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` SE REVIEW / SYNTHE

mTOR function in skeletal muscle: a focal point for overnutrition and exercise
Donato A. Rivas, Sarah J. Lessard, and Vernon G. Coffey

Abstract: The mammalian target of rapamycin (mTOR) is a highly conserved atypical serinethreonine kinase that controls numerous functions essential for cell homeostasis and adaptation in mammalian cells via 2 distinct protein complex formations. Moreover, mTOR is a key regulatory protein in the insulin signalling cascade and has also been characterized as an insulin-independent nutrient sensor that may represent a critical mediator in obesity-related impairments of insulin action in skeletal muscle. Exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial metabolic adaptation in skeletal muscle. Thus, the metabolic effects of nutrients and exercise have the capacity to converge at the mTOR protein complexes and subsequently modify mTOR function. Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition and the capacity for exercise to enhance mTOR activity in skeletal muscle. Key words: nutrients, insulin action, metabolism, training. ` res (mTOR) est une se rinethre onine kinase atypique ultra bien sume : La cible de la rapamycine chez les mammife Re e qui exerce de nombreuses fonctions essentielles a ` lhome ostasie cellulaire et a ` ladaptation des cellules des conserve ` res par la formation de 2 complexes prote iques distincts. De plus, la mTOR est une prote ine re gulatrice essenmammife ristiques, elle est un capteur de le ments nutritifs insutielle dans la voie de signalisation de linsuline; entre autres caracte pendant qui semble jouer le ro le de me diateur critique dans le de re ` glement de laction de linsuline dans le lino-inde ` se. Lexercice physique constitue une modalite the rapeutique, car il ame liore lactivite de la muscle squelettique de lobe quemment des adaptations me taboliques be ne fiques au muscle squelettique. Ainsi, les nutriments mTOR et favorise subse tabolisme et agissent sur les complexes prote iques de la mTOR pour en modifier la et lexercice physique modifient le me ` se est de pre ciser le ro le de la mTOR dans la re gulation de laction de linsuline en fonction. Lobjet de cet article-synthe action a ` la suralimentation et dindiquer comment lexercice physique ame liore lactivite de la mTOR dans le muscle re squelettique. tabolisme, entra nement. s : nutriments, action de linsuline, me Mots-cle daction] [Traduit par la Re

Introduction
The discovery and isolation of the immunosuppressant drug rapamycin in 1972 has led to the establishment of an essential biological pathway and has produced a revolution in the understanding and treatment of cancer (Sabatini 2006; Garber 2001). In the 1990s, rapamycins molecular target was identified, cloned, and termed the mammalian target of rapamycin (mTOR) (Sabatini et al. 1994; Heitman et al. 1991). mTOR is a highly conserved atypical serine threonine kinase that controls numerous functions essential

for cell homeostasis and adaptation (Coffey and Hawley 2007; Hay and Sonenberg 2004). Early studies examining the roles of mTOR were facilitated by the use of rapamycin, a compound that acutely disrupts and partially inhibits mTOR function (Yang and Guan 2007). However, using RNA interference technology, it was discovered that rapamycin did not inhibit all mTOR functions (Sarbassov et al. 2005a). This observation led to the discovery that mTOR exists in 2 structurally and functionally distinct multiprotein complexes. mTOR is a key regulatory protein for a multiplicity of

Received 29 March 2009. Accepted 26 May 2009. Published on the NRC Research Press Web site at apnm.nrc.ca on 6 October 2009. D.A. Rivas1 and V.G. Coffey.2 Exercise Metabolism Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia. S.J. Lessard. The Research Division, Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
1Present

address: Nutrition, Exercise Physiology and Sarcopenia Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111, USA. 2Corresponding author (e-mail: vernon.coffey@rmit.edu.au).
Appl. Physiol. Nutr. Metab. 34: 807816 (2009) doi:10.1139/H09-073 Published by NRC Research Press

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cell processes including, but not limited to, cell growth and differentiation, protein synthesis, and actin cytoskeletal organization. The specific roles for mTOR in these cellular functions have been reviewed elsewhere (Sabatini 2006; Hay and Sonenberg 2004; Sarbassov et al. 2005a; Bhaskar and Hay 2007). Of note, mTOR has also been established as an integral component of the insulin receptor substrate (IRS)phosphatidylinositol 3-kinase (PI3K) insulin signalling cascade and as an insulin-independent nutrient sensor in skeletal muscle (Tremblay et al. 2005; Martin and Blenis 2002; Potier et al. 2009). In addition, mTOR activity is modulated by repeated contraction (i.e., exercise) and has emerged as an important mediator of exercise-induced adaptation in skeletal muscle (Coffey and Hawley 2007). Thus, given its role in mediating the effects of nutrients and the capacity for contractile activity to enhance insulin sensitivity and cell growth, mTOR represents an important regulator of skeletal muscle insulin action and metabolism (Tremblay et al. 2005; Tremblay and Marette 2001; Krebs et al. 2007; Krebs et al. 2002; Patti et al. 1998). Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition, and the capacity for exercise to enhance mTOR function.

Overnutrition and skeletal muscle insulin action

Obesity and elevated levels of circulating nutrients are strongly associated with the development of insulin resistance and type 2 diabetes (Dresner et al. 1999; Kahn and Flier 2000). These conditions are exacerbated by excess nutrient intake and a sedentary lifestyle (Hawley and Lessard 2008). Increased dietary fat consumption has long been associated with the metabolic abnormalities that are linked with overnutrition (Hawley and Lessard 2008). However, increases in the intake of other dietary nutrients, such as protein and carbohydrates, also have negative effects on whole-body insulin action (Daly 2003; Um et al. 2006). The so-called western diet is characterized by the consumption of large quantities of processed meats, refined sugars, and fats, and is closely linked with obesity and insulin resistance (McCullough et al. 2002; van Dam et al. 2002). Skeletal muscle is responsible for the majority of insulin-mediated glucose disposal, and the muscles sensitivity to insulin is highly responsive to changes in circulating nutrients. Accordingly, significant efforts have been devoted to elucidating the mechanisms by which overnutrition leads to impaired insulin signal transduction in skeletal muscle. The postprandial uptake of glucose into muscle cells is initiated by the binding of insulin to its receptor, which results in insulin receptor (IR) bsubunit autophosphorylation and the subsequent tyrosine phosphorylation of the IRS. Phosphorylated IRS then binds and activates PI3K, leading to the activation of Akt (also known as protein kinase B). These initial signalling events converge on several downstream effectors that promote the metabolic actions of insulin, including the translocation of glucose transporters (GLUTs) to the myocellular membrane, allowing for the disposal of plasma glucose. When one or more of the steps that potentiate insulin signal transduction in muscle are defective, insulin action is reduced, a condition known as insulin

resistance. Skeletal muscle insulin resistance can lead to whole-body metabolic consequences such as type 2 diabetes mellitus and cardiovascular disease. The model used most frequently to examine nutrientinduced impairments to skeletal muscle insulin signal transduction is the high-fat diet compared with normal diet design. An increase in the storage of lipid species such as triglyceride, diacylglycerol, and ceramide in muscle, resulting from a high-fat diet, induces impairments to the insulin signalling cascade and the glucose transport system through multiple mechanisms (Hawley and Lessard 2008). Studies on lipid-induced insulin resistance have shown decreases in the phosphorylation, activation, and expression of insulin signalling components such as IR, IRS, PI3K, and Akt (Hawley and Lessard 2008; Lessard et al. 2007; Bjornholm and Zierath 2005). Hyperglycemia is also known to impair whole-body glucose disposal and glycogen synthesis (Krebs and Roden 2004). The exposure to chronically high glucose levels has been termed glucose toxicity and has a role in the insulin resistance of skeletal muscle, in part through the inhibition of glucose transportphosphorylation (Cline et al. 1997; Robertson et al. 1994). However, the treatment of hyperglycemia in type 2 diabetics only partially improves insulin sensitivity, indicating that insulin resistance might be a cause rather than a consequence of hyperglycemia (Krebs and Roden 2004). The results from recent studies have also implicated a role for high protein consumption in the inhibition of insulin signalling (Tremblay et al. 2005; Patti et al. 1998; Um et al. 2006). Almost 40 years ago, it was known that obese, insulin-resistant subjects had high circulating levels of plasma amino acids (AA) and that the infusion of AA inhibited muscle glucose uptake as measured across the forearm muscle (Felig et al. 1969; Pozefsky et al. 1969). Although it is becoming clear that nutrient excess results in skeletal muscle insulin resistance, there are several missing links in establishing a causative relationship between nutrient excess and the subsequent down-regulation of skeletal muscle glucose metabolism. It is thought that molecular sensors identify the nutrient excess and attenuate insulin signalling. Moreover, there have been recent implications that overnutrition directly inhibits insulin signalling at the level of IRS1 through the hyperactivation of the mTOR pathway (Tremblay and Marette 2001; Patti et al. 1998).

Metabolic functions of mTOR in skeletal muscle

mTOR regulates the activity and function of multiple cellular targets in skeletal muscle via 2 putative protein complex formations (Bhaskar and Hay 2007). The rapamycinsensitive mTOR complex 1 (mTORC1) is mTOR bound with G protein bsubunit-like protein (GbL, also called mLST8), regulatory-associated protein of mTOR (raptor), and proline-rich Akt substrate of 40 kDa (PRAS40) (Fig. 1) (Hay and Sonenberg 2004; Yang and Guan 2007; Hara et al. 2002; Kim et al. 2002). A primary phosphorylation target of mTORC1 is the threonine (Thr)389 site of ribosomal protein p70 S6 kinase 1 (S6K1) (Jastrzebski et al. 2007; Hornberger et al. 2007). Given the reputed capacity of S6K1 to both rePublished by NRC Research Press

Rivas et al. Fig. 1. Schematic representation of the molecular components that form each of the dual mTOR complexes and putative factors regulating mTOR complex activity and function. Bars represent inhibition, arrows represent activation. AICAR, 5-aminoimidazole-4carboxamide-b-D-ribofuranoside; mTORC, mammalian target of rapamycin complex; GbL, G protein bsubunit-like protein; mTOR, mammalian target of rapamycin; PRAS40, proline-rich Akt substrate of 40 kDa; mSIN1, mammalian stress-activated protein kinase-interacting protein 1; raptor, regulatory-associated protein of mTOR; rictor, rapamycin-insensitive companion of mTOR; S6K1, ribosomal protein p70 S6 kinase 1; Akt, also known as protein kinase B; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1.

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press IRS1 activity and enhance translation efficiency, mTORC1 may ultimately direct insulin signalling and translational processes in skeletal muscle (Tremblay and Marette 2001). In contrast, mTOR complex 2 (mTORC2) is rapamycin insensitive and composed of mTOR bound to GbL, rapamycin-insensitive companion of mTOR (rictor), and mammalian stress-activated protein kinase-interacting protein 1 (Fig. 1) (Bhaskar and Hay 2007; Sarbassov et al. 2004; Frias et al. 2006; Jacinto et al. 2006). mTORC2 has been implicated in Akt regulation via phosphorylation at serine (Ser)473 (Sarbassov et al. 2005b; Hresko and Mueckler 2005). Akt has been described as a critical node of the insulin signalling pathway and its putative metabolic roles include glucose transport and hypertrophy (Taniguchi et al. 2006). Thus, mTORC2 appears to have a crucial role in regulating insulin-stimulated glucose uptake in skeletal muscle. Paradoxically, mTORC2 seems to be a positive regulator of insulin signalling transduction, whereas mTORC1 has the capacity to negatively regulate insulin action via its inhibitory actions on IRS1. Much of our current knowledge regarding mTORC structure and assembly has been derived from mechanistic investigation of tumor growth. Disparity in the quantity of raptor (mTORC1)-bound mTOR and rictor (mTORC2)-bound mTOR has been observed in a variety of tumor cells (Sarbassov et al. 2004). However, analysis has been limited to quantifying intact mTOR complexes via coimmunoprecipita-

tion or copurification assays. The use of mTORC inhibitors in these experiments has revealed altered ATP-binding and subsequent kinase activity, but not complex assemblyformation (Guertin and Sabatini 2009). The possibility exists that mTOR inhibitors may bind to free mTOR and inhibit specific complex formation by perturbing assembly or protein interactions but this has yet to be established (Guertin and Sabatini 2009). As a result, there is currently a distinct lack of knowledge concerning the structure assembly of each mTOR complex. Thus, at present, it is presumed that mTORCs exist in their multiprotein complex formations, with the physiological activity determined by changes in phosphorylation of the mTOR kinase and (or) other proteins within each specific complex. Mechanistic understanding of the processes by which skeletal muscle cells sense nutrient statusavailability and subsequently convey molecular responses has received intense scientific scrutiny. In this regard, mTOR is emerging as a primary intracellular nutrient sensor involved in regulating energy homeostasis, fuel metabolism, and cell growth (Tzatsos and Kandror 2006; Krebs et al. 2007; Zinzalla and Hall 2008). Moreover, in addition to its pivotal role in insulin-mediated glucose uptake, mTOR has been shown to be a nutrient sensor for AA and lipids (Um et al. 2006; Powers 2008). The mechanism by which mTOR responds to AA has yet to be established clearly but recent evidence indicates modulation of the extracellular and (or) intracellular AA pools initiates Rag GTPase protein and Ca2+/calmodulin signalling that translocates and activates mTORC1 (Hundal and Taylor 2009; Gulati et al. 2008; Sancak et al. 2008). This cascade of events may promote the activity of an additional putative intermediary, the class 3 PI3K vacuolar protein sorting mutant 34, which is activated by AA and has been associated with prolonged mTORC1 activation (Nobukuni et al. 2005; MacKenzie et al. 2009). The pathway through which lipids may modify mTORC signalling is currently undefined but a recent study by Porstmann et al. (2008) revealed mTORC1-mediated control of sterolresponsive binding protein activity and regulation of lipogenesis. Regardless, a normal, healthy diet with adequate nutrient supply acutely stimulates mTORC1 and mTORC2 activity for their essential roles in cell growth and glucose homeostasis in skeletal muscle. Conversely, with chronic nutrient excess, the contrasting responsesfunctions of each mTOR complex appear incompatible and ultimately result in skeletal muscle insulin resistance (Fig. 2A). mTORC1: a negative regulator of insulin signal transduction with overnutrition Results from previous studies demonstrate that, in line with its proposed role as a nutrient sensor, the activation of mTORC1 is a process sensitive to growth factors (e.g., insulin, insulin-like growth factors), AA, glucose, fatty acids (FA), and ATP levels (Kim et al. 2002; Dennis et al. 2001; Fang et al. 2001; Lang 2006; Sun et al. 2008; Yeshao et al. 2005; Crozier et al. 2003; Mordier and Iynedjian 2007). Increases in AA availability have been shown to promote the interaction between raptor and mTOR (mTORC1), thereby enhancing the ability of mTORC1 to phosphorylate its wellcharacterized downstream substrates, S6K1 and eukaryotic
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810 Fig. 2. Simplified putative mTOR signalling pathways in response to (A) overnutrition and (B) exercise, which promote negative and positive effects on skeletal muscle insulin sensitivity and metabolic adaptation. Bars represent inhibition, arrows represent activation. (A) Chronic nutrient excess stimulates persistent mTORC1-S6K activity and subsequent attenuation of IRS activity, inhibiting mTORC2-mediated insulin-dependent glucose uptake. (B) Exercise promotes mTOR activation: question marks (?) denote our current lack of knowledge regarding the specific exercise-induced complex activation for enhanced translation (mTORC1) and glucose uptake (mTORC2), respectively. IR, insulin receptor; GLUT4, glucose transporter 4; IRS, insulin receptor substrate; PDK1, phosphoinositide-dependent kinase 1; Akt, also known as protein kinase B; AS160, AKT substrate of 160 kDa; PI3K, phosphatidylinositol 3-kinase; TSC2, tuberous sclerosis complex-2; mTORC, mammalian target of rapamycin complex; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein p70 S6 kinase 1; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1.

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translation initiation factor 4E-binding protein 1 (4E-BP1) (Hay and Sonenberg 2004; Um et al. 2006; Kim et al. 2002). The ability of AA to rapidly activate the mTORC1 substrates S6K1 and 4E-BP1 in the absence of insulin or other growth factors has been shown in various studies (Fox et al. 1998a, 1998b; Gulati and Thomas 2007; Hara et al. 1998; Wang et al. 1998). Work in cell culture by Patti and colleagues (1998) showed AA activation of S6K1 was comparable to that of insulin and these researchers also observed an additive effect with insulin plus AA treatment (Patti et al. 1998). Phosphorylation and activation of S6K1 following AA treatment results in inhibition of insulin-stimulated PI3K activity (Patti et al. 1998). Moreover, the inhibition of PI3K activity by AA is likely the result of S6K1-mediated serine phosphorylation and inhibition of IRS1, preventing its tyrosine phosphorylation (Tremblay and Marette 2001; Tremblay et al. 2007). Because rapamycin treatment has been shown to reverse these events, it is hypothesized that chronic AA-induced S6K1 hyperactivation is mTORC1 dependent (Tremblay and Marette 2001; Pullen et al. 1998). Furthermore, research utilizing tuberous sclerosis complex (TSC)1-null and TSC2-null cells has revealed that AAinduced activation of S6K1 is unaffected, despite TSC2 being associated with insulin-dependent regulation of mTORC1 (Nobukuni et al. 2005; Garami et al. 2003; Smith et al. 2005). Collectively, these data provide evidence for the capacity of AA to activate S6K1 in an mTORC1-

dependent manner. In addition, other studies indicate that mTORC1 may alter insulin action in response to increased FA availability (Fig. 2A) (Crozier et al. 2003; Mordier and Iynedjian 2007; Tremblay et al. 2007; Um et al. 2004; Khamzina et al. 2005; Korsheninnikova et al. 2006). There is evidence that mTORC1 activation may induce insulin resistance in skeletal muscle and that inhibition of mTORC1 or its downstream targets may improve insulin action (Tremblay and Marette 2001). In support of such a contention, mTORC1 activation is elevated in the skeletal muscle of high-fat-fed, insulin-resistant rats (Khamzina et al. 2005). Khamzina et al. (2005) observed, in response to high-fat feeding, increased mTORC1 activation associated with elevated phosphorylation of the downstream target, S6K1, and consequently, elevated inhibitory Ser636639 phosphorylation of IRS1 in skeletal muscle. The notion that mTORC1 activation is involved in FA-induced insulin resistance is also supported by the observation that transgenic S6K1 knockout mice (S6K/) are protected against high-fat-diet-induced insulin resistance (Um et al. 2004). Enhanced insulin sensitivity in S6K/ mice is accompanied by reduced Ser636639 phosphorylation of IRS1 in skeletal muscle (Um et al. 2004). In addition, studies using rapamycin treatment show that S6K1 phosphorylation and its inhibition of insulin signal transduction was decreased, even in the presence of increased nutrient availability (Tremblay and Marette 2001; Krebs et al. 2007; Berg et al. 2002). Moreover, the down-regulation of mTORC1 activation with acute rapamycin treatment prevented the insulin resistance induced by chronically high insulin levels (Berg et al. 2002) and AA exposure in L6 myotubes (Tremblay and Marette 2001). The maintenance of insulin sensitivity in rapamycin-treated cells has been attributed to increased IRS1-associated PI3K activation (Tremblay and Marette 2001). Such an observation is of potential clinical significance because attenuated PI3K activity is associated with impairments in skeletal muscle from type 2 diabetic and obese insulin-resistant rodents (Lessard et al. 2007) and humans (Bjornholm and Zierath 2005), implicating mTORC1 as a pivotal regulator of insulin action. mTORC2: a positive regulator of insulin signal transduction via Akt It is apparent that mTORC1 has the ability to negatively regulate insulin action via its control of inhibitory actions on IRS1. The precise effect that mTORC2 may have on insulin signal transduction is not completely understood. However, studies utilizing a loss-of-function approach to inhibit mTOR complex activity by suppressing the activity of mTOR and raptor (mTORC1), but not rictor (mTORC2), prevented the activation of the mTORC1 substrate S6K1 (Thoreen et al. 2009). These results demonstrate that mTORC1 and mTORC2 are distinct complexes that have diverse cellular functions. An established role for mTORC2 is as an effector of actin cytoskeleton organization through a downstream substrate protein kinase C a (Sarbassov et al. 2004; Jacinto et al. 2004). In addition, another role for mTORC2 was revealed in its modulation of insulin action. Sarbassov and colleagues (2005b) were the first to demonstrate that mTORC2 is the kinase that phosphorylates and
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activates Akt on Ser473. In support of this finding, Hresko and Mueckler (2005) utilized siRNA-induced knockdown of rictor in 3T3-L1 adipocytes and revealed that mTORC2 is responsible for the insulin-induced Akt Ser473 phosphorylation (Hresko and Mueckler 2005). Furthermore, when rictor was deleted in skeletal muscle, there was a decrease in insulin-stimulated muscle glucose uptake, likely due to decreases in Ser473 phosphorylation and subsequent activation of Akt and its downstream substrate Akt substrate of 160 kDa (AS160) (Kumar et al. 2008). Thus, mTORC2Akt interactions are critical to our current understanding of glucose homeostasis and are an influential component of insulin action. Akt is part of the protein kinase A-protein kinase G-protien kinase C (AGC) family expressed in 2 isoforms in skeletal muscle. It has been proposed that Akt2 (in conjunction with upstream regulation by IRS1) plays a predominant role in the regulation of insulin-stimulated glucose uptake, whereas Akt1 is involved in the regulation of muscle hypertrophy and subsequent protein synthesis degradation (Cleasby et al. 2007; Kim et al. 2000). In a study examining isoform-specific activation of Akt, Cleasby and colleagues (2007) electroporated constitutively active-Akt (ca-Akt) isoforms into skeletal muscle. These researchers have shown a 200% increase in the Thr389 phosphorylation of S6K1 in the ca-Akt1 electroporated rats vs. 53% in ca-Akt2 skeletal muscle (Cleasby et al. 2007). Of note, we have used a high-fat-fed rat model of insulin resistance and observed increases in Ser473 phosphorylation of the Akt1, but not the Akt2 isoform, and subsequent elevation in Thr389 phosphorylation of S6K1 (Rivas et al. 2009). Conversely, when the high-fat-fed rats were exercise trained (treadmill running, 5 daysweek1 for 4 weeks), the increased phosphorylation of Akt1 Ser473 and S6K1 Thr389 were ameliorated (Rivas et al. 2009). Additionally, other studies have shown that Akt1, via upstream signalling events initiated by IRS2, is also an important regulator of lipid metabolism in skeletal muscle. Specifically, Bouzakri et al. (2006) identified a role for the IRS2Akt1 pathway in the regulation of basal palmitate uptake and the insulininduced suppression of b-oxidation. Taken collectively, these results indicate that mTORC2 may mediate effects on FA metabolism and glucose transport in skeletal muscle via its ability to activate Akt1 and Akt2 independently. In this regard, a dual role for mTORC2 in the regulation of both FA and glucose metabolism was proposed by Sipula et al. (2006), who found that high-dose (2472 h) treatment of L6 myotubes with rapamycin (which inhibits both mTORC1 and mTORC2) (Sarbassov et al. 2006) decreased insulin-stimulated glucose uptake and increased FA oxidation. These researchers suggested that excessive rapamycin treatment attenuated the ability of insulin to increase glucose uptake and suppress FA oxidation by inducing an inappropriate fasting metabolic phenotype in these cells (Sipula et al. 2006). However, the effects of rapamycinmediated mTORC2 inhibition on Akt activation were not examined and it is currently unknown whether mTORC2 differentially regulates activation of the Akt1 and Akt2 isoforms to mediate its effects on FA and glucose metabolism, respectively.

mTOR and exercise-induced adaptation in skeletal muscle

Exercise is one of the most effective therapeutic interventions for enhancing skeletal muscle insulin sensitivity (Hawley and Lessard 2008). A single bout of exercise increases insulin-stimulated glucose uptake for up to 48 h, predominantly in the specific muscle groups recruited during exercise (Wojtaszewski and Richter 2006). An important mechanism by which exercise training improves wholebody glucose uptake is through improved insulin action in skeletal muscle (Hawley and Lessard 2008; Wojtaszewski and Richter 2006). The exercise-induced enhancements of insulin action in skeletal muscle may occur through multiple mechanisms, including improvements in the activation and expression of components of the insulin signalling cascade, increased GLUT expression and translocation to the plasma membrane, and increased mitochondrial biogenesis. These mechanisms have been reviewed recently elsewhere and the capacity for chronic exercise training to ameliorate the harmful effect of overnutrition on skeletal muscle insulin action is undeniable (Hawley and Lessard 2008). Accordingly, we focus on the effects of exercise on mTOR signalling and its potential to enhance metabolism and promote beneficial adaptation in skeletal muscle. Given the putative roles of mTORC1 and mTORC2 in initiating mRNA translation and glucose transport in skeletal muscle, respectively, it is not surprising that mTOR activation is altered by contractile activity. Importantly, our current understanding of exercise-induced modulation of mTOR activity is limited by the technical difficulties associated with quantifying the individual mTOR complexes (12) in in vivo skeletal muscle. Consequently, much of the available data regarding responses to exercise are restricted to mTOR Ser2448 phosphorylation to infer changes in activity and function. Adding to the complexity of interpreting the effect of exercise on mTOR complex activity is an additional nutrient-independent and growth-factor-independent pathway mediated by phospholipase Dphosphatidic acid that stabilizes mTOR complex formation and is proposed to regulate the mTORC1 response to mechanical stimuli (Hornberger et al. 2006a, 2006b; Toschi et al. 2009). Moreover, when nutrients (e.g., AA) are ingested during or immediately following training, the possibility exists that nutrient-exercise pathways converge at the mTOR complexes to alter the adaptation response in skeletal muscle. Regardless, available evidence indicates that mTOR activation is associated with the exercise-induced adaptation response to a variety of contractile stimuli (Coffey and Hawley 2007). Endurance-type and resistance-type exercise has been shown to enhance insulin sensitivity and training-specific adaptation in skeletal muscle (Coffey and Hawley 2007; Hawley and Lessard 2008; Wojtaszewski and Richter 2006; Holten et al. 2004; Ishii et al. 1998; Yaspelkis 2006; Houmard et al. 2004). Similarly, acute increases in mTOR activation are observed in the hours following bouts of diverse contractile activity associated with both aerobic endurance and heavy resistance exercise (Mascher et al. 2007, 2008; Dreyer et al. 2006; Bolster et al. 2003). Indeed, we have observed similar responses in mTOR activation during the
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postexercise recovery period following bouts of endurance and resistance exercise (Coffey et al. 2009). There is a paucity of data regarding changes in mTOR activity and total protein content with chronic exercise. Benziane and colleagues (2008) have shown that the acute exercise-induced increase in mTOR activity is preserved following prolonged endurance training, but chronic endurance adaptation may suppress basal mTOR activity and subsequently enhance insulin sensitivity (Glynn et al. 2008). Conversely, basal mTOR activity may be elevated following chronic resistance exercise to promote maintenance of skeletal muscle mass and (or) attenuate degradation and (or) atrophy (Leger et al. 2006). Regardless, endurance and resistance exercise appear capable of enhancing mTORC activity, and the diverse contractile stimuli may initiate both reciprocal and independent pathways to promote specific adaptation in skeletal muscle. However, there are limitations to our understanding of mTOR complex assembly, and the extent of complexspecific activation in response to skeletal muscle contraction is unknown. It may be reasonable to suggest that differences in duration and (or) intensity, and mode of contractile activity, likely generate altered activation of mTORC1 and mTORC2 for their regulation of translation and glucose transport, respectively. Modulation of energy and fuel status with regular bouts of exercise generates elevated GLUT protein content and activ ckl et al. 2008). Moreover, a prinity in skeletal muscle (Ro cipal contraction-induced pathway important for glucose transport that is enhanced following exercise is altered GLUT4 activity via activation by the AS160 (Fig. 2B) (Kramer et al. 2006; Arias et al. 2007). In addition, Akt interactions with adaptor protein containing pleckstrinhomology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) may also regulate GLUT4 in skeletal muscle (Saito et al. 2007). Notably, despite the critical role of mTORC2 in regulating Akt activity, a causative link with subsequent Akt-AS160-mediated or Akt-APPL-mediated glucose transport following exercise has not been fully elucidated. However, given that rictor deletion impairs glucose transport (Kumar et al. 2008) and that the mTOR pathway may also regulate GLUT1-mediated glucose uptake (Buller et al. 2008), mTORC2 represents an important putative mediator of enhanced insulin action in response to exercise. Indeed, resistance exercise shown to increase acute leg glucose uptake is associated with elevated mTOR activation and Akt-AS160 phosphorylation (Dreyer et al. 2006, 2008a). Likewise, changes in Akt-AS160 signal transduction following acute and chronic endurance exercise closely correlate with enhanced glucose transport and uptake (Lessard et al. 2007; Arias et al. 2007; Frosig et al. 2007; Howlett et al. 2008). Thus, increased mTORC2 activity with resistance and endurance exercise likely promotes enhanced glucose kinetics and metabolic status. Exercise of sufficient intensity and duration disrupts homeostasis, initiating adaptive processes to generate new functional protein in skeletal muscle (Coffey and Hawley 2007). An essential process in the regulatory steps controlling protein synthesis is mRNA translation (Coffey and Hawley 2007). In this regard, mTOR has been implicated as

an upstream mediator of protein synthesis via putative control of ribosomal biogenesis and Cap-dependent translation (Nader et al. 2005; Hannan et al. 2003; Besse and Ephrussi 2008). Moreover, the translation repressor eukaryotic translation initiation factor 4E-BP1 is a direct phosphorylation target of mTORC1, which derepresses 4E-BP1 inhibition of translation initiation (Fig. 2B) (Besse and Ephrussi 2008). Intuitively, both endurance and resistance exercise would be expected to switch on translation following exercise and generate skeletal muscle adaptation, yet clearly identifying increased mTORC1 activation and subsequent 4E-BP1 phosphorylation during recovery from exercise has proved elusive. Indeed, there is limited evidence with regard to these exercise-induced phosphorylation events being associated with increased protein fractional synthetic rate (Fujita et al. 2007), likely because of the energy-consuming, catabolic state of skeletal muscle during, and immediately following, exercise in the fasted state. Nonetheless, as a nutrient sensor, it is not surprising that AA ingestion has been shown to augment exercise-induced mTOR activation and 4E-BP1 phosphorylation, and subsequent fractional synthetic rate in skeletal muscle (Koopman et al. 2007; Dreyer et al. 2008b; Drummond et al. 2008). It is apparent that chronic endurance and resistance training generate specificity of adaptation and subsequent divergent phenotypes (Coffey and Hawley 2007). As such, the concomitant increase in mTORC14E-BP1-mediated translation initiation with exercise likely contributes to the specificity of training adaptation. In support of this contention, novel findings by Wilkinson and colleagues (2008) showed increased translational signalling and fractional synthetic rate following both endurance and resistance training. Notably, these researchers observed specificity of adaptation with chronic endurance exercise only elevating the mitochondrial protein synthetic response, whereas resistance training increased the myofibrillar but not mitochondrial fractional synthetic rate (Wilkinson et al. 2008). Therefore, exerciseinduced mTORC1 translation initiation following endurance and resistance exercise may enhance skeletal muscle metabolism via alternate adaptation that promotes muscle quality (mitochondria) and quantity (cross-sectional area), respectively. Regardless, the apparent capacity of mTORC1 to promote global protein synthesis through translational processes in response to exercise is undoubtedly beneficial for the metabolic status of skeletal muscle.

Summary and future directions

The intricate, multifactorial functions of the mTOR complexes highlight the capacity of mTOR to integrate a variety of adaptive signals that alter metabolic status in skeletal muscle. It appears that mTOR may represent a good cop bad cop nutrient sensor that fine tunes the skeletal muscle metabolic response to nutrients via the dual mTORC12 complex formations. In this regard, there is accumulating evidence that mTORC1 is implicated in skeletal muscle insulin resistance with chronic overnutrition through disruption of the insulin signalling cascade, whereas mTORC2 may be a critical positive regulator of glucose uptake. Moreover, exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial
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metabolic adaptation in skeletal muscle. However, it is apparent that mTOR has undergone less scientific scrutiny than other cellular machinery regulating insulin signal transduction and metabolic health in skeletal muscle. Thus, our current understanding of the mTOR-mediated responses to overnutrition and exercise is deficient. Indeed, we acknowledge that various aspects of this review may be considered speculative given the paucity of data regarding mTOR complex assembly and function in response to overnutrition and exercise. The challenge for researchers is to elucidate the mechanistic underpinning of nutrient-excess-induced and exercise-induced responses of mTOR complex function that further our understanding of the metabolic consequences and potential countermeasures for obesity-related diseases.

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