stimuli
are insufficient to induce increases in muscle protein synthesis and
thus are not considered part of a short-term training paradigm. Thus
the penultimate exercise session is considered to be an example of
acute resistance exercise.
Exercise sessions occurred under red light in the late afternoon. On
all four training days, sedentary control animals were placed in the
cage and given five mild shocks to simulate the stress experienced by
the exercised animals. One of the shock control sessions took place
J Appl Physiol • VOL
3 h before death. Although the shock received by the control group
only correlates to the effect of the foot shock on the earliest time point
examined, shock-matched controls were previously performed for
animals killed 16 h after the final bout of resistance exercise and no
effects were observed on any of the end points examined. Additionally, shock dose-response curves showed that there was no correlation
between the number of shocks administered and postexercise rates of
protein synthesis (unpublished data). Exercised animals were killed at
3, 24, or 48 h after the last acute resistance exercise session depending
on their inclusion in specific treatment groups. The animals were
fasted for 5 h before death. At the time of death, all rats were
anesthetized with isoflurane, and gastrocnemius tissue was excised
and immediately snap frozen in liquid nitrogen.
Isolation of total RNA. Gastrocnemius tissue was frozen between
aluminum blocks cooled to the temperature of liquid nitrogen, pulverized with a mortar and pestle under liquid nitrogen, and stored at
⫺80°C until processing. Total RNA was isolated by using the RNeasy
mini kit (Qiagen, Valencia, CA) according to the manufacturer’s
protocol specific for muscle, heart, and skin tissue. To reduce contamination by genomic DNA, samples were treated on the spin
columns with RNase-free DNase I (Qiagen). Samples were analyzed
for RNA abundance and quality via spectrophotometry on a Beckman
Coulter DU 640 (Fullerton, CA). Additional quality control analyses
were conducted by use of an Agilent Technologies 2100 bioanalyzer
lab chip system (Palo Alto, CA) in the Genomics Core Facility of The
Pennsylvania State University College of Medicine. All samples were
adjusted to a concentration of 1 ␮g RNA/␮l.
Real-time PCR for eIF2B⑀, eIF2␣, and eIF2␤. The real-time PCR
reactions for eIF2B⑀, eIF2␣, and eIF2␤ were conducted at the Nucleic
Acid Core Facility at The Pennsylvania State University (University
Park, PA). Real-time PCR was conducted by using the Perkin-Elmer/
Applied Biosystems Division 7700 sequence detector (Foster City,
CA). The methodology used at Penn State’s Nucleic Acid Facility has
been previously reported (15). Briefly, total RNA samples were
reverse transcribed with the use of the following reaction mix: 0.5 ␮l
RNase inhibitor (40 U/␮l), 2.0 ␮l 10X TaqMan universal master mix
buffer, 3.6 ␮l MgCl2 (25 mM), 1.0 ␮l specific reverse primer (gene of
interest or GAPDH), 1.0 ␮l random hexamers (50 ␮l), 1.0 ␮l of each
nucleotide triphosphate (adenine, thymidine, cytosine, and guanine)
(10 mM), and 5.0 ␮l deionized, diethyl pyrocarbonate-treated H2O.
This mixture was used in the no-template control wells after addition
of murine leukemia virus reverse transcriptase (0.44 ␮l; 50 U/␮l).
Total RNA samples (40 ng/␮l) were heated at 65°C for 5 min,
centrifuged, and placed on ice. A sample of the reaction mix was then
added to the microamplification tube and run in a thermocycler using
the following conditions: 1 h at 42°C, 5 min at 72°C, and 2 min
at 25°C.
The cDNA product of this RT reaction (20 ng/␮l) was then used in
the real-time PCR reaction. PCR reactions were optimized for Mg2⫹
concentration and primer concentration for each specific primer/probe
set. The reaction mix for the PCR reaction included the following: 5.0
␮l 10⫻ TaqMan universal master mix buffer, MgCl2 (25 mM),
gene-specific upper primer (10 ␮M), gene-specific lower primer (10
␮M), gene-specific probe, 1.0 ␮l of each nucleotide triphosphate (10
mM), 0.25 ␮l Taq Gold (5 U/␮l), 8.0 ␮l cDNA, and deionized H2O.
Specific quantities of some reaction components were omitted because of variation based on optimization of the reactions for individual primer/probe sets.
The primer/probe sets used for each of the respective eukaryotic
initiation factor genes were as follows: eIF2B⑀ upper primer, 5⬘TTCCCCATCTCCAAGGACC-3⬘; lower primer, 5⬘-TCGATCAGCGCGACATTG-3⬘; probe, 5⬘-CCTCGGGTCCTATTGCCCCTGG3⬘; eIF2␣ upper primer, 5⬘-GGGCTTGCGGTTTTCATTT-3⬘; lower
primer, 5⬘-TGGAGCCCCTGGTAATTCTCT-3⬘; probe, 5⬘-TGATGTCTCTGGATTGGCACCCTTA-3⬘; eIF2␤ upper primer,
5⬘-AACTGGCTTCCAGGCTGTCA-3⬘, lower primer, 5⬘-ATGGTGATTAGCAAATTAGTTAG-3⬘, probe, 5⬘-AGGCAAGCGAGCA-
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to exercise is entirely absent. To gain a more complete understanding of protein synthesis in response to exercise, a more
dynamic view of eIF2B regulation is needed, including experiments designed to address transcriptional regulation of the
initiation factor itself and its proposed upstream signaling
molecules.
We suggest that, although enhanced translational efficiency
appears to be the initial response to acute resistance exercise,
alterations in transcription of genes that code for translationally
relevant proteins will be an essential component of the adaptive
process associated with exercise training. Booth and Kirby (5)
suggest that mRNA species relevant to training adaptations
will accumulate during exercise training. However, it has also
been shown that alterations in mRNA levels for these trainingrelated species are often altered after a single bout of acute
exercise (7). The present study addresses this issue by examining alterations in mRNA expression of the aforementioned
eIF subunits, and their upstream kinases, during the 48 h after
an acute bout of resistance exercise. Second, we hypothesized
that total protein levels of eIF2B and eIF2 would be altered in
unison with directional changes in their respective relative
mRNA abundance.
RESISTANCE EXERCISE AND EIF2B REGULATION
J Appl Physiol • VOL
EDTA, 2 mM EGTA, 50 mM sodium fluoride, 50 mM ␤-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM dithiothreitol, and 0.5 mM sodium vanadate. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. The resulting
supernatant was combined with an equal volume of SDS sample
buffer and then subjected to protein immunoblot analysis as described
previously (26). Both eIF2B and eIF2 total proteins were run on
12.5% SDS-PAGE gels, with 50 ␮g of total protein (Bio-Rad assay)
loaded per well on all blots. The antibodies against eIF2B⑀ (24, 42)
and eIF2␣ (43) have been previously described.
Statistical analysis. Treatment comparisons were conducted by
using a one-way ANOVA. If the ANOVA reached statistical significance at a 95% confidence level, a Dunnett multiple-comparison test
was applied to assess significant differences between the various
treatment groups and the sedentary control animals. All results are
expressed as percent of control and are shown as means ⫾ SE.
RESULTS
eIF2B mRNA levels before and after resistance exercise.
There was no significant difference in relative mRNA expression levels of GAPDH in the time course examined, thus
validating the use of this housekeeping gene to normalize
expression of other mRNA species in the present study (Fig. 1).
There was a significant increase in the relative abundance of
eIF2B⑀, -␥, and -␦ mRNAs in the gastrocnemius muscle after
resistance exercise. The relative level of eIF2B⑀ mRNA in the
48-h postexercise group was significantly greater than that in
the sedentary group (33% increase; P ⬍ 0.01; Fig. 2A). Similar
to transcripts coding for the ⑀-subunit, relative mRNA levels of
eIF2B␥ were also significantly elevated compared with the
control only 48 h into the recovery period (26% increase; P ⬍
0.05; Fig. 2B). Relative expression of eIF2B␦ mRNA was also
increased in the recovery period after exercise. This increase
became statistically significant 24 h after the final exercise bout
(46% increase; P ⬍ 0.01; Fig. 2C) and remained elevated 48 h
into the recovery period (44% increase; P ⬍ 0.01; Fig. 2C).
Relative mRNA expression of eIF2B⑀ kinases. The mRNAs
encoding each of the protein kinases examined in this study
that have eIF2B⑀ as a common substrate decreased significantly over the time course studied. The relative level of
Fig. 1. Relative expression of GAPDH mRNA during the time course after an
acute bout of resistance exercise. Data are the combined results from all
real-time PCR analysis conducted in sedentary animals (n ⫽ 110) compared
with animals killed 3 h (n ⫽ 110), 24 h (n ⫽ 104), or 48 h (n ⫽ 99)
postexercise. Values are means ⫾ SE.
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CAGCTCCGT-3⬘. All probes were purchased from Biosearch Technologies and labeled at the 5⬘ end with the 6-carboxyfluorescein
aminohexyl amidite and at the 3⬘ end with Black Hole Quencher
(Biosearch Technologies, Novato, CA). The thermocycler conditions
for the PCR reactions were 2 min at 50°C, 10 min at 95°C, and then
40 cycles of 15 s at 95°C followed by 1 min at 60°C.
After the PCR run, Sequence Detector version 1.7 software (Applied Biosystems) was used to analyze the fluorescence data. A
suitable threshold was set above the noise, and the cycle number at
which the fluorescence exceeded the threshold was determined for
each well. Data for eukaryotic initiation factor mRNA expression
were normalized to GAPDH expression, which was determined using
a primer/probe kit from Applied Biosystems.
Real-time PCR for eIF2B␥, eIF2B␦, and the initiation factor
kinases. First-strand cDNA synthesis was conducted with the use of 1
␮g of total RNA from each sample. Total RNA was reverse transcribed by using the Invitrogen SuperScript One-Step RT-PCR kit
with platinum Taq polymerase and oligo(dT) primers (Invitrogen,
Carlsbad, CA). The RT-PCR was conducted for 1 h at 42°C. Realtime PCR was conducted with the Qiagen QuantiTech SYBR green
PCR kit. Again, expression of the genes of interest were normalized
to GAPDH expression. Because of the possibility of different amplification efficiencies between the reference gene and the gene of
interest, a standard curve was generated via seven twofold serial
dilutions (1:2–1:128) of one of the cDNA samples. A 1:16 dilution
was used for the other samples, and expression values were obtained
by using the slope of the equations from the appropriate standard
curve after linear regression analysis.
The reactions were performed in duplicate and run in 96-well
optical plates (Applied Biosystems). Each well contained 5 ␮l of the
cDNA dilution (as described above) and 45 ␮l of a mix containing 25
␮l of 2X QuantiTect SYBR green PCR Master Mix, 0.15 ␮l of each
of the gene-specific primers (100 pmol/␮l), and 19.7 ␮l of nucleasefree water (Ambion, Austin, TX).
The following primers were used: GAPDH upper primer, 5⬘GGGCTGCCTTCTCTTGTGA-3⬘; lower primer, 5⬘-TGAACTTGCCGTGGGTAGA-3⬘; eIF2B␥ upper primer, 5⬘-AGCAGCGTGACTTCATTGG-3⬘; lower primer, 5⬘-CACAAGGCCTGTCTGGAAT-3⬘;
eIF2B␦ upper primer, 5⬘-TACACAACACCTCCCAATGA-3⬘; lower
primer, 5⬘-AAAGAACTTGATGGCGTTACA-3⬘; GSK3␤ upper
primer, 5⬘-ACCAAATGGGCGAGACACA-3⬘; lower primer, 5⬘CGTTTGCAGGCGGTGAAG-3⬘; CKII␤ upper primer, 5⬘CCCAACCAGAGTGACTTGA-3⬘; lower primer, 5⬘-TCGAGGACAGTAGCCAAAG-3⬘; CKI␥3 upper primer, 5⬘-CGAGGCCGAAGGATGTTTG-3⬘; lower primer, 5⬘-TTACTGCCGCCGCTGAGA-3⬘;
heme-regulated inhibitor upper primer, 5⬘-TTGAACGAGTCGCCTCTCA-3⬘; lower primer, 5⬘-GGTACCGCGCCTCTGTCT-3⬘;
PKR upper primer, 5⬘-GAAATTGGCTCGGGTGGAT-3⬘, lower
primer, 5⬘-CACGCTTTGCCTTCTTGGT-3⬘; PKA type II regulatory
subunit upper primer, 5⬘-GCTGCAGCAGGAGAACGA-3⬘; lower
primer, 5⬘-GCGAAGTTGACACCCTTACT-3⬘.
The temperature profile was 95°C for 15 min, followed by 94°C for
15 s, 53°C for 30 s, and 72°C for 30 s for 50 cycles. Three hold steps
were added at the end; they were at 95°C for 15 s, 60°C for 20 s, and
95°C for 15 s. A ramp time of 19:59 min was applied to the final
temperature transition to collect data for melting point analysis. After
the PCR run, Sequence Detector version 1.7 software (ABI Prism)
was used to analyze the fluorescence data. A suitable threshold was set
above the noise, and the cycle number at which the fluorescence
exceeded the threshold was determined for each well. These values
were compared with the standard curves (as discussed above), and
normalized expression values for each gene of interest were obtained.
Buffer blanks and no-template controls were run on every 96-well
PCR plate.
Analysis of total eIF2B and eIF2 protein. Gastrocnemius muscles
were excised, weighed, and homogenized by polytron in 7 volumes of
buffer containing 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.2 mM
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RESISTANCE EXERCISE AND EIF2B REGULATION
0.01; Fig. 3B). CKII␤ mRNA, which codes for the regulatory
subunit of this kinase, was also was decreased 48 h postexercise vs. the sedentary control group (42% decrease; P ⬍ 0.01;
Fig. 3C).
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Fig. 2. Real-time PCR analysis of mRNA species coding for various subunits
of the eukaryotic initiation factor 2B (eIF2B) protein, normalized to GAPDH
mRNA content and expressed relative to control values. Sedentary animals
(n ⫽ 10) are compared with animals killed 3 h (n ⫽ 10), 24 h (n ⫽ 9), or 48 h
(n ⫽ 9) after acute resistance exercise. Values are means ⫾ SE. A: eIF2B⑀.
*Values for the 48-h postexercise group are significantly greater than the
sedentary group (P ⬍ 0.01). B: eIF2B␥. *Values for the 48-h postexercise
group are significantly greater than for the sedentary group (P ⬍ 0.05). C:
eIF2B␦. *Values for the 24- and 48-h postexercise groups are significantly
greater than for the sedentary group (P ⬍ 0.01).
GSK-3␤ mRNA was significantly decreased 48 h after the
exercise bout compared with the sedentary group (49% decrease; P ⬍ 0.01; Fig. 3A). The mRNA coding for CKI␥3, one
of the isoforms of this kinase found in skeletal muscle, was
significantly decreased in the 48-h postexercise group compared with the sedentary control group (48% decrease; P ⬍
J Appl Physiol • VOL
Fig. 3. Real-time PCR analysis of mRNA species coding for eIF2B⑀ kinases
in the gastrocnemius muscle. A: glycogen synthase kinase-3 (GSK3)-␤. B:
casein kinase I (CKI)␥3. C: casein kinase II (CKII)␤. All values are normalized to GAPDH and expressed relative to control values. Values are means ⫾
SE. Sedentary animals (n ⫽ 10) are compared with animals killed 3 h (n ⫽ 10),
24 h (n ⫽ 10), or 48 h (n ⫽ 9) after a bout of acute resistance exercise. *Values
for the 48-h postexercise group are significantly lower than for the sedentary
control group (P ⬍ 0.01).
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RESISTANCE EXERCISE AND EIF2B REGULATION
683
of resistance exercise. This increase peaked at 3 h postexercise
(2.2-fold vs. control, P ⬍ 0.01) and remained significantly
elevated at both 24 and 48 h postexercise (Fig. 6A). It is
important to note that this increase in total eIF2B protein
occurred before any alteration in expression of mRNAs coding
for eIF2B subunits. In contrast to these findings, there was no
significant change in total eIF2 protein in the time course
examined after acute resistance exercise as determined by
Western blot analysis of the ␣-subunit (Fig. 6B).
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Fig. 4. Real-time PCR analysis of mRNA species coding for eIF2␣ (A) and
eIF2␤ (B), normalized to GAPDH mRNA content and expressed as a fraction
of control values. Sedentary animals (n ⫽ 10) are compared with animals
killed 3 h (n ⫽ 10), 24 h (n ⫽ 9), or 48 h (n ⫽ 9) after acute resistance exercise.
Values are means ⫾ SE. *Values for the 24-h postexercise group are significantly lower than for the sedentary control group (P ⬍ 0.05).
Relative mRNA expression of eIF2␣ and eIF2␤. In contrast
to eIF2B subunit mRNAs, there was a statistically significant
decrease in the relative eIF2␣ mRNA transcript levels over the
time course examined. This decrease was transient and statistically significant only 24 h after the completion of the exercise
bout (28% decrease; P ⬍ 0.05; Fig. 4A). No statistically
significant alteration in eIF2␤ mRNA was observed over the
time course examined (Fig. 4B).
Relative mRNA expression of eIF2␣ and eIF2␤ kinases. The
relative mRNA quantity for the eIF2␣ kinase PKR was significantly decreased during the time course after the exercise bout
(33% decrease 48 h postexercise vs. control; P ⬍ 0.01; Fig.
5A). The relative mRNA quantities for the eIF2␣ kinase HCR
(Fig. 5B) were unchanged throughout the time course examined. Likewise, the mRNA levels of eIF2␤ kinase PKA (Fig.
5C) were not significantly altered by resistance exercise during
the time course examined.
Relative protein expression of eIF2B⑀ and eIF2␣ total proteins. On the basis of Western blot analysis of the ⑀-subunit,
there was a significant increase in the total amount of eIF2B
protein present in the gastrocnemius muscle after an acute bout
J Appl Physiol • VOL
Fig. 5. Real-time PCR analysis of mRNA species coding for eIF2␣ and eIF2␤
kinases, normalized to GAPDH mRNA expression and expressed as a fraction
of control values. A: RNA-activated protein kinase (PKR). B: heme-regulated
inhibitor (HCI). C: protein kinase A (PKA). Sedentary animals (n ⫽ 10) are
compared with animals killed 3 h (n ⫽ 10), 24 h (n ⫽ 9), or 48 h (n ⫽ 9) after
acute resistance exercise. Values are means ⫾ SE. *Values for the 48-h
postexercise group are significantly lower than for the sedentary control group
(P ⬍ 0.01).
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RESISTANCE EXERCISE AND EIF2B REGULATION
DISCUSSION
The most immediate effect observed in the present study was
a significant increase in the quantity of eIF2B⑀ protein 3 h after
resistance exercise (2.2-fold). This increase remained statistically significant at both 24 and 48 h postexercise. We focused
on the ⑀-subunit because this subunit catalyzes the guanine
nucleotide exchange reaction on eIF2; however, previous work
suggests that all subunits of eIF2B holoprotein are present in a
stoichiometric complex (25, 35). Therefore, we speculate that
these results are indicative of an increase in the total protein
expression of the total eIF2B complex. This approach has been
used previously to quantify total eIF2B protein (25).
The increase in protein appeared before an increase in the
relative quantity of the mRNA species coding for subunits of
eIF2B. This result suggests that eIF2B is translationally regulated after an acute bout of resistance exercise and that,
temporally, transcriptional regulation of eIF2B is a secondary
response in the postexercise period. It is important to note that
we cannot eliminate decreased degradation of eIF2B protein as
an alternative explanation for the present results; however,
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Fig. 6. Western blot analysis of eIF2B⑀ and eIF2␣ total proteins in gastrocnemius muscle after an acute bout of resistance exercise. Samples were loaded
according to total protein and values are based on absorbance densitometry. A:
total eIF2B⑀ protein. B: eIF2␣ total protein. Values are expressed as a fraction
of control and shown as means ⫾ SE. Sedentary animals (n ⫽ 4) are compared
with animals killed 3 h (n ⫽ 4), 24 h (n ⫽ 4), or 48 h (n ⫽ 4) after acute
resistance exercise. *Values for the 3 h (P ⬍ 0.01), 24 h (P ⬍ 0.05), and 48 h
(P ⬍ 0.05) postexercise groups are significantly greater than those for the
sedentary control group.
because of the long half-life of this protein under basal conditions, this explanation is considered less likely.
Unlike the alterations in eIF2B, eIF2 protein did not change
significantly in the postexercise period examined. Therefore,
translational regulation of eukaryotic initiation factors appears
to be specific to particular proteins after resistance exercise.
Although the mechanism of such discrimination is not known,
this hypothesis is teleologically attractive because eIF2B is
present in limiting quantities relative to the number of ribosomes present in some cell types (18, 40), whereas eIF2 is
believed to be more abundant. Oldfield et al. (35) found that
eIF2 was 5.6 times more abundant than eIF2B in rabbit
reticulocytes and ⬎10 times more abundant in the heart.
Furthermore, computer predictions of secondary structure in
the rat eIF2B⑀ 5⬘-untranslated region (free energy ⫽ ⫺30.79
kcal/mol, RNA structure 3.71, unpublished results) (32) suggest the possibility of translational regulation of this mRNA
sequence. Thus, because eIF2B activity may be the most
important determinant of increased translational initiation after
an acute bout of resistance exercise, an increase in available
eIF2B in the gastrocnemius could lead to an increased capacity
of skeletal muscle to elevate postexercise global protein synthesis. Interestingly, the initial increase in eIF2B total protein
occurred before the time of the increase in protein synthesis
previously reported (17). Thus the increase in eIF2B could play
a role in acute increases in protein synthesis after load-bearing
activity; however, the increase in eIF2B protein alone is
insufficient to explain such a response.
Whereas changes in mRNA translation appear to be the
dominant process for modulating eIF2B abundance immediately after exercise, transcriptional processes could potentially
be important later in the postexercise period. There was a
significant increase in mRNAs coding for the ⑀-, ␥-, and
␦-subunits of eIF2B in the postexercise time course examined.
The five subunits of eIF2B are categorized into two families on
the basis of amino acid sequence homology. The ␣-, ␤-, and
␦-subunits comprise one family, whereas the ⑀- and ␥-subunits
make up the other family. Therefore, mRNA species encoding
proteins belonging to both families were increased by the
resistance exercise protocol.
The changes observed in the mRNAs encoding eIF2B⑀
kinases were also in line with the initial hypotheses. GSK-3␤
mRNA levels were significantly reduced in the 48-h postexercise group compared with the sedentary group and the 3 h
postexercise group. Although Dholakia and Wahba (9) found
increases in eIF2B activity with phosphorylation of the ⑀-subunit by GSK-3, the majority of published studies have found
the exact opposite to be true. Price and Proud (39) and Webb
and Proud (49) proposed that the activity of eIF2B is increased
by the dephosphorylation of eIF2B⑀, therefore suggesting that
phosphorylation of eIF2B⑀ at the GSK-3 site would actually
inhibit exchange activity. There is some evidence to suggest
that this hypothesis may be correct in the case of exercise.
First, Markuns et al. (31) showed that GSK-3␣ and GSK-3␤
activity were both reduced after treadmill running, an exercise
protocol that has been shown to induce skeletal muscle hypertrophy (23). Furthermore, a recently published study by Vyas
et al. (48) showed that GSK-3␤ activation negatively regulated
skeletal muscle hypertrophy in myotubes. The present results
predict that the quantity of GSK-3 protein in the cell may be
limited via downregulation of mRNA transcription, thereby
RESISTANCE EXERCISE AND EIF2B REGULATION
J Appl Physiol • VOL
in the acute resistance exercise model employed in the present
study.
The results of the present study do not preclude the possibility that these mRNA transcripts are regulated in an alternative manner as well. It is possible that existing mRNA transcripts are preferentially shifted into polysomes after an acute
bout of resistance exercise. Resistance exercise (17), electrical
stimulation (3), and stretch (4) have been shown to increase the
activity of p70S6K, which is known to upregulate the translation
of mRNA sequences that contain a 5⬘ oligopyrimidine tract (5⬘
TOP) (20, 21, 29, 46). Such sequences are present in mRNAs
encoding other members of the translational apparatus, including elongation factors and ribosomal proteins, but 5⬘-oligopyrimidine tract sequences have not currently been identified in
eukaryotic initiation factors.
The arguments presented above with regard to the functional
consequences of the observed alterations in mRNA expression
are all contingent on transcriptional contributions to total
protein expression. It was not particularly surprising that the
increases or decreases in gene expression were seemingly
minor (⬍2-fold). Exercise is a unique physiological perturbation in that to obtain a “training” effect many bouts must be
performed with an appropriate amount of recovery in between
each bout. Therefore, our results provide direction for future
inquiries. In particular, it will be interesting to determine
whether the completion of several acute sessions of resistance
exercise results in an additive increase or decrease in the
mRNA species examined. Dissecting the relative contribution
of translational regulation vs. transcriptional regulation of
these species in acute vs. chronic exercise paradigms will be
essential to understanding regulation of eIF2B protein capacity
and eIF2B upstream signaling capacity. Because the acute
protein synthetic response is attenuated by 8 wk of resistance
exercise training (11), it will be interesting to explore the
temporal nature of the alterations in the context of short- vs.
long-term exercise training protocols. Overall the results demonstrate the need for a more clear understanding of the effect
of eIF2B abundance (and subsequent posttranslational control)
on eIF2B activity and the effect of prolonged training on gene
expression of translationally relevant mRNA species.
In summary, there was a rapid increase in the amount of total
eIF2B protein 3 h after an acute bout of resistance exercise.
Furthermore, a modest but significant increase in mRNA levels
of species coding for the ⑀-, ␥-, and ␦-subunits of the eIF2B
protein was observed after an acute bout of resistance exercise,
but after the postexercise time when an increase in eIF2B
protein was observed. To our knowledge, this is the first
evidence that gene expression of mRNAs coding for proteins
involved in translation initiation is altered by exercise. These
results suggest a rapid increase in eIF2B mRNA translation
followed by a slower elevation in eIF2B gene transcription.
Furthermore, mRNAs coding for the eIF2B⑀ kinases GSK3,
CKI, and CKII were all decreased over this time course. These
results implicate both translational and transcriptional regulation of eIF2B subunit mRNAs and its regulatory kinases as a
potentially important mechanism of protein synthesis regulation after resistance exercise. Transcriptional regulation may
be especially important late in the recovery period, or perhaps
on the completion of subsequent bouts of exercise (i.e., exercise training). Further studies will be required to assess the
functional significance of these findings in rat skeletal muscle.
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creating a decreased potential for suppression of eIF2B activity. Because GSK-3 has other actions (8, 14) in skeletal
muscle, it is also possible that the kinase contributes to alterations in eIF2B activity through less direct mechanisms.
The argument presented above can be extended to the
interpretation of the decrease in mRNA levels of CKI and CKII
observed in this study. Both CKI␥3 and CKII␤ mRNA were
significantly reduced 48 h postexercise compared with the
control sedentary animals. The casein kinases phosphorylate
sites on eIF2B⑀ that are different from one another and different from the GSK-3 site. CKI is believed to phosphorylate
Ser464, whereas CKII phosphorylates sites in the extreme
COOH terminus (Ser712, 713). This pattern of phosphorylation creates the possibility of differential effects of these
phosphorylation events on eIF2B activity. There is no consensus on the effects of casein kinase phosphorylation of the
eIF2B⑀ subunit on eIF2B activity. According to Singh et al.
(44), phosphorylation by CKI and CKII activates eIF2B; however, other groups have shown no change in eIF2B activity
associated with these phosphorylation events. As with the
GSK-3 data, the results concerning the mRNA levels of the
casein kinases suggest that the protein abundance of these
kinases may be reduced with resistance exercise training.
Because both CKI and CKII are kinases with a wide range of
cellular functions, including Wnt signaling, circadian rhythm,
nuclear import, and cell survival (10, 30, 33, 38, 47), it is
certainly possible that these alternate functions might also play
a role in skeletal muscle adaptations to resistance exercise.
Surprisingly, mRNA coding for eIF2␣ was significantly
decreased 24 h after our resistance exercise protocol relative to
control mRNA expression levels. This result is contrary to the
hypothesis that transcriptional alterations after resistance exercise support an increase in translation capacity. Although
phosphorylation of the ␣-subunit is known to decrease eIF2B
activity, an increase in the eIF2 protein (potentially regulated
via increased transcription of mRNAs coding for subunits of
the protein) would be expected under conditions of increased
capacity of the translation apparatus. It is possible that the
transient decrease in eIF2␣ mRNA is not additive on multiple
bouts of resistance exercise and therefore does not have any
effect on the available pool of eIF2 protein. Second, it is
possible that eIF2 is not limiting relative to the number of
ribosomes present in the gastrocnemius and/or to the amount of
eIF2B present during the time course studied. Finally, this
result might represent an experimental artifact because the
mRNA coding for eIF2␤ and the amount of eIF2␣ protein did
not change in this study.
The alteration in expression of mRNA coding for the eIF2␣
kinase PKR was consistent with the initial hypothesis. Because
eIF2␣ phosphorylation results in negative regulation of eIF2B
activity, decreases in transcription of PKR could blunt signaling through this pathway, thus preserving increases in eIF2B
activity after resistance exercise. These findings are the first
implication that eIF2␣ phosphorylation may play a role in
regulating eIF2B activity after exercise. Because PKR is activated in response to proinflammatory signals, the effects of
proinflammatory cytokines on translational initiation in acute
vs. chronic resistance exercise could be a potentially interesting area of future investigation. PKR has also been shown to be
a positive regulator of muscle differentiation in vitro (28, 41).
However, extensive satellite cell invasion is unlikely to occur
685
686
RESISTANCE EXERCISE AND EIF2B REGULATION
ACKNOWLEDGMENTS
We are grateful to Dr. Deborah Grove and the staff of the Penn State
University Nucleic Acid Facility for assistance in optimizing and running the
real-time PCR assays for eIF2B⑀, eIF2␣, and eIF2␤. We thank Kevin Finnerty
for assistance in developing the SYBR green real-time PCR assay. We thank
JC Kostyak, Brian Krawick, and Fritz Kraemer for assistance in completing the
resistance exercise protocol.
GRANTS
This study was supported by National Institutes of Health Grants AR-43127
(P. A. Farrell) and DK-15658 (L. S. Jefferson).
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