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Eur J Appl Physiol DOI 10.1007/s00421-007-0588-3 INV ITED R EVIEW Mechanical stimuli of skeletal muscle: implications on mTOR/p70s6k and protein synthesis Nelo Eidy Zanchi · Antonio Herbert Lancha Jr Accepted: 25 September 2007 © Springer-Verlag 2007 Abstract The skeletal muscle is a tissue with adaptive properties which are essential to the survival of many species. When mechanically stimulated it is liable to undergo remodeling, namely, changes in its mass/volume resulting mainly from myoWbrillar protein accumulation. The mTOR pathway (mammalian target of rapamycin) via its eVector p70s6k (ribosomal protein kinase S6) has been reported to be of importance to the control of skeletal muscle mass, particularly under mechanical stimulation. However, not all mechanical stimuli are capable of activating this pathway, and among those who are, there are diVerences in the activation magnitude. Likewise, not all skeletal muscle Wbers respond to the same extent to mechanical stimulation. Such evidences suggest speciWc mechanical stimuli through appropriate cellular signaling to be responsible for the Wnal physiological response, namely, the accumulation of myoWbrillar protein. Lately, after the mTOR signaling pathway has been acknowledged as of importance for remodeling, the interest for the mechanical/chemical mediators capable of activating it has increased. Apart from the already known MGF (mechano growth factor), some other mediators such as phosphatidic acid (PA) have been identiWed. This review article comprises and discusses relevant information on the mechano-chemical transduction of the pathway mTOR, with especial emphasis on the muscle protein synthesis. N. E. Zanchi (&) · A. H. Lancha Jr Laboratory of Applied Nutrition and Metabolism, Physical Education and Sport School, University of São Paulo, Av. Prof. Mello Moraes, 65, PO Box 05508-900, São Paulo, SP, Brazil e-mail: [email protected] Keywords mTOR · p70s6k · Mechanical stimulation · Hypertrophy · Protein synthesis Introduction The skeletal muscle is a highly plastic tissue, namely, capable of adapting to adverse conditions. In nature, this intrinsic adaptive feature is crucial and ensures the survival of many species, once a vast number of environmental impositions (e.g. contractile activity and mechanic overload) can be overcome by means of adaptive responses from the muscles. There are two main changes promoted by mechanical stimulation upon the skeletal muscle: (1) remodeling (namely, adaptation which induces changes in the muscle mass/volume); (2) alterations in the molecular engine of the muscle Wber, e.g. phenotypical changes in myosin isoforms expression, important constituents of the skeletal muscle (Booth and Baldwin 1966). Taken together, these changes allow the muscle to adapt its contractile properties so that both power and strength are incremented. Until lately, little was known about the way the skeletal muscle increases its mass in order to respond to mechanical stimulation. However, important recent studies have demonstrated protein kinase mammalian target of rapamycin (mTOR) to be a key regulator of the muscle size and that the remodeling response of the skeletal muscle submitted to mechanical stimulation depends on mTOR (Bodine et al. 2001; Reynolds et al. 2002; Pallafacchina et al. 2002; Hornberger et al. 2003). Corroborating its speciWc role in size control, the mTOR pathway does not seem to control the skeletal muscle Wber speciWcation (Pallafachina et al. 2002). Adult skeletal muscle cells are not liable to undergo division (mitosis). As a consequence, adaptive mecha- 123 Eur J Appl Physiol nisms which induce contractile Wber protein accumulation through either increased protein synthesis or reduced protein degradation on pre existing Wbers are the most likely candidates to remodeling control of the skeletal muscle. The mTOR pathway seems to control mechanisms of protein synthesis at several levels (e.g. translation capacity, translation eYciency), through increases of translation of speciWc mRNAs, which culminate in skeletal muscle Wber enlargement (Wullschleger et al. 2006; Nader et al. 2005). Nonetheless, so that this process occurs, it is essential that beyond mechanical stimuli (e.g. overload) a biological interpretation (e.g. cell signaling) happens. Taken together, these events are called mechanotransduction. It is generally agreed that mTOR is activated via its upstream signaling PI3K (phosphatidylinositol 3 kinase, which is considered crucial to Akt activation) (Stokoe et al. 1997), and Akt (phosphatidilinositol regulated protein kinase), culminating in the activation of its eVector p70s6k (ribosomal protein kinase S6) under overload conditions (Bodine et al. 2001). This indicates that mechanical stimuli are capable of stimulating the muscle anabolic mediators, via the mTOR pathway (PI3K/Akt/mTOR/p70s6k). Although it may seem repetitive to mention, these mediators remain not entirely understood. The autocrine/paracrine factors, which are released by the muscle in response to mechanical stimuli, such as the Insulin-like growth factor 1 (IGF1) and the mechanical growth factor (MGF), deWnitely represent strong hypertrophy stimuli via the PI3K/Akt/mTOR/p70s6k pathway (Rommel et al. 2001; Ohanna et al. 2005; Nave et al. 1999; Scott et al. 1998). Nevertheless, these mechanisms, seem to be responsible for the late activation response of this pathway (Rennie and Wackerhage 2003; Haddad and Adams 2002). In dramatic contrast, the mTOR pathway can be activated almost immediately after the muscle overload stimulation (Bolster et al. 2003). This indicates that more immediate signaling mechanisms induced by per se muscle stimulation are also regulatory targets. All of them activate potential mechanisms that either control or positively inXuence muscle remodeling via the mTOR pathway. Acute mechanical stimulation inducing mTOR activation seems to be more complex than that mediated by growth factors, once elements inside the mTOR pathway seem to respond nonlinearly (e.g. not exactly via the PI3K/ Akt/mTOR/p70s6k pathway) to diVerent types of mechanical stimuli. Therefore, the purpose of the present review is that of presenting and discussing the recent advances concerning mechanotransduction of the mTOR pathway. Apart from that, questions thus far not yet clariWed will be brought into discussion, with a permanent focus on the muscle protein synthesis control mechanisms. 123 mTOR, p70s6k and mechanical stimulation The mTOR is a key signaling pathway in the diVerentiation process and control of cell size (Schmelzle and Hall 2000) activated by nutritional, chemical and mechanical stimuli (Reiling and Sabatini 2006). Studies in yeast have demonstrated that the target of rapamycin (TOR) performs two essential functions in this organism; one regulates the moment when a cell grows and the other determines the place where a cell grows (Wullschleger et al. 2006). Therefore, growth control seems to be a crucial function performed by the TOR/mTOR. In the skeletal muscle cells, the rapamycin (a potent inhibitor of the mTOR) attenuates proliferation, survival, diVerentiation and hypertrophy, thus indicating that the mTOR participates in the muscle developmental programme at diVerent levels (Ohanna et al. 2005). Corroborating this, the delivery of rapamycin to 2-weak-old rat pups for 11 days resulted in a 30% reduction in body weight in both male and female pups (Bodine 2006). Surprisingly, the treatment with rapamycin (1.5 mg/kg) in young adult rats, produced no change in the muscle mass, but its concomitant administration under overload conditions almost completely blocked the hypertrophy process (Bodine 2006; Bodine et al. 2001). This indicates that, at least in the adult skeletal muscle, increased mechanical stimuli exert speciWc activation of the mTOR pathway which is not attained by normal life mechanical stimulation. Thus, it has been proposed that the mechanically induced tension plays a critical role in the regulation of the skeletal muscle mass (Hornberger and Chien 2006; Hornberger et al. 2006a). However, the events linking mechanical stimuli to the anabolic signaling pathways remain still not thoroughly understood. Increased muscle tension seems to stimulate skeletal muscle growth in two diVerent manners: either via increased growth factors production (e.g. IGF-1, MGF) or via contractions per se. While growth factors regulating the mTOR pathway have been well characterized, the characterization of the regulation of the mTOR pathway in response to mechanical stimuli remains still at its very beginning. It is generally agreed that growth factors stimulate the mTOR pathway via PI3K/Akt dependent mechanisms (Rommel et al. 2001; Nave et al. 1999; Ohanna et al. 2005). However, under acute mechanical stimulation, the importance of PI3K/Akt dependent mechanisms in order to activate mTOR calls for clariWcation. As a matter of fact, recent studies have suggested mTOR, when under mechanical stimulation, to be liable to be activated via PI3K/Akt independent mechanisms (Hornberger et al. 2004). Independently of the requirement of mechanical stimuli as a means to activate Akt or not, contractile stimuli seem to be capable of activating the mTOR pathway. Eur J Appl Physiol Several phosphorylation sites have been proposed as capable of activating mTOR. For example, phosphorylation on its serine 2481 residue was suggested to be of importance for the regulation of its eVector p70s6k under a treatment with rapamycin (Peterson et al. 2000). Likewise, the treatment of several cellular types with IGF-1 seems to activate mTOR on its serine residue 2481, correlating with the activation of p70s6k (Thiimmaiah et al. 2005). Even though the mTOR regulation mediated by phosphorylation on serine 2481 seems to be of importance for its activity regulation, a very small number of studies seem to have assessed it. To our knowledge, only three studies have so far assessed the eVects of mechanical stimulation on phosphorylation of mTOR (Ser 2481) and no alterations have been observed by its authors (Eliasson et al. 2006; Mascher et al. 2007; Hornberger and Chien 2006a). Contrariwise, it has been acknowledged that under mechanical stimuli the mTOR protein is phosphorylated in its 2448 serine end (Reynolds et al. 2002). This event activates several eVector pathways, like the eukaryotic initiation factor 4 (EIF4)binding protein 1 (4E-BP1) and p70s6k, which culminates in increased protein synthesis. It has been widely assumed that the activation of mTOR signaling leads to increased protein synthesis via (1) activation of p70s6k, subsequent phosphorylation of mRNAs containing a 5⬘- terminal oligopyrimidine tract (5⬘TOP) (encoding elongation factors and ribosomal proteins) and, ultimately, an increase in the translation capacity and (2) phosphorylation of 4E-BP1, increased eukaryotic initiation factor 4E (eIF4E) availability, and eukaryotic initiation factor 4F (eIF4F) complex assembly, leading to increased rates of translation initiation (Kubica et al. 2005). Thus, the p70s6k activation under the eVect of mechanical stimuli seems to be critical for the hypertrophy response. It is unquestionable that p70s6k has been one of the most widely studied of mTOR eVectors. Its phosphorylation in the 389 threonine end has been considered a mTOR activation mirror (Jacinto and Hall 2003; Chen 2004; Sarbassov et al. 2005) and a marker of increased protein synthesis induced by mechanical stimulation (Baar and Esser 1999). Consequently, a great amount of information on mTOR is available in the literature, owing to studies conducted with p70s6k. However, recent studies with human subjects focusing on mechanical stimulation of the skeletal muscle have demonstrated that the activation of p70s6k (phosphorylation in its threonine 389 residue) is not always correlated with the activation of mTOR (phosphorylation on serine 2448) (Eliasson et al. 2006; Fujita et al. 2007). Moreover, the activation of mTOR seems to be not always capable of activating p70s6k (Mascher et al. 2007). In fact, recent “in vitro” studies have questioned the dependence of mTOR on p70s6k activation (Chiang and Abraham 2005; Holz and Blenis 2005). The signiWcance of this Wnding remains unknown given that it is still unclear whether mTOR phosphorylation Ser 2448 is a positive, negative or inconsequential regulatory phosphorylation (Holz and Blenis 2005), at least in the “in-vitro” context. That is why, in this review article, the mTOR pathway will be analyzed with a focus not only on mTOR but also on p70s6k. We believe that this particular aspect of the mTOR signaling review will allow a closer approach to the mTOR pathway study. EVects of acute mechanical stimuli on mTOR/p70s6k: dependence on PI3K/Akt in signaling Increased mechanical stimulation is a powerful stimulus toward protein synthesis in the skeletal muscle. This relation has been clearly demonstrated in a number of diVerent species, using diVerent models of mechanical stimulation (Timson 1990). In the last years, several studies have demonstrated the involvement of the mTOR pathway in such a process (Bodine et al. 2001; Reynolds et al. 2002; Pallafacchina et al. 2002; Hornberger et al. 2003). More speciWcally, important previous studies using synergist ablation as a model of mechanical stimulation have demonstrated mTOR to be liable of being activated by Akt dependent mechanisms (Bodine et al. 2001). Synergist ablation is a very interesting mechanical stimulation model, since it allows the overloaded muscle to undergo robust hypertrophy. However, it remains unclear, from this model of mechanical stimulation, whether activation of mTOR via Akt is acutely or chronically accomplished. In other words, under continuous mechanical stimulation, it remains unclear whether the pathways are directly activated by either mechanical stimulation or by circulating factors released by the skeletal muscle, such as MGF. Likewise, it remains unclear whether diVerent mechanical stimuli are able to diVerently activate these signaling elements. In the skeletal muscle, increased protein synthesis is mediated by a continuum time course initiated by mechanical stimuli, which can be modulated by diVerent anabolic inputs, such as mechanical and circulating factors. Such anabolic stimuli can exert distinct contributions to signaling pathways, although both reXect on protein synthesis. As an example, growth factors (e.g. IGF, MGF) released by the skeletal muscle in response to mechanical stimulation are generally accepted to promote increased protein synthesis through PI3K/Akt/mTOR pathway (Rommel et al. 2001; Bodine et al. 2001). Moreover, growth factors leading to PI3K/Akt/mTOR activation seem to be part of a late component of protein synthesis signaling, since MGF mRNA expression increases can be observed around 2.5 h following a resistance training session or even later (Hameed et al. 2003; Haddad and Adams 2002). Contrariwise, 123 Eur J Appl Physiol acute skeletal muscle stimulation seems to be not always dependent on PI3K/Akt as a means to activate mTOR (Hornberger et al. 2004, 2006c). Acute exercise studies may help to explain the way this element is diVerently regulated. Nonetheless, as stated by Tidball (2005) in a recent review article tackling the theme, “The general picture that is emerging from these investigations is that there are multiple processes through which muscle adaptation loading can occur. However, the current understanding is fragmentary”. Therefore, in face of these comments, a deWnite answer calls for research. In relation to dependence of Akt in order to stimulate mTOR/p70s6k through mechanical stimulation, the Wrst point to be evaluated is: Is acute mTOR/p70s6k activation always a result of acute Akt activation? To respond this, a recent study carried out by Hornberger et al. (2004) has shown intermittent stretch induced signaling through mTOR/p70s6k not to be disrupted in muscles from Akt1¡/ ¡ mice. Moreover, the same mechanical stimuli performed in the presence of Wortmannin (an inhibitor of PI3K) were capable of activating p70s6k through PI3K/Akt independent pathway (Horngerger et al. 2006). In line with this, recent studies have shown activation of mTOR/p70s6k even in the absence of Akt activation (Fujita et al. 2007; Eliasson et al. 2006; Parkington et al. 2003; Blomstrand et al. 2006; Mascher et al. 2007). Taken together, these recent Wndings suggest that under acute stimuli mTOR/ p70s6k is activated not always by Akt but via alternative pathways. Contrarily, several studies carried out in animals and human subjects have shown that Akt is activated under acute contractile stimuli (Sakamoto et al. 2003; Nader and Esser 2001; Bolster et al. 2003; Dreyer et al. 2006). These studies have been performed under diVerent contractile stimuli, such as resistance and endurance types. Thus, diVerent contraction patterns are capable of activating Akt in a distinct manner, still not thoroughly understood. In face of these Wndings, a number of questions still remain. One of the main concerns related to Akt activation is: Why some mechanical stimuli are capable of activating Akt whereas others are not? What are the implications on the activation of mTOR/p70s6k? Presently, an accurate answer calls for further research. However, some speculations have taken place in order to explain these diVerential responses. One important key to elucidate the dependence of PI3K/ Akt in order to activate mTOR/p70s6k is the investigation of Akt related to the intensity of mechanical stimulation. Higher mechanical stimuli intensity have been advocated to be necessary in order to activate Akt, as observed by Sakamoto et al. (2002), and conWrmed by Atherton et al. (2005) and Dreyer et al. (2006). Nonetheless, such assumption is not a consensus. For example, Sakamoto et al. (2003) carried out studies with diVerent aerobic exercise 123 intensities, and observed that Akt activation was not dependent on the exercise intensity. In agreement with these results, three recently published studies with human subjects have reported no changes in Akt phosphorylation after high intensity resistance exercise (CoVey et al. 2005; Eliasson et al. 2006; Deshmukh et al. 2006). The reason for the divergent changes, though, remains unclear. It is likely to be related to either temporary activation of Akt after exercise (Fujita et al. 2007), to diVerent patterns of contraction elicited by voluntary or electrical stimulus (Fry 2004), or even related to diVerent species. An additional possibility is that diVerent intensity/duration of mechanical stimulation can exert divergent responses on insulin level, which can, in turn, work against a contraction induced activation of Akt, as suggested by Sakamoto et al. (2004). In fact, Mascher et al. (2007) reported a reduced level of insulin immediately after cycling exercise in human subjects, which correlated with Akt inhibition. Another possibility to explain these discrepancies of Akt signaling on the mTOR pathway are likely to be related to the fact that multiple isoforms of Akt are present in the skeletal muscle (Akt1, Akt2, Akt3), and seem to have distinct physiological roles. Of the three isoforms, Akt 1 is the most active and is the isoform that seems to respond to mechanical stimuli (Turinsky and Damrau-Abney 1999). Apart from the above Wnding, little is known about the eVect of mechanical stimuli on the other isoforms of Akt. Furthermore, variables such as Wnal activation of mTOR/p70s6k (specially related to phosphorylation on residues responsible for the full activation of the enzymes) and protein synthesis or indirect markers of protein synthesis (e.g. phosphorylation of ribosomal protein S6, polisome proWle, etc.) are said to provide detailed information on the importance of mechanical stimuli on the PI3K/Akt/mTOR/ p70s6k pathway. As an example, a recent study has indicated that acute activation of Akt and ribosomal protein S6 is not necessarily correlated with mTOR activation (Bolster et al. 2003). Therefore, it is highly advisable that future studies consider investigating all the key elements related to this signaling pathway and its consequences on protein synthesis. Finally, redundant systems of mTOR activation are in all likelihood to be activated speciWcally by mechanical stimuli. As an example, in “in vitro” studies, lisophospatidic acid (LPA) and platelets derived growth factor (PDGF) seem to activate mTOR via diVerent PI3K/Akt mechanisms (Kam and Exton 2004). Mechanotransduction related to mTOR/p70s6k Skeletal muscle is intrinsically a mechanical stimulation responsive tissue being, therefore, named “mechanocyte” Eur J Appl Physiol (Goldspink and Booth 1992). It suggests that mediators sensitive to mechanical stimuli are likely to promote signal transduction processes, mediating remodeling responses and metabolic adaptive responses in the contractile machinery. The understanding of mechanotransduction (the mechanism by which cells convert mechanical signals into biological responses) controlled by pathways sensitive to rapamycin is still beginning (Hornberger and Chien 2006a). Nevertheless, it is interesting to speculate the way mechanical stimuli acutely activate physical/chemical mediators that are capable of modulating mTOR pathway. Phosphatidic acid (PA) One of the substances capable of activating mTOR is phosphatidic acid (PA), a well-known lipid based second messenger which plays a role in several signaling systems, whose activation inXuences mitogenesis, secretory processes and cytoskeleton reorganization, among others. PA activation depends on phospholipase D (PLD) enzyme activity, which hydrolyses phosphatidilcholine in PA and choline (Hong et al. 2001). Once synthesized, PA binds the FRB domain of mTOR and activates p70s6k activity (Fang et al. 2003). Therefore, PA is unquestionably a strong candidate to mechanotransducer in skeletal muscle. The question which has remained unanswered until recently is: Are mechanical stimuli enough to increase PA production in skeletal muscle? To address this question, Hornberger and Chien (2006) submitted skeletal muscle “in vitro” to stretching and observed PA and its precursor enzyme PLD behavior. In this study, stretching is reported to have caused increments of PA and its precursor enzyme PLD concentration, besides having activated p70s6k. Additionally, treating muscles with a PLD inhibitor, neutralized stretching eVects over p70s6k in a dose dependent way. These data provided consistent information that mechanical stimulation acutely activates mTOR pathway through increments in intracellular concentration of PA. At which level this activation occurs is a question that remains open to investigation. PI3K activation through PA increments was observed in a Wbroblast cells culture (Kam and Exton 2004). In the skeletal muscle, however, mechanical stimulation does not seem to be dependent on PI3K activation (Hornberger and Chien 2006a; Hornberger et al. 2004). Another relevant question is the understanding of the processes which link the muscle contraction with PA intracellular increments. Even though no study has thus far addressed question, some speculations have taken place. One of them is based on PLD and structural protein -actinin proximity (Hornberger et al. 2006a). It has been stated, for instance, that -actinin protein and -actin inhibit PLD activity through physical interaction (Lee et al. 2001). The observation that PLD is located near this structural proteins and Z discs, which are essential components to force transmission in skeletal muscle, generates speculation that proximity of these two regulatory elements might constitute one of the ways of mechanical activation of PA (Park et al. 2000). Additionally, Hornberger and Chien (2006b), speculate that mechanical activation produces a physical dissociation between PLD and -actinin and/or -actin, which would further reinforce the concept of PA as an acute mechano-chemical mediator. However, this speculation calls for experimental evidences to conWrm it. Finally, PA concentrations control in skeletal muscle also depends on several synthesis and degradation regulatory mechanisms. It is possible that mechanical stimulation modulates these mechanisms leading to increments in PA production. Further details on this issue can be found in the excellent paper by Hornberger et al. (2006b). Integrin signaling Several systems of mechanotransduction use membrane protein/receptors sensitive to physical stimulation to convert mechanical information into molecular events (Ingber 2006). Thus, it is likely that skeletal muscle have also some similar control mechanisms. In skeletal muscle, structures known as costameres play an important role in lateral force transmission (Bloch and Gonzales-Serratos 2003; Ervasti 2003). SpeciWcally, costameres are composed by several structural proteins that are overexpressed at regular intervals in skeletal muscle Wber, overcoming Z lines. Given the central role that extracellular matrix plays on force transmission in tissues, structures connecting extracellular matrix to the muscle cell membrane (and z discs) have been regarded as possible mechanoreceptors (Ingber 2006). Not coincidentally, one of the costameres constitutive proteins which connect extracellular matrix elements to the cell membrane are integrins (Grounds et al. 2005), which have been speculated to participate in the mechanical stimulation that activates mTOR. Concerning this, Boppart et al. (2006), showed that exercise carried out on a treadmill with some decline degree is able to activate mTOR and p70s6k. Nonetheless, in genetically modiWed mice that over expressed 71-integrin protein (a membrane glucoprotein that belongs to a major family of surface adhesion cellular receptors), mTOR activation did not occur. Interestingly, overexpression of 71-integrin, which might be induced by physical exercise, attenuates muscular damage appearance as much as mTOR activation pathway, suggesting that integrins act as negative regulators of mTOR. What implications would lie behind integrin regulation? Apparently, structural proteins protective eVect would be the Wrst option. It is already known that integrins play an important role in structural conservation of skeletal muscle and that several myopathies are related to the absence of 123 Eur J Appl Physiol 71-integrins (Mayer 2003). Therefore, it is possible that its expression increase works as a chronic protective mechanism against damage caused by muscle contraction in skeletal muscle cell. However, it is interesting to notice that trained individuals compared to untrained ones show acute reduction in protein synthesis after strength training (Phillips et al. 2002). Furthermore, it is equally observed that trained individuals submitted to strength training show less activation of the mTOR pathway in comparison with untrained ones (CoVey et al. 2005), a phenomenon which must be approached with caution, once there is the possibility that mTOR pathway is chronically increased in trained individuals. Anyway, it is possible that there is some connection between acute decrease in protein synthesis, mTOR activation decrease and integrin overexpression as chronic eVects of mechanical stimuli. Despite the former information, it is important integrin as a negative regulator of mTOR pathway to be assessed with caution. For instance, Malik and Parsons (1996) observed that p70s6k activation was positively related to the integrin presence, at least in cell culture. Additionally, King et al. (1997) observed that PI3K/Akt activation is dependent on the integrin presence. Although these cells were not submitted to mechanical stimuli and were not even skeletal muscle cells, this data suggest that the integrin presence is likely to act as positive mTOR regulators. Capacity of skeletal muscle cell to sense divergent mechanical stimuli A Wnal topic on mechanotransduction concerns the kind of mechanical force to which the muscle responds. This theme is of utmost importance to other areas of knowledge. For instance, in endothelial cells, laminar blood Xow imposes mechanical forces totally diVerent from oscillatory blood Xow. This results in very diVerent biochemical responses, even imitating pathological conditions (De Keulenaer et al. 1998). In order to elucidate this question, Hornberger et al. (2005) observed that uniaxial stretching of myotubes (C2C12) activates Akt and ERK (extracellular regulated protein kinase). However, only multiaxial stretching of myotubes seems to activate p70s6k. That indicates muscle cells are also capable of identifying and respond in a speciWc manner to diVerent mechanical stimuli. Nevertheless, its physiological implications and possible correlations with the diVerent types of muscle actions remain unclear. mTOR/p70s6k stimulated by diVerent actions/patterns of muscle activation According to studies performed with “in vitro” skeletal muscle culture cells, diVerent mechanical stimuli (e.g. 123 actions/patterns) of in-vivo skeletal muscle contractions are capable of signaling hypertrophy pathways diVerently. A classical example to illustrate this is the diVerential hypertrophy process which cardiac muscle exhibits when submitted to diVerent overload patterns of activation. Similarly to cardiac muscle, skeletal muscle also adapts in a peculiar manner. However, while for the cardiac muscle eccentric stimuli seem to stretch the Wbers sometimes weakening them, on the skeletal muscle, the same stimuli promotes a robust hypertrophy process (Russel et al. 2000). Beyond muscle arrangement diVerences and the type of mechanical tension to which Wbers are submitted, it is possible that under speciWc conditions of skeletal muscle contraction, mechano sensitive activators respond with diVerent magnitudes, as observed in MGF (Goldspink 1999; Mckoy et al. 1999). As an example, Eliasson et al. (2006) conducted a study with human subjects observing that maximal eccentric contractions are more eYcient in activating p70s6k than maximal concentric contractions. Nader and Esser (2001) observed the same in a study carried out with rodents. One of the hypotheses to explain the stronger activation is based on the type of mechanical activity induced by eccentric contractions in skeletal muscle. During eccentric contractions, the muscle is submitted to both, an increase in its tension and in its length, while for concentric contractions, the stretching stimuli either does not occur or occurs with small intensity. Thus, the added response to both stimuli (e.g. stretching and tension) during eccentric contraction seems to act on p70s6k. In fact, it has been recently demonstrated that chronic stretching of skeletal muscle is capable of increasing its mass/length through activation of mTOR pathway (Aoki et al. 2006). It is worth saying, though, that this response is not a consensus among scholars. Cuthbertson et al. (2006) observed equal magnitude of responses in p70s6k activation induced either by dynamic concentric or eccentric contractions, although in the last study, subjects were supplemented with amino acids and sucrose, which are likely to have inXuenced the observed response. Apart from the type of muscle action performed, other interactive factors such as intensity/duration and frequency of the Wber stimulation are also likely to promote speciWc signaling responses. Atherton et al. (2005), carried out a study which tested electric long term and low intensity stimuli, similar to those promoted by aerobic training, against electric high intensity and short term stimuli, similar to resistance strength training. It was observed that aerobic exercise stimuli promoted speciWc adaptive responses toward mitochondrial biogenesis besides slow twitch Wbers phenotype, not activating mTOR and p70s6k. Resistance strength training stimuli, however, intensely activated mTOR and p70s6k pathway. Interestingly, Nader and Esser (2001) found that both high intensity and low intensity Eur J Appl Physiol electrical stimuli promoted activation of p70s6k, with a less prolonged response promoted by the low intensity electrical stimuli. The diVerent responses related to low intensity electrical stimulation in the two studies can be related to the fact that Atherton et al. (2005) carried out his study in “in vitro” conditions, while Nader and Esser (2001) carried out their study in in-vivo conditions. Furthermore, Atherton et al. (2005) submitted skeletal muscles to contraction during 3 h, while in the Nader and Esser study, skeletal muscle underwent contraction during only 30 min. These diVerences could generate distinct patterns of substrates depletion (e.g. glycogen content) culminating in the diVerential activation of energetic sensors inside the muscle cell, such as AMP kinase (AMPK), which has been shown to suppress protein synthesis and downregulate mTOR signaling (Bolster et al. 2002). Although not measured in the Nader and Esser’s (2001) study, Atherton et al. (2005) found that the increased activation of AMPK was strictly related to the inhibition of mTOR and p70s6k, which would be likely to explain the null activation of p70s6k. There is hardly any information in literature on the eVect of endurance exercise in the activation of signaling pathways controlling protein synthesis. However, several studies showed an acute increase in muscle protein synthesis after a single session of aerobic exercise (Carraro et al. 1990; SheYeld-Moore et al. 2004). These eVects are probably mediated by the activation of anabolic signaling pathway. In order to verify the involvement of the mTOR pathway in this response, Mascher et al. (2007) carried out a study with human subjects, using cycling exercise. They observed that cycling exercise was capable of acutely phosphorylating the mTOR (Serine 2448) and p70s6k (Serine 424/Threonine 421). Nevertheless, p70s6k was not activated since phosphorylation of threonine 389 did not occur (Pullen and Thomas 1997). The lack of p70s6k activation despite an increase in mTOR phosphorylation suggests another role for mTOR following endurance exercise. Therefore, as proposed by Mascher et al. (2007), mTOR could promote increases in protein synthesis mediated not only by p70s6k but also via deactivation of eukaryotic elongation factor 2 kinase (eEF2k). Inactivating eEF2k causes a dephosphorilation of eEF2 in threonine 56 and eEF2 becomes activated, thus increasing the protein synthesis (Redpath et al. 1993). Actually, it was observed in Mascher et al. (2007) study, that eEF2 becomes activated following aerobic exercise. In support of this notion, there is the fact that the muscle protein synthesis increased in association with a decrease in the phosphorylation of eEF2 after resistance exercise (Dreyer et al. 2006). Thus far, the way these pathways can be diVerently activated through diVerent types of exercise (e.g. resistance or aerobic exercise) remains unknown. Aerobic exercise is not likely to cause any marked hypertrophy response. Thus, Mascher et al. (2007) speculated that, at least in humans, the acute activation of mTOR and the activation of GSK-3 (glycogen synthase kinase-3) without the activation of p70s6k could be related to an increased rate of the mitochondrial protein synthesis. The activation of p70s6k (phosphorylation in Threonine 389) was reported to occur only in response to high force contractions in rodents (Nader and Esser 2001; Haddad and Addams 2002; Atherton et al. 2005) as well as in human subjects (Dreyer et al. 2006; Eliasson et al. 2006) and this event has been associated with increased protein synthesis after resistance exercise (Hernandez et al. 2000; Cuthbertson et al. 2006). However, a recent study carried out in human subjects showed a marked increase in p70s6k during low intensity resistance exercise, combined with a moderate reduction of blood Xow to the working muscle (Fujita et al. 2007). Furthermore, in this study, increased protein synthesis was correlated to p70s6k activation, which occurred only in response to exercise performed with vascular occlusion (e.g. it did not occur in the low intensity exercise control group). In the light of the present knowledge, these Wndings suggest that speciWc activation of p70s6k mediated by diVerent combinations of contraction patterns continues to be a “marker for the acute characterization of growth stimuli”, as proposed by Baar and Esser (1999). Additionally, it suggests that other factors unrelated to mechanical stimuli (e.g. hypoxia, metabolic stress) are capable of activating it. Nonetheless, whether the divergent responses related to the activation of mTOR without the activation of p70s6k can be responsible for the diVerential translation of speciWc mRNA mediated by diVerent modes of exercise remains, thus far, unclear. DiVerential activation of mTOR/p70s6k mediated by diVerent skeletal muscle Wber types Traditionally, Wber type discrimination and its contractile and metabolic properties have been one of the main subjects of research in exercise physiology. Biochemical and physiological information discerning these features are widely found in books which discuss adaptive aspects of strength training/overload (Fleck and Kraemer 1997). However, molecular diVerentiations responsible for such features are still under investigation. An important observation about the remodeling process is related to the lower susceptibility that the slow twitch Wber (type I) shows in comparison with the fast twitch Wber (type II) in the hypertrophy process (Widrick et al. 2002). Besides the classical concepts involving diVerent motor recruitment patterns, molecular mechanisms begin to be elucidated. Among such mechanisms, a decreased activation of the mTOR pathway in the skeletal muscle containing 123 Eur J Appl Physiol slow twitch Wbers appears. For example, Baar and Esser (1999) reported the phosphorylation of p70s6k to be more dramatic in the muscle groups containing a greater proportion of type II vs. type I muscle Wbers (e.g. tibialis anterior vs. soleus). More recently, in a study designed to investigate responsiveness of the skeletal muscle group to mTOR and p70s6k, it has been observed the skeletal muscle groups predominantly containing fast twitch Wber (tibialis anterior, plantaris) to be responsible for activating/phosphorylating mTOR in its 2448 serine end (especially type II Wber), exhibiting similar pattern of activation of p70s6k (Parkington et al. 2003). Contrariwise, soleus muscle, known to have predominantly slow twitch Wbers, does not show activation/phosphorylation of mTOR. In that study, the authors used electrical stimulation directly upon the sciatic nerve, which recruits all the motor units inside the stimulated muscle. Hence, the observed response does not seem to result from diVerential activation of motor units. Although coherent, the preferential activation of the mTOR pathway is not always demonstrated. As an example, Koopman et al. (2006) conducted a study with human subjects submitted to resistance exercise and reported increased phosphorylation of p70s6k in the type II Wbers when compared to the type I Wbers, measured in threonine 421/serine 424 residue. However, although the authors did not measure phosphorylation of p70s6k (threonine 389) and ribosomal protein S6 in a Wber type speciWc manner, phosphorylation of S6 was not increased in homogenates of the vastus lateralis muscle, suggesting that the greater phosphorylation of p70s6k (thr421/ser424) in the fast Wber type does not account for its activation. Whether maximal lengthening contractions are capable of diVerentially activating mTOR or p70s6k in human subjects, though, remains unclear. Therefore, these results demonstrate, at least in rodents, the reduced susceptibility that the type I skeletal muscle Wber presents in the mTOR pathway activation, thus partly explaining its reduced hypertrophy capacity induced by mechanical stimulation. Nevertheless, the fact that the type I skeletal muscle Wber also increases its size when induced by mechanical stimulation should be bore in mind. This suggests that diVerential time courses of activation or diVerential extents of signaling responses might explain the diVerential responses observed in human subjects versus those observed in studies with rodents. Besides, diVerent forms of the skeletal muscle stimulation were also used in the above-mentioned studies (voluntary stimulation vs. electrical stimulation) which might have diVerently aVected the activated muscles. Another relevant hypothesis which might explain muscle Wber type-speciWc eVects on hypertrophy response is that contracting skeletal muscle Wbers are less eVective of increasing the protein synthesis and activating the mTOR pathway (Atherton et al. 2005; Rennie 2005; Goldberg 123 1967). Thus, because the muscle groups containing predominantly slow twitch Wbers are mainly postural or tonic, their continuous mechanical stimulation does not allow contractile proteins accumulation similar to phasic or fast twitch Wbers, which usually show much more intermittent mechanical stimulation. However, Mittendorfer et al. (2005) observed in human subjects neither diVerences of anatomical location nor Wber type composition in the protein synthesis. The observed response was under basal conditions, a situation which neutralizes diVerential activation of tonic or phasic Wbers (although the authors did not investigate this response under overload conditions, which is obviously diYcult). It is possible diVerent responses related to Wber types to be speciWc under diVerent mechanical stimulation. mTOR/p70s6k and the protein synthesis According to the previously discussed information, the pathways activated by mTOR are highly complex. However, this knowledge is of the utmost importance, once depending on the cellular target activated by mTOR, diVerent translational processes will be recruited. For example, activation (phosphorylation) of p70s6k seems to be involved in translation of the mRNA containing oligopirimidinic terminations (5⬘ TOP mRNA) important to the mRNA translation coding ribossomal proteins as well as to increases in translational eYciency (Ruvinsky and Meyuhas 2006), whereas the 4EBP-1 activation seems to be involved in all kinds of eukariotes mRNA (Lawrence and Abraham 1997). Collectively, the activation of these eVectors seems to determine increased skeletal muscle protein synthesis induced by mechanical stimuli. However, skeletal muscle hypertrophy selectively occurs through the accumulation of myoWbrillar proteins and not by sarcoplasmic proteins (Balagopal et al. 2001; Hasten et al. 2000; Rasmussen and Phillips 2003). As an example, a recently published study has demonstrated that, in basal conditions, the sarcoplasmic protein synthesis was higher than the myoWbrillar protein synthesis. However, after resistance exercise, the stimulation of rates of sarcoplasmic protein synthesis was »20% less than that of myoWbrillar protein synthesis (Cuthbertson et al. 2006). Likewise, during atrophic state mediated by denervation, negative remodeling occurs predominantly due to contractile protein losses (Almon and Dubois 1990). This indicates that the correct understanding of the translational process of speciWc mRNA (e.g. coding myoWbrillar proteins) seems to be a crucial aspect of mechanical stimulation inducing skeletal muscle remodeling. Surprisingly, the way the mRNA coding contractile proteins are recruited by mTOR under mechanical stimuli remains still not thoroughly understood. The current knowledge is that the general muscle protein synthesis is regulated via increases in translational eYciency (e.g. ini- Eur J Appl Physiol tiation, elongation and termination) or capacity, since muscular mRNA abundance has been observed even in atrophy states (Welle et al. 1999; Meyuhas 2000; Goldspink 2002; Goldspink 1999). Once again, however, messages coding myoWbrillar mRNA are the main responsible for the remodeling process induced by mechanical stimulation. Thus, as a recommendation to future studies, measurements of both changes in the rates of protein synthesis (or speciWc protein synthesis) and signaling proteins alike (including mTOR and p70s6k), as already observed recently (Dreyer et al. 2006; Cuthbertson et al. 2006), will help gain insight on the speciWc role of the protein signaling in speciWc muscular adaptations induced by diVerent mechanical stimuli. Final considerations Day after day, new mechanistic studies unmask important physiological responses. Thirty years ago it was speculated whether the skeletal muscle was capable of generating biochemical events induced by contraction that would culminate in the hypertrophy of the skeletal muscle (Goldberg et al. 1975). However, owing to the lack of knowledge as well as to the lack of the necessary technological tools, three decades went by until the discovery of these mechanochemical mediators took place. Mechanical stimulation is capable of activating several growth factors including MGF, a potent stimulator of hypertrophy in the skeletal muscle. Besides, new mediators responsible for the acute conversion of the mechanical information into molecular signals have been studied. One of these mediators is PA. Although several advances have been attained, other questions remain open to investigation, particularly those related to the pathway mediated by mTOR. For example, stimulation induced by nutrients seems to activate common pathways mediated by growth factors, which, in turn, activate the mTOR/p70s6k (Horberger et al. 2006c). Nonetheless, overexpression of growth factors results in a strong hypertrophic response (Lai et al. 2004; Barton-Davis et al. 1998; Goldspink and Yang 2001), whereas stimulation induced by nutrients seems to be much more important in the inhibition of the atrophic process (Almon and Dubois 1988). This response is further supported by the fact that overfeeding does not induce additional skeletal muscle protein synthesis (Glick et al. 1982). Likewise, although nutrient induced stimulation does not induce expressive increase in the skeletal muscle size, its utilization during strength exercise emphasizes the p70s6k activation and increases the protein synthesis (Karlsson et al. 2004; Wackerhage and Rennie 2006). This indicates, once again, that parallel or synergic pathways can be activated through diVerent stimuli combinations. As a result, the characterization and additional detailing regarding the magnitude and duration of these pathways induced by diVerent combinations (e.g. diVerent mechanical stimuli and/or nutritional stimuli) might be important aspects to be considered for a better understanding of the mTOR signaling pathway. Acknowledgments We thank Dr. Érico Chagas Caperuto for his critical reading of the manuscript and Maria Cristina Fioratti Florez for the careful specialized language revision of the present review article. References Almon RR, Dubois DC (1988) Adrenalectomy eliminates both Wbertype diVerences and starvation eVects on denervated muscle. Am J Physiol 225:E850–E856 Almon RR, Dubois DC (1990) Fiber-type discrimination in disuse and glucocorticoid induced atrophy. Med Sci Sports Exerc 22:304–311 Aoki MS, Myiabara EH, Soares AG, Saito ET, Moriscot AS (2006) mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching. Cell Tissue Res 324:149–156 Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, Wackerhage H (2005) Selective activation of AMPK-PGC-1alpha or PKBTSC2-mTOR signaling can explain speciWc adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19:786–798 Baar K, Esser K (1999) Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol 45:C120-C127 Balagopal P, Schimke JC, Ades PA, Adey DB, Nair KS (2001) Age eVect on transcript levels and synthesis rate of muscle MHC and response to resistance exercise. Am J Physiol 280:E203–E208 Barton-Davis E, Shoturma DI, Musaro A, Rosenthal N, Sweeney Hl (1998) Viral mediated expression of insulin-like growth factor-1 blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci USA 95:15603–15607 Bloch RJ, Gonzalez-Serratos H (2003) Lateral force transmission across costameres in skeletal muscle. Exerc Sport Sci Rev 31:73–78 Blomstrand E, Eliasson J, Karlsson HK, Kohnke R. (2006) Branched chain amino acids activate key enzymes in protein synthesis after physical exercise. J Nutr 136:269S–273S Bodine SC (2006) mTOR signaling and the molecular adaptation to resistance exercise. Med Sci Sports Exerc 38:1950–1957 Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD (2001) Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3:1014–1019 Bolster DR, Crozier SJ, Kimball SR, JeVerson LS (2002) AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through downregulated mTOR signalling J Biol Chem 277:23977–23980 Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA, Kimball SR, JeVerson LS (2003) Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 15:213–220 Booth FW, Baldwin KM (1966) Muscle plasticity: energy demand and supply processes. In: Rowell LB, Shepard JT (eds) Handbook of physiology. Oxford University Press, New York, pp 1074–1123 Boppart MD, Burkin DJ, Kaufman SJ (2006) Alpha7beta1-integrin regulates mechanotransduction and prevents skeletal muscle injury. Am J Physiol 290:C1660–C1665 Carraro F, Stuart CA, Hartl WH, Rosenblatt J, Wolfe RR (1990) EVect of exercise and recovery on muscle protein synthesis in human subjects. Am J Physiol 259:E470–E476 123 Eur J Appl Physiol Chen J (2004) Novel regulatory mechanisms of mTOR signaling. Curr Top Microbiol Immunol 279:245–257 Chiang GC, Abraham RT (2005) Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70s6 kinase. J Biol Chem 280:25485–25490 CoVey VG, Zhong Z, Shield A, Canny BJ, Chibalin AV, Zierath JR, Hawley JA (2005) Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans. FASEB J 20:190–200 Cuthberson DJ, Babraj J, Smith K, Wilkes E, Fedele MJ, Esser K, Rennie M (2006) Anabolic signaling and protein synthesis in human skeletal muscle after dynamic shortenin or lengthening exercise. Am J Physiol 290:E731–E738 De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK (1998) Oscillatory and steady laminar shear stress diVerentially aVect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res 82:1094–101 Deshmukh A, CoVey VG, Zhong Z, Chibalin AV, Hawley JÁ, Zierath JR (2006) Exercise-induced phosprolylation of the novel Akt substrates AS 160 and Wlamin A in human skeletal muscle. Diabetes 55:1776–1782 Dreyer HC, Fujita S, Cadenas J, Chinkes DL, Volpi E, Rasmussen BB (2006) Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 576:613–624 Eliasson J, Elfegoun T, Nilsson J, Kohnke R, Ekblom B, Blomstrand E (2006) Maximal lengthening contractions increase p70s6k kinase phosphorylation in human skeletal muscle in the absence of nutritional supply. Am J Physiol 291:E1197–E1205 Ervasti JM (2003) Costameres: the Achilles’ heel of Herculean muscle. J Biol Chem 278: 13591–13594 Fang Y, Park IH, Wu AL, Du G, Huang P, Frohman MA, Huang P, Frohman MA, Brown HA, Chen J (2003) PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1. Curr Biol 13:2037–2044 Fleck SJ, Kraemer WJ (1997) Muscle physiology. In: Designing resistance training programs. Human kinetics, Ilinois, pp 45–63 Fry AC (2004) The role of resistance exercise intensity on muscle Wbre adaptations. Sports Med 34:663–679 Fujita S, Abe T, Drummond MJ, Cadenas JG, Dreyer HC, Sato Y, Volpi E, Rasmussen BB (2007) Blood Xow restriction during lowintensity resistance exercise increases S6K1 phosphorylation and muscle protein synthesis. J Appl Physiol 103:903–910 Glick Z, Mcnurlan MA, Garlick PJ (1982) Protein synthesis rate in liver and muscle of rats following four days of overfeeding. J Nutr 112:391–397 Goldberg AL (1967) Protein synthesis in tonic and phasic skeletal muscles. Nature 216:1219–1220 Goldberg AL, Etlinger JD, Goldspink DF, Jablecky C (1975) Mechanism of workinduced hypertrophy of skeletal muscle. Med Sci Sports 7:185–198 Goldspink G (1999) Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload. J Anat 194:323–334 Goldspink G (2002) Gene expression in skeletal muscle. Biochem Soc Trans 30:285– 290 Goldspink G, Booth F (1992) General remarks: mechanical signals and gene expression in muscle. Am J Physiol 262:R327–R328 Goldspink G, Yang SY (2001) Method of treating muscular disorders. United States Patent. Patent N° US 6,221,842 B1, April 24 Grounds MD, Sorokin L, White J (2005) Strength at the extracellular matrix–muscle interface. Scand J Med Sci Sports 15:347–358 Haddad F, Adams GR (2002) Selected contribution: acute cellular and molecular responses to resistance exercise. J Appl Physiol 93:393–403 123 Hameed M, Orrell RW, Cobbold M, Goldspink G, Harridge SDR (2003) Expression of IGF-1 splice variants in young and old human skeletal muscle after high resistance exercise. J Physiol 547:247–254 Hasten DL, Pak-Loduca KA, Obert KE, Yarasheski KE (2000) Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in 78–84 and 23–32 yr olds. Am J Physiol 278:E620–E626 Hernandez JM, Fedele MJ, Farrell PA (2000) Time course evaluation of protein synthesis and glucose uptake after acute resistance exercise in rats. J Appl Physiol 88:1142–1149 Holz MK, Blenis J (2005) IdentiWcation of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)- phosphorylating kinase. J Biol Chem 280:26089–26093 Hong JH, Oh SO, Lee M, Kim YR, Kim DU, Hur GM, Lee JH, Lim K, Hwang BD, Park SK (2001) Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid. Biochem Biophys Res Commun 281:1337–1342 Hornberger TA, Chien S (2006) Mechanical stimuli and nutrients regulate rapamycin-sensitive signaling through distinct mechanisms in skeletal muscle. J Cell Biochem 97:1207–1216 Hornberger TA, McLoughlin TJ, Leszczynski JK, Armstrong DD, Jameson RR, Bowen PE, Hwang ES, Hou H, Moustafa ME, Carlson BA, HatWeld DL, Diamond AM, Esser KA (2003) Selenoprotein-deWcient transgenic mice exhibit enhanced exercise-induced muscle growth. J Nutr 133:3091–3097 Hornberger TA, Armstrong DD, Koh TJ, Burkholder TJ, Esser K (2005) Intracellular signaling speciWcity in response to uniaxial vs. multiaxial stretch: implications for mechanotransduction. Am J Physiol 288:C185–C194 Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML, Chin ER, Esser KA (2004) Mechanical stimuli regulate rapamycin sensitive signaling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochem J 380:795–804 Hornberger TA, Mak YW, Hsiung JW, Huang SA, Chien S (2006a) The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle. Proc Natl Acad Sci USA 103:4741–4746 Hornberger TA, Sukhija KB, Chien S (2006b) Regulation of mTOR by mechanically induced signaling events in skeletal muscle. Cell Cycle 13:1391–1396 Ingber DE (2006) Cellular mechanotransduction: putting all the pieces together again. FASEB J 20:811–827 Jacinto E, Hall MN (2003) Tor signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol 4:117–126 Kam Y, Exton JH (2004) Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid. FASEB J 18:311–319 Karlsson HK, Nilsson PA, Nilsson J, Chibalin AV, Zierath JR, Blomstrand E (2004). Branched-chain amino acids increase p70s6k phosphosphorylation in human skeletal muscle after resistance exercise. Am J Physiol 287:E1–E7 King WG, Mattaliano MD, Chan TO, Tsichlis PN, Brugge JS (1997) Phosphatidylinositol 3-kinase is required for integrin-stimulated akt and raf-1/mitogen-activated protein kinase pathway activation. Mol Cell Biol 17:4406–4418 Koopman R, Zorenc AHG, Gransier RJJ, Cameron-Smith D, Van Loon LJC (2006) Increase in S6K1 phosphorylation in human skeletal muscle following resistance exercise occurs mainly in type II muscle Wbers. Am J Physiol 290:E1245–E1252 Kubica N, Bolster D, Farrell PA, Kimball SR, JeVerson L (2005) Resistance exercise increases muscle protein synthesis and translation of eukaryotic initiation factor 2Be mRNA in a mammalian target of rapamycin-dependent manner. J Biol Chem 280:7570– 7580 Eur J Appl Physiol Lai KM, Gonzalez M, Poueymirou WT, Kline WO, Na E, Zlotchenko E, Stitt TN, Economides AN, Yancopoulos GD, Glass DJ (2004) Conditional activation of Akt in adult skeletal muscle induces rapid hypertrophy. Mol Cell Biol 24:9295–9304 Lawrence JC, Abraham RT (1997) PHAS/4E-BPs as regulators of mRNA translation and cell proliferation. Trends Biochem Sci 22:345–349 Lee S, Park JB, Kim JH, Kim Y, Kim JH, Shin KJ, Lee JS, Ha SH, Suh PG, Ryu SH (2001) Actin directly interacts with phospholipase D, inhibiting its activity. J Biol Chem 276:28252–28260 Malik RK, Parsons JT (1996) Integrin-dependent activation of the ribosomal s6 kinase signaling pathway. J Biol Chem 271:29785–29791 Mascher H, Andersson H, Nilsson PA, Ekblom B, Blomstrand E (2007) Changes in signaling pathways regulating protein synthesis in human muscle in the recovery period after endurance exercise. Acta Physiol 191:67–75 Mayer U (2003) Integrins: redundant or important players in skeletal muscle? J Biol Chem 278:14587–14590 McKoy G, Ashley W, Mander J, Yang SY, Williams N, Russell B, Goldspink G (1999) Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. J Physiol 516:583–592 Meyuhas O (2000) Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 267:6321–6330 Mittendorfer B, Andersen JL, Plomgaard P, Saltin B, Babraj JA, Smith K, Rennie MJ (2005) Protein synthesis rates in human muscles: neither anatomical location nor Wbre-type composition are major determinants. J Physiol 563:203–211 Nader GA, Esser K (2001) Intracellular signaling speciWcity in skeletal muscle in response to diVerent modes of exercise. J Appl Physiol 90:1936–1942 Nader GA, McLoughlin TJ, Esser KA (2005) mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators. Am J Physiol 289:C1457–C1465 Nave BT, Ouwens DJ, Withers DR, Shepherd A, Shepherd PR (1999) Mammalian target of rapamycin is a direct target for protein kinase B: identiWcation of a convergence point for opposing eVects of insulin and amino-acid deWciency on protein translation. Biochem J 344:427–431 Ohanna M, Sobering AK, Lapointe T, Lorenzo L, Praud C, Petroulakis E, Sonenberg N, Kelly PA, Sotiropoulos A, Pende M (2005) Atrophy of S6k1 ¡/¡ skeletal muscle cells reveals distinct mTOR eVectors for cell cycle and size control. Nat Cell Biol 7:286–294 Pallafacchina G, Calabria E, Serrano A, Kalhovde JM, SchiaVino S (2002) A protein kinase B-dependent and rapamycin sensitive pathway controls skeletal muscle growth but not Wber type speciWcation. Proc Natl Acad Sci USA 99:9213–9218 Park JB, Kim JH, Kim Y, Há SH, Yoo JS, Du G, Frohman MA, Suh PG, Ryu SH (2000) Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner. J Biol Chem 275:21295–21301 Parkington JD, Siebert AP, Lebrasseur NK, Fielding RA (2003) DiVerential activation of mTOR signaling by contractile activity in skeletal muscle. Am J Physiol 285:R1086–R1090 Peterson RT, Beal PA, Comb MJ, Schreiber SL (2000) FKBP12-Rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J Biol Chem 275:7416–7423 Phillips SM, Parise G, Roy BD, Tipton KD, Wolfe RR, Tarnopolsky MA (2002) Resistance-training-induced adaptations in skeletal muscle protein turnover in fed state. Can J Physiol Pharmacol 80:1045–1053 Pullen N, Thomas G (1997) The modular phosphorylation and activation of p70s6k. FEBS Lett 410:78–83 Rasmussen BB, Phillips SM (2003) Contractile and nutritional regulation of human muscle growth. Exerc Sports Sci Rev 31:127–131 Redpath NT, Price NT, Severinov KV, Proud GC (1993) Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem 213:689–699 Reiling JH, Sabatini DM (2006) Stress and mTORture signaling. Oncogene 25:6373–6383 Rennie MJ (2005) Why muscle stops building when it’s working. J Physiol 569:3 Rennie MJ, Wackerhage H (2003) Connecting the dots for mechanochemical transduction in muscle. J Physiol 553:1 Reynolds TH, Bodine SC, Lawrence Jr JC (2002) Control of Ser 2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J Biol Chem 277:17657–17662 Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, Yancopoulos GD, Glass DJ (2001) Mediation of IGF-1 induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/ Akt/GSK3 pathways. Nat Cell Biol 3:1009–1013 Russell B, Motlagh D, Ashley W (2000) Form follows function: how muscle shape is regulated by work. J Appl Physiol 88:1127–1132 Ruvinsky I, Meyuhas O (2006) Ribossomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci 31:342–348 Sakamoto K, Hirshman MF, Aschenbach WG, Goodyear LJ (2002) Contraction regulation of Akt in rat skeletal muscle. J Biol Chem 277:11910–11917 Sakamoto K, Aschenbach WG, Hirshman MF, Goodyear LJ (2003) Akt signaling in skeletal muscle: regulation by exercise and passive stretch. Am J Physiol 285:E1081–E1088 Sakamoto K, Arnolds DE, Ekberg I, Thorell A, Goodyear LJ (2004) Exercise regulates Akt, glycogen synthase kinase-3 activities in human skeletal muscle Biochem Biophys Res Commun 319:419–425 Sarbassov D, Ali SM, Sabatini DM (2005) Growing roles for the mTOR pathway. Curr Opin Cell Biol17:596–603 Schmelzle T, Hall MN (2000) TOR, a central controller of cell growth. Cell 103:253–262 Scott PH, Brunn GJ, Konh AD, Roth RA, Lawrence Jc-JR (1998) Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc Natl Acad Sci USA 95:7772–7777 SheYeld-Moore M, Yeckel CW, Volpi et al\ (2004) Post exercise protein metabolism in older and younger men following moderate intensity aerobic exercise. Am J Physiol 287:E513–E522 Stokoe D, Stephens LR, Copeland T, GaVney PR, Reese CB, Painter GF, Holmes AB, Mccormick F, Hawkins PT (1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277:567–701 Thiimmaiah KL, Easton JB, Germain GS, Morton CL, Kamath S, Buolamwini JK, Houghton PJ (2005) IdentiWcation of N10-substituted phenoxazines as potent and speciWc inhibitors of Akt signaling. J Biol Chem 280:31924–31935 Tidball JG (2005) Mechanical signal transduction in skeletal muscle growth and adaptation. J Appl Physiol 98:1900–1908 Timson BF (1990) Evaluation of animal models for the study of exercise-induced muscle enlargement. J Appl Physiol 69:1935–1945 Turinsky J, Damrau-Abney A (1999) Akt kinases and 2-deoxyglucose uptake in rat skeletal muscles in vivo: study with insulin and exercise. Am J Physiol 276:R277–R282 Wackerhage H, Rennie MJ (2006) How nutrition and exercise maintain the human musculoskeletal mass. J Anat 208:451–458 Welle S, Bhatt K, Thornton CA (1999) Stimulation of myoWbrillar synthesis by exercise is mediated by more eYcient translation of mRNA. J Appl Physiol 86:1220–1225 Widrick JJ, Stelzer JE, Shoepe TC, Garner DP (2002) Functional properties of human muscle Wbers after short-term resistance exercise training. Am J Physiol 283:R408–R416 Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484 123