Resistance Exercise Increases Muscle Protein Synthesis and Translation of Eukaryotic Initiation Factor 2Bϵ mRNA in a Mammalian Target of Rapamycin-dependent Manner
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m413732200
ISSN1083-351X
AutoresNeil Kubica, Douglas R. Bolster, P. A. Farrell, Scot R. Kimball, Leonard S. Jefferson,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoThe contribution of mammalian target of rapamycin (mTOR) signaling to the resistance exercise-induced stimulation of skeletal muscle protein synthesis was assessed by administering rapamycin to Sprague-Dawley rats 2 h prior to a bout of resistance exercise. Animals were sacrificed 16 h postexercise, and gastrocnemius protein synthesis, mTOR signaling, and biomarkers of translation initiation were assessed. Exercise stimulated the rate of protein synthesis; however, this effect was prevented by pretreatment with rapamycin. The stimulation of protein synthesis was mediated by an increase in translation initiation, since exercise caused an increase in polysome aggregation that was abrogated by rapamycin administration. Taken together, the data suggest that the effect of rapamycin was not mediated by reduced phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein 1 (BP1), because exercise did not cause a significant change in 4E-BP1(Thr-70) phosphorylation, 4E-BP1-eIF4E association, or eIF4F complex assembly concomitant with increased protein synthetic rates. Alternatively, there was a rapamycin-sensitive decrease in relative eIF2Bϵ(Ser-535) phosphorylation that was explained by a significant increase in the expression of eIF2Bϵ protein. The proportion of eIF2Bϵ mRNA in polysomes was increased following exercise, an effect that was prevented by rapamycin treatment, suggesting that the increase in eIF2Bϵ protein expression was mediated by an mTOR-dependent increase in translation of the mRNA encoding the protein. The increase in eIF2Bϵ mRNA translation and protein abundance occurred independent of similar changes in other eIF2B subunits. These data suggest a novel link between mTOR signaling and eIF2Bϵ mRNA translation that could contribute to the stimulation of protein synthesis following acute resistance exercise. The contribution of mammalian target of rapamycin (mTOR) signaling to the resistance exercise-induced stimulation of skeletal muscle protein synthesis was assessed by administering rapamycin to Sprague-Dawley rats 2 h prior to a bout of resistance exercise. Animals were sacrificed 16 h postexercise, and gastrocnemius protein synthesis, mTOR signaling, and biomarkers of translation initiation were assessed. Exercise stimulated the rate of protein synthesis; however, this effect was prevented by pretreatment with rapamycin. The stimulation of protein synthesis was mediated by an increase in translation initiation, since exercise caused an increase in polysome aggregation that was abrogated by rapamycin administration. Taken together, the data suggest that the effect of rapamycin was not mediated by reduced phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein 1 (BP1), because exercise did not cause a significant change in 4E-BP1(Thr-70) phosphorylation, 4E-BP1-eIF4E association, or eIF4F complex assembly concomitant with increased protein synthetic rates. Alternatively, there was a rapamycin-sensitive decrease in relative eIF2Bϵ(Ser-535) phosphorylation that was explained by a significant increase in the expression of eIF2Bϵ protein. The proportion of eIF2Bϵ mRNA in polysomes was increased following exercise, an effect that was prevented by rapamycin treatment, suggesting that the increase in eIF2Bϵ protein expression was mediated by an mTOR-dependent increase in translation of the mRNA encoding the protein. The increase in eIF2Bϵ mRNA translation and protein abundance occurred independent of similar changes in other eIF2B subunits. These data suggest a novel link between mTOR signaling and eIF2Bϵ mRNA translation that could contribute to the stimulation of protein synthesis following acute resistance exercise. Understanding the molecular basis of muscle hypertrophy is critical to the development of targets for exercise, nutritional, and pharmaceutical intervention in muscle wasting conditions such as sarcopenia, cachexia, diabetes, exposure to microgravity, and extended bed rest. Regulation of muscle hypertrophy has recently been the subject of intense investigation (1Glass D.J. Nat. Cell Biol. 2003; 5: 87-90Crossref PubMed Scopus (534) Google Scholar). In particular, there has been a great deal of attention focused on the role of signaling through the mammalian target of rapamycin (mTOR) 1The abbreviations used are: mTOR, mammalian target of rapamycin; PKB, protein kinase B; ERK, extracellular signal-regulated kinase.1The abbreviations used are: mTOR, mammalian target of rapamycin; PKB, protein kinase B; ERK, extracellular signal-regulated kinase. protein kinase in the hypertrophic response following load-bearing contractile activity. Several models of resistance exercise, muscle contraction, and/or muscle loading suggest that mTOR signaling is activated in the recovery period following these perturbations (2Hernandez J.M. Fedele M.J. Farrell P.A. J. Appl. Physiol. 2000; 88: 1142-1149Crossref PubMed Scopus (65) Google Scholar, 3Nader G.A. Esser K.A. J. Appl. Physiol. 2001; 90: 1936-1942Crossref PubMed Scopus (284) Google Scholar, 4Bolster D.R. Kubica N. Crozier S.J. Williamson D.L. Farrell P.A. Kimball S.R. Jefferson L.S. J. 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Zlotchenko E. Stitt T.N. Economides A.N. Yancopoulos G.D. Glass D.J. Mol. Cell. Biol. 2004; 24: 9295-9304Crossref PubMed Scopus (332) Google Scholar). The effect of PKB on muscle hypertrophy has been suggested to signal through mTOR, since muscle growth induced by synergistic ablation can be completely prevented by chronic administration of the mTOR inhibitor rapamycin (6Bodine S.C. Stitt T.N. Gonzalez M. Kline W.O. Stover G.L. Bauerlein R. Zlotchenko E. Scrimgeous A. Lawrence J.C. Glass D.J. Yancopoulos G.D. Nat. Cell Biol. 2001; 3: 1014-1019Crossref PubMed Scopus (1916) Google Scholar). Furthermore, hypertrophy in response to chronic sciatic nerve stimulation is highly correlated to phosphorylation of the 70-kDa ribosomal protein S6 kinase, S6K1 (8Baar K. Esser K. Am. J. Physiol. 1999; 276: C120-C127Crossref PubMed Google Scholar), a well defined downstream target of mTOR signaling. The role of PKB/mTOR signaling in the regulation of acute increases in translation initiation and protein synthesis is less clear. It has been widely assumed that activation of mTOR signaling leads to increased protein synthesis via 1) activation of S6K1, subsequent phosphorylation of ribosomal protein S6, enhanced translation of mRNAs containing a 5′-terminal oligopyrimidine tract (5′-TOP) (encoding elongation factors and ribosomal proteins), and ultimately an increase in translation capacity (9Meyuhas O. Eur. J. Biochem. 2000; 267: 6321-6330Crossref PubMed Scopus (432) Google Scholar) and 2) phosphorylation of 4E-BP1, increased eIF4E availability, and eIF4F complex assembly, leading to increased rates of translation initiation (10Lawrence J. John C. Fadden P. Haystead T.A.J. Lin T.A. Adv. Enzyme Regul. 1997; 37: 239-267Crossref PubMed Scopus (63) Google Scholar, 11Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.P. Kasuga M. Nishimoto I. Avruch J. J. Biol. 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Alternatively, it has been established that eIF2B activity, the other well established rate-controlling step in translation initiation, is elevated concomitant with the stimulation of protein synthesis in the recovery period following resistance exercise (21Farrell P.A. Fedele M.J. Vary T.C. Kimball S.R. Lang C.H. Jefferson L.S. Am. J. Physiol. 1999; 276: E721-E727Crossref PubMed Google Scholar, 25Kostyak J.C. Kimball S.R. Jefferson L.S. Farrell P.A. J. Appl. Physiol. 2001; 91: 79-84Crossref PubMed Scopus (19) Google Scholar). eIF2B is a complex, five-subunit guanine nucleotide exchange factor that exchanges GTP for GDP bound to eIF2, thus allowing eIF2 to deliver Met-tRNAi to the 40 S ribosomal subunit during each round of translation initiation (26Price N. Proud C.G. Biochimie (Paris). 1994; 76: 748-760Crossref PubMed Scopus (89) Google Scholar, 27Webb B.L.J. Proud C.G. Int. J. Biochem. Cell Biol. 1997; 29: 1127-1131Crossref PubMed Scopus (77) Google Scholar, 28Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (637) Google Scholar). Previous work has shown that the mRNA abundance of a number of eIF2B subunits is increased subsequent to the observed stimulation of protein synthesis following an acute bout of resistance exercise, suggesting a role for transcriptional regulation on eIF2B availability and/or activity with chronic exercise training (29Kubica N. Kimball S.R. Jefferson L.S. Farrell P.A. J. Appl. Physiol. 2004; 96: 679-687Crossref PubMed Scopus (23) Google Scholar). Importantly, that study also reported a rapid increase in eIF2Bϵ protein expression observed prior (3 h postexercise) to the stimulation of protein synthesis in the recovery period following exercise. Theoretically, this post-transcriptional increase in the catalytic ϵ-subunit of eIF2B could participate in acute protein synthetic regulation. Interestingly, previous work in the model of resistance exercise employed in those studies has established a rapid but transient increase in mTOR signaling within the first 1 h into the recovery period (4Bolster D.R. Kubica N. Crozier S.J. Williamson D.L. Farrell P.A. Kimball S.R. Jefferson L.S. J. Physiol. (Lond.). 2003; 553: 213-220Crossref Scopus (178) Google Scholar). Since phosphorylation of ribosomal protein S6 and increased eIF4E availability (functional consequences of mTOR signaling) are both purported to be associated with translation of specific messages, it is possible that this early signaling through mTOR could lead to an increase in translation of progrowth mRNAs, such as eIF2Bϵ, that could ultimately play a role in the protein synthetic response observed later in the recovery time course. The ϵ-subunit of eIF2B is known to be phosphorylated on numerous residues by several kinases (e.g. GSK-3, CKI, CKII, and DYRK). Among these kinases, GSK-3 is the only signaling protein with a well established effect on eIF2B activity. GSK-3 phosphorylates eIF2Bϵ on Ser-535, thus reducing the activity of this positive regulator of translation initiation (30Jefferson L.S. Fabian J.R. Kimball S.R. Int. J. Biochem. Cell Biol. 1999; 31: 191-200Crossref PubMed Scopus (89) Google Scholar). Activated GSK-3 inhibits myotube hypertrophy in vitro (31Vyas D.R. Spangenburg E.E. Abraha T.W. Childs T.E. Booth F.W. Am. J. Physiol. 2002; 283: C545-C551Crossref PubMed Scopus (127) Google Scholar) and cardiac hypertrophy both in vitro (32Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (335) Google Scholar) and in vivo (33Antos C.L. McKinsey T.A. Frey N. Kutschke W. McAnally J. Shelton J.M. Richardson J.A. Hill J.A. Olson E.N. Proc. Natl. Acad. 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J. 1993; 296: 15-19Crossref PubMed Scopus (755) Google Scholar, 39Sutherland C. Cohen P. FEBS Lett. 1994; 338: 37-42Crossref PubMed Scopus (192) Google Scholar), thus inhibiting GSK-3 activity and theoretically derepressing eIF2B activity. Despite the clear link between eIF2B activity and initiation of translation, the role of this protein as a mediator of both skeletal muscle protein synthesis and muscle hypertrophy downstream of the PKB/mTOR pathway has been understudied. Importantly, the mechanism by which eIF2B activity is regulated following resistance exercise remains unknown, and the phosphorylation status of the ϵ-subunit following load-bearing exercise has not been reported. The study described herein was designed to assess the contribution of PKB/mTOR signaling to the stimulation of gastrocnemius protein synthesis following an acute bout of resistance exercise. In particular, the roles of the well described mTOR targets S6K1 and 4E-BP1 in regulation of skeletal muscle protein synthesis following exercise were reexamined, and novel connections between PKB/mTOR signaling and eIF2B subunit abundance and/or phosphorylation status were explored. 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 (∼250–300 g) were housed in temperature- and humidity-controlled holding facilities on a 12:12 h light-dark cycle. They were fed a standard rodent diet (Harlan-Teklad Rodent Chow, Madison, WI), and both food and water were provided ad libitum. Animals were randomly assigned to treatment groups and fasted for 5 h prior to tissue procurement. Acute Resistance Exercise Protocol—Details of the exercise protocol have been described previously (22Fluckey J.D. Vary T.C. Jefferson L.S. Farrell P.A. Am. J. Physiol. 1996; 270: E313-E319Crossref PubMed Google Scholar). Briefly, male Sprague-Dawley rats were operantly conditioned to stand on their hind limbs and touch an illuminated bar located high on the wall of a plexiglass 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-g (day 1), 0.4-g (days 2 and 3), and 0.6-g (day 4) weighted vest/g of body weight, respectively. On training days, sedentary control animals were placed in the cage and given five mild shocks to simulate the stress experienced by the exercised animals. Sixteen hours following the last acute resistance exercise session, all animals were anesthetized by breathing a 95% O2, 5% CO2 gas mixture via a nose cone connected to an isoflurane vaporizer (∼3.0–4.0% isoflurane). Animals were deemed deeply anesthetized only if they did not respond to numerous tactile stimuli (e.g. tail pinch response and eye reflex response). These assessments were made frequently during the surgical protocol, and animals remained in a deeply anesthetized state during all described procedures. Once the appropriate anesthetic state was achieved, both gastrocnemius muscles were excised for subsequent analyses (see below). Animals were then killed by guillotine while still deeply anesthetized with isoflurane. Administration of Rapamycin—Rapamycin (NCI, National Institutes of Health, Rockville, MD) was dissolved in 100% ethanol (1 mg/20 μl) and then added to 980 μl of sterile saline. Animals in the rapamycin treatment groups received a tail vein injection containing 0.75 mg/kg of body weight. This dosage has been previously used in rodents and demonstrated to inhibit signaling downstream of mTOR (40Anthony J.C. Yoshizawa F. Anthony T.G. Vary T.C. Jefferson L.S. Kimball S.R. J. Nutr. 2000; 130: 2413-2419Crossref PubMed Scopus (616) Google Scholar). Based on the timing of the injections, exercised animals received rapamycin 2 h prior to the final resistance exercise bout. Untreated animals received an injection containing an equal volume of ethanol/saline vehicle. Assessment of Protein Synthesis—The rate of global protein synthesis was assessed in the gastrocnemius muscle by the flooding dose method previously reported (41Garlick P.J. McNurlan M.A. Preedy V.R. Biochem. J. 1980; 192: 719-723Crossref PubMed Scopus (755) Google Scholar) with several modifications (42Fedele M.J. Hernandez J.M. Lang C.H. Vary T.C. Kimball S.R. Jefferson L.S. Farrell P.A. J. Appl. Physiol. 2000; 88: 102-108Crossref PubMed Scopus (48) Google Scholar). Briefly, rats were anesthetized as described, and the left carotid artery and right jugular vein were catheterized. A flooding dose of l-[2,3,4,5,6-3H]phenylalanine (1 mCi/rat; Amersham Biosciences) was delivered in unlabeled phenylalanine (150 mm; 1 ml/100 g of body weight) via injection into the venous catheter over a 15-s period. Arterial blood was obtained 10 min after infusion of the flooding dose and subsequently centrifuged at 1800 × g for 10 min at 4 °C to obtain serum. The specific radioactivity of [3H]phenylalanine in the serum was assessed by measuring the amount of radioactivity in the serum via liquid scintillation spectrometry (10 μl in 10 ml of 989 scintillation fluid; Packard) and the concentration of phenylalanine via high pressure liquid chromatography following amino acid extraction from the serum. Immediately following the blood collection, the left gastrocnemius was excised and frozen between aluminum blocks cooled to the temperature of liquid nitrogen. The muscle was then powdered, and 0.5 g was homogenized in 7 volumes of homogenization buffer (20 mm HEPES (pH 7.4), 100 mm potassium chloride, 0.2 mm EDTA, 2 mm EGTA, 50 mm sodium fluoride, 50 mm β-glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 1 mm dithiothreitol, and 0.5 mm sodium vanadate). A 500-μl aliquot of homogenate was then added to 2.5 ml of 1 n perchloric acid and boiled for 15 min. Following boiling, the samples were centrifuged at 3200 × g for 10 min at 4 °C. The resulting supernatant was aspirated, and the pellet was subjected to the following wash protocol: twice in 0.5 n perchloric acid, twice in chloroform/ethanol/ether (1:2:1), and once in ether. The pellet was then dried overnight in a fume hood, resuspended in 3 ml of 0.1 n NaOH and boiled for 15 min with occasional vortexing. The final sample was counted via liquid scintillation in duplicate (1-ml sample in 10 ml of 989 scintillation fluid), and 5 μl were assayed for total protein concentration using the Bio-Rad protein assay. Rates of protein synthesis (nmol of Phe/mg of protein/h) were then calculated as described previously (43Kimball S.R. Vary T.C. Jefferson L.S. Biochem. J. 1992; 286: 263-268Crossref PubMed Scopus (66) Google Scholar). Skeletal Muscle Polysome Aggregation and eIF2Bϵ mRNA Distribution—Sucrose density gradient centrifugation was employed to separate the subpolysomal from the polysomal ribosome fractions following resistance exercise with and without pretreatment with rapamycin. Gastrocnemius tissue was powdered, and connective tissue was carefully removed. Powdered tissue was stored at –80 °C until the time of analysis. Powdered muscle tissue (∼0.2 g) was homogenized for 20 s in 10 volumes of resuspension buffer (10 mm Tris (pH 7.5), 250 mm KCl, 10 mm MgCl2, 0.5% Triton X-100, 2 mm dithiothreitol, 100 μg/ml cycloheximide, and 100 units/ml SUPERase·In™ RNase inhibitor (Ambion, Austin, TX)) using a Polytron homogenizer PT10–35. Homogenates were incubated on ice for 5 min, and then 150 μl of Tween-deoxycholate mix (1.34 ml of Tween 20, 0.66 g of deoxycholate, 18 ml of sterile water) were added per 1 ml of resuspension buffer, and the samples were briefly vortexed. Samples were incubated on ice for 15 min and then centrifuged at 10,000 × g for 10 min at 4 °C. The resulting supernatant (600 μl) was layered on a 15–50% linear sucrose gradient (20 mm Tris (pH 7.5), 250 mm KCl, 10 mm MgCl2) and centrifuged in a SW41 rotor at 40,000 rpm for 165 min at 4 °C. Following centrifugation, the gradient was displaced upward (2 ml/min) using Fluorinert (Isco, Lincoln, NE) through a spectrophotometer, and OD at 254 nm was continuously recorded (chart speed, 150 cm/h). Two sucrose fractions representing the subpolysomal and polysomal portions of the gradient were collected directly into an equal volume of TRIzol Reagent (Invitrogen). In order to improve recovery of RNA from the dense sucrose portions of the gradient, the polysomal fraction was diluted 2× with RNase-free water (Ambion), and the appropriate amount of TRIzol was employed. RNA was extracted using the standard manufacturer's protocol and resuspended in RNA Storage Solution (Ambion). RNA samples were analyzed for quality using the Agilent 2100 Bioanalyzer microfluidics platform (Agilent Biotechnologies, Palo Alto, CA). An equal quantity of RNA in each fraction was converted to cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Finally, the resulting cDNA was used to quantify the relative abundance of eIF2Bϵ, eIF2Bδ, and glyceraldehyde-3-phosphate dehydrogenase transcripts using the QuantiTect SYBR Green real time PCR kit (see manufacturer's protocol; Qiagen, Valencia, CA). Western Blotting Protocols—The gastrocnemius was excised, cleaned of connective tissue, and homogenized in 7 volumes of homogenization buffer (described above) on ice using a Polytron PT10–35 homogenizer. The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C. An aliquot of the resulting supernatant was combined with an equal volume of 2× SDS sample buffer and then subjected to protein immunoblot analysis as described previously (12Kimball S.R. Jurasinski C. Lawrence J.C.J. Jefferson L.S. Am. J. Physiol. 1997; 272: C754-C759Crossref PubMed Google Scholar). All gels were loaded according to protein concentration (as assessed by the Bio-Rad protein assay), with 75–100 μg of protein/lane. Hyperphosphorylation status of S6K1 and 4E-BP1 was assessed using antibodies (catalog numbers A300–510A and A300–501A, respectively) from Bethyl Laboratories (Montgomery, TX). Phospho-ERK1/2(Thr-202/Tyr-204) (number 9101), ERK1/2 total (number 9102), phospho-eIF4E(Ser-209) (number 9741), phospho-S6 ribosomal protein(Ser-235/236) (number 2211), phospho-S6 ribosomal protein(Ser-240/244) (number 2215), phospho-4E-BP1(Thr-37/46) (number 9459), phospho-4E-BP1(Thr-70) (number 9455), and phospho-GSK-3β(Ser-9) (number 9336) antibodies were obtained from Cell Signaling Technologies (Beverly, MA). GSK-3β antibody (number 361528) was from Calbiochem. Phospho-eIF2Bϵ(Ser-535 antibody) (number 44-530G) was obtained from BIOSOURCE International (Camarillo, CA). eIF2β antibody (sc-9978) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies raised against the β-, δ-, and ϵ-subunits of eIF2B (44Fabian J.R. Kimball S.R. Jefferson L.S. Protein Expression Purif. 1998; 13: 16-22Crossref PubMed Scopus (27) Google Scholar), eIF4E and eIF4G (45Yoshizawa F. Kimball S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1997; 240: 825-831Crossref PubMed Scopus (73) Google Scholar), and eIF2α (46Kimball S. Everson W. Myers L. Jefferson L. J. Biol. Chem. 1987; 262: 2220-2227Abstract Full Text PDF PubMed Google Scholar) used in the current study were developed in this laboratory and have been described previously. Total actin was assessed using an antibody (A-5060) from Sigma. Statistical Analysis—Treatment comparisons were conducted using a one-way analysis of variance (GraphPad Prism 4, San Diego, CA). If the analysis of variance reached statistical significance at a 95% confidence level, a Student-Newman-Keuls multiple comparison test was applied to assess significant differences (p < 0.05) between the various treatment groups. All data sets were assessed for potential outliers using a Grubbs test. All results are expressed as a fraction of control and presented as mean ± S.E. Effect of Resistance Exercise and Rapamycin Treatment on mTOR-dependent Signaling—Previous work has established that mTOR signaling is rapidly but transiently increased within 1 h following the model of acute resistance exercise employed in this study (4Bolster D.R. Kubica N. Crozier S.J. Williamson D.L. Farrell P.A. Kimball S.R. Jefferson L.S. J. Physiol. (Lond.). 2003; 553: 213-220Crossref Scopus (178) Google Scholar). The results presented herein suggest that there is also a small but significant increase in the hyperphosphorylation status of the mTOR targets S6K1 (+19%; Fig. 1A) and 4E-BP1 (+30%; Fig. 1B) 16 h into the recovery period following acute resistance exercise compared with sedentary control values. In both cases, hyperphosphorylation status was assessed by dividing the combined densitometry of the slower migrating phosphorylated β and γ bands by the total densitometry signal from all of the bands. Previously published results established that tail vein administration of rapamycin (0.75 mg/kg) is sufficient to severely blunt phosphorylation of the aforementioned mTOR targets 3 h following injection (40Anthony J.C. Yoshizawa F. Anthony T.G. Vary T.C. Jefferson L.S. Kimball S.R. J. Nutr. 2000; 130: 2413-2419Crossref PubMed Scopus (616) Google Scholar) even following an oral gavage of leucine (a known stimulator of mTOR signaling) 1 h prior to sacrifice. In the present study, this dose of rapamycin significantly decreased hyperphosphorylation of both S6K1 (Fig. 1A) and 4E-BP1 (Fig. 1B) in both sedentary control and exercised animals, even 18 h postinjection, thus confirming the efficacy of the inhibitor. Whereas muscle contractions are known to transiently increase phosphorylation of members of the mitogen-activated protein kinase pathway, phosphorylation of ERK1/2(Thr-202/Tyr-204) (Fig. 1C) and its downstream target eIF4E(Ser-209) (Fig. 1D) were unchanged 16 h following resistance exercise. Importantly, rapamycin administration had no nonspecific effects on the phosphorylation of these proteins. Assessment of Protein Synthesis following Resistance Exercise with and w
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