Corresponding author L. S. Jefferson: Penn State College of Medicine, Department of Cellular and Molecular Physiology, H166, 500 University Drive, Hershey, PA 17033, USA. Email: firstname.lastname@example.org
The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282 % of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292 %, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336 %, P= 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647 %, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292 %, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2α phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2α. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.
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An influx of research investigating the regulation of skeletal muscle hypertrophy has recently enhanced our understanding of the cellular mechanisms by which muscle anabolism occurs under growth conditions. Various research models (resistance exercise, stretch, insulin-like growth factor (IGF) stimulation, electrical stimulation, synergistic ablation, etc.) have been used to examine signalling events that are necessary for mediating, as well as maintaining, skeletal muscle hypertrophy. Among these signalling cascades, select intracellular proteins have been highlighted (e.g. glycogen synthase kinase 3-β (GSK3β), PKB, mTOR, and the 70 kDa ribosomal protein S6K1) as crucial for muscle growth, but an integrative response appears to be essential for maximizing the hypertrophic response (Baar & Esser, 1999; Bodine et al. 2001; Rommel et al. 2001; Reynolds et al. 2002; Vyas et al. 2002).
A crucial aspect to mediating a growth response is the upregulation of the PKB-mTOR signalling pathway, which is indispensable for inducing skeletal muscle hypertrophy under chronic loading conditions. Recent work provides compelling in vivo evidence for the sustained input of this pathway enhancing long-term increases in muscle fibre size (Bodine et al. 2001; Pallafacchina et al. 2002). Furthermore, inhibition of this pathway through treatment with rapamycin (a specific inhibitor of mTOR) completely blocks muscle hypertrophy under growth conditions (Bodine et al. 2001; Pallafacchina et al. 2002), directly linking this pathway with muscle growth.
Several of the downstream targets of mTOR are proteins that function in the control of mRNA translation, including S6K1, eIF4G, and the eIF4E-binding protein, 4E-BP1 (Pain, 1996). During the initiation phase of mRNA translation, the eIF4E-mRNA complex reversibly associates with eIF4G, a protein that mediates association of the eIF4E-mRNA complex with the 40S ribosomal subunit. The binding of eIF4E to eIF4G is regulated in part by the association of eIF4E with 4E-BP1. Binding of eIF4E to 4E-BP1 is controlled by phosphorylation of 4E-BP1 whereby hyperphosphorylation of 4E-BP1 prevents it from binding to eIF4E, thus liberating eIF4E to enter the eIF4F complex (Pause et al. 1994). Phosphorylation of 4E-BP1 is partially under the control of the protein kinase referred to as the mammalian target of rapamycin (mTOR), which serves as a convergence point for signalling by growth factors and amino acids to the mRNA-binding step of translation initiation (Gingras et al. 2001). An additional downstream protein under the control of mTOR is S6K1. S6K1 is vital to regulating a subset of mRNAs containing 5′-terminal polypyrimidine tracts (TOP sequences) and these encode ribosomal proteins and factors essential to the translational machinery (e.g. elongation factors) (Terada et al. 1994).
In addition to the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit, regulation can occur through modulation of the binding of the initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit to form the 43S preinitiation complex. In this step, the eIF2-GTP-met-tRNAi complex binds to the 40S ribosomal subunit to form a ternary complex. The GTP bound to eIF2 is subsequently hydrolysed to GDP, and the eIF2-GDP complex is released from the 40S ribosomal subunit. eIF2 must then exchange GDP for GTP in order to participate in a subsequent round of initiation and form a new ternary complex. A second translation initiation factor, eIF2B, mediates the guanine nucleotide exchange on eIF2 and the inhibition of eIF2B activity reduces the amount of eIF2-GTP available for ternary complex formation. In part, the activity of eIF2B is regulated by phosphorylation of the α-subunit of eIF2, which becomes a competitive inhibitor of eIF2B when phosphorylated on the α-subunit (Kimball & Jefferson, 1994).
The intent of the present investigation was to determine at what point mTOR-mediated signalling and associated regulatory proteins of translation initiation are activated in skeletal muscle following acute resistance exercise. Although activation of the PKB pathway has been shown to increase during muscle contractions (Turinsky & Damrau-Abney, 1999; Sakamoto et al. 2002), the detailed nature of signal transduction through this pathway immediately following resistance exercise is unknown (Baar & Esser, 1999; Farrell et al. 2000; Nader & Esser, 2001; Haddad & Adams, 2002). We hypothesized that signalling through this growth pathway would be upregulated during the immediate phase of exercise recovery.
Animal facilities and the experimental protocol were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of The Pennsylvania State University College of Medicine. Male Sprague-Dawley rats were kept on a 12 h-12 h light-dark cycle with food (Harlan-Teklad Rodent Chow, Madison, WI, USA) and water provided freely.
The following protocol has previously been described in detail (Fluckey et al. 1995). Briefly, rats were operantly conditioned to touch an illuminated bar low on a Plexiglass exercise cage and then were taught to stand and touch an illuminated bar located high on the opposite wall of the cage. These movements required the animals to complete concentric and eccentric muscle contractions of the hindlimb musculature. Electrical foot shock (< 1 mA, 60 Hz) was utilized to reinforce these movements. Once the learning process was completed (3-4 sessions), weighted vests were strapped over the scapulae and the rats were required to touch the high bar 50 times during one acute exercise session. We defined ‘acute’ resistance exercise as four separate sessions with 1 day of rest between sessions. Rats performed 50 repetitions each day with 0.2 (on day 1), 0.4 (on days 2 and 3) and 0.6 (on day 4) g weighted vest (g body weight)−1. Day 4 is considered as the experimental day. We are aware of metabolic adaptations, which occur after only a few bouts of exercise. However, previous work has shown that these acute sessions were essential, given that rats naive to the lifting procedure would not lift 0.6 g (g body weight)−1 on the first day weights were applied to the vest. This protocol can be considered as acute because it does not result in changes in body or muscle weight (Fluckey et al. 1995). Control non-exercised rats were placed in the lifting cages at least three times during the week of acute exercise and were given five electric shocks to simulate some of the stress experienced by the exercised group. One of the shock control sessions occurred on the day of the experiment. Food was withdrawn from animals approximately 5-8 h prior to exercise on experimental Day 4.
Following the acute bout of resistance exercise on day 4 animals were anaesthetized at various time points during recovery. Animals breathed a 95 % O2-5 % CO2 gas mixture via a nose cone connected to an isoflurane vaporizer (≈2.0-4.0 %). Subsequently, the gastrocnemius muscles were rapidly excised and processed. The animals were then killed by decapitation with a guillotine (Kent Scientific, Torrington, CT, USA). Our initial time course study design included 0, 15, 30 and 60 min time points following exercise and these data prompted us to add 5 and 10 min groups.
Analysis of eukaryotic initiation factors (eIFs)
Gastrocnemius muscles were weighed and homogenized in 7 volumes of buffer containing 20 mm Hepes (pH 7.4), 100 mm potassium chloride, 0.2 mm EDTA, 2 mm EGTA, 50 mm sodium fluoride, 50 mmβ-glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm benzamidine, 1 mm dithiothreitol (DTT) 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 (Kimball et al. 1997).
The phosphorylation of PKB on Ser-473, a marker for PKB activation (Alessi et al. 1996), was assessed using an anti-phospho-PKB antibody (Cell Signaling Technology, Beverly, MA, USA). Phosphorylation of mTOR on Ser-2448, a site directly phosphorylated in vitro by PKB (Navéet al. 1999), was measured using an anti-phospho-mTOR antibody (Cell Signaling Technology). Phosphorylation of S6K1 on Thr-389, a phosphorylation site associated with maximal activation of the kinase (Burnett et al. 1998), was determined using an anti-phospho-S6K1 (Thr-389) antibody (Cell Signaling Technology). Ribosomal protein S6 phosphorylation on Ser 235-236/240-244 was evaluated using anti-phospho-S6 antibodies (Cell Signaling Technology). Cell Signaling antibody dilutions were all 1:1000. eIF2α phosphory-lation, an event responsible for regulating ternary complex formation (binding of met-tRNAi to eIF2-GTP) and binding of met-tRNAi to ribosomes, was assessed using an anti-phospho-eIF2α antibody at a dilution of 1:4000 (BioSource International, Hopkinton, MA, USA). Total eIF2α was measured by Western blot analysis using a monoclonal antibody that recognizes both the phosphorylated and unphosphorylated forms of the protein (Rowlands et al. 1988). Blots were loaded according to protein concentration (≈100-150 µg). No change in the total protein content was observed for any of the variables during these experiments.
Determination of phosphorylation state of 4E-BP1
An aliquot of the 10 000 g supernatant was boiled for 10 min and then centrifuged at 10 000 g for 30 min at 4 °C. The resulting supernatant was mixed with an equal volume of SDS sample buffer and then subjected to protein immunoblot analysis as described previously (Kimball et al. 1997).
Quantification of eIF4G-eIF4E complex
For quantification of the amount of eIF4G present in the eIF4G-eIF4E complex, eIF4E was immunoprecipitated from 10 000 g supernatants using a monoclonal antibody (Kimball et al. 1997). Samples were subjected to immunoblot analysis using a polyclonal antibody to eIF4G to assess the association of eIF4G with eIF4E (Kimball et al. 1997). The results were normalized to the amount of eIF4E in the immunoprecipitates.
Measurement of ODC activity
l-Ornithine decarboxylase (ODC) catalyses the conversion of l-ornithine to putrescine and CO2. Tissue was homogenized in ODC assay buffer (50 mm TrisHCl, pH 7.5, 2.5 mm dithiothreitol, 0.1 mm EDTA). ODC activity was determined by measuring the release of 14CO2 from l-[1-14C]ornithine as described previously (Coleman et al. 1993). ODC activity was used in the present study as representative of a translationally regulated protein, which is induced under growth conditions (Pegg et al. 2003). The activity of ODC was determined at 10, 30 and 60 min of recovery.
Measurement of eIF2B activity
The guanine nucleotide exchange activity of eIF2B in skeletal muscle was measured by the exchange of [3H]GDP bound to eIF2 for non-radioactively labelled GDP as described previously (Kimball & Jefferson, 1988). eIF2B activity was measured only at 5 and 10 min of recovery as well as in control animals due to tissue limitations.
Statistical differences between sedentary and exercised groups were analysed by using a one-way ANOVA with Dunnett's post hoc test (GraphPad InStat version 3.05, GraphPad Software, San Diego, CA, USA). Statistical significance was set at P < 0.05.
Following completion of the acute resistance exercise protocol animals were killed at various times to determine whether or not PKB-mTOR signalling events were affected during the immediate phase of exercise recovery. In general, mTOR-mediated signalling and regulatory proteins of translation initiation displayed a step-wise, yet transient increase in phosphorylation, with maximal activation occurring between 10 and 15 min of recovery.
The most proximal step in the mTOR signalling cascade examined during this investigation involved PKB phosphorylation. PKB phosphorylation on Ser-473 increased to 282 % of control values (P < 0.05) between 5 and 10 min post-exercise and returned to control values by 30 min (Fig. 1). mTOR phosphorylation on Ser-2448 (Fig. 2) was elevated in a similar pattern to PKB during exercise recovery, but the magnitude of the increase varied considerably among the untrained animals such that mean changes were not significant.
The two predominate downstream targets of mTOR, 4E-BP1 and S6K1, were affected during recovery. The regulation of eIF4E and 4E-BP1 association involves reversible phosphorylation of 4E-BP1, whereby hyperphosphorylation of 4E-BP1 leads to a diminished affinity for eIF4E. 4E-BP1 phosphorylation can be visualized using SDS-PAGE, during which 4E-BP1 resolves into distinct electrophoretic forms (α, β and γ) representing the unique phosphorylated forms of the protein. The γ-form represents the hyperphosphorylated form and does not bind eIF4E. Although the data for 4E-BP1 are expressed as a percentage of control values the amount of 4E-BP1 in the γ-form for the sedentary animals was less than 10 %. Subsequent increases in 4E-BP1 phosphorylation during recovery from exercise peaked at 10 min (280 % of control, P < 0.01) and remained significantly elevated through 30 min (Fig. 3). The significant increases in 4E-BP1 phosphorylation imply that eIF4E availability was enhanced and in turn should augment eIF4F complex formation. Indeed, eIF4E association with eIF4G (Fig. 4) was increased to a maximal level at around 10 min (292 % of control, P < 0.05). Additionally, eIF4E has been shown to be involved in regulating the translation of specific mRNAs containing highly structured 5′-untranslated regions (5′-UTRs) such as the mRNA encoding ODC (Manzella et al. 1991). For this reason, ODC activity (Fig. 5) was evaluated as an index of translational control and cellular growth. However, the change in ODC activity did not reach statistical significance during this period of recovery.
In the present study site-specific phosphorylation of S6K1 on Thr-389 was determined because this phosphorylation event is required for maximal activation of this kinase (Burnett et al. 1998). As with 4E-BP1, a strong trend for peak phosphorylation on Thr-389 (Fig. 6) occurred at 10 min of recovery (336 % of control, P= 0.06). Ribosomal protein S6 phosphorylation (Fig. 7) was significantly elevated at 10-30 min (P < 0.05) with maximal phosphorylation noted at 15 min of recovery (647 % of control, P < 0.05).
Components regulating the binding of met-tRNAi to the 40S ribosomal subunit during translation initiation were examined by measuring eIF2B activity and eIF2α phosphorylation. eIF2B activity (Fig. 8) and eIF2α phosphorylation on Ser-51 (Fig. 9) were not significantly different from control values.
The current investigation was pursued in an attempt to provide a detailed time course of PKB/mTOR-dependent signalling events in response to resistance exercise during the acute phase of recovery. No previous investigation has examined signalling components involved with translational control mechanisms during this period, and therefore such changes may have been overlooked following resistance exercise. The results herein provide the first evidence of a rapid induction of the PKB-mTOR pathway and associated downstream proteins of translation initiation in response to acute resistance exercise. Although the exact implication of these findings is presently unknown, we submit that the transient nature of these responses may serve as a priming event to promote a coordinated growth response by the cell during exercise recovery.
The work of Bodine et al. (2001) provides the strongest direct evidence to date regarding the importance of the PKB-mTOR pathway for mediating the chronic adaptations of muscle hypertrophy or, conversely, where input is down regulated through this signalling pathway during muscle atrophy. High frequency electrical stimulation, capable of inducing skeletal muscle hypertrophy, has been shown to significantly increase PKB phosphorylation (266 % over controls) in the tibialis anterior immediately following acute exercise (Nader & Esser, 2001). This response is completely absent when examined at 3 and 6 h post-exercise. Our in vivo exercise protocol corroborates these findings as peak response was elicited between 5 and 10 min of recovery. In turn, PKB has been shown to phosphorylate mTOR on Ser-2248 in vitro (Navéet al. 1999), and this phosphorylation event correlates with muscle hypertrophy under chronic overloading conditions (Reynolds et al. 2002). In contrast, our results do not support an acute response of this site-specific phosphorylation of Ser-2448 during this time period, but this finding does not preclude the involvement of mTOR-mediated signalling. Previous research has demonstrated that mutation of this phosphorylation site to a non-phosphorylated alanine residue does not prevent the insulin or PKB stimulation of S6K1 and 4E-BP1 phosphorylation in rapamycin-treated HEK 293 cells (Sekulic et al. 2000). Thus, the functional consequence of Ser-2448 phosphorylation on mTOR activity remains unclear.
No prior studies examining acute resistance exercise have investigated translational steps regulating the recruitment of the mRNA to the 43S ribosomal subunit in the early hours of recovery. Work by Farrell et al. (2000) constitutes the only acute exercise study in which this step of translation initiation was examined. They demonstrated a trend towards increased eIF4E to eIF4G association at 16 h, but no changes were found in 4E-BP1 phosphorylation. In contrast, we provide evidence for increased 4E-BP1 phosphorylation that presumably facilitated an enhanced eIF4E to eIF4G association during the early minutes of recovery. This sequence of events permits eIF4F complex formation, which allows the preferential translation of mRNA encoding proteins vital to enhancing components involved in translational control mechanisms (Gingras et al. 2001).
A paucity of data exists regarding the aspects of polyamine metabolism in relation to physical exercise. Its relevance is coupled to eIF4F complex formation that subsequently promotes the translation of ODC under growth conditions. Mastri et al. (1982) found that chronic electrical stimulation dramatically increased ODC activity between 18 and 48 h, whereas rats performing acute resistance and endurance exercise resulted in maximal ODC activity 2 h following exercise (Turchanowa et al. 2000). In the present study, our data offer the earliest time frame where ODC activity has been measured following exercise but ODC activity did not reach significance over 60 min. Quite possibly, 60 min may be too soon to detect an appreciable increase in ODC activity during exercise recovery.
Baar & Esser (1999) were the first to report changes in S6K1 phosphorylation following an exercise stimulus in skeletal muscle. They noted that S6K1 phosphorylation was unchanged in extensor digitorum longus (EDL) and tibialis anterior (TA) muscles when compared to controls immediately following resistance exercise contractions. Between 3 and 6 h, maximal levels of phosphorylation were reported (TA, 363 % of control; EDL, 353 % of control), while elevated phosphorylation was maintained through 36 h of recovery. Hernandez et al. (2000) determined S6K1 activity at 1, 3, 6, 12 and 24 h of recovery using the same exercise protocol as the present study and did not detect increases until 6 h of recovery. Our results agree with those of Baar & Esser (1999), such that no increases in S6K1 phosphorylation were found immediately following exercise, as if a brief refractory period exists. However, in contrast we examined site-specific phosphorylation on Thr-389 and noted increases in phosphorylation within minutes of exercise recovery.
Changes in S6K1 phosphorylation following acute resistance-type exercise have been proposed as a surrogate marker for the long-term increases of skeletal muscle mass (Baar & Esser, 1999). However, the anabolic response of skeletal muscle growth is highly complex and although constitutively active forms of S6K1 in vitro are able to induce muscle growth, the increases are blunted when compared to upstream activation of PKB (Rommel et al. 2001). The importance of S6K1 phosphorylation lies in its ability to phosphorylate ribosomal protein S6 when activated, which has not previously been reported with resistance exercise. Indirect evidence suggests that S6 phosphorylation is associated with enhanced translation of mRNAs encoding proteins involved in translational (e.g. ribosome biogenesis and elongation factors) as well as cell cycle control (e.g. cyclin D1; Peterson & Schreiber, 1998; Jirmanova et al. 2002). Thus, we predict the involvement of S6 will increase the cell's capacity to synthesize protein, which serves an obvious function for mediating muscle hypertrophy.
A common finding with acute resistance exercise in both animal and human experimental models is the increase in skeletal muscle protein synthesis during recovery (Chesley et al. 1992; Biolo et al. 1995; Phillips et al. 1997; Farrell et al. 1999; Welle et al. 1999), but the precise point in time (2-4 h) for this induction remains unresolved (Rasmussen & Phillips, 2003). Elevated rates of protein synthesis at early time points during recovery from acute exercise involve enhanced mRNA translation (efficiency) as opposed to, for example, an increase in ribosome content (capacity) because total RNA content remains unchanged (Chesley et al. 1992; Farrell et al. 1999; Welle et al. 1999). Rates of protein synthesis were not analysed in the present study due to technical limitations of radioactive labelling over a short period, but changes are not likely during this immediate phase of recovery. This is supported by the fact that global rates of protein synthesis are tied more closely with eIF2B. Our data suggest that eIF2B activity remains unchanged, but eIF2B activity has been shown to coincide with increased protein synthesis several hours following resistance exercise (Farrell et al. 1999). Changes with eIF4F complex formation and S6K1 phosphorylation are associated with upregulating select mRNAs containing highly structured 5′-UTRs and TOP sequences, respectively, which encode proteins essential for increasing the translational capacity of the cell (Sonenberg et al. 1980; Jefferies et al. 1997). The preferential translation of these specific mRNAs would remain undetected by analysing the incorporation of radiolabelled amino acid into total protein. Moreover, several studies support the concept that eIF4F assembly and S6K1 phosphorylation do not affect global rates of protein synthesis under acute conditions (Kimball et al. 1998, 1999, 2003).
Haddad & Adams (2002) have postulated that only when data with ‘higher temporal resolution’ can be provided with acute resistance exercise can we better understand the integrated responses required for adaptive skeletal muscle hypertrophy. This investigation represents the first in vivo resistance exercise study to incorporate greater scrutiny regarding the sequential events of mTOR-mediated signalling and regulatory proteins of translation initiation during exercise recovery. Collectively, our data imply a coordinated regulation of mRNA translation and this response may comprise the most proximal steps mediating an anabolic response after resistance exercise. A precedence occurs for this type of rapid and transient signalling through PKB, which has previously been shown during muscle contraction (Sakamoto et al. 2002). This however, is the first evidence for such regulation during the recovery phase following acute resistance exercise.
The nearly identical response observed with the majority of the variables measured in this study exhibits a ‘bell-shaped’ curve pattern and suggests that crucial events of the PKB-mTOR signalling pathway may be phosphorylated in an oscillatory fashion during the early phase of recovery. These rapid, yet transient increases in translation initiation may signify a growth response of the cell whereby translational efficiency is dramatically upregulated allowing for the synthesis of specific mRNAs essential to growth. This plausible intermittent regulation over a brief period could allow for more precision when inducing a growth response, and it may be the summation of these responses that invokes muscle hypertrophy with chronic training.
Certainly much work remains to be done to elucidate the mechanisms regulating skeletal muscle hypertrophy. Specifically, we hope to determine whether a link exists between these early translational events and muscle growth with chronic exercise training. Establishing these connections will provide insight into the exquisite control by the cell in which distinct intracellular signals lead to alterations in gene expression and eventual phenotype of the muscle.
This study was supported in part by National Institutes of Health grant DK15658 and T32-GM08619. D.R.B. was supported by a Mentor Based Postdoctoral Fellowship grant from the American Diabetes Association. The authors would like to thank Lynne Hugendubler, Sharon Rannels, Susan Nguyen and Jamie Crispino for their expert technical assistance, and Lisa Shantz for graciously determining ODC activity.