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. 2008 Nov 17;587(Pt 1):253–260. doi: 10.1113/jphysiol.2008.159830

mVps34 is activated following high-resistance contractions

Matthew G MacKenzie 1, D Lee Hamilton 1, James T Murray 2, Peter M Taylor 1, Keith Baar 1
PMCID: PMC2670038  PMID: 19015198

Abstract

Following resistance exercise in the fasted state, both protein synthesis and degradation in skeletal muscle are increased. The addition of essential amino acids potentiates the synthetic response suggesting that an amino acid sensor, which is involved in both synthesis and degradation, may be activated by resistance exercise. One such candidate protein is the class 3 phosphatidylinositol 3OH-kinase (PI3K) Vps34. To determine whether mammalian Vps34 (mVps34) is modulated by high-resistance contractions, mVps34 and S6K1 (an index of mTORC1) activity were measured in the distal hindlimb muscles of rats 0.5, 3, 6 and 18 h after acute unilateral high-resistance contractions with the contralateral muscles serving as a control. In the lengthening tibialis anterior (TA) muscle, S6K1 (0.5 h = 366.3 ± 112.08%, 3 h = 124.7 ± 15.96% and 6 h = 129.2 ± 0%) and mVps34 (3 h = 68.8 ± 15.1% and 6 h = 36.0 ± 8.79%) activity both increased, whereas in the shortening soleus and plantaris (PLN) muscles the increase was significantly lower (PLN S6K1 0.5 h = 33.1 ± 2.29% and 3 h = 47.0 ± 6.65%; mVps34 3 h = 24.5 ± 7.92%). HPLC analysis of the TA demonstrated a 25% increase in intramuscular leucine concentration in rats 1.5 h after exercise. A similar level of leucine added to C2C12 cells in vitro increased mVps34 activity 3.2-fold. These data suggest that, following high-resistance contractions, mVps34 activity is stimulated by an influx of essential amino acids such as leucine and this may prolong mTORC1 signalling and contribute to muscle hypertrophy.

Mechanical stimulation plays an essential role in the maintenance and growth of skeletal muscle. The connection between mechanical stimuli and muscle growth has been recognized for millennia; however, the molecular mechanisms underlying this connection have yet to be fully elucidated (Vandenburgh, 1987). Across all species studied to date, the first physiological response to resistance exercise is an increase in protein synthesis (Wong & Booth, 1988; Booth & Thomason, 1991). In the fasted state, the increase in protein synthesis is concomitant with an increase in protein degradation. In fact, in humans the increase in muscle protein synthesis and degradation correlate 3 h following resistance training in the fasted state (Phillips et al. 1999). From these data, one could hypothesize that, in the fasted state, increased protein degradation drives the increase in protein synthesis.

The rapid up-regulation of protein synthesis after an acute bout of resistance exercise is the result of the activation of the mammalian target of rapamycin complex (mTORC)1 and its down-stream targets p70 S6 kinase (S6K1), the eukaryotic initiation factor 4E binding protein (4EBP), initiation factor 2Bɛ and the proto-oncogene myc (Bolster et al. 2003; Kubica et al. 2005; Nader et al. 2005). The activation of mTORC1 following resistance exercise is well correlated with the degree of hypertrophy following training in both rats and humans (Baar & Esser, 1999; Terzis et al. 2008). Furthermore, this correlation extends from 30 min to 6 h following the bout of exercise, suggesting that prolonged activation of mTORC1 is important in growth.

mTORC1 is also activated in response to other growth stimuli including growth factor treatment and amino acids. In response to growth factors, receptor activation of the class 1 phosphatidylinositol 3OH-kinases (PI3K)–protein kinase B (PKB/akt) pathway leads to the activation of mTORC1 promoting protein synthesis. However, growth factor signalling is not required for hypertrophy or the activation of mTORC1 following resistance exercise. Mutation of the insulin-like growth factor 1 receptor does not prevent muscle hypertrophy (Spangenburg et al. 2008) and neither the accumulation of PtdInsP3 nor PKB/akt activity is required for the activation of mTORC1 following stretch in isolated muscle (Hornberger et al. 2004).

Unlike growth factors, amino acids do not signal through PKB/akt to activate mTORC1. Instead, amino acids signal through other kinases to mTORC1. MAP4K3, a Ste20-related kinase that is involved in the activation of mTORC1 but not mTORC2, is inactivated when amino acids are removed and rapidly reactivated when amino acids are returned, making it a candidate for amino acid control of mTORC1 (Findlay et al. 2007). Another candidate is the class 3 PI3K, vacuolar protein sorting mutant 34 (Vps34). In human cells, Vps34 can signal the amino acid and glucose levels to mTORC1 (Byfield et al. 2005; Nobukuni et al. 2005). First characterized for its role in vesicular trafficking (Herman & Emr, 1990; Stack et al. 1993) and autophagy in response to nutrient deprivation (Kihara et al. 2001b) in Saccharomyces cerevisiae, Vps34 is one of the oldest kinases in the human kinome. When associated with Vps15, Vps34 regulates intracellular protein trafficking (Stack et al. 1993). With a further associated protein, beclin 1 (Liang et al. 1998; Kihara et al. 2001a), Vps34 has been implicated in mammalian autophagy (Petiot et al. 2000; Eskelinen et al. 2002). Human Vps34 (hVps34) is required for insulin stimulation of S6K1 phosphorylation (Byfield et al. 2005) but is not regulated by insulin itself and does not affect insulin-stimulated activation of PKB or inactivation of TSC2 (Byfield et al. 2005).

Because of its role in both sensing amino acid levels and regulating protein degradation, we hypothesized that Vps34 is involved in the activation of mTORC1 by resistance exercise and amino acids. To test this hypothesis, the activity of mammalian Vps34 and S6K1 was determined in three different muscles (TA, PL and Sol) 0.5, 3, 6 and 18 h after an acute bout of high-resistance contractions in rats. In addition, free amino acid levels were determined in the TA muscle 1.5 h after exercise, and the effect of raising the leucine concentration on mVps34 was determined in C2C12 cells.

Methods

Materials

Anti-Vps34 and anti-S6K antibodies, and Vps34 peptide were obtained from the Division of Signal Transduction Therapy (DSTT; University of Dundee). All other chemicals were from Sigma-Aldrich unless stated otherwise.

Animals and acute high-resistance contraction protocol

All procedures were approved by the University of Dundee research ethics committee and performed under UK Home Office project licence number 60/3441. Adult female Wistar rats weighing ∼200 g were obtained from Charles River Laboratories (Tranent, UK). All surgical and collection procedures on animals took place under inhalation anaesthetic using a 2.5% concentration of isoflurane throughout the procedure. Rats were allowed to recover for the appropriate time period post stimulation and were terminated after muscle collection under anaesthesia. The high-resistance contraction protocol was performed as previously described (Baar & Esser, 1999). Briefly, electrodes were attached to the right sciatic nerve anterior to the anatomical branching point. Tetanic contractions were performed using a Grass stimulator at a frequency of 100 Hz, 6–12 V, 1 ms duration, 9 ms delay for 10 sets of 6 repetitions. Each repetition lasted 2 s, a 10 s recovery was permitted between repetitions, and a 1 min recovery was allowed between sets resulting in a 20 min stimulation protocol. At certain periods (0.5, 3, 6 and 18 h) following the acute bout of contractions, stimulated and contralateral muscles were rapidly removed, snap-frozen in liquid nitrogen and stored at −80°C until processed. In order to assess tissue distribution of Vps34, brain, kidney, lung, liver, fat and heart tissue were removed and treated as described above.

Tissue lysis

Frozen tissue was powdered using a mortar and pestle and homogenized using a Polytron PT3000 (Kinematica, Lucerne, Switzerland) at 18 000g in 10-fold mass excess of ice-cold Cantley lysis buffer (10 mm Tris pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton-X 100, 10% glycerol, 100 mm NaF). This was briefly vortexed and centrifuged at 4°C for 5 min at 13 000g to remove insoluble material. Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA, USA).

Western blot

Immunoprecipitated proteins were boiled for 5 min and separated on a 10% gel by SDS-polyacrylamide gel electrophoresis (PAGE) in 1 × Laemmli sample buffer (LSB). Following electrophoresis, proteins were transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) at 100 V for 1 h. The membrane was blocked for 1 h in 3% milk in TBST (Tris-buffered saline + 0.1% Tween). Membranes were incubated overnight at 4°C with appropriate primary antibody in TBST at 1 : 1000. The membrane was then washed 3 times in TBST before incubation for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-sheep IgG secondary antibody in 0.5% milk in TBST at 1 : 10000 (Perbio Science, Cramlington, UK). Antibody binding was then detected using an enhanced chemiluminescence HRP substrate detection kit (Millipore, Watford, UK). Imaging and band quantification were carried out using a Chemi Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK).

S6K activity assay

Endogenous S6K1 was immunoprecipitated for 2 h at 4°C with 1 μg of rabbit anti-S6K1 antibodies using lysates (3 mg protein) prepared as described above, then immobilized on protein-G sepharose for 1 h. Immunocomplexes were washed one time in high detergent buffer A (1% NP-40, 100 mm NaCl, 10 mm Tris pH 7.2, 1 mm EDTA), two times in high salt buffer B (1 m NaCl, 0.1% NP-40, 10 mm Tris pH 7.2), and one time in ST buffer (150 mm NaCl, 50 mm Tris pH 7.2). The immunocomplexes were resuspended in 20 μl of 1.5 × kinase buffer (200 mm Hepes pH 7.2, 100 mm MgCl2, 1 mg ml−1 BSA. 1 : 3000 2-mercaptoethanol). Kinase activity towards a recombinant GST-S6 peptide was assayed for 10 min at 30°C. Reactions were initiated and terminated using 10 μl ATP mix (50 μm unlabelled ATP, 5 μCi of [γ-32P] ATP, 1 μl GST-S6) and 4 × LSB (0.5 m Tris pH 6.8, 0.8% SDS, 20% 2-mercaptoethanol, 30% glycerol), respectively. Reactions were subjected to 10% SDS-PAGE and incorporation of 32P into GST-S6 was assessed by autoradiography and quantified using a Chemi Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK).

Vps34 activity assay

Endogenous Vps34 was immunoprecipitated overnight at 4°C with 2 μg of sheep anti-Vps34 antibodies using 3 mg of total lysate. mVps34 protein was then immobilized on protein-G sepharose for 1 h. Immunocomplexes were washed three times in Cantley lysis buffer, one time in Tris-LiCl (10 mm Tris, pH 7.5, 5 mm LiCl, 0.1 mm Na2VO4) and two times in TNE (10 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1 mm Na2VO4). The immunocomplexes were resuspended in 60 μl TNE+ (TNE, 0.5 mm EGTA, pH 8.0, 1 : 1000 2-mercaptoethanol) and incubated at room temperature for 10 min with 20 μg Vps34 antigen peptide. Substrates for the assay were prepared by adding 10 μl of 30 mm MnCl2 and 10 μl of 2 mg ml−1 phosphoinositol (PI) (bovine liver, Avani Polar Lipids) to each sample. PI was sonicated for 5 min in 10 mm Tris, pH 7.5–1 mm EGTA prior to addition to the assay. Reactions were performed at 30°C with shaking throughout the assay and initiated with the addition of 5 μl of ATP mix (400 μm unlabelled ATP, 12.5 μCi of [γ-32P] ATP, 4.3 μl water). After 10 min, reactions were terminated by the addition of 20 μl of 8 m HCl and phase separated with 160 μl 1 : 1 chloroform : methanol and centrifuged for 5 min at 18 000g. The lower organic phase was spotted on an aluminium-backed 60 Å silica TLC plate (Merck, Damstadt, Germany) and run in a TLC chamber solvent system (60 ml chloroform, 47 ml methanol, 11.2 ml water and 2 ml ammonium hydroxide). The specificity of the assay was determined by using other lipids (PI4P and PI4,5P2) in the assay as substrates. The absence of phosphorylation of these lipids from either muscle or HeLa cell lysates indicated that the assay is specific for a 3-phosphoinositol kinase, presumably Vps34 (data not shown).

2D cell culture

C2C12 cells (ATCC, Middlesex, UK) were grown in a growth media consisting of DMEM supplemented with 10% FBS and 1% penicillin–streptomycin and passaged using trypsin–EDTA. C2C12 cells were seeded on 10 cm plates (500 k cells per plate). When 90% confluent, cells were moved to differentiation medium consisting of DMEM supplemented with 2% horse serum and 1% penicillin–streptomycin. After 5 days of differentiation, medium was removed and cells were incubated with serum-free medium (EBSS) containing no amino acids, physiological amino acids or 320 μm leucine.

HPLC

A 25 mg aliquot of powdered muscle was homogenized in 250 μl ice-cold 12% PCA and centrifuged at 20 000g for 30 min at 4°C. The supernatant was collected and neutralized with 100 μl 2 m K2CO3 and centrifuged for a further 30 min, 20 000g at 4°C and the supernatant collected. Samples were then dried overnight in a vacuum concentrator at 45°C and resuspended in 10 μl of a 2 : 2 : 1 solution of ethanol : 1 m sodium acetate : triethylamine (TEA). Excess reagent was removed by vacuum at 45°C. The samples were then reacted with 20 μl of a 7 : 1 : 1 : 1 derivatizing mixture of ethanol : dH2O : TEA : phenylisothiocyanate (PITC) (Pierce, Rockford, IL, USA) for 20 min at room temperature and dried by vacuum. The resultant phenylthiocarbamyl peptides were dissolved in 200 μl of 50 mm sodium acetate (pH = 6.5) containing 10% (v/v) acetonitrile, and the solution was filtered through a 0.45 μm filter. After derivatization, amino acids were separated by a Hewlett Packard 1050 HPLC system (Minnesota, USA) using standard protocols. The column was maintained at 50°C, and the absorbance at 254 nm monitored. Individual amino acids were identified by their retention times using standard amino acids and relative changes in peak size were measured.

Statistics

Data are given as mean ±s.e.m. values with a minimum n of 4. Statistical significance (P < 0.05) of the data was determined by a Student's t test or single factor ANOVA with post hoc analysis using Tukey's honestly significant difference test.

Results

mVps34 tissue distribution

To determine the distribution of mVps34 in rats, mVps34 protein was analysed by immunoprecipitation followed by Western blotting. mVps34 protein was found in all of the tissues investigated with the highest levels found in brain and muscle (Fig. 1).

Figure 1. Levels of mVps34 immunoprecipitated from 3 mg of total protein from rat brain, kidney, lung, liver, fat, heart, soleus and tibialis anterior muscle lysates.

Figure 1

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mVps34 is approximately 115 kDa.

mVps34 and S6K1 activity following an acute bout of high-resistance contraction

The high-resistance contraction model used results in ∼16% hypertrophy of the TA and EDL muscles, ∼7% hypertrophy in the plantaris muscle and no hypertrophy in the soleus following 6 weeks of training allowing a dose-dependent analysis of the hypertrophic response (Baar & Esser, 1999). In the TA muscle, mVps34 activity increased 3 h after acute high-resistance contraction (68.8 ± 15.1%), was maintained high out to 6 h (36.01 ± 8.79%) and returned to baseline values by 18 h (Fig. 2A). In the PLN muscle, mVps34 activity increased only at the 3 h time point (24.5 ± 7.92%), while in the soleus muscle Vps34 activity did not change after the acute exercise bout (Fig. 2B). The increase in Vps34 activity was not due to an increase in Vps34 protein since no difference in total Vps34 was seen between the control and stimulated legs (Fig. 2C).

Figure 2. mVps34 activity following high resistance contractions.

Figure 2

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A, difference in mVps34 activity between the exercised and control TA muscles 0.5, 3, 6 and 18 h after high-resistance contractions expressed as the percentage difference between the high-resistance contraction and the contralateral control leg. Inset shows representative assay of mVps34 activity in the TA at all time points. Significant difference (P < 0.05) between the stimulated and control muscle is denoted with †. B, difference in mVps34 activity 3 h following high-resistance contractions in the TA, PLN and SOL muscles. Significant difference (P < 0.05) between the stimulated and control muscle is denoted with †. C, representative images of the total amount of mVps34 immunoprecipitated from control and stimulated TA muscles 3 h following high-resistance contractions.

In the TA muscle, high-resistance contractions resulted in a large increase in S6K1 activity 30 min after cessation of the exercise bout (Fig. 3A; 466.3 ± 112.08%) and this increase in activity was maintained out to 18 h following the completion of exercise (3 h = 124.7 ± 15.96%; 6 h = 129.2 ± 17.18%; and 18 h = 297.5 ± 95.73%). In the PLN muscle, S6K1 activity was elevated at 0.5 h and 3 h (0.5 h = 33.1 ± 2.29% and 3 h = 47.0 ± 6.65%; Fig. 3B), but had returned to control levels by 6 h (data not shown). In the soleus muscle there was no significant change in S6K1 activity at any time point.

Figure 3. S6K1 activity following high resistance contractions.

Figure 3

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A, difference in S6K1 activity between the exercised and control TA muscles 0.5, 3, 6 and 18 h after high-resistance contractions expressed as the percentage difference between the high-resistance contraction (S) and the contralateral control leg (C). Inset shows representative assay of S6K1 activity in the TA at all time points. Significant difference (P < 0.05) between the stimulated and control muscle is denoted with †. B, difference in S6K1 activity 3 h following high-resistance contractions in the TA, PLN and SOL muscles. Significant difference (P < 0.05) between the stimulated and control muscle is denoted with †.

Intramuscular leucine increases 90 min following high-resistance contraction

To determine whether amino acid levels within the active muscle were altered following high-resistance contraction, the concentration of the branched chain amino acids and the ammonia shuttling amino acid glutamine were measured 1.5 h following an acute bout of high-resistance contractions using HPLC. While there was a trend towards increased glutamine levels, only isoleucine and leucine increased significantly (Fig. 4; 28.6 ± 12.8% and 24.7 ± 2.9%). Control levels of leucine, valine, glutamine and isoleucine (0.46 ± 7.1, 0.27 ± 2.7, 9.6 ± 4.0 and 0.024 ± 7.5 nmol mg−1, respectively) were within the normal range for rat muscle. To initially test whether the measured increase in intramuscular amino acids was the result of proteolysis due to activation of Vps34, we determined the activity of Vps34 at this time point. Ninety minutes following completion of a bout of high-resistance contractions there was no change in Vps34 activity (−15.9 ± 15.03%). Furthermore, at 3 h when Vps34 activity is increased, we saw no difference in intramuscular amino acid level between the stimulated and control muscles (data not shown).

Figure 4. Difference in leucine (Leu), valine (Val), glutamine (Gln) and isoleucine (Ile) levels between the exercised and control TA muscles 1.5 h after high-resistance contractions expressed as the percent difference between the high-resistance contraction and the contralateral control leg.

Figure 4

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Significant differences (P < 0.05) between stimulated and control muscles are denoted with †.

mVps34 is activated by leucine in C2C12 cells

Since leucine plays a key role in the activation of mTORC1 and the increase in intracellular amino acids preceded the activation of Vps34, the effect of increased leucine on mVps34 activity was determined in C2C12 cells. The addition of leucine alone to amino acid-starved C2C12 muscle cells for 3 h resulted in the activation of mVps34 (Fig. 5; control = 179.67 ± 23.21 AU; Leu = 632.06 ± 98.75 AU), whereas the addition of physiological levels of all amino acids had no effect on mVps34 activity.

Figure 5. Effect of leucine addition on mVps34 activity in C2C12 cells.

Figure 5

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C2C12 cells were treated by either amino acid starvation, all of the amino acids at physiological levels or addition of 320 μm leucine. Significant differences are denoted with † with P < 0.05.

Discussion

An acute bout of high-resistance contractions increases leucine concentration in the active muscle and activates the mammalian class III PI3K, mVps34 and the ribosomal protein S6 kinase. This is the first demonstration that high-resistance contraction increases the leucine concentration in the working muscle. Furthermore, high-resistance contraction is the first physiological stimulus that is shown to increase the activity of Vps34. The time course of activation of S6K1 suggests that mVps34 is not involved in the early activation of S6K1 but may play a role in the prolonged activation of mTORC1 following high-resistance contractions.

The stimulation protocol used in the current study results in contraction of all distal hind limb muscles. The larger muscle mass in the posterior of the leg (gastrocnemius, soleus and plantaris muscles) undergoes powerful shortening contractions causing antagonistic lengthening contractions of the TA and extensor digitorum longus (EDL) muscles in the anterior of the leg. Previous work has shown that both the activation of mTORC1 and the training-induced skeletal muscle hypertrophy are greater in the lengthening muscles than in the shortening muscles. The increase in mVps34 activity 3 h following an acute bout of high-resistance contractions follows the same pattern. In fact, the activities of the mVps34 and S6K1 correlate 3 h after exercise (Fig. 6), the time point where both the degree of protein synthesis and degradation are highest (Phillips et al. 1997). While the activity of S6K1 and Vps34 correlate at 3 and 6 h, they do not correlate at either 30 min or 18 h. These data suggest that mVps34 may play a limited role in the activation of mTORC1 following high-resistance contractions.

Figure 6. Correlation between mVps34 and S6K1 activity 3 h following high-resistance contractions.

Figure 6

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The activity of Vps34 and S6K1 are plotted for the SOL, PLN and TA muscles 3 h following an acute bout of high-resistance contractions.

If mVps34 is involved in signalling to mTORC1 and S6K1 following an acute bout of high-resistance contractions, what aspect of the exercise bout activates mVps34? Our original hypothesis was that, following high-resistance contractions, the rate of protein synthesis increased resulting in a diminished free amino acid pool. In the muscle, we thought that mVps34 would then be activated to replenish the intramuscular amino acid pool through autophagic protein degradation (Tee et al. 2005). In fact, when amino acid levels were determined, there was a modest but significant increase in intramuscular leucine levels 1.5 h after the acute bout of high-resistance contractions. This increase in intracellular amino acid levels is consistent with the increase in leucine, lysine and alanine influx observed 3 h after a bout of resistance exercise in humans (Biolo et al. 1995). Further, this same study found that the increase in amino acid influx was associated with both increased protein synthesis and degradation. With this background, it is important to note that the plasma amino acid levels in the current study would be similar in the exercised and control limbs. This means that the increase in amino acid concentration following resistance exercise is a direct result of the exercise bout. Interestingly, as little as a 17.5% increase in leucine has been shown to result in maximal phosphorylation of S6K1 in oocytes (Christie et al. 2002). To determine whether the modest increase in leucine measured following high-resistance contractions was sufficient to activate mVps34, 320 μm leucine was added to C2C12 muscle cells. Even with this relatively small increase in leucine levels, mVps34 was activated 3.2-fold. This suggests that mVps34 may be activated in muscle secondary to an increase in intramuscular leucine resulting either from a surge in protein breakdown or accelerated skeletal muscle uptake following high-resistance contractions. Vps34 activation could then result in the stimulation of protein synthesis through mTORC1 and protein degradation through its binding partners Vps15 and beclin-1.

If mVps34 is activated by leucine and is also important in maintaining the high level of mTORC1 activity after resistance exercise, this could well explain the benefits of amino acid ingestion concomitant with resistance exercise. In the fasted state, net protein balance remains negative following resistance exercise with protein breakdown exceeding protein synthesis (Biolo et al. 1995; Phillips et al. 1997; Tipton & Wolfe, 1998). Ingestion of an essential amino acid–carbohydrate mix immediately before (Tipton & Witard, 2007) or soon after (Koopman et al. 2005) an exercise bout enhances the degree of protein synthesis and reduces breakdown thereby maximizing muscle growth (Tipton et al. 2001). It is important to note that the animals in this study were in the fasted state and that this might have resulted in an increase in Vps34 activity that is greater than would have been seen in the fed state. It will be important to determine the differential effect of resistance exercise in the fed versus fasted state on Vps34 and mTORC1 activity.

Leucine appears to have the most significant effect on protein balance since the fractional synthetic rate following a protein + CHO + leucine supplement is greater than protein + CHO alone (Koopman et al. 2005). This could be partly explained by the Vps34 data presented here. Returning all of the amino acids to the C2C12 cells had no effect on Vps34 whereas the addition of leucine to C2C12 cells activates mVps34, suggesting that Vps34 may play a role in the anabolic effects of leucine. However, protein, CHO and leucine supplementation in vivo reduces protein breakdown whereas Vps34 activation should increase autophagy. The explanation for this apparent paradox may lie in the Vps34 binding partner. In the absence of amino acids, the association of Vps34 with beclin-1 may increase, resulting in increased autophagy. When amino acid levels are high, Vps34 may exist in a distinct complex that is better able to activate mTORC1 but has little autophagic activity.

It is important to note that Vps34 is not the only kinase that could link amino acids to mTORC1. MAP4K3 is another kinase that is sensitive to amino acids, activates S6K1 but not PKB/akt, and is not activated by insulin (Findlay et al. 2007). Further, Ste20 kinases like MAP4K3 control ion transport and cell size (Strange et al. 2006). The ion transport activity is interesting since stretch of muscle cells increases amino acid transport in a ouabain-sensitive manner and this increase in amino acids is required for the stretch-induced increase in protein synthesis (Vandenburgh & Kaufman, 1982). Lastly, knockdown of MAP4K3 in HeLa cells has the same effect on cell size as rapamycin treatment (Findlay et al. 2007) suggesting that the two affect the same pathway, namely mTORC1.

While the activity of Vps34 and S6K1 correlate 3 h following high-resistance contractions, it is clear from comparing Figs 2 and 3 that S6K1 is initially activated in the absence of Vps34 activation. This initial activation of S6K1, and by extension mTORC1, is probably a direct result of the mechanical stimulus. Since the activation of mTORC1 by mechanical force occurs independent of PKB/akt (Hornberger et al. 2004), and results in the activation of phospholipase D and the production of phosphatidic acid (Hornberger et al. 2006), this suggests that the mechanical effects are probably entering the mTORC1 pathway at either TSC2 or Rheb. When Rheb is activated, either by an as yet unidentified GTP exchange factor or inactivation of TSC2, it increases its association with PLD1 and this promotes the PA-dependent activation of mTORC1 (Sun et al. 2008). Vps34 also cannot be responsible for the activation of mTORC1 at 18 h. This stage of the response might be the result of growth factors such as Wnt that are in some way required for the response to resistance exercise (Armstrong & Esser, 2005; Armstrong et al. 2006).

In conclusion, the results from the present study suggest that mVps34 is activated after an acute bout of high-resistance contractions. The increase in Vps34 activity may be the result of increased uptake of amino acids, specifically leucine, and correlates with the prolonged activation of mTORC1. This suggests that Vps34 may play a role in the maintained activation of mTORC1 and skeletal muscle hypertrophy following exercise.

Acknowledgments

This work was supported by a project grant from the Wellcome Trust (077426). J.T.M. was supported by an Intermediate Fellowship from the British Heart Foundation (FS/05/059).

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