Resistance-exercise training results in a progressive increase in muscle mass and force production. Following an acute bout of resistance exercise, the rate of protein synthesis increases proportionally with the increase in protein degradation, correlating at 3 h in the starved state. Amino acids taken immediately before or immediately after exercise increase the post-exercise rate of protein synthesis. Therefore a protein that controls protein degradation and amino acid-sensitivity would be a potential candidate for controlling the activation of protein synthesis following resistance exercise. One such candidate is the class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting mutant 34). Vps34 controls both autophagy and amino acid signalling to mTOR (mammalian target of rapamycin) and its downstream target p70 S6K1 (S6 kinase 1). We have identified a significant increase in mVps34 (mammalian Vps34) activity 3 h after resistance exercise, continuing for at least 6 h, and propose a mechanism whereby mVps34 could act as an internal amino acid sensor to mTOR after resistance exercise.
- mammalian target of rapamycin (mTOR)
- protein degradation
- protein synthesis
- resistance exercise
- vacuolar protein sorting mutant 34 (Vps34)
The cellular response to resistance exercise
Resistance exercise results in an acute increase in muscle protein synthesis that, when repeated in the presence of proper nutrition, results in an increase in muscle mass and strength . The mTOR [mammalian TOR (target of rapamycin)] and its downstream target p70 S6K1 (S6 kinase 1) regulate protein synthesis  and ultimately cell size and proliferation . Indeed, Baar and Esser  identified a tight correlation between the degree of hypertrophy and S6K1 phosphorylation following resistance exercise.
As well as the protein synthesis response to resistance exercise, there is a simultaneous increase in protein breakdown . Phillips et al.  identified a significant correlation between the muscle protein synthesis and breakdown rate 3 h after resistance exercise in the starved state; however, it is as yet not established which precedes the other. A model proposed by Tipton and Wolfe  suggested that the initial amino acid availability is the ultimate determinant of whether degradation precedes synthesis or vice versa. When amino acids within the muscle are high, synthesis can precede degradation, whereas when amino acids are low, degradation must occur first to supply amino acids. Amino acids, in particular leucine, regulate the formation of the nutrientsensing complex TORC1 [TOR complex 1; mTOR, raptor (regulatory associated protein of mTOR) and GβL (G-protein β-protein subunit-like)] and pass nutrient information to the protein synthesis machinery through phosphorylation of S6K1 and its downstream target, the ribosomal protein S6 . S6K1 phosphorylation at Thr-389 by mTOR is also regulated by insulin through a complex pathway involving class I PI3Ks (phosphoinositide 3-kinases) and the downstream kinases PKB (protein kinase B; also called Akt) and PDK1 (phosphoinositide-dependent kinase 1) (reviewed in ). Upstream regulators of mTOR mainly function through the tumour suppressors TSC1 (tuberous sclerosis complex 1) and TSC2 (reviewed in ), which negatively regulate mTOR through inhibition of Ras-like GTPase, Rheb (Ras homologue enriched in brain) . Amino acid input is thought to enter the pathway during Rheb activation of mTOR ; however, the potential influence of upstream TSC1/TSC2 on amino acid activation of mTOR is not without controversy .
Vps34 (vacuolar protein sorting mutant 34) and S6K1
Since resistance exercise increases both protein synthesis and degradation, and amino acids are required for maximal adaptations in response to training, a protein that is sensitive to amino acids, plays a role in protein degradation and signals to mTOR would be a prime candidate for regulating the adaptations due to resistance exercise. One such candidate is Vps34.
Vps34 is the class III PI3K that was first characterized for its role in vesicular trafficking in Saccharomyces cerevisiae . The associated protein kinase Vps15  functionally interacts with Vps34 and is required for its activity in regulating intracellular protein trafficking through protein and lipid phosphorylation events . Vps34 has a further role in yeast, being required for autophagy in response to nutrient deprivation . The human homologue hVps34 (human Vps34) was first identified and characterized by Volinia et al.  and shown to be highly conserved from yeast to humans and to function in protein trafficking. It has since been implicated in the regulation of early endosomal sorting and mitogenesis  and to have an involvement in mammalian autophagy , with a further associated protein, beclin 1 . Byfield et al.  proposed a mechanism whereby the human class III PI3K hVps34 integrated the signalling of amino acids and glucose to mTOR and S6K1. Similar findings were reported by Nobukuni et al. . hVps34 has been shown to be required for insulin stimulation of S6K1 phosphorylation but is not regulated by insulin and does not affect insulin-stimulated activation of PKB or TSC2 . This suggests that hVps34 is downstream of TSC2 in the activation of mTOR. Byfield et al.  also identified amino acid starvation and AMPK (AMP-activated protein kinase) as inhibitors of hVps34 activity.
Vps34 and resistance exercise
mVps34 (mammalian Vps34) is a widely distributed protein found in most tissues. Of particular interest is its surprising variation of abundance in muscle tissues, with very little in cardiac muscle compared with skeletal muscle. Protein levels are also higher in muscles with a higher percentage of slow oxidative fibres, with the tibialis anterior (predominately fast fibres) showing less mVps34 than the soleus (predominantly slow fibres) muscle (M.G. MacKenzie, D.L. Hamilton, J.T. Murray and K. Baar, unpublished work). We proposed that mVps34, as both a kinase involved in protein degradation in the form of autophagy and a nutrient-sensitive signaller to S6K1, might have a role in controlling muscle protein turnover following resistance exercise. We identified an increase in mVps34 activity 3 h after exercise that was maintained for up to 6 h. The increase in mVps34 activity was greater in muscles undergoing lengthening contractions, suggesting a role closely related to the degree of hypertrophy following resistance exercise. Although there was a correlation between the activities of S6K1 and mVps34 at the 3 h time point, S6K1 activity is high 30 min after exercise and maintained for up to 18 h (M.G. MacKenzie, D.L. Hamilton, J.T. Murray and K. Baar, unpublished work). The fact that S6K1 activity is high earlier and continues after mVps34 activity has returned to basal levels suggests that mVps34 is not the main mechanism of activating mTOR. However, in light of the proposed correlation of protein synthesis and breakdown 3 h after resistance exercise identified by Phillips et al. , it is possible that mVps34 may play a permissive role in activating mTOR at this time point. We (M.G. MacKenzie, D.L. Hamilton, J.T. Murray and K. Baar, unpublished work) and others  have identified PKB as a possible initial activator of mTOR; however, PKB phosphorylation returns to basal levels after 30 min, allowing another mechanism for the continued activation.
Resistance exercise, mVps34 and amino acids
Perhaps the most likely mechanism allowing mVps34 activity to stimulate mTOR is through autophagy and increasing internal amino acid concentration. This is particularly relevant after the resistance exercise stimulus when muscle damage is occurring. Lengthening contractions cause a major disruption to the normal protein architecture of the muscle, with the breakdown of various components of the sarcomere. This is characterized by several metabolic events including release of muscle enzymes such as creatine kinase , soreness and reduction in muscle function , and influx of Ca2+ . Although little research has described the role of autophagy in skeletal muscle following eccentric contractions, it has been identified as the major amino acid-dependent proteolytic pathway in skeletal-muscle myotubes . It seems logical that, during protein synthesis, internal amino acid concentration would decrease, thereby activating protein degradative processes to replenish internal amino acid pools. This is supported by Legar et al.  who found that the atrogenes MuRF1 (muscle ring finger-1) and atrogen-1 were increased following a bout of resistance exercise in humans. The resulting increase in internal amino acids could be a mechanism by which mVps34 activates mTOR and S6K1. We are currently investigating this further by using the glucocorticoid hormone Dex (dexamethasone) to increase protein degradation. Early indications suggest that mVps34 activity in C2C12 cells is activated in response to low concentrations of Dex, possibly as a mechanism to increase protein degradation, and is inhibited by higher concentrations (M.G. MacKenzie, D.L. Hamilton, J.T. Murray and K. Baar, unpublished work). This inhibition may be due to the subsequent increase in internal amino acids following high Dex treatment. We propose a mechanism whereby mVps34 activity is controlled by internal amino acid concentration and may play a role in amino acid sensing after resistance exercise (Figure 1).
mVps34 plays a role in activation of S6K1 and thus protein synthesis under certain conditions. However, it is unlikely to be a direct influence, particularly in the case of resistance exercise. It is plausible that depletion of internal amino acid pools following lengthening contractions activates an mVps34-containing complex that regulates autophagy as a mechanism to replenish the amino acid pool, thereby maintaining mTOR activity. Research into this area is of vital importance in understanding muscle growth and how muscle wasting might be avoided or reversed in the diseased and elderly.
Exercise: A Focus Topic at Life Sciences 2007, held at SECC Glasgow, U.K., 9–12 July 2007. Edited by C. Downes (Dundee, U.K.), P. Greenhaff (Nottingham, U.K.) and P. Taylor (Dundee, U.K.).
Abbreviations: Dex, dexamethasone; TOR, target of rapamycin; mTOR, mammalian TOR; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; Rheb, Ras homologue enriched in brain; S6K1, S6 kinase 1; TSC1, tuberous sclerosis complex 1; Vps34, vacuolar protein sorting mutant 34; hVps34, human Vps34; mVps34, mammalian Vps34
- © The Authors Journal compilation © 2007 Biochemical Society