An important component of the hypertrophic response of skeletal muscle to resistance exercise is an increase in protein mass per tissue. The increase in protein mass is the result of a net increase in protein synthesis relative to degradation. The upregulation of protein synthesis results in part from modulation of mechanisms involved in the translation of mRNA into protein. Translation of mRNA involves the processes of initiation, elongation, and termination with the initiation process being particularly important as a regulatory site. Modulation of translation initiation during resistance exercise upregulates the translation of the bulk of mRNAs, resulting in a global increase in protein synthesis. Moreover, and perhaps more importantly, resistance exercise activates discriminatory mechanisms that mediate selection of mRNAs for translation, thus modulating the gene expression profile of the tissue. In this manner, translational control mechanisms actually play a key role in initiation of the hypertrophic response of skeletal muscle to resistance exercise. The goal of this review is to summarize briefly recent studies that provide evidence in support of the role of translational control mechanisms in skeletal muscle hypertrophy.
The process of translation initiation in mammals is mediated by a group of proteins referred to as eukaryotic initiation factors (eIF; reviewed in (7)). Of the several steps in initiation, two have been shown to be subject to regulation in vivo: the binding of initiator methionyl-tRNAi (met-tRNAi) to the 40S ribosomal subunit and the binding of mRNA to the 40S ribosomal subunit (see Fig. 1). Met-tRNAi binds to the 40S ribosomal subunit as a ternary complex consisting of met-tRNAi, guanosine triphosphate (GTP), and eukaryotic initiation factor 2 (eIF2). During a later step, the GTP bound to eIF2 is hydrolyzed to guanosine diphosphate (GDP) and the eIF2·GDP complex is released from the 40S ribosomal subunit. Before undergoing another cycle of initiation, the GDP bound to eIF2 must be exchanged for GTP. The GDP–GTP exchange reaction is mediated by another initiation factor, eIF2B. The activity of eIF2B can be regulated directly through phosphorylation of the ε-subunit of the protein, or indirectly through phosphorylation of the α-subunit of its substrate, eIF2. Phosphorylation of eIF2α increases the affinity of eIF2B for eIF2, converting eIF2 from a substrate into a competitive inhibitor of the guanine nucleotide exchange reaction. Phosphorylation of eIF2Bε can either stimulate or inhibit the guanine nucleotide exchange activity of the protein, depending on the residue(s) phosphorylated. In general, inhibition of eIF2B activity results in a repression of translation of most mRNAs. However, a few mRNAs with certain structural characteristics continue to be translated, and in fact some are preferentially translated under conditions promoting inhibition of eIF2B. Such mRNAs typically have multiple open reading frames in the 5′-leader region of the message, and those identified to date, for example activating transcription factor (ATF)-4, encode proteins involved in recovery from stress conditions.
The binding of mRNA to the 40S ribosomal subunit is mediated by three initiation factors collectively referred to as eIF4F (reviewed in (7)). The eIF4F complex is comprised of eIF4A, an RNA helicase, eIF4E, the protein that binds to the m7GTP cap located at the 5′-end of the mRNA, and eIF4G, a scaffolding protein that, in addition to binding eIF4A and eIF4E, associates with eIF3 and the poly(A) binding protein (PABP). One of the best-studied mechanisms for regulating the mRNA binding step involves sequestration of eIF4E by the eIF4E binding protein, 4E-BP1, into a complex unable to bind to eIF4G. Because association of the eIF4G component of eIF4F with eIF3 is a prerequisite for binding of mRNA to the 40S ribosomal subunit, the binding of eIF4E to 4E-BP1 inhibits the process. The interaction between eIF4E and 4E-BP1 is regulated by phosphorylation of 4E-BP1, whereby hypophosphorylated forms of 4E-BP1 bind to eIF4E but hyperphosphorylated forms do not. Although it may seem that sequestration of eIF4E by 4E-BP1 would lead to a reduction in global rates of translation, in many cases large changes in the amount of eIF4G bound to eIF4E are observed under conditions where only small changes in incorporation of radiolabeled amino acids into protein occur. The reason for this apparent discrepancy likely relates to the differences in the mechanisms involved in the initial binding of mRNA to the 40S ribosomal subunit (i.e., initiation) and the process of reinitiation where the mRNA is already in polysomes. A discussion of such differences is beyond the scope of the present discussion. However, another function for changes in eIF4F assembly relate to modulation of translation of mRNAs with extensive secondary structure in the 5′-leader sequence. Examples of such mRNAs include some encoding proteins involved in proliferation and cell cycle control.
A second mechanism whereby binding of mRNA to the 40S ribosomal subunit is regulated involves changes in phosphorylation of ribosomal protein S6 (rpS6, reviewed in (6)). In initial studies, phosphorylation of rpS6 was reported to stimulate protein synthesis globally. Later studies revealed that rather than promote the translation of all mRNAs, rpS6 phosphorylation enhances the translation of a subset of mRNAs that have as a common structural feature an uninterrupted stretch of pyrimidine residues (referred to as TOP mRNAs) immediately downstream of the 5′-cap. mRNAs belonging to the TOP family include, among others, the ribosomal proteins, translation elongation factors 1A and 2, eIF4G, and PABP. How phosphorylation of rpS6 promotes the translation of TOP mRNAs is as yet a mystery. However, it is interesting to note that rpS6 is positioned near the mRNA binding site on the 40S ribosomal subunit and is thus located in a position that may permit a role in mRNA selection.
INTRACELLULAR SIGNALING PATHWAYS THAT REGULATE TRANSLATION INITIATION
Studies in flies and mice have shown that genetic disruption of genes encoding proteins in the phosphatidylinositol-3 kinase (PI-3 kinase)–mammalian target of rapamycin (mTOR) signal transduction pathway, including PI-3 kinase, phosphoinositide-dependent protein kinase (PDK1), protein kinase B (PKB, aka Akt), mTOR, and S6K1, results in a decrease in cell size with no reduction in cell number (reviewed in (8)). In contrast, activation or exogenous overexpression of these proteins enhances cell size. Several of the protein kinases in the pathway regulate translation initiation (see Fig. 2). For example, PKB is downstream of PI-3 kinase in the insulin and insulin-like growth factor (IGF)-1 signal transduction pathway. PKB phosphorylates and inactivates another protein kinase, glycogen synthase kinase (GSK)-3. Similarly, GSK-3 phosphorylates eIF2B, resulting in inhibition of its guanine nucleotide exchange activity. Thus, activation of PKB indirectly results in enhanced eIF2B activity through inhibition of GSK-3.
In addition to GSK-3, PKB also phosphorylates mTOR on residue Ser2448 (12). mTOR subsequently phosphorylates Thr389 in the 70 kDa rpS6 protein kinase (S6K1), a key event in its activation. Although most studies have concluded that phosphorylation of mTOR by PKB activates mTOR, evidence refuting the idea has been reported (12). Thus, rather than directly regulating mTOR activity, PKB may instead regulate signaling through mTOR by phosphorylating tuberous sclerosis complex (TSC)-2, a protein that recently has been shown to bind to mTOR and to repress phosphorylation of downstream targets such as S6K1 and 4E-BP1 (reviewed in (8)). In this regard, in both flies and mammalian cells, artificial reduction in TSC-2 expression enhances cell size, whereas exogenous overexpression of TSC-2 and its binding partner TSC-1 reduces cell size. Expression of a TSC-2 variant wherein the PKB phosphorylation sites are changed to alanine to prevent phosphorylation is even more effective than expression of the wild-type protein in repressing growth, demonstrating the importance of PKB phosphorylation in regulating TSC-2 activity.
REGULATION OF mRNA TRANSLATION INDUCES SKELETAL MUSCLE HYPERTROPHY
The preponderance of available evidence suggests that resistance exercise induces skeletal muscle hypertrophy by enhancing protein synthesis. The evidence also strongly suggests that the intracellular signaling pathway that mediates the exercise-induced increase in muscle protein synthesis is the PI-3 kinase–mTOR pathway. The paragraphs below review recent studies supporting these conclusions, starting with evidence that translation initiation is rate controlling for muscle hypertrophy (see Table 1).
For mRNA to be translated into protein, it must first associate with ribosomes to form translationally competent structures referred to as polysomes. Under conditions that result in reduced translation initiation, polysomes disaggregate, releasing the mRNA and ribosomal subunits into inactive forms. In contrast, stimulation of translation initiation results in an increase in the proportion of ribosomal subunits in polysomes. In an attempt to define the step in translation that is stimulated by exercise, Baar and Esser (1) examined the distribution of ribosomal subunits between polysomal and nonpolysomal fractions after high frequency (100 Hz) electrical stimulation of rat hindlimb muscles. They found that in the extensor digitorum longus (EDL) muscle, but not the soleus, ribosomal subunits redistribute into polysomes 6 h after electrical stimulation, indicating that translation initiation is enhanced under these conditions. Moreover, hypertrophy is observed in EDL muscles stimulated twice a wk for 6 wk, suggesting that enhanced translation initiation leads to skeletal muscle hypertrophy.
The results of several recent studies link increased protein synthesis and muscle hypertrophy to activation of PKB. For example, Bodine et al. (2) used a rat model of increased chronic workload involving synergist muscle ablation to induce hypertrophy of the plantaris muscle. In this model, PKB phosphorylation is increased as early as 3 d after ablation and is maintained for at least 2 wk. In another study, IGF-1 was used to induce hypertrophy of C2C12 myotubes in culture (11). In that study, IGF-1 both enhanced phosphorylation of PKB as well as promoted hypertrophy, suggesting that activation of the PI-3 kinase signaling pathway by the hormone is important in the hypertrophic response.
More direct evidence linking PKB to muscle hypertrophy is provided by studies wherein a constitutively active form of the kinase (caPKB) is exogenously expressed in rat skeletal muscle. In tibialis anterior muscle expressing a hybrid protein consisting of caPKB linked to enhanced green fluorescent protein (EGFP) muscle fiber size is increased 1.6-fold compared with muscle expressing EGFP alone (2). Moreover, expression of caPKB-EGFP significantly reduces tibialis anterior atrophy associated with denervation. The effect is not unique for tibialis anterior because expression of caPKB in EDL muscle also attenuates the decrease in muscle fiber cross-sectional area associated with bupivacaine-induced muscle injury (9).
As noted above, PKB serves as a branch point in the PI-3 kinase signaling pathway. One branch is involved in regulation of GSK-3 activity. Thus, it may be expected that in response to resistance exercise, GSK-3 phosphorylation would correlate with hypertrophy. Indeed, Bodine et al. (2) found that in plantaris muscle of mice subjected to synergistic ablation, GSK-3 phosphorylation is enhanced. Likewise, in IGF-1-treated C2C12 myotubes, GSK-3 phosphorylation is also dramatically increased (13). Results from Farrell et al. (5) indirectly suggest that the inhibition of GSK-3 associated with resistance exercise results in enhanced eIF2B activity. In that study, rats were operantly conditioned to reach an illuminated bar that requires the animals to extend fully the hindlimbs while completing concentric and eccentric movements. It was found that both skeletal muscle protein synthesis and eIF2B activity are increased 16 h after completion of the exercise protocol. Unfortunately the phosphorylation state of eIF2B was not measured in that study, which prevents a definitive conclusion about the role of GSK-3 in regulating eIF2B activity in response to exercise from being reached. However, based on previous studies demonstrating that insulin regulates eIF2B activity through a GSK-3–dependent process (15), it seems likely that resistance exercise may use a similar mechanism for regulating eIF2B activity.
In addition to phosphorylating GSK-3, PKB also phosphorylates Ser2448 on mTOR (12). Evidence implicating mTOR in muscle hypertrophy is provided by a study showing that in plantaris muscle of rats subjected to synergistic ablation, hypertrophy is associated with enhanced Ser2448 phosphorylation (10). In contrast, in gastrocnemius muscle of rats subjected to hindlimb suspension, mTOR phosphorylation is reduced and muscle atrophy occurs. Further evidence linking mTOR and hypertrophy is provided by studies using a specific inhibitor of mTOR, rapamycin. Treatment with rapamycin almost completely prevents the hypertrophic increase in plantaris muscle weight and fiber size after synergistic ablation, but has no effect on nonoverloaded muscles (2). Rapamycin also significantly reduces muscle growth associated with recovery from hindlimb suspension. Importantly, rapamycin also prevents the muscle hypertrophy that occurs in tibialis anterior (2) or EDL (9) muscle expressing caPKB. The latter result provides strong evidence for a link between PKB and mTOR in activation of the hypertrophic response.
mTOR phosphorylates at least two proteins that may be involved in increasing protein synthesis after exercise, 4E-BP1 and S6K1. Indeed, Baar and Esser (1) found that hypertrophy induced by electrical stimulation of hindlimb muscle is associated with phosphorylation of S6K1. A more recent study (2) extends the finding of Baar and Esser to show that after synergist ablation, phosphorylation of S6K1 is increased, the amount of 4E-BP1 bound to eIF4E is reduced, and the amount of eIF4G bound to eIF4E is increased in plantaris muscle. Similar changes are observed during muscle growth associated with recovery from hindlimb suspension. Hyperphosphorylation of 4E-BP1 and S6K1 also is observed in C2C12 myotubes during IGF-1-induced hypertrophy (11). Each of these effects is prevented by treatment with rapamycin, suggesting that mTOR activity is required for the observed changes. The relative importance of the exercise-induced changes in S6K1 activity compared with assembly of the eIF4F complex have not yet been deciphered. However, expression of a constitutively active form of S6K1 partially, but not completely, recapitulates the growth-promoting effects of caPKB overexpression (11), indicating that some other downstream target of PKB-mediated signaling, perhaps involving phosphorylation of 4E-BP1, is required for maximal hypertrophic response.
HYPERTROPHY AND GENE EXPRESSION
Recent evidence from a number of experimental model systems shows that modulation of transcription and translation contribute to altered gene expression profiles observed in response to various stimuli. In a recent study by Chen et al. (3), altered profiles of gene expression were examined by microarray analysis 1 and 6 h after resistance exercise in rats. The transcriptional component of the response was assessed by measuring total tissue mRNA content of specific mRNAs whereas the translational component was assessed by measuring the proportion of individual mRNAs in nonpolysomal (i.e., inactive) compared with polysomal (i.e., active) fractions. Approximately 65% of the mRNAs whose abundance was altered were detected in both the total and polysomal analyses, indicating that such gene products are regulated through transcriptional mechanisms. There were also 25 mRNAs whose expression in the total mRNA analysis was not altered, but whose abundance in the polysomal fraction changed with exercise. Thus, these mRNAs are regulated solely through alterations in mRNA translation and would not have been detected using the conventional approach to microarray analysis where only total RNA is analyzed. Of interest is the finding that a cluster of mRNAs that were identified encode proteins characterized as being involved in the growth response (i.e., c-fos, c-jun, Atf3, Egr1/2, and c-myc) and are typically associated with cell proliferation. However, an additional cluster of mRNAs encoding proteins that function as antiproliferative agents or play roles in tumor suppression (i.e., PC3 and GADD45) were differentially expressed, which may provide a compensatory response to the cell cycle-promoting changes or a reciprocal reaction to the stress associated with resistance exercise. Overall, the results of this study provide compelling evidence that resistance exercise leads to selective changes in gene expression through both transcriptional and translational mechanisms.
Although pioneering work by S. M. Phillips, K. D. Tipton, and R. R. Wolfe has characterized extensively the net protein response as well as the importance of nutritional state with regard to the response to resistance exercise in humans, to date, no investigations have examined key steps in translation initiation. One of the earliest human studies suggesting a posttranscriptional regulation of protein synthesis subsequent to acute resistance exercise was reported by Chesley et al. (4) in which they demonstrated no change in RNA content. However, they did find that translational efficiency, that is, synthesis rate per content of RNA, was significantly elevated. Later work examining elderly individuals (14) confirmed earlier findings that despite a 30% increase in myofibrillar synthesis of the vastus lateralis after resistance exercise, total RNA remained unchanged when compared with sedentary controls. This finding provides additional evidence of a translational mechanism being regulated in the response to resistance exercise. Since the report by Welle et al. (14), no further human studies have corroborated previously reported animal and in vitro studies where changes in translation initiation with resistance exercise have been observed. An obvious potential limitation of conducting this work in humans may involve tissue sample size obtained from skeletal muscle biopsies. However, the sensitivity and accuracy of current molecular techniques and reagents makes this now a tangible goal.
Collectively, this review highlights a growing body of evidence that inextricably links mRNA translation with the growth and maintenance of skeletal muscle mass along with modulation of gene expression that occurs in a variety of hypertrophy models. Although many studies have provided evidence supporting a role for one or more protein kinases in regulating muscle hypertrophy, it is not yet possible to establish a hierarchy whereby the contribution of select signaling pathways can be quantified. However, individually, each signaling protein is essential and the mechanisms regulating skeletal muscle hypertrophy should be viewed as an integrated response. Moreover, the exercise-induced changes in translation initiation factor phosphorylation state as well as the changes in protein kinase activity and phosphorylation state would be expected to lead not only to global changes in protein synthesis, but also to selective alterations in gene expression through modulation of translation of mRNAs encoding particular proteins. The study by Chen et al. (3) is an important first step in identifying the proteins that are subject to such regulation. Future studies should expand our knowledge of the number and identity of mRNAs that are translationally regulated in response to endurance exercise as well as extend such analysis to human models of resistance exercise.
The obvious arenas in which this research has the opportunity to impact humans includes aging and sarcopenia, sports performance, clinical disorders, and space flight, whereby therapeutic countermeasures, nutritional supplementation, pharmacologic interventions, or a combination thereof can be used for optimizing skeletal muscle mass.
The authors thank Dr. David L. Williamson for comments and suggestions during preparation of the manuscript. The studies described in this article that were performed in the laboratories of the authors were supported by research grants DK13499 and DK15658 from the National Institutes of Health.
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