Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.     

Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats

Acute administration of leucine and norleucine activates the mammalian target of rapamycin (mTOR) cell-signaling pathway and increases rates of protein synthesis in a number of tissues in fasted rats. Although persistent stimulation of mTOR signaling is thought to increase protein synthetic capacity, little information is available concerning the effects of chronic administration of these agonists on protein synthesis, mTOR signal transduction, or leucine metabolism. Hence, we developed a model of chronic leucine/norleucine supplementation via drinking water and examined the effects of chronic (12 days) supplementation on protein synthesis in adipose tissue, kidney, heart, liver, and skeletal muscle from ad libitum-fed rats. The relative concentration of proteins involved in mTOR signaling and the two initial steps in leucine oxidation were also examined. Leucine or norleucine supplementation was accompanied by increased rates of protein synthesis in adipose tissue, liver, and skeletal muscle, but not in heart or kidney. Supplementation was not associated with increases in the anabolic hormones insulin or insulin-like growth factor I. Chronic supplementation did not cause apparent adaptation in either components of the mTOR cell-signaling pathway that respond to leucine (mTOR, ribosomal protein S6 kinase, and eukaryotic initiation factor 4E-binding protein-1) or the first two steps in leucine metabolism (the mitochondrial isoform of branched-chain amino acid transaminase, branched-chain keto acid dehydrogenase, and branched-chain keto acid dehydrogenase kinase), which may be involved in terminating the signal from leucine. These results suggest that provision of leucine or norleucine supplementation via the drinking water results in stimulation of postprandial protein synthesis in adipose tissue, skeletal muscle, and liver without notable adaptive changes in signaling proteins or metabolic enzymes.     

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5-2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.     

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.     

Leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats

The branched-chain amino acid leucine stimulates muscle protein synthesis in part by directly activating the mTOR signaling pathway. Furthermore, leucine, if given in conjunction with resistance exercise, enhances the exercise-induced mTOR signaling and protein synthesis. Here we tested whether leucine can activate the mTOR anabolic signaling pathway in uremia and whether it can enhance work overload (WO)-induced signaling through this pathway. Chronic kidney disease (CKD) and control rats were studied after 7 days of surgically induced unilateral plantaris muscle WO and a single leucine or saline load. In the basal state, 4E-BP1 phosphorylation was modestly depressed in non-WO muscle of CKD rats, whereas rpS6 phosphorylation was nearly completely suppressed. After oral leucine mTOR, S6K1 and rpS6 phosphorylation increased similarly in both groups, whereas the phospho-4E-BP1 response was modestly attenuated in CKD. WO alone activated the mTOR signaling pathway in control and CKD rats. In WO CKD, muscle leucine augmented mTOR and 4E-BP1 phosphorylation, but its effect on S6K1 phosphorylation was attenuated. Taken together, this study has established that the chronic uremic state impairs basal signaling through the mTOR anabolic pathway, an abnormality that may contribute to muscle wasting. However, despite this abnormality, leucine can stimulate this signaling pathway in CKD, although its effectiveness is partially attenuated, including in skeletal muscle undergoing sustained WO. Thus, although there is some resistance to leucine in CKD, the data suggest a potential role for leucine-rich supplements in the management of uremic muscle wasting.     

Mechanical loading stimulates hypertrophy in tissue-engineered skeletal muscle: Molecular and phenotypic responses

Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size. Studies on humans are limited by the need for repeated sampling, and studies on animals have methodological and ethical limitations. In this investigation, three-dimensional skeletal muscle was tissue-engineered utilizing the murine cell line C2C12, which bears resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The work aimed to determine if mechanical loading induced an anabolic hypertrophic response, akin to that described in vivo after mechanical loading in the form of resistance exercise. Specifically, we temporally investigated candidate gene expression and Akt-mechanistic target of rapamycin 1 signalling along with myotube growth and tissue function. Mechanical loading (construct length increase of 15%) significantly increased insulin-like growth factor-1 and MMP-2 messenger RNA expression 21 hr after overload, and the levels of the atrophic gene MAFbx were significantly downregulated 45 hr after mechanical overload. In addition, p70S6 kinase and 4EBP-1 phosphorylation were upregulated immediately after mechanical overload. Maximal contractile force was augmented 45 hr after load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Overall, mechanical loading of tissue-engineered skeletal muscle induced hypertrophy and improved force production.     

Ubiquitin-specific peptidase 9, X-linked (USP9X) modulates activity of mammalian target of rapamycin (mTOR)

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that responds to extracellular environment to regulate a number of cellular processes. These include cell growth, proliferation, and differentiation. Although both kinase-dependent and -independent functions of mTOR are known to be critical modulators of muscle cell differentiation and regeneration, the signaling mechanisms regulating mTOR activity during differentiation are still unclear. In this study we identify a novel mTOR interacting protein, the ubiquitin-specific protease USP9X, which acts as a negative regulator of mTOR activity and muscle differentiation. USP9X can co-immunoprecipitate mTOR with both Raptor and Rictor, components of mTOR complexes 1 and 2 (mTORC1 and -2), respectively, suggesting that it is present in both mTOR complexes. Knockdown of USP9X leads to increased mTORC1 activity in response to growth factor stimulation. Interestingly, upon initiation of differentiation of C2C12 mouse skeletal myoblasts, knockdown of USP9X increases mTORC2 activity. This increase in mTORC2 activity is accompanied by accelerated differentiation of myoblasts into myotubes. Taken together, our data describe the identification of the deubiquitinase USP9X as a novel mTORC1 and -2 binding partner that negatively regulates mTOR activity and skeletal muscle differentiation.     

Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O₂; 21–1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O₂; with culture at 5% and 1% O₂ causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O₂, myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O₂ levels. At the molecular level we observed increases in mRNA expression of MuRF‐1 only at 1% O₂ whereas MAFbx expression was elevated at 10%, 5%, and 1% O₂. In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O₂, with no significant changes seen above this O₂ level. Overall, these data suggest that engineered muscle exposed to O₂ levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599–2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

A novel functional assessment of the differentiation of micropatterned muscle cells

Duchenne muscular dystrophy (DMD) is a lethal disease characterized by rapid, progressive atrophy of muscle tissues. Timely screening of therapeutic interventions is necessary for the development of effective treatment approaches for DMD. We have developed an in vitro model using a combination of micropatterning of C2C12 skeletal muscle cells and cell traction force microscopy (CTFM). In this model, C2C12 cells were micropatterned on a highly elongated adhesive island such that the cells assumed a shape typical of a myotube. During differentiation, these cells gradually fused together and began expressing dystrophin, a structural protein of myotubes, meanwhile, their contractile forces, represented by cell traction forces, continually increased until the myotubes reached maturation. In addition, the high-degree alignment of cells favored myotube differentiation and dystrophin expression. Since the fundamental structural unit of muscle tissue is myofiber, which is responsible for muscle contraction, such a technology that can directly quantify the contractile forces of the myotube, a precursor of myofiber, may constitute a fast and efficient screening approach for DMD therapies.     

Leucine facilitates the insulin-stimulated glucose uptake and insulin signaling in skeletal muscle cells: involving mTORC1 and mTORC2

Leucine, a branched-chain amino acid, has been shown to promote glucose uptake and increase insulin sensitivity in skeletal muscle, but the exact mechanism remains unestablished. We addressed this issue in cultured skeletal muscle cells in this study. Our results showed that leucine alone did not have an effect on glucose uptake or phosphorylation of protein kinase B (AKT), but facilitated the insulin-induced glucose uptake and AKT phosphorylation. The insulin-stimulated glucose uptake and AKT phosphorylation were inhibited by the phosphatidylinositol 3-kinase inhibitor, wortmannin, but the inhibition was partially reversed by leucine. The inhibitor of mammalian target of rapamycin complex 1 (mTORC1), rapamycin, had no effect on the insulin-stimulated glucose uptake, but eliminated the facilitating effect of leucine in the insulin-stimulated glucose uptake and AKT phosphorylation. In addition, leucine facilitation of the insulin-induced AKT phosphorylation was neutralized by knocking down the core component of the mammalian target of rapamycin complex 2 (mTORC2) with specific siRNA. Together, these findings show that leucine can facilitate the insulin-induced insulin signaling and glucose uptake in skeletal muscle cells through both mTORC1 and mTORC2, implicating the potential importance of this amino acid in glucose homeostasis and providing new mechanistic insights.     

Antagonistic control of muscle cell size by AMPK and mTORC1

Nutrition and physical activity have profound effects on skeletal muscle metabolism and growth. Regulation of muscle mass depends on a thin balance between growth-promoting and growth-suppressing factors. Over the past decade, the mammalian target of rapamycin (mTOR) kinase has emerged as an essential factor for muscle growth by mediating the anabolic response to nutrients, insulin, insulin-like growth factors and resistance exercise. As opposed to the mTOR signaling pathway, the AMP-activated protein kinase (AMPK) is switched on during starvation and endurance exercise to upregulate energy-conserving processes. Recent evidence indicates that mTORC1 (mTOR Complex 1) and AMPK represent two antagonistic forces governing muscle adaption to nutrition, starvation and growth stimulation. Animal knockout models with impaired mTORC1 signaling showed decreased muscle mass correlated with increased AMPK activation. Interestingly, AMPK inhibition in p70S6K-deficient muscle cells restores cell growth and sensitivity to nutrients. Conversely, muscle cells lacking AMPK have increased mTORC1 activation with increased cell size and protein synthesis rate. We also demonstrated that the hypertrophic action of MyrAkt is enhanced in AMPK-deficient muscle, indicating that AMPK acts as a negative feedback control to restrain muscle hypertrophy. Our recent results extend this notion by showing that AMPKα1, but not AMPKα2, regulates muscle cell size through the control of mTORC1 signaling. These results reveal the diverse functions of the two catalytic isoforms of AMPK, with AMPKα1 playing a predominant role in the control of muscle cell size and AMPKα2 mediating muscle metabolic adaptation. Thus, the crosstalk between AMPK and mTORC1 signaling is a highly regulated way to control changes in muscle growth and metabolic rate imposed by external cues.     

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.     

Optically controlled contraction of photosensitive skeletal muscle cells

As the skeletal muscle cell is an efficient force transducer, it has been incorporated in bio-microdevices using electrical field stimulation for generating contractile patterns. To improve both the spatial and temporal resolutions, we made photosensitive skeletal muscle cells from murine C2C12 myoblasts, which express channelrhodopsin-2 (ChR2), one of archaea-type rhodopsins derived from green algae Chlamydomonas reinhardtii. The cloned ChR2-expressing C2C12 myoblasts were made and fused with untransfected C2C12 to form multinucleated myotubes. The maturation of myotubes was facilitated by electrical field stimulation. Blue LED light pulse depolarized the membrane potential of a ChR2-expressing myotube and eventually evoked an action potential. It also induced a twitch-like contraction in a concurrent manner. A contraction pattern was thus made with a given pattern of LED pulses. This technique would have many applications in the bioengineering field, such as wireless drive of muscle-powered actuators/microdevices.     

Branched-Chain Amino Acids Enhance Premature Senescence through Mammalian Target of Rapamycin Complex I-Mediated Upregulation of p21 Protein

Branched-chain amino acids (BCAAs) have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR) links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer''s ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.     

Insulin-like growth factor-1 (IGF-1) and leucine activate pig myogenic satellite cells through mammalian target of rapamycin (mTOR) pathway

Myogenic satellite cells are adult stem cells and have important roles in skeletal muscle growth, repair, and regeneration. Both insulin-like growth factor-1 (IGF-1) and leucine stimulate skeletal muscle growth, which link to the activation and proliferation of myogenic satellite cells in skeletal muscle. Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis and cell proliferation. Thus, IGF-1 and leucine may stimulate activation of myogenic satellite cells through mTOR signaling. In this study, myogenic satellite cells were isolated from 6-month-old pigs and subjected to IGF-1 and leucine treatments. Both IGF-1 and leucine upregulated mTOR signaling in myogenic satellite cells. The phosphorylation of mTOR at Ser(2448) increased 83.8 +/- 7.7% by IGF-1 (P < 0.05) and 83.4 +/- 5.7% by leucine (P < 0.05). The downstream targets of mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) were also phosphorylated due to IGF-1 and leucine treatments. Treatment with IGF-1 and leucine induced the phosphorylation of tuburin (TSC2), a key mediator upstream of mTOR signaling, by 272.8 +/- 26.4% and 94.2 +/- 28.7%, respectively. Treatment of cells with both IGF-1 and leucine did not show synergistic effect on mTOR signaling. Inhibition of mTOR by rapamycin abolished the protein synthesis and cell proliferation stimulated by both IGF-1 and leucine. In summary, our data showed that in preliminary cultured myogenic satellite cells mTOR signaling was activated due to IGF-1 and leucine treatments, and this mTOR activation is necessary for the activation of myogenic satellite cells.     

Soluble miniagrin enhances contractile function of engineered skeletal muscle

Neural agrin plays a pleiotropic role in skeletal muscle innervation and maturation, but its specific effects on the contractile function of aneural engineered muscle remain unknown. In this study, neonatal rat skeletal myoblasts cultured within 3-dimensional engineered muscle tissue constructs were treated with 10 nM soluble recombinant miniagrin and assessed using histological, biochemical, and functional assays. Depending on the treatment duration and onset time relative to the stage of myogenic differentiation, miniagrin was found to induce up to 1.7-fold increase in twitch and tetanus force amplitude. This effect was associated with the 2.3-fold up-regulation of dystrophin gene expression at 6 d after agrin removal and enhanced ACh receptor (AChR) cluster formation, but no change in cell number, expression of muscle myosin, or important aspects of intracellular Ca(2+) handling. In muscle constructs with endogenous ACh levels suppressed by the application of α-NETA, miniagrin increased AChR clustering and twitch force amplitude but failed to improve intracellular Ca(2+) handling and increase tetanus-to-twitch ratio. Overall, our studies suggest that besides its synaptogenic function that could promote integration of engineered muscle constructs in vivo, neural agrin can directly promote the contractile function of aneural engineered muscle via mechanisms distinct from those involving endogenous ACh.     

Distinct anabolic signalling responses to amino acids in C2C12 skeletal muscle cells

The essential amino acids (EAA) activate anabolic signalling through mechanisms, which are unclear in detail but include increased signalling through the mammalian target of rapamycin complex 1 (mTORC1). Of all the EAA, the branched chain amino acid (BCAA) leucine has been suggested as the most potent in stimulating protein synthesis, although there have been no studies investigating the effects of each EAA on anabolic signalling pathways. We therefore undertook a systematic analysis of the effect of each EAA on mTORC1 signalling in C2C12 myotubes whereby cells were serum (4 h) and amino acid (1 h) starved before stimulation with 2 mM of each amino acid. Immunoblotting was used to detect phosphorylated forms of protein kinase B (Akt)/mTORC1 signalling enzymes. The phosphorylation of Akt was unchanged by incubation with EAA. Phosphorylation of mTOR and 4E binding protein-1 (4EBP1) were increased 1.67 ± 0.1-fold and 2.5 ± 0.1-fold, respectively, in response to leucine stimulation but not in response to any other EAA. The phosphorylation of ribosomal s6 kinase (p70S6K1) was increased by stimulation with all EAA with the exceptions of isoleucine and valine. However, the increase with leucine was significantly greater, 5.9 ± 0.3-fold compared to 1.6–2.0-fold for the non-BCAA EAA. This pattern of activation was identical in ribosomal protein s6 (RPS6) with the additional effect of leucine being 3.8 ± 0.3-fold versus 1.5–2.0-fold. Phosphorylation of eukaryotic initiation/elongation factors eIF2α and eEF2 were unaffected by EAA. We conclude that leucine is unique amongst the amino acids in its capacity to stimulate both mTOR and 4EBP1 phosphorylation and to enhance p70S6K1 signalling.     

Suppressing mTORC1 on the lysosome

The mechanistic target of rapamycin, mTOR, is a protein kinase that integrates environmental and nutritional inputs into regulation of cell growth and metabolism. Key outputs of mTOR signalling occur from the lysosome membrane in the form of the multi‐subunit mTOR complex 1 (mTORC1), which phosphorylates multiple targets. While class I phosphoinositide kinase (PI3K‐I) is a well‐known activator of mTORC1, a recent paper (Marat et al, 2017) shows that a class II PI3K with a different substrate specificity, PI3K‐C2β, serves to inhibit mTORC1 on lysosomes under conditions of growth factor deprivation.     

The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.