Abstract

mTOR SIGNALING AND PROTEIN SYNTHESIS

mTOR is a serine/threonine kinase of the phosphatidylinositol kinase-related kinase family and is highly conserved from yeast to mammals (61). mTOR signaling pathway has a wide range of functions including regulation of protein synthesis, cell proliferation, apoptosis, and autophagy. In mammalian cells, mTOR exists in two distinct multi-protein complexes, mTOR complex 1 (TORC1) and mTOR complex 2 (TORC2). TORC1 is a complex comprised of mTOR, regulatory associated protein of mTOR (Raptor), and G protein β-subunit-like (Gβl/also known as mLST8). The TORC2 complex is comprised of mTOR, rapamycin-insensitive companion of mTOR (Rictor), stress-activated-protein-kinase-interacting protein 1 (SIN1), and Gβl. For the purpose of this review, we will focus on mTOR/TORC1 signaling and regulation of protein synthesis during hypertrophy in skeletal muscle.

To date, numerous studies have shown that mTOR plays a critical role in regulating protein synthesis and cell growth/hypertrophy in skeletal muscle (11, 36, 54, 74, 86). Consistent with its role in regulation of protein synthesis, signaling through mTOR occurs in muscle after high force contractions/mechanical loading and is maintained for many hours (18–36 h) after a single bout of loading (10). In contrast, mTOR signaling is only transiently (<6 h) upregulated after low force contractions that do not elicit growth of skeletal muscle (10, 73). Also considered in this review is that mTOR receives and integrates many different upstream signals that can function as either activators or inhibitors of activity and many of these upstream regulators are known to be active in muscle following mechanical loading. For example, activators of mTOR include growth factors, amino acid availability, and mechanical strain, while factors that could inhibit mTOR function include energy stress (via AMPK-dependent regulation) and other stress response molecules (induction of REDD1/REDD2). In the following sections we provide a brief summary of known components of the mTOR signaling pathway and these are depicted in Fig. 1. More detailed reviews of mTOR regulation can be found elsewhere (80, 84, 108).

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Fig. 1.

Simplified scheme depicting a model through which both positive and negative factors can contribute to mTOR/TORC1 signaling and protein synthesis in skeletal muscle. Activators, growth factors, amino acids, and mechanical stretch are labeled in blue while inhibitory signals are labeled in red. Solid lines depict defined interactions among molecules, dotted lines indicate suggested interactions.

GROWTH FACTOR/INSULIN SIGNALING AND REGULATION OF mTOR ACTIVITY

The most well-defined pathway regulating mTOR activity in skeletal muscle is the IGF-1/insulin pathway. Stimulation of muscle with growth factors (e.g., insulin and/or IGF-1) leads to activation of phosphoinositide 3-kinase (PI3K), triggering multiple downstream signaling events associated with the control of cell cycle progression, differentiation, survival, and cell growth [see Frost and Lang (31) for a more comprehensive review on this topic]. Serine/threonine kinase Akt/PKB is a well-established target of PI3K, and Akt/PKB phosphorylation of glycogen synthase kinase 3 (GSK-3) leads to its inhibition and an increase in global protein synthesis through increase in the activity of eukaryotic initiation factor 2B (eIF2B; Ref. 106). Additionally Akt/PKB is a known upstream regulator of mTOR signaling through regulation of the tuberous sclerosis complex (TSC; Refs. 20, 58, 70, 79). The TSC protein complex is a heterodimer of TSC1 and TSC2 and functions as a negative regulator of mTOR activity. Cells null for TSC1 or TSC2, cells depleted of TSC1 or TSC2 by RNA interference, and human and mouse tissues deficient in TSC1 or TSC2, all have high mTOR activity (33, 43, 58, 79). Together with its partner TSC1, TSC2 functions as a guanosine triphosphate (GTP)ase activating protein (GAP) for a small G protein named Ras homolog enriched in brain (Rheb). It has been reported that Akt/PKB directly phosphorylates TSC2 on multiple residues (at least two sites, Ser939 and Thr1462; Refs. 55, 58, 70). This phosphorylation of TSC2 by Akt/PKB is thought to inhibit its GAP activity, allowing Rheb to accumulate in its active GTP-bound form. GTP-bound Rheb strongly stimulates mTOR activity, thus TSC2 functions as a negative regulator of mTOR through increasing the intrinsic rate of GTP hydrolysis to GDP on Rheb (34, 57, 60, 92). This leads to the well-established growth factor associated signaling cascade through PI3K-Akt/PKB-TSC1/2-Rheb-mTOR seen in muscle as well as other cell types.

To date there have been very few studies that have incorporated analysis of TSC1/TSC2 or Rheb in the regulation of protein synthesis during skeletal muscle growth/hypertrophy. Consistent with the role of TSC complex as a negative regulator of muscle growth Wan et al. (104) reported that overexpression of human TSC1 in mouse skeletal muscle leads to 20% reduction of muscle mass and decreased fiber cross-sectional area in the tibialis anterior and EDL muscles compared with controls. In response to overexpression of TSC1, TSC2 was stabilized and this was associated with significant inhibition of mTOR signaling. Other studies in rodents have reported that TSC2 is modified by phosphorylation at several different sites (Ser1345 and Thr1462 sites) after ex vivo high-frequency electrical stimulation (8) or in vivo mechanical overload model (71). Experiments evaluating the mRNA levels of TSC1, TSC2, and Rheb were recently reported from human muscle biopsies following one bout of high-resistance contractions and nutritional supplements. Drummond et al. (27) found that mRNA levels of TSC1 and TSC2 (negative regulators of mTOR) were decreased and Rheb (positive regulator of mTOR) was increased at 6 h after single bout of resistance exercise in young subjects. While limited, these findings support the likely contribution of the TSC complex and Rheb to regulation of muscle protein synthesis and size.

mTOR AND AMINO ACID AVAILABILITY

In addition to growth factors, mTOR is also known to be regulated by the availability of amino acids (33, 46, 63, 68, 88). In mammalian cells, amino acid deprivation leads to decreased mTOR signaling and to decreased rates of protein synthesis, effects that are rapidly reversed by readdition of amino acids (30, 46). Among the amino acids, changes in leucine levels alone are sufficient to regulate the phosphorylation state and activity of the mTOR pathway (46, 69). Several studies in rodents and human have shown that administration/ingestion of essential amino acids (specifically leucine) leads to increased/enhanced protein synthesis in skeletal muscle primarily through activation of mTOR signaling (5, 24, 25, 32, 102). Currently, it is widely accepted that amino acid availability affects mTOR signaling and protein synthesis rate; however, how amino acids regulate the mTOR signaling pathway remains poorly understood. For instance, it has been proposed that amino acids activate mTOR in both a TSC1/TSC2-dependent (33) and TSC1/TSC2-independent (88) manner. Some studies suggest that Rheb plays an essential role in regulation of the mTOR activity in response to amino acids (57) and that nutrient deprivation may decrease Rheb binding to mTOR (68). However, other work implies that Rheb may not be involved in nutrient-induced mTOR signaling, but rather that nutrient poor conditions lead to alterations in the proteins associated in the mTOR complex and this results in decreased activity (63). Recently, a class III PI3K, hVps34, has been implicated in the amino acid-induced activation of mTOR signaling and this is independent of Akt/PKB-TSC-Rheb pathway (15, 76). In addition, two research groups [Kim et al. (64) and Sancak et al. (87)] reported that a heterodimeric complex of the Rag proteins, a family of four related small GTPases, plays a fundamental role in amino acid-induced regulation of mTOR/TORC1 activity both in Drosophila and mammalian cells. It is still unclear, however, what the relationship is between hVps34 and the Rag proteins in activating mTOR/TORC1 in response to amino acids. The lack of consensus in the understanding of the mechanisms by which amino acids regulate mTOR suggests that other molecules, not yet identified, are involved in the signaling and/or the mechanisms for signaling might be varied among different cell types.

MECHANOTRANSDUCTION OF mTOR SIGNALING

Work by Bodine et al. (11) established that mechanical overload-induced signaling through mTOR was necessary for skeletal muscle hypertrophy. Since then, many studies have confirmed the association between mechanical loading and activation of mTOR signaling in mammalian muscle. However, there is still very little known about the actual biochemical events that link the mechanical loading of a muscle/muscle fiber to mTOR signaling. Ex vivo muscle stretch and in vitro muscle cell stretch models of mechanical loading have defined that 1) passive mechanical loading is sufficient to activate mTOR signaling in skeletal muscle, 2) stretch-induced activation of mTOR occurs in a PI3K-independent manner, and 3) mechanical stretch can activate mTOR in the absence of amino acids or growth factors (49, 50, 54). Our lab has found that treatment of the myotubes with cytochalasin D to disrupt the microfilament system (49) will block stretch-induced signaling to mTOR even though insulin activation of mTOR was still intact. These results suggest that there is a cytoskeletal requirement for communication of signaling pathway from mechanical stretch to mTOR. Other intracellular targets of stretch include phospholipase D1 (PLD1) and the lipid second messenger phosphatidic acid (PA) as the mediators of stretch-induced mTOR signaling in skeletal muscle (51). The PLD1 pathway has been proposed to contribute to the activation of mTOR through generating PA from the hydrolysis of phosphatidylcholine. PA binds specifically to the FRB domain of the mTOR in a manner competitive with inhibitor rapamycin/FKBP12 complex (28, 29). Recently, studies from Dr. Y. Chen's laboratory (91) identified that PLD1 is required for Rheb activation of mTOR signaling, and their experiments place PLD1 downstream of Rheb and upstream of mTOR. Thus mechanical loading/stretch clearly acts independently of amino acids and growth factors to activate mTOR signaling in skeletal muscle. We believe that this mechanical pathway likely acts synergistically with changes in amino acid uptake and growth factor availability to contribute to the prolonged activation of mTOR signaling following high resistance contractions/mechanical overload.

CELLULAR ENERGY STATUS AND mTOR ACTIVITY

In addition to the availability of growth factors and amino acids, the rate of protein synthesis is regulated by the cellular energy status in muscle and non-muscle cells (13, 81). It has been shown that glucose deprivation, through use of a non-hydrolyzable glucose analog (2-deoxyglucose), results in decreased mTOR signaling (21). It is currently suggested that mTOR functions as a sensor of cellular energy state through input from the AMP-activated protein kinase (AMPK) pathway (60). AMPK is well known as a sensor of cellular energy status, which is regulated by changes in the cellular levels of AMP-to-ATP ratio (62). Inoki et al. (60) identified that, under low cellular energy conditions, activated AMPK phosphorylates TSC2 on Thr1227 and Ser1345 residues (these residues correspond to Thr1271 and Ser1387, respectively, in human TSC2) and enhances its inhibitory function leading to decreased mTOR activity. This research group has also reported that AMPK-dependent phosphorylation of TSC2 on Ser1387 primes TSC2 for further phosphorylation by GSK-3 on multiple residues. This series of phosphorylation steps leads to subsequent inhibition of mTOR activity under energy-deprived conditions (59). Additionally, a recent study by Gwinn et al. (45) reported that AMPK can directly phosphorylate the mTOR binding partner Raptor on two well-conserved serine residues (Ser722/Ser792), and Raptor phosphorylation is required to inhibit mTOR activity through cellular energy stress-induced AMPK activation. Consistent with these observations, it was demonstrated that activation of AMPK, by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR), results in decreased protein synthesis and a repression of mTOR-mediated signaling in skeletal muscle in both in vitro and in vivo experimental models (12, 82, 107). Deshmukh et al. (22) reported that preincubation with AICAR completely inhibited insulin-induced activation of mTOR signaling using an ex vivo organ bath system. Thomson et al. (93) also reported that AICAR treatment inhibited mTOR activation, which is induced by high-frequency electrical stimulation of rat EDL muscle. Finally Thomason and Gordon (94) found a correlation between increased phosphorylation of AMPK and reduced muscle hypertrophy in aging rats.

These studies indicate that activation of AMPK in skeletal muscle will contribute to diminished protein synthesis through inhibiting mTOR signaling. However, it is still controversial whether an AMPK-dependent regulatory mechanism plays an active role in exercise/contraction-induced skeletal muscle growth responses. To address the potential role of AMPK in muscle growth, McGee et al. (71) used the compensatory hypertrophy model with mice lacking LKB1, the primary upstream kinase for AMPK. The hypothesis for this study was that decreased phosphorylation of AMPK, via loss of LKB1, would enhance mTOR signaling and lead to a greater degree of muscle hypertrophy in response to functional overload. To their surprise, loss of LKB1 did not alter the growth of skeletal muscle after 7 or 28 days of functional overload. Normal hypertrophic responses were accompanied in skeletal muscle of LKB1−/− mice compared with the wild-type strain. However, these authors found that while α2-AMPK activity was reduced in the muscles of these mice, α1-AMPK was still activated following overload in both the LKB1−/− mice as well as the wild-type mice. Thus the conclusion was that α2-AMPK is likely not an important molecule in the regulation of mechanical load signaling to mTOR and growth.

STRESS RESPONSE GENES REDD1/REDD2 AND mTOR SIGNALING

Another negative regulator of mTOR activity are two factors that were originally identified from a genetic screen for negative regulators of the Drosophila TOR pathway (83). The mammalian orthologs are called REgulated in Development and DNA damage responses 1 (REDD1) and REDD2 (also called RTP801/DDIT4 and RTP801L/DDIT4L, respectively). REDD1 is ubiquitously expressed in all cell types while expression of REDD2 is significantly enriched in human and mouse skeletal muscle (http://symatlas.gnf.org/SymAtlas/). Cros et al. (19) identified REDD2 (also named SMHS1) as a highly upregulated gene in soleus muscle undergoing atrophy following 14 days of hindlimb unloading (78). Biochemical studies of both REDD1 and REDD2 found that they inhibit mTOR kinase activity in non-muscle and muscle cells downstream of Akt/PKB but upstream of TSC2 (14, 18, 89, 105). In skeletal muscle, REDD1 has been shown to inhibit mTOR signaling and protein synthesis in response to dexamethasone and alcohol intoxication (67, 105). In addition, Kimball et al. (65) found that rapid degradation of REDD1 in muscle was associated with enhanced mTOR signaling. Recent studies in human skeletal muscle have also suggested that expression of REDD1 and REDD2 plays a role in regulating mTOR activity and protein synthesis (26, 27). These observations suggest the possibility that REDD1 and REDD2 are stress responsive negative regulators of mTOR signaling with implications for controlling skeletal muscle size.

SUMMARY

In summary, there is a long history of research in understanding the contribution of mechanical loading/resistance exercise to regulation of protein synthesis in skeletal muscle and growth. The goal of this short review was to provide a general overview linking what is currently known using well-established animal models and cell culture models of mechanical loading and hypertrophy with regulation of mTOR, a key kinase involved in protein synthesis and growth. One of the unique features seen across all models of skeletal muscle hypertrophy in humans and rodents is that rates of protein synthesis and signaling through mTOR stay elevated for hours to days after one bout of high-resistance contractions. We propose in this review that the prolonged activation of mTOR signaling, and thus rates of protein synthesis, is the cumulative result of synergism among several independent positive signals including mechanical loading, amino acids, and growth factors. We also suggest that resistance exercise/mechanical loading can act to dampen the inhibitory contributors to mTOR signaling via downregulation of the TSC complex and REDD1/REDD2 molecules. Thus to understand the critical molecular and cellular steps involved in resistance exercise-induced regulation of protein synthesis will require carefully designed experiments that will consider balancing both the positive and negative inputs to mTOR signaling in skeletal muscle.

GRANTS

This work was supported by National Institutes of Health AR-045617 to K. A. Esser and American Heart Association Postdoctoral Fellowship 0825668D to M. Miyazaki.

REFERENCES