journal of biological and chemical sciences

ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 The role of mTORC1 in controlling the growth and synthesis of skeletal muscle Oelasunkanmi A.J. Eadegoke Biomechanics Laboratory, Department of Physiology Abstract: Skeletal muscle mass and strength are critical for substrate metabolism and overall health of the organism. Diabetes, RA, and cancer are just a few of the many illnesses that have their roots in or are made worse by problems with the metabolism and functioning of the muscles. There are many environmental elements that might influence muscle health, but the two most essential ones are diet and physical exercise. Muscle mass regulation is controlled at the molecular level by the mammalian target of rapamycin complex 1, or mTORC1. Gains in muscle mass and mTORC1 activation happen simultaneously in response to dietary changes and resistance training. We review the latest research on mTORC1 and how it is regulated in skeletal muscle by resistance training, amino acid consumption, and other factors. Drugs that target mTORC1 are currently being researched or are undergoing clinical trials. This is due to the fact that increased complex activity is linked to the development of muscle insulin resistance, obesity, and certain malignancies (such as ovarian and breast tumors). On the other hand, malnutrition and tumor load contribute to the widespread muscular atrophy that is seen in many malignancies. Possible adverse effects of anticancer medications include this atrophy of muscular tissue. We discuss the potential effects on skeletal muscle of long-term mTORC1 inhibition, particularly in cases of muscle wasting, as this condition is linked with metabolic anomalies and dose-limiting toxicity. Key words: mTORC1, protein synthesis, proteolysis, resistance exercise. with protein or amino acid intake. Obesity, muscle insulin resistance, and certain cancers (e.g., ovarian, breast, and kidney) are all linked to increased mTORC1 activity, which is shown by increased phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and decreased abundance of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (Tremblay et al. 2007a; Dowling et al. 2010a; Guertin and Sabatini 2009). Consequently, complex inhibitors are either already in use or are being considered for use in the treatment of certain conditions. We go over the potential consequences of using these inhibitors over the long run, as mTORC1 is essential for muscle development. This review primarily focuses on mTORC1, mRNA translation, and muscle mass, while it has also been shown that mTORC1 regulates lipogenesis (Laplante and Sabatini 2009b). mTOR complexes Research with cells other than muscle has provided the bulk of our knowledge on the make-up and mechanisms of mTORC1 activity regulation (Fig. 1). While these systems are still crucial to current results, the fact that discoveries in other tissues and cell types have been replicated in muscle indicates that the discoveries in the past probably apply to many other cell types as According to Laplante and Sabatini (2009a), mTOR is a conserved serine threonine kinase that forms two separate complexes, mTORC1 and mTORC2. Although they do seem to communicate with one another, the two complexes are distinct in their subunit composition, regulatory mechanism, substrate selectivity, and cellular roles (Dibble et al. 2009). mTORC1 is a homodimer that includes mTOR, Raptor, PRAS40, demetors, and mLST8, which stands for mammalian lethal with Sec 13 Introduction Skeletal muscle mass and strength are crucial indicators of overall health. According to Ferrucci and Studenski (2009), congestive heart failure (CHF) is caused by or made worse by problems with skeletal muscle development and metabolism. Mafra et al. 2008), RA (Roubenoff 2009), CHD (Mafra et al. 2008), PAD (Mafra et al. 2008), cancer (Little and Phillips 2009), and HIV (Little and Phillips 2008). The skeletal muscle is essential for the transport of glucose, amino acids, and fatty acids throughout the body (DeFronzo and Tripathy 2009; Marliss and Gougeon 2002; Franklin and Which, when accumulated, may aggravate metabolic status (Marliss and Gougeon, 2002; Kanaley, 2009). According to many studies (Marliss and Gou-geon, 2002; Pilz et al., 2006), a patient's muscle mass is an indicator of their illness prognosis, hospital stay duration, and treatment results. Muscle mass and function are controlled by two major factors: food and exercise. Proteins and branched-chain amino acids stimulate muscle protein anabolism more than other macronutrients (Kimball and Jefferson 2010; Little and Phillips 2009). There is a lot of evidence that resistance training increases muscle hypertrophy and protein production (Little and Phillips 2009). The molecular level anabolic impact of resistance exercise and amino acids seems to be mediated by the nutrition or energy sensor mammalian target of rapamycin complex 1 (mTORC1) (Tanti and Jager 2009; Wacke- rhage and Ratkevicius 2008). The first part of this review is a summary of the most current research on mTORC1 and how it is regulated. We continue by going over some new information on how the complex affects the anabolism of skeletal muscles brought about by strength training, whether that's done alone or in conjunction well. Page | 1

protein 8 (also called GL) (Fig. 1). Alternatively, mTORC2 includes not only mTOR but also mSin1, Rictor, Deptor, mLST8, and the adaptor protein rapamycin insensitive companion of mTOR (Rictor). As opposed to the other components The two mTOR complexes are negatively regulated by PRAS40 (Laplante and Sabatini 2009a) and Deptor (Peterson et al. 2009). In contrast to mTORC1, which is susceptible to the immunosuppressant rapamycin, mTORC2 complexes do not exhibit this sensitivity (Dowling et al. 2010a). In addition, unlike animals without Rictor (mTORC2), those lacking Raptor (i.e., mTORC1) show decreased muscle mass (Bentzinger et al. 2008). Goodman et al. (2010) found that over-expressing the Ras homologue enriched in brain (Rheb), a mTORC1 activator (more on this later), in the tibialis anterior muscle of mice activates mTORC1 but not mTORC2. This overexpression also boosts cap-dependent mRNA translation and the cross-sectional area of the fibers. Based on these findings, mTORC1 is the primary regulator of muscle metabolism. New evidence has made it harder to distinguish between the two complexes. Even though rapamycin is said to be insensitive to mTORC2, Sarbassov et al. (2006) found that mTORC2 assembly and function may be suppressed by extended incubation with the medication. Both mTORC1 and 2 are favorably regulated by a variety of mTOR interacting partners, such as Tel2 in-teracting protein 1 (Tti1) (Kaizuka et al., 2010) and Ras-related C3 botulinum toxin substrate 1 (Rac 1) (Saci et al., 2011). Also, contrary to what was once thought, there is evidence that amino acids activate mTORC2 and mTORC1 (Tato et al., 2011; Oh et al., 2010; Tato et al., 2011). In addition, both mTORC1 and mTORC2 are activated when insulin or amino acid stimulation targets mTOR to intracellular membranes (Saci et al. 2011). As a result, mTORC2 probably isn't solely responsible for the multiple roles that mTORC1 is thought to perform. Regulation of mTORC1 signalling The control of the complex by amino acids and growth hormones (insulin-IGF-1) has been the most extensively investigated, however mTORC1 activity may be regulated by DNA damage, energy (ATP), and oxygen levels (Sengupta et al. 2010; Wang and Proud 2011). Step one: insulin or IGF-1 activation Insulin and IGF-1 bind to their receptors, which trigger the activation of the AKT (protein kinase B, PKB) pathway). When AKT is activated, it phosphorylates the tuberous sclerosis complex (TSC1/2), which in turn promotes ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 mTORC1. The phosphorylation of TSC1/2 blocks its ability to inhibit Rheb, a mTORC1 activator (Laplante and Sabatini 2009a). Additionally, cyclic AMP (cAMP), a second messenger, promotes Rheb-mTOR interaction, which in turn activates mTORC1 (Kim et al. 2010). The processes by which Rheb stimulates mTORC1 when linked to GTP are unclear. Sengupta et al. (2010) found that AKT activates mTORC1 via phos-phorylating PRAS40, which inhibits the inhibitory binding of PRAS40 to mTOR. Not to mention In contrast to the traditional PI3K-AKT growth factor stimulation of mTORC1, Rac1 may activate mTORC1 and mTORC2 without PI3K involvement (Saci et al. 2011). In conclusion, Mitogen-activated protein kinase 1 (RAS) is another protein that has been linked to mTORC1 activation. Raptor is phosphorylated on serine 8, 696, and 863 by the extracellular signal regulated kinase (ERK1/2). Raptor Figure 1: The two mTOR complexes. In paths where the mechanisms of action are unclear, dashed arrows are used. Forms with solid lines indicate components with several lines of evidence or shown activity in skeletal muscle or muscle cells; forms with dashed lines do not have established importance in muscle. Inhibitors of mTORC1 or mRNA translation are indicated by gray backgrounds. The material delves into more topics related to mTORC1 activators or substrates. 4E-BP1, death-associated protein 1, DEP- domain-containing mTOR-interacting protein, eIF4A, eIF4B, eIF4E, eIF4F, eIF4G, G L, and other acronyms for eukaryotic translation initiation factors acronyms for hypoxia inducible factor 1a, inositol polyphosphate multikinase, insulin receptor substrate 1, mitogen-activated protein 4- kinase 3, mSin1, programmed cell death protein 4, and proline-rich PKB substrate 40 kDa; Proctor-1, a protein associated with Rictor-1; Rac 1, a gene for recombination activation; and Rag, a protein connected to C3 botulinum toxin substrate 1. Rheb, a Ras homologue abundant in the brain; Raptor, a protein linked with mTOR regulation; The acronyms STAT-3, ribosomal protein S6, ribosomal protein S6 kinase 1, rapamycin insensitive partner of mTOR, tuberous sclerosis complex, and a host of other acronyms The abbreviations Tti1, Ulk1, Vps34, and YY-1 stand for yin yang protein 1, vacuolar protein sorting 34, and Tel2 interacting protein 1, respectively. As out by Carriere et al. (2011), mutant at these places cannot communicate with mTORC1 substrata 4E-BP1 and S6K1. Hence, insulin may activate mTORC1 via many mechanisms at once. Amyotrophic activation There are six distinct pathways via which amino acids, particularly arginine and leucine (Hara et al., 1998), communicate with mTORC1. The binding of calmodulin to hVps34, a member of the class III PI3K family, is enhanced Page | 2

when amino acids enter cells, which causes a rise in intracellular Ca2+. An upregulation of phosphatidylinositol 3-phosphate (PI3P) levels follows, which activates mTORC1 via processes that are not yet fully understood (Gulati et al. 2008). Additionally, according to Sengupta et al. (2010), mTORC1 may be activated by amino acids via attaching to the RAG protein family, which is a tiny guanosine triphosphatase (GTPase). Through a heterotrimeric complex, RAG proteins bring mTORC1 to lysosomal membranes. The Ragulator is a protein complex. The complex is activated because mTORC1 activator Rheb is also present on this membrane. Third, researchers have shown that mTORC1 is connected with mostly perinuclear lysosomes and that the intracellular pH rises from 7.0 to 7.7 when growth factors and amino acids are removed from cells that are not muscle cells. Nutrient addition reduces pH and reintroduces mTORC1 to plasma and other peripheral membranes. Growth factors and amino acids may control mTORC1 via altering intracellular pH, as altering intracellular pH impacts mTORC1 interaction with peripheral membranes and mTORC1 activity, even when no changes are made to the surrounding diet (Korolchuk et al., 2011). Fourth, in order for amino acids to signal to mTORC1 in human embryonic kidney cells, they must phosphorylation and activation (Yan et al. 2010). It is worth noting that the fifth point pertains to the lipid kinase inositol poly- By maintaining the connection between Raptor and mTOR, phosphate multikinase (IPMK) may facilitate amino acid signaling to mTORC1 (Kim et al. 2011). Loss of IPMK in cells impairs their ability to signal to mTORC1 in response to amino acids. The Ras superfamily member Ra1A can finally modulate the signaling of amino acids to mTORC1 (Maehama et al. 2008). When exposed to amino acids, hela cells that lack Ra1A are unable to properly phosphorylate S6K1 and 4E-BP1. Therefore, there may be more than one mechanism at work when it comes to the impacts of amino acids. The six hypothesized processes seem to be related to one another. Amino acid stimulation of mTORC1 via the hVps34, Rag-Ragulator, and Ra1A pathways is reliant on Rheb. Additionally, mTOR is localized to an endomembrane compartment via the hpVs34 and Rag-Ragulator pathways. Last but not least, MAP4K3 signaling to mTORC1 is inhibited by RAG protein reduction, suggesting that the RAG proteins are essential for MAP4K3 signaling (Yan et al. 2010). The relevance of these processes in skeletal muscle metabolism is unclear since many of these results were obtained in cells or tissues that are not muscle. without ambiguity. Research on piglets has shown that increased skeletal muscle protein synthesis is linked to higher levels of RagB and RagB's binding to Raptor. Suryawan and Davis (2010) found that neither insulin nor ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 amino acid infusion affected RagB or its interaction with Raptor, suggesting that the observed changes in this RAG protein might not be mediating the effect of these factors on muscle protein synthesis. In a different research, Kazi et al. (2011) found that myotubes protein production and 4E-BP1 and S6K1 phosphorylation were both enhanced when Deptor was knocked down. Consequences of mTORC1 activation The activation of mTORC1 by amino acids or growth factors leads to several cellular events, such as the promotion of ribosome biogenesis, prevention of apoptosis, and stimulation of mRNA translation (Laplante and Sabatini 2009a). Cell size and cell number are both enhanced as a result of this. According to Laplante and Sabatini 2009a and Ma and Blenis 2009, these outcomes are caused by mTORC1 acting on its substrates, particularly 4E-BP1 and S6K1. One of mTORC1's other functions is to control the transcription of genes that code for ribosomes. To inhibit RNA polymerase III in reaction to serum deprivation, mTORC1 is necessary for the inactivation of MAF1. Genes involved in ribosome production cannot be transcribed without RNA polymerase III. As previously shown by Kantidakis et al. (2010), Michels et al. (2010), and Shor et al. (2010), mTORC1 inhibits MAF1 in the nucleus by phosphorylating it on several serine residues, with particular emphasis on serine 75. Both growth factor and amino acid stimulation may phosphorylate MAF1 (Shor et al., 2010) and recruit mTORC1 to areas inside genes that code for promoters (Shor et al., 2010; Tsang et al., 2010). The role of mTORC1 in controlling miRNA is the last point to be discussed. In order to control gene expression, this family of short RNAs binds to the 3 end of potential messenger RNAs, which either stops translation or makes the target mRNA unstable (Braun and Gautel 2011). One such microRNA that has been linked to myogenesis regulation is miR-1, which is exclusive to muscles. The transcriptional regulator mTORC1 treatment with rapamycin decreases the quantity of this miRNA in myotubes, as shown by Sun et al. (2010). The autophagy inhibitor death-associated protein 1 (DAP1) is inactivated when mTORC1 phosphorylates it, regulating autophagy in human embryonic kidney and Hela cells (Ko-ren et al. 2010). Activation of DAP1 in the absence of mTORC1 is consistent with the cell's attempt to restrict autophagy in times of inadequate energy or nutrient supply, however this seems to contradict the anti-autophagy actions of mTORC1. A second substrate of mTORC1 that is associated with autophagy is Unc- 51-like kinase 1 (Ulk1). Ulk1 is phosphorylated at many serine residues, including serine 638 and 758, when nutrients are abundant, by mTORC1. Nevertheless, Ulk1 is dephosphorylated in the midst of food withdrawal, allowing it to initiate autophagy in murine fibroblasts (Shang et al. 2011). Restoring lysosomal homeostasis (i.e., re-generation of free lysosomes to a pre- autophagy level) is essential even while starvation persists, as first trigger MAP4K3 Page | 3

shown by Yu et al. (2010), even if autophagy in kidney cells is enabled by mTORC1 inhibition during starvation. Reactivation of mTORC1 after prolonged (>4 h) deprivation is caused by autophagy breakdown products, which reactivate the complex. In conclusion, autophagy is enhanced by mTORC1 inactivation, but equilibrium is restored when products of this cellular degradation process reactivate mTORC1. According to Cunningham et al. (2007), Yang 1 (YY1) is a transcriptional factor that controls the expression of genes related to mitochondria and their oxidative activities. Hypoxia inducible factor 1 alpha (HIF-1a), serum-and glucocorticoid- induced protein kinase 1 (SGK1), and signal transducer and activator of transcription 3 (STAT3) are other proposed substrates of this complex (Lap-lante and Sabatini 2009a). The control of these additional mTORC1 substrates in skeletal muscle has not been published yet, with the exception of YY1, whose regulation was first characterized in muscle cells (Cunningham et al. 2007), and HIF-1a, which has been shown to block IGF-1-induced myogenesis (Ren et al. 2010). However, mTORC1's effects on 4E-BP1 and S6K1 have been the most thoroughly studied in relation to its skeletal muscle activities (Ma and Blenis phosphorylates 4E-BP1, it frees eIF4E from 4E-BP1's inhibition, which in turn promotes eIF4F complex formation and mRNA translation. According to Dowling et al. 2010b and Risson et al. 2009, mTORC1-induced hypertrophy seems to be S6K1 dependent, with 4E-BPs mediating the influence on cell proliferation. Phosphorylation of eIF4B, which enhances eIF4A's helicase activity (Holz et al., 2005), ribosomal protein S6, which is believed to promote translation of a specific class of mRNAs (Jefferies et al., 1997), and phosphorylation of the mRNA translation inhibitor programmed cell death 4 (PDCD4) (reviewed in Laplante and Sabatini 2009a) are all potential pathways through which phosphorylation of S6K1 causes increased translation. Though much of what is known about PDCD4 comes from cells other than muscle, it has been shown that PDCD4 protein accumulates in starving rats' skeletal muscle. According to Zargar et al. (2011), its level decreases when animals are re-fed. Taken together, these findings suggest that mTORC1 effectors are involved in their stimulation of muscle protein synthesis, although the specifics of how they work are yet unclear. mTORC1 and skeletal muscle intracellular proteolysis The control of protein synthesis and proteolysis, two opposing continuous processes, leads to a change in muscle protein mass. The skeletal muscle intracellular proteolysis process includes (i) the ubiquitin system, which is reliant on ATP and proteasomes, (ii) the autophagy-lysosomal route, which is dependent on Ca2+ proteases, and (iv) apoptosis. Among them, research on the ubiquitin system predominates. The activity of three enzymes ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin protein ligases (E3) sequentially occurs in this pathway to conjugate protein substrates to ubiquitin (Ciechanover 2005; Hershko 2005). The 26S proteasome complex subsequently polyubiquitinated substrate and degrades it. Proteolysis increases in tandem with ubiquitination levels and increases in the expression (mRNA, protein) of the E2, E3, and other proteasome subunits (Jagoe and Goldberg 2001). Muscle Atrophy F-box (MAFbx), also known as Atrogin-1, and Muscle RING Finger 1 (MuRF1) are two muscle-specific ubiquitin protein ligases; their existence highlights the ubiquitin system's role in controlling skeletal muscle proteolysis (reviewed in Glass 2005). According to Cohen et al. (2009), MuRF1 ubiquitinates myofibrillar proteins in muscle cells and targets them for destruction in skeletal muscle that is atrophying. Additionally, blocking the ubiquitin system attenuates muscle proteolysis in various catabolic circumstances (Jagoe and Goldberg 2001). Additionally, in catabolic conditions, there is an increase in the activity of lysosomal enzymes (cathepsins) and autophagy (Ventadour and At-taix, 2006; Mammucari et al., 2008). Further, decreased muscle mass may be caused by an increase in apoptosis (Ferreira et al. 2008). The subject of whether mTORC1 controls proteolysis in muscles is a significant one. The capacity of activated mTORC1 to suppress autophagy is a major piece of evidence that implicates the complex (Jung et al. 2010). Even while autophagy plays a crucial role in lysosomal proteolysis, it is proteolysis of muscle tissue seen under hypoxia, malnutrition, and gluco- with the administration of corticosteroids to rats, autophagy activation is only weakly rapamycin-sensitive (Mammu-cari et al. 2008; Ogata et al. 2010; Schak-man et al. 2009). The function of mTORC1 in ubiquitin system regulation has been the subject of little research. Research using C2C12 myotubes found that arginine, atrogin-1, and isoleucine all worked together to decrease MuRF1 and Atrogin-1 production. According to Herningtyas et al. (2008), rapamycin reduces the suppression of Atrogin-1 but has no effect on MuRF1. Lean mass loss, decreased muscle protein synthesis, and increased mRNA expression of Atrogin-1 and MuRF1 are all seen in mTOR heterozygous mice (Lang et al. 2010). To determine if mTORC1 is necessary for the control of proteolysis in skeletal muscle, further evidence is obviously required. mTORC1 and the process of resistance exercise-induced anabolism We shall address this issue by asking three questions: During skeletal muscle anabolism generated by resistance recognizes the 2009). When mTORC1 Page | 4

training, are the mTORC1 activators and downstream effectors modulated? (ii) When these regulators or effectors are blocked, pulled down, or eliminated, what happens to the exercise-induced anabolism? (iii) Can resistance exercise induce skeletal muscle anabolism be abolished by inhibiting mTORC1 or by knocking down or knocking out mTOR? While human subjects are included in the following discussion, the majority of our knowledge on the functions of mTORC1 in exercise-induced muscle hypertrophy originates from research conducted on rats and in cell cultures. Studies that examine the necessity of mTORC1 for a certain trait frequently involve genetic techniques, which are not possible in humans, unless mTORC1 inhibitors are used. Although cell culture and rodent models cannot replace human subjects, they may nonetheless provide testable hypotheses. Strength training and mTORC1 upstream activators In rodents, hypertrophy caused by functional overload occurs at the same time as an increase in phosphorylation of AKT serine 473 and mTOR serine 2448 (Spangenburg et al. 2008; Reynolds et al. 2002). Resistance training in boosts AKT phosphorylation and, within one to two hours after exercise, increases protein synthesis in human muscles (Dreyer et al. 2008). Nevertheless, when rats' EDLs are stimulated electrically, it causes according to O'Neil et al. (2009), Wortmannin, an inhibitor of the PI3K-AKT pathway, has no effect on the phosphorylation of the mTORC1 substrate p70S6K1. Therefore, whereas resistance training does enhance AKT activation, whether or not this is essential for mTORC1 activation remains unclear. According to Fang et al. (2001), phosphatidic acid (PA), which is produced by phospholipase D (PLD), acts as an upstream positive regulator of insulin-activated signaling to mTORC1. At different periods after stimulation begins, mechanically stimulated muscles in rats activate PLD and collect PA (Hornberger (O'Neil et al., 2009; et al., 2006). Researchers Hornberger et al. (2006) and O'Neil et al. (2009) found that blocking PLD with 1- butanol or neomycin prevented PA buildup and inhibited mechanically induced mTORC1 signaling. The experiments listed did not measure protein synthesis, hence it is unclear whether inhibiting PA generation during recycling Reducing the effects of exercise on protein synthesis and muscle hypertrophy is another benefit of resistance training. The AMP-activated protein kinase (AMPK) is another mTORC1 upstream (negative) regulator that is activated by exercise. In a research conducted on humans, it was shown that vastus lateral muscle showed an increase in AMPKa2 activity and a suppression of protein synthesis and mTOR phosphorylation immediately after resistance exercise. Protein synthesis and mTOR phosphorylation were both increased immediately after exercise, whereas AMPK activity persisted for at least another hour (Dreyer et al. 2006). The modulation of ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 this kinase was not shown in another investigation including resistance training in individuals who engage in leisure physical activity (Hulmi et al. 2009). Recent investigations have shown that AMPK is involved in exercise-induced muscle anabolism, and that this impact is at least partially connected to mTORC1. the number of myotubes is larger when AMPK is absent or when the muscle of AMPK-deficient mice is examined (Mounier et al., 2009; Lantier et al., 2010). When rapamycin is present, the AMPK-deficient myotubes' enlarged size is reduced. According to Lantier et al. (2010), once AMPK is knocked out or knocked down, continued activation of AKT and, by extension, mTORC1, does not result in any additional anabolism. Along with larger muscles, the research found that muscles from AMPK knock-out animals had resistance activity- induced muscular anabolism that was about 20% higher. This was accompanied by stronger activation of mTORC1, as reported by Mounier et al. in 2009. Another protein that acts as a negative regulator of mTORC1 is hypoxia inducible protein REDD1 or RTP801 (Brugarolas et al. 2004). The role of REDD1 in mediating resistance exercise- induced muscle hypertrophy has not been shown, despite the fact that REDD1 levels are elevated in humans after low- intensity resistance training (Drummond et al. 2008a). Working out with resistance and the molecules that mTORC1 regulates An increase in phosphorylation of S6K1, 4E-BP1, and mTOR occurs during resistance exercise in humans, which may be accompanied by an increase in muscle protein synthesis and fiber size (Tannerstedt et al. 2009; Witard et al. 2009). Thirty to sixty minutes after recovery is when phosphorylation of these proteins peaks. from a fully extended leg position with a maximum of one repetition (Camera et it will be premature to see changes in protein syn-thesis (al. 2010). Muscles made mostly of oxidative fibers also exhibit a reaction, according to rat studies (Agata et al. 2009; Miyazaki et al. 2008), however the effects are more pronounced in muscles composed of type II fibers (Par-kington et al. 2003). Mayhew et al. (2009) found that acute and chronic resistance workouts boosted muscle protein synthesis and phosphorylation of AKT, mTOR, S6, and 4E-BP1 in a group of young and elderly volunteers, whose ages ranged from 28 to 64 years. In untrained people, resistance training increased protein synthesis by around 68% for myofibrillar and sarcoplasmic proteins, but it increased protein synthesis for myofibrillar protein family by 36% after training. In endurance exercise, on the other hand, had no effect on training status and merely increased mitochondrial protein production. Wilkinson et al. (2008) also found that mTORC1 signaling pathway components were phosphorylated, but that training type had no influence on this parameter. Consumption of whey proteins, a solution containing leucine- enriched essential amino acids and carbohydrates, or a diet Page | 5

containing casein protein and carbohydrates all enhance resistance exercise-induced increases in human muscle protein synthesis and regulation of mTORC1 and its sub-strates. Drummond et al. 2008b investigated the rates of protein synthesis in muscle at various intervals following resistance exercise and amino acid consumption in human volunteers of varying ages. There was no change in performance one hour after exercise. Protein synthesis was enhanced by exercise and amino acid consumption in both groups 3 hours after exercise, but after 6 hours, the effect was the same in the young people. Results showed that treatment-induced phosphorylation of Protein synthesis variations were not correlated with mTOR, S6K1, or 4E-BP1. Therefore, the exact relationship between changes in muscle protein synthesis and signaling proteins is not well understood, even though they often occur simultaneously. Is mTORC1 essential for the anabolism of skeletal muscle that resistance training induces? Research that aimed to answer this topic employed two main methods: using drugs that block mTOR or mTORC1, and (ii) using knockouts of mTOR or mTORC1 components in entire animals or particular tissues. Rapamycin, an allosteric inhibitor of mTOR, is the medication of choice for inhibition experiments. Research conducted by Fluckey et al. (2006) and Kubica et al. (2005) in rats has shown that resistance exercise-induced stimulation of muscle protein synthesis requires mTORC1 activation. Consistent with these results, rapamycin inhibits the effects of 2-week repetitive stretching on rat denervated soleus muscle atrophy and on S6K1 phosphorylation (Agata et al., 2009). In addition, Wortmannin or rapamycin pre-treatment of myotubes reduces the hypertrophy induced by passive cyclic stretching in cell culture (Sasai et al. 2010). Surprisingly, rapamycin, a mTORC1 inhibitor, exhibits more inhibition than PI3K, an inhibitor. There was no measurement of protein synthesis in those two experiments, but the findings still point to mTORC1 as a key signaling node in the hypertrophy caused by contractions. In line with the results of the rapamycin research, it has been shown that muscle or muscle cell size is reduced when S6K1 is not present (Ohanna et al., 2005) and when Raptor is specifically knocked out in muscle (Bentzinger et al., 2008). Furthermore, fast-twitch muscles in particular show atrophy due to muscle-specific mTOR deletion in mice, with weights around 20% lower than control. Loss of muscular mass completely explains why knockout animals are much leaner. A smaller cross-sectional area of muscle fibers is another consequence of reducing muscle mass (Risson et al. 2009). Muscle specific mTOR knockouts are more severe than Raptor knockouts or combined knockouts of Raptor and Rictor, which raises the intriguing possibility that contributions to muscle metabolism beyond mTORC1 and 2. An finding that was not observed in the muscle-specific Raptor knockdown was a reduction in force output in muscles missing ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 mTOR, which is relevant to contractile activities. The role of mTOR or mTORC1 in controlling muscle size and function is so evident. There hasn't been a research that we're aware of that investigates how resistance exercise affects muscle mass and metabolism in animals deficient in mTOR or other mTORC1 components or substrates. The only one that comes to mind is the investigation of responses to electrical stimulation. mTORC1 and the regulation of intracellular proteolysis in response to resistance exercise Consistent withearlierreports(Glass2005),during4 8 days of rat leg immobilization, increased activities of the ubiquitin system and apoptosis were recorded, which were reversed as the animals were allowed to move around from days 9 to 40 (Vazeille et al. 2008). The time course of changes in the component of the ubiquitin system, rather than of apoptosis, mirrored the changes in muscle mass during both immobilization and recovery phases. In another study, during muscle recovery following 14-day hind limb unload- ing, soleus muscle weight and fibresize were restoredby day 5 of recovery. In that study, mRNA levels of m-calpain, atrogin-1 or MAFbx, and MuRF1, which were elevated 50% 70% during unweighting, returned to baseline by 1 day of re- covery (Andrianjafiniony et al. 2010). Results from human studies are divided, perhaps because of the different end- points measured. Some labs did not see substantial changes in markers of proteolysis during leg immobilization (Glover et al. 2010; Phillips et al. 2009). In contrast, but consistent with rodent studies (Bodine et al. 2001; Glass 2005; Reid 2005), another lab showed 400% increase in 3-methyl histi- dine release (a measure of myofibrillar proteolysis) and in Atrogin-1 and MuRF1 mRNA in vastus lateral muscle fol- lowing 72 h of lower limb suspension (Gustafsson et al. 2010; Tesch et al. 2008). Because of lack of agreement amongst data from different laboratories, more studies are needed to ascertain the contribution of altered proteolysis to human protein anabolism during muscle re-loading following a period of unloading. In addition, studies that employ inhib- itors of mTORC1 will be needed to ascertain the significance of this pathway in regulating proteolysis in response to un- loading and resistance exercise. mTORC1 inhibitors metabolism In spite of its significance in regulating muscle growth, overactivation of mTORC1 is implicated in human and ro- dent models of obesity, in muscle insulin resistance (Newgard et al. 2009; Tremblay et al. 2007b), and in cancer(Guertin and Sabatini 2009). The link to insulin resistance is because activated mTORC1 S6K1 phosphorylates insulin receptor substrate 1 (IRS-1) on multiple serine residues, an event that destabilizes IRS-1 and negatively impinges on its signalling to PI3K. In addition, the growth factor receptor-bound pro- tein 10 (Grb-10) is phosphorylated by activatedmTORC1, and phosphorylated Grb-10 mediates inhibition of PI3K and ERK- elevations in and skeletal muscle mTOR mediates additional Page | 6

ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 MAPK pathways (Hsu et al. 2011; Yu et al. 2011). Hu- man cancers, including ovarian, breast, and kidney, are asso- ciated withaberrantactivationofPI3K mTORC1pathway (Guertin and Sabatini 2009). Several inhibitors of mTORC1 are either in clinical trials or are approved for treatment of mainly cancers. These inhibitors include rapamycin and its derivatives (Fig. 2): sirolimus, everolimus, and AP-23573 (Dancey 2005). While some beneficial anti-tumour effects have been described, the efficacy of rapamycin and rapalogs has been less than anticipated (Guertin and Sabatini 2009). This suboptimal performance is attributed to the loss of neg- ative feedback control that activation of mTORC1 confers on the IRS-1 PI3K AKT pathway. Upon rapamycin treatment, mTORC1 is inhibited and IRS-1 signalling is restored, lead- ing to the activation of IRS-1 PI3K AKT signalling, a path- way that is implicated in abnormal cell proliferation. To circumvent this, a number of active site inhibitors of mTOR have been developed (Fig. 2). This class targets the kinase activity of mTOR and therefore will inhibit both mTORC1 and 2. These second generation inhibitors, including Torin 1 Fig. 2. Inhibitors of mTOR. The inhibitors fall into 2 classes: allos- teric and active site inhibitors. Note that the latter class inhibits the 2 mTOR complexes. Please see text for a complete list of the inhibi- tors. See Fig. 1 for definitions. mTORC2 Rac 1 G L mTOR Deptor PRAS Raptor 40 (Thoreen et al. 2009), PP242 and PP30 (Feldman et al. 2009), Ku-0063794 (Garc a-Mart nez et al. 2009), AZD8055 (Chresta et al. 2010), and WYE-354 (Yu et al. 2009), inhibit the phosphorylation of substrates of mTORC1 (S6K1 and 4E-BP1) and of mTORC2 (AKT and SGK). They also sup- press global protein synthesis in tumour cells with better po- tency than rapamycin, and WYE-354 inhibits tumour growth in rodents while AZD8055 inhibits growth and (or) promotes regression in xenografts. For the reasons already mentioned, inhibition of mTORC1 will likely lead to an impairment of the regulation of skeletal muscle mass and metabolism. In this regard, mice over- expressing human TSC1 (a negative regulator of mTORC1) are smaller in size, have TA and EDL muscles that are 23% smaller, and an 18% reduction in muscle fibre com- pared with wild-type animals (Wan et al. 2006). This sug- gests that prolonged mTORC1 inhibition may negatively affect skeletal muscle mass, especially given the fact that muscle protein metabolism is impaired in many cancers. Extensive skeletal muscle wasting is a feature of cachexia, a condition often seen in cancer patients (Dodson et al. 2011; Prado et al. 2011). Cachexia can be attributed to malnutrition and hypermetabolism (Prado et al. 2011), and elevated circu- lating levels of catabolic inflammatory factors (Dodson et al. 2011; Eley et al. 2008). In addition, muscle wasting is an un- desirable side effect of chemotherapy. For example, renal cancerpatientstreatedwithsorafenib,the antiangiogenic multikinase inhibitor that targets PI3K AKT pathway, lose 5% to 8% of their skeletal muscle mass, compared with pa- tients in the placebo group (Antoun et al. 2010b). Another study by the same group shows that dose-limiting toxicity (DLT) highly correlates with loss of lean mass inpatients with metastic renal cancer patients treated with sorafenib, with DLT rate being 28% higher in sarcopenic vs. non-sarcopenic patients (Antoun et al. 2010a). In addition, lean body mass is a good predictor of adverse drug toxicity in cancer patients Allosteric Inhibitors Rapamycin Everolimus Sirolimus Tti 1 Rac 1 mSin 1 G L Rictor Tti 1 mTOR Deptor Proctor-1 mTORC1 Active site Inhibitors Torin 1 PP242 PP30 examined the long-term effect of mTOR inhibitors on muscle metabolism. Given the data from rodent mTORC1 knockout studies, muscle wasting is likely a consequence of long-term use of rapamycin and rapalogs. In this respect, doses of ever- olimus that inhibit tumour growth in mice models of human tumours cause weight loss (O Reilly et al. 2011). treated with diverse chemotherapies (Prado et al. 2007, 2009). Thus, chemotherapy increases loss of lean mass and the resulting sarcopenia increases the risk of drug toxicity in diverse cancer groups treated with different drugs. Given the established significance of mTORC1 in regulat- ing muscle mass and the frequency with which impairments are observed in muscle protein content and mass in cancer patients, whatever successes that are achieved in reducing tu- mour burden by the use of mTOR inhibitors will possibly be associated with loss of muscle mass. No patient studies have Summary Recent findings on the composition and regulation of mTORC1 have broadened our understanding of the link be- tween the activation of this complex and specific cellular out- Page | 7

ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 comes. However, many of these studies were conducted in non-muscle cells. Recent studies on the effect of resistance exercise suggest a link between the activation of mTORC1 and exercise-induced regulation of muscle protein synthesis. The significance of intracellular proteolysis during muscle loading and unloading is less clear: the role of mTORC1 in this process has received little attention. The few studies that are available suggest that long-term inhibition of mTORC1 may lead to undesirable side effects in skeletal muscle. References The authors of this 2009 work are Agata, Sasai, Kawakami, Hayakawa, Kobayashi, and Sokabe, with the assistance of Inoue-Miyazu. Rats' soleus muscles are protected against denervation-induced atrophy when subjected to repetitive stretching. The article may be found in the journal Muscle Nerve with the citation 39(4): 456-462. Published online: 19260063, This sentence is a citation for a 2010 paper by Andrianjafiniony, Dupre-Aucouturier, Letexier, Couchoux, and Desplanches. Repair of skeletal muscles during deloading: oxidative stress, cell death, and proteolysis. Published in the American Journal of Physiology, Cell Physiology, 299(2), pages C307 C315. Accessed 20505039 times. doi:10.1152/ajpcell. 00069.2010. doi:10.1002/mus.21103. Concluding comments Since some functions of mTORC1 are rapamycin-insensitive (Thoreen et al. 2009), studies using drugs thatcaninhibit the kinase activity of mTOR are needed to answer questions relating to the essentiality of mTORC1 in regulating muscle anabolic response to resistance exercise. In addition, de- tailed studies are needed to examine the implication of long-term inhibition of mTOR (in the context of mTORC1 or 2) on muscle mass, as this will have implication for treatment outcome and overall patient health. An alternative might be to target inhibition of mTORC1 to tissues and cells other than skeletal muscle. It will also be useful to ex- amine whether resistance exercise amelioratesmusclewast- ing associated with chemotherapy. Data from studies employing genetic approaches suggest that inhibiting mTORC2 may block tumour development without the side effects associated with mTORC1 inhibition. For example, deletion of Rictor has no effect on normal pros- tate cells, but Rictor is required for transformation induced by PTEN loss (Guertin and Sabatini 2009). If the observa- tions made in prostate cancer hold true in other cancers, and since deletion of Rictor, unlike that of Raptor, has no effect on muscle metabolism (Guertin and Sabatini 2009), inhibit- ing mTORC2 may represent an attractive intervention that will limit or prevent tumour growth while having no negative effect on muscle metabolism. In their 2010a publication, Antoun et al. cite the work of Baracos, Birdsell, Escudier, and Sawyer. Sarcopenia and low body mass index linked to sorafenib's dose-limiting effects in renal cell cancer patients. Notice of Cancer 21(8): 1594-1598. Publication date: 20089558. DOI:10.1093/annonc/ mdp606. Researchers Antoun, Birdsell, Sawyer, Venner, Escudier, and Baracos published their findings in 2010b. Link between sorafenib and skeletal muscle atrophy in advanced renal cell carcinoma patients: findings from a randomized, placebo- controlled trial. The citation for this article is "Journal of Clinical Oncology" (JCO.2009.24.9730), and it is located at 28(6): 1054-1060. 20085939 the number. PMID: In 2008, a group of researchers including Bentzinger, Romanino, Cloetta, Lin, Mascarenhas, and Oliveri published a paper. Muscle degeneration and metabolic alterations are the outcomes of raptor-specific skeletal muscle ablation, as opposed to rictor-specific ablation. Metabolic Cell 8(5): 411 424. Publication date: 19046572, cmet.2008.10.002. In 2001, Bodine et al. were joined by Baumhueter, Latres, Lai, Nunez, and Clarke. Skeletal muscle atrophy-related ubiquitin ligases have been identified. Article published in Science, 294(5547): 1704 1708. PMID: 11679633. doi:10.1126/science.1065874. Regulatory pathways of transcription in skeletal muscle development, maturation, and homeostasis. Braun, T., & Gautel, M. 2011. Nature Reviews: Molecular Cell Biology, 12(6), 349-361. PMID: 21602905, doi:10.1038/nrm3118. In 2004, Brugarolas et al. were joined by Lei, Hurley, Manning, Reiling, Hafen, and Brugarolas. The hypoxia- responsive REDD1 and TSC1/TSC2 tumor suppressor complex regulates mTOR activity. Publication: Genes Dev., volume 18, issue 23, pages 2893 2904. DOI: 10.1101/gad. DOI: 10.1016/j. Page | 8

ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 In 2010, the authors Camera, Edge, Short, Hawley, and Coffey published a paper detailing their findings. Time series analysis of Akt phosphorylation after resistance and endurance training. Medical Science and Exercise 42(10): 1843 1852. doi:10.1249/MSS.0b013e3181d964e4. In 2011, Carriere, Romeo, Acosta-Jaquez, Moreau, Bonneil, Thibault, and Thibault were among the authors. mTOR complex 1 (mTORC1) may be activated Ras-dependently by ERK1/2 phosphorylation of Raptor. Scientific American 286(1): 567-577. PMID: M110.159046. A 2009 publication by Dibble, C.C., Asara, J.M., and Manning, B.D. Investigating the phosphorylation locations on Rictor showed that S6K1 directly controls mTOR complex 2. Published in the journal Mol. Cell. Biol., volume 29, issue 21, pages 5657 5670. doi:10.1128/MCB.00735-09. PMID:20195183; PubMed:19720745; In 2011, Dodson, Baracos, Jatoi, Evans, Cella, Dalton, and Steiner published a paper. The loss of muscle mass due to cancer cachexia: new diagnostic tools, therapeutic options, and potential clinical consequences. Annual Review of Medicine 62(1): 265-279. 2014. PMID: 20731602; doi:10.1146/annurev-med-061509-131248. 21071439. doi:10.1074/jbc. In 2010, Chresta et al. had the following affiliations: Davies, B.R., Hickson, I., Harding, T., Cosulich, S., Critchlow, S.E. AZD8055 has anticancer effects in both vitro and in vivo; it is an ATP-competitive mammalian target of rapamycin kinase inhibitor; and it is orally accessible. The citation is from Cancer Research, volume 70, issue 1, pages 288 298. The publication (doi:10.1158/0008-5472.CAN-09-) MEDLINE: 20028854 (1751). In 2010a, Dowling, Topisirovic, Fonseca, and Sonenberg published a paper. Analyzing mTOR's function: insights from mTOR inhibitors. Publishing information: Biochim. Biophys. Acta, 1804(3): 433-439. PubMed: 20005306. The 4E-BPs regulate mTORC1-mediated cell proliferation but not cell growth (Dowling et al., 2010b). Publication: Science, volume 328, issue 5982, pages 1172-1176. PMID:20508131. doi:10.1126/science.1187532. "Proteolysis: from the lysosome to ubiquitin" (Ciechanover, 2005). together with proteasomes. Scientific Reports, Volume 6, Issue 1, Pages 79 87, DOI:10.1038/nrm1552, PubMed: 15688069. In 2009, Cohen et al. were joined by Brault, Gygi, Glass, Valenzuela, Gartner, and others. Muscle atrophy is accompanied by the degradation of thick filament components by MuRF1-dependent ubiquitylation, but not thin filament components. Citation: J. Cell Biol. 185(6): 1083-1095. DOI:10.1083/jcb. 200901052. PMID: 19506036. In skeletal muscle from humans, resistance training raises AMPK activity while decreasing 4E-BP1 phosphorylation and protein synthesis (Rasmussen, B.B. 2006). Journal of Physiology, 576(2), 613 624. In: Journal of Physiology, Volume 206, Issue This was published in 2008 by Dreyer et al. and was authored by Drummond, Pennings, Fujita, Glynn, Chinkes, and Drummond. Improving mTOR signaling and protein synthesis in human muscle is achieved by consuming carbohydrates and necessary amino acids with a leucine enrichment after resistance exercise. A.M. J. Published in Physiology, Endocrinology, and Metabolism, volume 294, issue 2, pages E392-E400, 10.1152/ajpendo.00582.2007 and the PubMed ID: 18056791. In 2008a, Drummond, Fujita, Abe, Dreyer, Volpi, and Rasmussen wrote the paper. Effects of resistance training and blood flow restriction on gene expression in human muscles. Medical Science and Physical Education 40(4): 691 698. Publication date: doi:10.1249/MSS.0b013e318160ff84. 13, Page 16873412. This sentence is a citation for a 2007 paper by Cunningham, Rodgers, Arlow, Vazquez, Mootha, and Puigserver. By means of a YY1-PGC-1alpha transcriptional complex, mTOR regulates mitochondrial oxidative activity. The publication is located in Nature, volume 450, issue 7190, pages 736 740. PMID: 18046414. doi:10.1038/nature06322. with the DOI: 18317375. Inhibitors of the mammalian target of rapamycin was published by Dancey, J.E. in 2005. Reference: Expert Opinion on Investigative doi:10.1517/ 13543784.14.3.313, Tripathy, D., and DeFronzo, R.A. 2009. The Main Defect in Type 2 Diabetes Is Insulin Resistance in Skeletal Muscles. 32(Suppl. 2): S157-S163. Citation: Diabetes Care. PMID: 19875544; doi:10.2337/dc09-S302. In 2008b, a group of researchers including Drummond, Pennings, Fry, Dhanani, and Dillon published a study. As we become older, our skeletal muscles take longer to respond anabolitically to resistance training and vital amino acids. The reference is from the Journal of Applied Physiology, volume 104, issue 5, pages 752 451. PMID: 18323467; Drugs, 14(3), 313 328, 15833062. PubMed: Page | 9

ISSN: 2322-3537 Vol-01 Issue-01 Jan 2012 doi:10.1152/japplphysiol.00021.2008. This entry was published in 2008 by Eley, Russell, and Tisdale. The effects of lipopolysaccharide, tumor necrosis factor, and angiotensin II on muscle protein synthesis may be mitigated by b-hydroxy-b-methylbutyrate. In: Am. J. Physiol. Endocrinol. Metab. doi:10.1152/ajpendo.90530.2008. PMID:18854427. linking. The Science journal, doi:10.1126/science. S.M. (2008). Research paper. When young men engage in resistance exercise, their eIF2B3 phosphorylation drops and the activation of p70S6K1 and rpS6 by eating is amplified. The citation is from the American Journal of Physiology Regulatory Integrative and Comparative Physiology, volume 295, issue 2, pages R604 R610, 2008, with the DOI:10.1152/ajpregu.00097.2008 and the PubMed ID: 18565837. 295(6): E1409-E1416. 294(5548), 1942 1945, This work is a collaboration between Phillips, Abadi, Glover, Yasuda, and Tarnopolsky. In 2010, S.M. Humans experiencing skeletal muscular atrophy due to immobility show no changes in indicators of protein breakdown and oxidative stress. Nutrient and applied physiology 35(2): 125 133. Bibcode:2010H09-137. PMCID: 20383222. In 2009, Feldman, Apsel, Uotila, Loewith, Knight, Z.A., Ruggero, and Shokat wrote a paper. When rapamycin becomes resistant to mTORC1 and mTORC2, active-site mTOR inhibitors go after those outputs. published in PLoS Biology, volume 7, doi:10.1371/journal.pbio. 1000038, and with the following PMID:19209957. issue 2, pages e38, In 2010, Goodman, C.A., Miu, M.H., Frey, J.W., Mabrey, D.M., Lincoln, H.C., Ge, Y., and others published. Skeletal muscle hypertrophy may be induced via a signaling pathway that does not include phosphatidylinositol 3-kinase/protein kinase B. The authors of this 2008 work are Ferreira, R., Vitorino, R., Appell, H.J., Amado, F., and Duarte, J.A. Muscle atrophy in the soleus begins with signs of cell death in mice with their hindlimbs suspended. Physiological Research 57(4): 601-611. with the PubMed In 2009, Ferrucci and Studenski published a paper. Muscles, diabetes, and the misconception of Ulysses' bow Diabetes Care, vol. 32, no. 11, pp. 2136 2137, doi:10.2337/dic09- 1592. Citation: 19875609. ID: 17705678. In 2006, Fluckey, Knox, Smith, Dupont-Versteegden, Gaddy, Tesch, and Peterson published an article. A MAP kinase pathway is involved in the insulin-facilitated increase in muscle protein synthesis after resistance exercise. "American Journal of The article is published in Endocrinology and Metabolism and may be accessed online at doi:10.1152/ajpendo. 00593.2005. The article has a PubMed abstract number of 16418205. Physiology" In 2009, Franklin and Kanaley investigated the effects of exercise, obesity, and age on intramyocellular lipids. Physical therapy and sports medicine, volume 37, issue 1, pages 20 26, 2009, doi:10.3810/PSM.2009.04.1679. 20048484. Specifically, Ku-0063794 inhibits the mammalian target of rapamycin (mTOR), according to Garc a-Mart nez, Moran, Clarke, Alessi, and Cosulich (2009). Journal of Biochemistry 421(1): 29 42. doi:10.1042/BJ20090489. PMID: Years 1940 2821. PubMed: The signaling mechanisms that regulate skeletal muscle growth and atrophy (Glass, 2005). International Journal of Biochemistry and Cell Biology, 37(10), 1974 1984. PubMed: 16087388; doi:10. 1016/j.biocel.2005.04.018. Allen, J.E., Moore, D.R., Tarnopolsky, M.A., and Phillips, Page | 10