skeletal muscle mass is regulated by the balance between muscle protein synthesis (MPS) and muscle protein breakdown. Resistance exercise (RE) is known to induce muscle hypertrophy as a result of cumulative increases in MPS that occur in response to acute RE (2, 20). RE volume is recognized as an important factor that stimulates MPS (3–5) independently of RE intensity (15). For example, three sets of RE lead to a more pronounced increase in MPS than a single set of RE (4). However, the effect of further increase in muscle contractions on MPS remains unknown.

Although the molecular mechanisms underlying RE-induced MPS are not fully elucidated, members of the mammalian target of rapamycin (mTOR) signaling pathway, especially mTOR complex 1 (mTORC1), are known to play a critical role in the process. Previous studies reported that RE-induced p70S6K phosphorylation, a marker of mTORC1 activity, is RE volume-dependent (5, 23). Therefore, an increase in RE volume leads to mTORC1 activation and would significantly contribute to RE volume-dependent increase in MPS. However, similar to MPS, the effect of higher-volume RE (e.g., >10 sets of RE) on mTORC1 activity remains unclear.

In contrast to its positive regulatory effect on mTORC1 and MPS, muscle contraction, especially endurance-type contraction, additionally stimulates the activity of signaling molecules that inhibit mTORC1 and MPS, including regulated in development and DNA damage response 1 (REDD1) and AMP-activated protein kinase (AMPK; Refs. 7, 8, 11, 17, 19, 21). REDD1 and AMPK are activated by various cellular stressors such as energy depletion and hypoxia (11, 13). Therefore, we hypothesized that RE volume-dependent mTORC1 activation and MPS increase eventually reach a plateau and/or start to decrease with higher-volume RE. To test this hypothesis, we investigated the effects of low- to high-volume RE (1, 3, 5, 10, and 20 sets of RE) on mTORC1 signaling and MPS using a rodent model of RE.

Sample size was determined by a power analysis of preliminary data from our laboratory. The sample size of n = 5 per group has enough power to detect a 20% change in MPS. Moreover, n = 5 per group is large enough to detect statistical significance in p70S6K, 4E-BP1, and rpS6 before and after five sets of exercise ( n = 3–5 is needed to provide 80% power). Two-way ANOVA (RE × set) was used to evaluate changes in the phosphorylated and total proteins. Post hoc analyses were performed using t -tests with a Benjamini and Hochberg false discovery rate correction for multiple comparisons when significant interactions were found. The level of significance was set at P < 0.05.

Muscle protein synthesis was measured using the in vivo surface sensing of translation (IV-SUnSET) method ( 9 ). As previously reported ( 19 ), 0.04 μmol puromycin/g body wt (Wako) diluted in a 0.02 M PBS stock solution was injected intraperitoneally to the rats under anesthesia, and the gastrocnemius muscle was removed exactly 15 min after puromycin administration. Following homogenization and centrifugation at 2,000 g for 3 min at 4°C, the supernatant was collected and processed for Western blotting.

Western blotting analysis was performed as previously reported ( 18 ). Briefly, 20 mg of powdered muscle samples were homogenized in 10 volumes of homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). The homogenates were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected, and the protein concentration of each sample was determined using a Protein Assay Rapid Kit (Wako, Osaka, Japan). Samples were diluted in 3× sample buffer (Cell Signaling Technology) and boiled at 95°C for 5 min. Equal amounts of protein were then separated using electrophoresis in 5–20% sodium dodecyl sulfate polyacrylamide gradient gels. Proteins were subsequently transferred to polyvinylidene difluoride membranes, washed in Tris-buffered saline solution containing 0.1% Tween 20 (TBST), and blocked with RAPIDBLOCK SOLUTION (AMRESCO, Solon, OH) for 5 min at room temperature. Membranes were washed and incubated overnight at 4°C with the primary antibody, washed again in TBST, and incubated for 1 h at room temperature with the appropriate secondary antibody. Proteins were visualized using chemiluminescent reagents, and the bands were detected using the C-DiGit Blot Scanner (LI-COR, Lincoln, NE). Staining with Coomassie blue was performed to normalize protein loading. Phosphorylated proteins were normalized to the corresponding total proteins except for rpS6, 4E-BP1, and TSC2. Band intensities were quantified using Image Studio (LI-COR).

Unilateral isometric muscle contractions were stimulated in the gastrocnemius as previously described ( 19 ). Briefly, rats were anesthetized with isoflurane, and a patch of hair in the lower leg of each rat was shaved off. The rats were then positioned with their right foot on a footplate (the ankle joint angle was positioned at 90°) in the prone posture. Similar to our previous studies ( 18 , 19 ), after 12-h overnight fasting, the gastrocnemius muscle was stimulated percutaneously with electrodes (Vitrode V, Ag/AgCl; Nihon Kohden, Tokyo, Japan) cut into 10× 5-mm sections and connected to an electric stimulator and isolator (SS-104J; Nihon Kohden). The right gastrocnemius muscle was isometrically exercised (3-s stimulation × 10 contractions with a 7-s interval between contractions and 3-min rest interval between sets), whereas the left gastrocnemius muscle served as an internal control. The voltage (~30 V) and stimulation frequency (100 Hz) were adjusted to produce maximum isometric tension. Fasting was maintained for 6 h after 1, 3, 5, 10, and 20 sets of RE, and muscle samples were obtained ( n = 5 for each condition). Afterward, muscle samples were rapidly frozen in liquid nitrogen and stored at −80°C until use.

Antibodies against phosphorylated (phospho-) TSC2 (Ser1387, cat. no. 5584), total TSC2 (cat. no. 4308), phospho-Akt (Ser473, cat. no. 9271), total Akt (cat. no. 9272), phospho-mTOR (Ser2448, cat. no. 2971), total mTOR (cat. no. 2983), phospho-AMPK (Thr172, cat. no. 2535), total AMPK (cat. no. 2532), phospho-p70S6K (Thr389, cat. no. 9205), total p70S6K (cat. no. 2708), phospho-rpS6 (Ser240/244, cat. no. 2215), total rpS6 (cat. no. 2217), phospho-4E-BP1 (Thr37/46, cat. no. 9459), total 4E-BP1 (cat. no. 9452), LC3 (cat. no. 2775), and ubiquitin (cat. no. 3933) were obtained from Cell Signaling Technology (Danvers, MA). The total REDD1 (cat. no. 10638-1-AP) and puromycin (cat. no. MABE343) antibodies were obtained from Proteintech (Chicago, IL) and Millipore (Billerica, MA), respectively.

Male Sprague-Dawley rats (10 wk old) were obtained from Japan SLC (Hamamatsu, Japan). All of the rats were housed for 1 wk in an environment maintained at 22–24°C under a 12:12-h light-dark cycle and were allowed food (CE-2; CLEA Japan) and water ad libitum. Energy percentages from protein, fat, and carbohydrate intake were 29, 13, and 58%, respectively. The animal protocol and use of facilities were reviewed and approved by the Ethics Committee for Animal Experiments at Nippon Sport Science University.

We next investigated the signaling molecules that inhibit mTORC1. As shown in Fig. 6 , REDD1 levels did not change following 1 set of RE but increased significantly in all groups subjected to multiple sets of RE. However, no significant differences in the increase in REDD1 levels were observed between groups that performed 10 and 20 sets of RE. On the other hand, although AMPK phosphorylation did not change after RE, Ser1387 phosphorylation of TSC2, a downstream target of AMPK, decreased gradually with increasing number of RE sets and was accompanied by a decrease in total TSC2 levels.

Fig. 4. Phosphorylated (p-) protein and total (t-) protein levels of mTOR ( A ), Akt ( B ), p70S6K ( C ), rpS6 ( D and E ), and 4E-BP1 ( F and G ) after 1, 3, 5, 10, and 20 sets of resistance exercise. Values represent means ± SE. * P < 0.05 vs. control muscle, a P < 0.05 vs. exercised muscle after 1S, b P < 0.05 vs. exercised muscle after 1S and 3S, c P < 0.05 vs. exercised muscle after 1S, 3S, and 5S, and d P < 0.05 vs. exercised muscle after 1S, 3S, 5S, and 10S.

We first investigated Akt, which is known as an effector of insulin/IGF-1 signaling and can induce muscle hypertrophy via mTOR signaling ( 22 ). As shown in Fig. 4 , although a significant main effect of RE was observed, the number of RE sets (RE volume) or their interactions did not produce a significant effect on Akt phosphorylation. By contrast, significant increase in p70S6K phosphorylation after RE was observed in all of the conditions tested, and the degree of phosphorylation gradually increased with increasing number of RE sets. Similar results were observed in terms of rpS6 phosphorylation, a downstream target of p70S6K. On the other hand, Thr37/46 phosphorylation of 4E-BP1, a direct target of mTORC1, increased in response to RE (main effect of RE), but no main effect of RE volume or interactions were observed. The active form ratio of 4E-BP1 (γ/total α + β + γ), a marker of 4E-BP1 activity, also increased with RE (main effect of RE), and the observed increasing trend was more pronounced when a higher number of RE sets was performed (main effect of RE set). As shown in Fig. 5 , MPS response plateaued out at approximately five sets of RE, whereas the p70S6K phosphorylation response continued to increase with further stimulation. Approximately 30% of the p70S6K phosphorylation response was observed after five sets of RE.

We investigated the effect of increasing RE volume on LC3-I and -II proteins (autophagosome marker) and ubiquitinated proteins. As shown in Fig. 2 , no changes in the levels of both muscle catabolic markers were observed even after 20 sets of RE.

A single set of RE did not increase MPS, but multiple sets (3, 5, 10, and 20 sets) of RE significantly increased MPS ( Fig. 1 ). These observations are in accordance with those of a previous study in humans showing that 3 sets of RE increased MPS more than 1 set of RE ( 4 ). However, the increase in MPS reached a plateau after 3 or 5 sets of RE, and no further increase in MPS was observed in response to additional RE sets.

DISCUSSION

RE volume is known to be an important variable affecting MPS in response to RE and is considered, at least in part, through mTORC1 signaling-dependent mechanism (3–5, 23). However, the relationship between RE volume and MPS at relatively high-volume RE remains unclear. Results from the present study using sedentary rats demonstrate that MPS increases with increasing number of RE sets and eventually reaches a plateau. By contrast, both p70S6K phosphorylation at Thr389, a marker of mTORC1 activity, and phosphorylation of rpS6 at Ser240/244, a downstream target of p70S6, increased gradually with increasing RE volume.

The mechanisms responsible for MPS response to increasing RE volume are considered to be related, in part, to p70S6K phosphorylation (4). Similar to reports from previous studies (4, 23), multiple sets of RE led to a more pronounced increase in p70S6K phosphorylation and MPS than a single set of RE. However, although p70S6K phosphorylation increased linearly with the number of RE sets, the levels of phosphorylation did not correspond to MPS response. The reason for the mismatch in the relationship between p70S6K phosphorylation and MPS is currently unclear. Although multiple studies show a correlation between p70S6K phosphorylation and MPS (3, 4), a previous study reported that 500 eccentric muscle contractions increased p70S6K phosphorylation but significantly decreased MPS 1 h after performing muscle contractions (14). Moreover, the relationship between p70S6 phosphorylation and MPS is currently controversial, given that recent studies have reported that exercise increases MPS independently of p70S6K phosphorylation (18, 21, 24). Therefore, p70S6K or rpS6 phosphorylation may not always be a reliable marker of RE-induced MPS, and RE may regulate MPS via p70S6K phosphorylation-independent mechanisms.

Previous studies of nutrition and insulin signaling showed results similar to those of our study. Bilan et al. (1) summarized previous studies and reported that very low levels of insulin-stimulated Akt phosphorylation (only ~30% of maximal stimulation) are necessary to stimulate GLUT4 translocation fully. Crozier et al. (6) found that p70S6K phosphorylation had no plateau effect on increasing leucine stimulation, whereas MPS plateaued at a much lower dose. Greenhaff et al. (12) demonstrated a similar effect for insulin-stimulated MPS in human skeletal muscle. These observations indicate that small changes in signaling are physiologically relevant and required for a full MPS response. However, it should be noted that p70S6K phosphorylation and MPS may have different time courses after RE. Further studies are required to determine the regulatory role of mTOR/p70S6K signaling in RE-induced MPS and investigate the mechanisms of the observed mismatch between levels of p70S6K phosphorylation and MPS after RE. Additional studies are needed to understand the relationship between signaling responses and MPS.

In this study, we also measured phosphorylation of 4E-BP1 as marker of mTORC1 activity. Surprisingly, although RE increased 4E-BP1 phosphorylation, no differences among the different RE volumes were observed. The reason for the discrepancy remains to be investigated, but previous studies have also reported varying degrees of phosphorylation of p70S6K and 4E-BP1 in response to muscle contraction (16), suggesting that p70S6K and 4E-BP1 may be regulated differently or have different phosphorylation sensitivities. In this study, the active form ratio between the level of the γ-form of 4E-BP1 (the slowly migrating and heavily phosphorylated active form that dissociates from eIF4E and promotes translation initiation) and the total 4E-BP1 level was used as measure of 4E-BP1 activity. The calculated active form ratio also indicates that the response of 4E-BP1 to RE is different from that of p70S6K but is almost similar to MPS response. A similar result was reported that the cellular levels of γ-form are thought to be regulated by both mTORC1-dependent and -independent mechanisms (16) and therefore may more greatly integrate cellular anabolic signaling than p70S6K and 4E-BP1 phosphorylation.

Previous studies reported that cellular levels of REDD1 affect p70S6K phosphorylation (10, 11). More specifically, studies report that although REDD1 expression levels did not affect the degree of the contraction-induced increase in p70S6K phosphorylation, loss of REDD1 per se increased p70S6K phosphorylation (10). In this study, REDD1 expression was observed to increase significantly in response to multiple sets of RE. However, p70S6K phosphorylation levels increased gradually with increasing RE volume, suggesting that contraction-induced increase in REDD1 levels has a minor role in the regulation of contraction-induced p70S6K phosphorylation. By contrast, a decrease in both total and phosphorylated TSC2 level by RE was observed, particularly after large numbers of muscle contractions. TSC2 is known to inhibit mTORC1 activity; therefore, a decrease in TSC2 activates mTORC1 and increases p70S6K phosphorylation. Although the exact mechanisms responsible for the observed decrease in TSC2 at relatively higher volumes of RE remain unclear, TSC2 may contribute to elevated levels of p70S6K phosphorylation as observed in the present study. However, it should be noted that higher levels of p70S6K phosphorylation at higher-volume RE did not correspond to changes in 4E-BP1 and MPS.

We investigated the effect of RE volume on LC3-I and -II proteins (autophagosome marker) and ubiquitinated proteins. However, no changes in the levels of both muscle catabolic markers were observed even after 20 sets of RE. Thus the results suggest that acute RE, even at high volume, has little or no effect in increasing muscle protein breakdown via autophagosome formation and/or protein ubiquitination at least during the middle phase of the recovery period (e.g., 6 h after RE).

In summary, we demonstrated that levels of p70S6K phosphorylation increased linearly with increasing RE volume. However, the relationship between RE volume and MPS was not linear, suggesting that 1) the increase in MPS with increasing RE volume does not solely depend on p70S6K phosphorylation, 2) higher RE volume inhibits MPS via p70S6K phosphorylation-independent mechanisms, or 3) MPS response to RE reaches saturation with further increase in RE volume (small changes in signaling are physiologically relevant and required for a full MPS response). Future studies should elucidate the detailed mechanisms underlying the observed plateau phenomenon of RE-induced increase in MPS and are expected to serve as a basis for understanding the in vivo relationship between signaling responses and physiological output measures.