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Am J Physiol Endocrinol Metab 290: E1205-E1211, 2006. First published January 17, 2006; doi:10.1152/ajpendo.00593.2005
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Insulin-facilitated increase of muscle protein synthesis after resistance exercise involves a MAP kinase pathway

James D. Fluckey,1 Micheal Knox,3 Latasha Smith,3 Esther E. Dupont-Versteegden,2,3 Dana Gaddy,3 Per A. Tesch,5 and Charlotte A. Peterson2,3,4

1Department of Health and Kinesiology, Texas A&M University, College Station, Texas; Departments of 2Geriatrics and 3Physiology and Biophysics, University of Arkansas for Medical Sciences, and 4Central Arkansas Veterans Health Care System, Little Rock, Arkansas; and 5Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden

Submitted 29 November 2005 ; accepted in final form 11 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have implicated the mTOR-signaling pathway as a primary component for muscle growth in mammals. The purpose of this investigation was to examine signaling pathways for muscle protein synthesis after resistance exercise. Sprague-Dawley rats (male, 6 mo old) were assigned to either resistance exercise or control groups. Resistance exercise was accomplished in operantly conditioned animals using a specially designed flywheel apparatus. Rats performed two sessions of resistance exercise, separated by 48 h, each consisting of 2 sets of 25 repetitions. Sixteen hours after the second session, animals were killed, and soleus muscles were examined for rates of protein synthesis with and without insulin and/or rapamycin (mTOR inhibitor) and/or PD-098059 (PD; MEK kinase inhibitor). Results of this study demonstrated that rates of synthesis were higher (P < 0.05) with insulin after exercise compared with without insulin, or to control muscles, regardless of insulin. Rapamycin lowered (P < 0.05) rates of synthesis in controls, with or without insulin, and after exercise without insulin. However, insulin was able to overcome the inhibition of rapamycin after exercise (P < 0.05). PD had no effect on protein synthesis in control rats, but the addition of PD to exercised muscle resulted in lower (P < 0.05) rates of synthesis, and this inhibition was not rescued by insulin. Western blot analyses demonstrated that the inhibitors used in the present study were selective and effective for preventing activation of specific signaling proteins. Together, these results suggest that the insulin-facilitated increase of muscle protein synthesis after resistance exercise requires multiple signaling pathways.

mammalian target of rapamycin; rapamycin; extracellular signal-related kinases; PD-098059

THE AKT (PKB)/MTOR PATHWAY has recently been implicated as a primary regulator of skeletal muscle hypertrophy or recovery from atrophy in mammals. Bodine et al. (2) demonstrated that rapamycin, a specific inhibitor of the mTOR pathway, inhibited skeletal muscle hypertrophy 7 days after synergistic ablation in rodents. Others have demonstrated increased levels of mammalian target of rapamycin (mTOR) activity in skeletal muscle after a single bout of resistance exercise (3) or following high-frequency electrically-stimulated contractile activity (23) in rodents, suggesting that mTOR plays an important role in post exercise anabolic responses. In support of this notion, it appears that phosphorylation of mTOR at Ser2448 is indicative of chronic hypertrophy as observed during recovery from immobilization, and a lack of phosphorylation at Ser2448 was associated with chronic atrophy following limb immobilization (24). Together, these results suggest a significant role for mTOR in hypertrophy of skeletal muscle in a variety of conditions.

The Akt-mTOR pathway directly interacts with factors involved with peptide chain initiation (2, 19, 24) and therefore is involved in protein synthesis. We (11, 12) have demonstrated that insulin is necessary for the elevation of muscle protein synthesis after resistance exercise. It is thought that postexercise rates of muscle protein synthesis are a major contributor to the elevation of muscle mass in response to resistance exercise (27, 28, 30, 31). Although studies examining mTOR activity as it correlates with muscle protein mass are compelling, and likely required for overall hypertrophy via directing translation of specific mRNAs coding for proteins that are involved with the translational process, such as eukaryotic initiation factor (eIF)2B-{epsilon}, it is unclear whether mTOR activity is directly involved with signaling processes associated with the postresistance exercise elevations of muscle protein synthesis. For example, Bolster et al. (3) demonstrated increased mTOR activity 1 h after resistance exercise in rats, but a similar study (17) did not observe alterations of muscle protein synthesis for up to 12 h after exercise. This suggests that signaling through the mTOR pathway may not always be directly associated with increases of muscle protein synthesis.

Alternatively, the control of muscle hypertrophy after exercise may involve a mitogen activated protein (MAP) kinase signaling pathway (18). Exercise has been shown to activate the Ras-ERK (extracellular signal-regulated kinase) pathway (4, 15), and this activation correlates to peak tension developed in the muscle (25). Furthermore, the use of PD-098059, a selective inhibitor of the Ras-ERK pathway at the level of MAPK kinase (MEK1/2), effectively inhibits IGF-I-induced muscle hypertrophy (16). Although these results are consistent with the concept that MAPK pathways are involved with skeletal muscle hypertrophy, little is known about this signaling pathway as it relates to muscle protein synthesis, particularly after resistance exercise.

Therefore, the purpose of this study was to examine the effect of resistance exercise, a potent anabolic stimulant for skeletal muscle, on insulin signaling for the transient elevation of muscle protein synthesis following resistance exercise. We utilized a previously published model of resistance exercise, i.e., flywheel technology, because we have shown this methodology to be effective for elevating rates of protein synthesis in exercised muscle (9). We specifically focused on two major pathways of signaling that have been implicated in the posttranscriptional growth process, and involve rapamycin-sensitive and PD-098059-sensitive kinases. Furthermore, because it has been demonstrated that insulin is a necessary component for postresistance exercise elevations of muscle protein synthesis (12) and a potent agonist for signaling in these two pathways, we examined rates of synthesis either with or without insulin (18). We hypothesized that the insulin-mediated elevation of protein synthesis after 2 days of resistance exercise utilizes a MAP kinase pathway.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and operant conditioning. Male Sprague-Dawley rats (6 mo old) were used for this study, and methods were approved by the Institutional Animal Care and Use Committee of University of Arkansas for Medical Sciences. Resistance-exercised (RE) and age-matched control animals were singly housed in a controlled temperature and humidity environment with food and water provided ad libitum. Before experiments, all animals were operantly conditioned to engage in rat resistance exercise training, as described previously (9, 14, 29). Briefly, rats were taught to press an illuminated lever in a specially designed cage to avoid a brief foot shock stimulus (<1 mA, 60 Hz; 1–5 V). The movement facilitated by the entrainment process required full extension and flexion of the hindlimb, which resembles the "squat" as seen in traditional weight room settings, with the exception that animals using the flywheel are positioned in a prone, horizontal position to perform resistance exercise (see Resistance exercise training). Once appropriately entrained, the animals engaged in the flywheel training protocol with very little or no shock. Entrainment to reach this point required approximately four distinct sessions lasting 30–45 min, with each session separated by 48–72 h. All familiarization and resistance exercise sessions were conducted at the onset of the dark cycle.

Tail cast attachment. RE rats were fitted with a special tail cast for attaching the animal to the training apparatus. Briefly, following anesthesia (ketamine-xylazine, 60 and 10 mg/kg, respectively), we used a modified method (20) that may be used for hindlimb suspension whereby a surgical steel pin was placed cross-sectionally through a portion of the tail and subsequently casted into place. A braided cable was inserted through the pin and secured on the dorsal aspect of the cast, forming a loop to secure the resistance exercise tether. The advantage of using this methodology is that the tail cast apparatus remains in place during the rigorous resistance exercise protocol with little or no additional stress to the animal. The tail cast technique was performed 2 days before the resistance exercise studies. Resistance exercise was performed on days 1 and 3 of the study, and muscles were harvested on day 4.

Resistance exercise training. We have developed a modified version of the human flywheel resistance exercise apparatus (1, 9). Briefly, RE rats were tethered via a tail cast apparatus that was spooled around an inertia wheel located on the outside of the resistance exercise apparatus. The rat was allowed to place its feet on a shock grid at one end of the cage, and a bar capable of illumination was located in the apparatus opposite to the shock grid. After the entrainment period described above, the animals engaged in resistance exercise. For resistance exercise, upon illumination of the bar, the animal fully extended its hindlimbs in an effort to depress the bar. As a result of this extension, the rat pulled against the tether, using enough force to overcome the mass of the wheel (similar to the unwinding of a yo-yo). Once the tether was fully unspooled at the rat’s full extension, the momentum or inertia of the spinning wheel forced the tether to spool again. This spooling action facilitated movement of the animal back to the original starting point. Once the animal was back in the starting position, the bar was illuminated again, facilitating another repetition by the animal. The flywheel exercise facilitates movements that are similar to a squat, as performed by humans, and involves movement at the hip, knee, and ankle joints. When needed, shock was applied briefly (<1 s) to facilitate movement of the animal.

The resistance training protocol consisted of two exercise sessions over a 4-day period (on days 2 and 4), with two sets of a maximum of 25 repetitions (or point of failure; i.e., the animal would not respond to the illuminated bar even with a brief footshock) for each session. Each resistance exercise session required about 15 min to complete. Force measurements were recorded using a load cell (Entran Devices, Fairfield, NJ) attached to the flywheel apparatus and integrated to a personal computer. Data acquisition was obtained by software programmed by the investigative team, and collected data were obtained at a rate of 40 times per second throughout the 25-repetition period of each set.

In vitro assessment of muscle protein synthesis. Approximately 16 h after the last exercise session (on day 4), animals were anesthetized (50 mg/kg pentobarbital sodium), and soleus muscles were dissected from origin to insertion and weighed. After muscles were obtained, the animals were euthanized by lethal injection (70 mg/kg pentobarbital sodium ic). Soleus muscle was selected because it is very active during this type of exercise activity, responds with elevated rates of protein synthesis (13, 14), and is a suitable muscle for the study of muscle protein synthesis in vitro (9). Sixteen hours after exercise was chosen to study rates of synthesis, since previous studies have demonstrated elevated rates of synthesis during this period (12, 17). Rates of muscle protein synthesis were measured as described previously (8, 9, 12). Muscles were bisected longitudinally from origin to insertion and placed into specially designed clamps to maintain muscle length. Muscle strips were then placed into a Krebs-Henseleit buffer (KHB), maintained at 37°C, and gassed with humidified O2 (95%) and CO2 (5%). The medium contained all amino acids at concentrations mimicking physiological levels, 5 mM D-glucose, and 4.5% bovine serum albumin, dialyzed against 40 volumes of KHB for 48 h prior to inclusion in the buffer. After a 15-min incubation period, the muscle strips were transferred to wells containing fresh buffer that included a 2 mM concentration of cold (nonradioactive) phenylalanine plus 1 µCi/ml radiolabeled L-[2,3,4,5,6-3H]phenylalanine. After a 35-min incubation period in the presence of the radioisotope, strips were placed into liquid nitrogen, frozen at –70°C, and later assessed for incorporation of tritiated phenylalanine into TCA-precipitable extracts (26), corrected for the specific radioactivity of the incubation medium. Time was recorded for each muscle strip from the start of the radiolabeling period to the point of freezing so that rates of muscle protein synthesis might be expressed per unit time. Muscle protein determinations for all strips were conducted using the bicinchoninic acid assay (BCA) (Sigma, St. Louis, MO). Identical medium was used for all soleus muscle experiments. Rates of muscle protein synthesis were expressed as nanomoles of phenylalanine incorporated per gram of muscle per hour. Previous experiments have demonstrated that this incubation methodology is effective in maintaining ATP stores, indicative of muscle viability and adequate permeability, during the incubation period (8).

Activation/inhibition signal transduction studies. To determine the involvement of signal transduction pathways after resistance exercise, soleus muscles were incubated with or without insulin (20,000 µU/ml) and with or without PD-098029 (1 µM) and/or rapamycin (1 µM). PD-098029 is a specific inhibitor of the MAPK pathway (specifically the ERKs) at the level of MEK, and rapamycin is an inhibitor of mTOR. These inhibitors were used alone or together to determine the effect of MAPK and/or mTOR in the postexercise protein synthesis response. Furthermore, because insulin is a strong agonist of activity in these pathways and of muscle protein synthesis after resistance exercise, inhibition studies were also done with and without insulin in the incubation medium.

Phosphospecific kinase activity. As a surrogate marker of activity of specific target proteins in the rapamycin- or MEK-sensitive pathway, we chose to examine the phosphorylation states of specific kinases involved with insulin signaling and muscle protein synthesis through these pathways. Activity of these kinases is associated with the phosphorylation of specific residues on the protein. The specific kinases measured in this study were p70 S6 kinase (p70S6K; a downstream kinase of mTOR) and ERK1/2 (particularly at specific phosphorylation sites indicative of MEK activation). Kinase phosphorylation states were assessed in the presence or absence of rapamycin or PD-098059 to determine the effectiveness of our inhibitors on their target kinases in our in vitro system. Phosphorylation of the kinases was assessed by Western analysis performed as described previously (5). All primary antibodies were purchased from Cell Signaling (Beverly, MA).

For these studies, soleus muscles were incubated in the presence of insulin with or without rapamycin and/or PD-098059. After incubation, soleus muscles were homogenized on ice in 400 µl of buffer containing 25 mM HEPES, 4 mM EDTA, 25 mM benzamidine, 1 µM concentrations of leupeptin and pepstatin, 0.15 mM aprotinin, and 2 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 15,000 g at 4°C for 1 h, as described previously (10). After centrifugation, the supernatant containing the cytosolic fraction was taken, protein content in the supernatant was assayed using BCA, and 10 µg of protein were applied to a 4–8% discontinuous polyacrylamide gel. After electrophoresis, the gel was transferred to a PVDF membrane using a semidry method (Multiphor II; Amersham Pharmacia Biotech) and immunoblotted with the appropriate primary antibody.

The following primary antibodies were used: phosphospecific p44/p42 (Thr202/Tyr204); phosphospecific p70S6K (Thr389), and total eIF2B-{epsilon} (Cell Signaling, Beverly, MA). Membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce Endogen, Rockford, IL). Antibody binding was detected by incubating membranes for 5 min in the chemiluminescence substrate kit (ChemiGlow; Alpha Innotech, San Leandro, CA). Membranes were scanned using ChemiImager 5500 (Alpha Innotech) or placed on X-ray film, and the densities of the bands were analyzed using FluorChem software (Alpha Innotech). All treatments between groups were represented on each of the blots for qualitative comparisons, and each band density was corrected for total protein content using Ponceau S staining.

Statistics. One-way analysis of variance (ANOVA) was used to compare means of muscle protein synthesis rates among treatments (insulin and/or rapamycin and/or PD-098059) and between groups. One-way ANOVA was also used to determine differences among treatments and groups for Western Blot analyses. Force production variables were also compared with ANOVA when appropriate. Differences among means were considered significant when P < 0.05. When f-ratios were significant, a Student-Newman-Keuls test was used to compare relevant means when multiple comparisons were tested. All data are expressed as means ± SE.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
All RE rats completed two resistance exercise sessions. Because peak tension may be responsible for activation of MAPK signaling pathways (25), we compared force production for each animal across sets. Average forces (Fig. 1; top) were compared between sets 1 and 2 from session 1, and sets 3 and 4 from session 2. Although there was a tendency toward reduction of force production, perhaps indicative of fatigue, there were no statistical differences among sets (P > 0.05). Sets are presented individually for clarity. There were also no differences among sets for maximum forces produced (middle, P > 0.05) or repetitions completed across sessions (bottom, P > 0.05). This resistance exercise paradigm was sufficient to elicit an anabolic response demonstrated by an elevated expression of eIF2B-{epsilon} (Fig. 2), which may be an important surrogate marker of enhanced anabolism following resistance exercise (21).


Figure 1
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  		Fig. 1. Force profile for each of 2 sets of 2 flywheel resistance exercise sessions. Each bar represents the average force produced for the 1st or 2nd set for the 2 resistance exercise (RE) sessions combined (top). Bottom: average force produced for all sets combined and average maximum force for all sets. Rats (n = 14) completed an average of 22.9 ± 1.3 repetitions per set. Force was measured using a load cell from Entran Industries. Data are represented as means ± SE.

 

Figure 2
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  		Fig. 2. RE increases the eukaryotic peptide chain initiation factor eIF2B-{epsilon} implicative of the anabolic effect of RE. Western analyses and averaged density units corrected for Ponceau S staining of control (n = 10) and RE (n = 8) soleus muscles from control and RE rats. Inset: representative bands of immunodetected eIF2B-{epsilon}. *Different from control (P < 0.05).

 
Soleus muscle protein synthesis is augmented with insulin after two sessions of resistance exercise. Rates of protein synthesis in soleus muscle were significantly higher (P < 0.05) in the presence of insulin after two sessions of resistance exercise compared with resistance exercise without insulin or no resistance exercise with or without insulin (Fig. 3). No differences in rates of protein synthesis were observed among soleus muscles from control rats with or without insulin and resistance exercise without insulin. Previous work demonstrated that insulin is a necessary component for postexercise elevations of muscle protein synthesis (12), likely mediated via enhanced signaling through one or more pathways. These results suggest that the resistance exercise paradigm in the present study was sufficient to elicit a training response with two sessions of resistance exercise training over a 4-day period. Future studies will assess the efficacy prolonged training with this resistance exercise paradigm for muscle hypertrophy.


Figure 3
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  		Fig. 3. Elevated rates of soleus muscle protein synthesis with RE become less dependent on mTOR signaling after insulin administration. Rates of muscle protein synthesis with (filled bars; n = 7–9) and without (open bars; n = 6–8) 2 days of RE using flywheel technology, measured with and without 20K U of insulin (ins) and/or 1 µM rapamycin (rap). aDifferent from others within group (P < 0.05); bdifferent from "none" within group (P < 0.05); cdifferent from others among treatments and between group (P < 0.05); ddifferent within treatment between groups (P < 0.05). Data are represented as means ± SE.

 
Rates of synthesis with and without inhibitors of signal transduction. Rates of soleus muscle protein synthesis were measured with rapamycin, an mTOR inhibitor, in the incubation medium, with and without insulin (Fig. 3). Rates of protein synthesis with rapamycin and without insulin were significantly lower (P < 0.05) than without rapamycin (none) in both control and RE rats. The addition of insulin to rapamycin in control rats yielded similar rates of synthesis as rapamycin alone (P > 0.05). The addition of insulin to rapamycin yielded higher rates of synthesis after resistance exercise (P < 0.05), and these rates were similar to no treatment (none) within the RE group. These results suggest that rates of muscle protein synthesis following resistance exercise become less dependent on mTOR pathways following insulin administration.

To investigate the contribution of the MAPK pathway (Ras-ERK), rates of protein synthesis were also measured in the presence of PD-098029, a MAP kinase inhibitor, with and without insulin (Fig. 4). In control animals, the addition of PD-098029 had no influence on rates of muscle protein synthesis, with or without insulin, and these rates were similar to control values (none) from Fig. 2. However, when PD-098039 was added to the incubated muscles after resistance exercise, protein synthesis was significantly lower than control levels (P < 0.05), and the influence of PD-098029 could not be overcome by the addition of insulin. These results suggest that MAP kinase signaling pathways may be playing a substantial role in the postexercise anabolic response of muscle that is observed in the presence of insulin.


Figure 4
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  		Fig. 4. Insulin-dependent elevation of protein synthesis after flywheel-based RE is mediated by a MAP kinase signaling pathway. Rates of muscle protein synthesis with (filled bars) or without (open bars) 2 days of RE using flywheel technology, measured in the presence of 1 µM PD-098059 (PD) with and without 20K U of insulin and/or 1 µM rapamycin. Rates of protein synthesis were not different between "none" (as presented in Fig. 2), with or without insulin in control groups (Fig. 2) and PD in the control groups with or without insulin in this figure. aDifferent from PD only or PD/ins from control animals (P < 0.05); bdifferent within treatment between groups (P < 0.05). Data are represented as means ± SE.

 
We also incubated muscle with both inhibitors with and without insulin (Fig. 4). As expected, rates of synthesis were significantly lower (P < 0.05) in the presence of both inhibitors with or without resistance exercise, and this inhibition could not be rescued by insulin in either group.

Measurement of activity of specific kinases. To demonstrate that the inhibitors indeed influenced downstream effectors, phosphorylation state on specific amino acid residues that have been associated with kinase activity was measured. The kinases of interest were p70S6K and ERK1/2. Results from these analyses demonstrated that rapamycin and PD-098059 were effective and specific for the inhibition of p70S6K in control and RE groups (Fig. 5) and ERK1/ERK2 in the RE group only (Fig. 6), respectively. Furthermore, our results demonstrate that the ERK2 phosphorylation is higher after resistance exercise, suggesting that 2 days of resistance exercise facilitates activity of this kinase, at least in the presence of insulin (Fig. 6). Together, these results suggest that the inhibitors used in this study were selective and effective for inhibiting specific regions of insulin signaling, without apparent "cross talk" between the studied signaling pathways. Furthermore, our data demonstrate that the ERK-signaling kinases have a role in the insulin-mediated elevation of muscle protein synthesis after resistance exercise.


Figure 5
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  		Fig. 5. Rapamycin specifically inhibits phosphorylation of p70 S6 kinase-1 (p70S6K). Effects of PD-098059 and rapamycin on insulin-mediated p70S6K activity (as indicated by phosphorylation of Thr389) in soleus muscles of rats with (filled bars) and without (open bars) 2 days of flywheel RE. Averaged densities of bands were normalized to Ponceau S staining, as described in METHODS. Inset: representative bands immunodetected with phosphospecific antibodies directed toward p70S6K. *Different from other treatments within group (P < 0.05).

 

Figure 6
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  		Fig. 6. PD-098059 inhibits the phosphorylation of ERK1 and -2. Effects of PD-098059 and rapamycin on insulin-mediated ERK activity on p44 (A; as indicated by dual phosphorylation on Thr202/Tyr204) and p42 (bottom; as indicated by dual phosphorylation on Thr183/Tyr185) in soleus muscles of rats, with (filled bars) and without (open bars) 2 days of RE. Averaged densities of bands were normalized to Ponceau S staining, as described in METHODS. Inset: representative bands immunodetected with phosphospecific antibodies directed toward p42 and p44. *Different from other treatments within group and within protein (P < 0.05); #different from no exercise within treatment (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, the role of mTOR- and MAP kinase-specific signaling pathways was studied in vitro for their involvement in the elevation of insulin-mediated muscle protein synthesis after resistance exercise. The most important finding from this study is that insulin-mediated elevations of muscle protein synthesis after resistance exercise is dependent on signal transduction through an ERK signaling pathway. We observed a significant increase in soleus muscle protein synthesis in the presence of insulin after just two resistance exercise sessions, and this increase was concomitant with elevated ERK activity. Furthermore, inhibition of this MAP kinase pathway using PD-098059 not only abolished the postexercise elevation in rates of muscle protein synthesis but suppressed rates of synthesis compared with control values, and this suppression could not be rescued by insulin. Therefore, we conclude that insulin signaling through a MAP kinase pathway is, at least in part, a necessary response in rates of muscle protein synthesis after resistance exercise. These results are the first to demonstrate that a MAP kinase pathway is available for the insulin-facilitated rates of muscle protein synthesis after resistance exercise.

In support of our findings, several investigations have implicated the MAPK signaling pathways for the control of hypertrophy after muscle overload by using phosphorylation of the kinase as an indicator of activity (4, 15, 25). We expanded on that work by assessing rates of protein synthesis with and without resistance exercise, a potent stimulant of muscle hypertrophy, in the presence of insulin and/or PD-098059 or rapamycin (4, 15, 25). Surprisingly, the presence of PD-098059 had no effect on muscle protein synthesis in muscle without resistance exercise, suggesting that control of muscle protein synthesis in sedentary skeletal muscle is not dependent on the activation of ERK. Alternatively, we found that rates of protein synthesis are dramatically diminished in the presence of rapamycin in sedentary muscle, suggesting that mTOR may serve as a primary signaling pathway for the maintenance of normal muscle protein synthesis in skeletal muscle without prior exercise.

After 2 days of resistance exercise with flywheel technology, we observed an increase of muscle protein synthesis in response to insulin, which is consistent with prior work utilizing resistance exercise paradigms (6, 12, 17). This is in contrast to sedentary muscle, where changes in rates of muscle protein synthesis are not observed with acute administration of insulin using similar methodologies (10, 12), unless preceded by prolonged insulin deprivation (7). We speculated that the acute effect of insulin administration on muscle protein synthesis may represent a shift in insulin signaling. In support of this hypothesis, we demonstrated that the inhibitory effect of rapamycin on muscle protein synthesis was partially rescued by insulin after 2 days of resistance exercise but not in control muscles. Thus it appears that the dependence on a rapamycin-sensitive pathway is diminished following resistance exercise and may signify increased control of muscle protein synthesis through hormone signaling. To our knowledge, the present data are the first to demonstrate that insulin is sufficient to overcome the inhibitory effect of rapamycin after intermittent resistance exercise at the level of muscle protein synthesis, at least in soleus muscle.

Further support for the hypothesis that multiple pathways may be required for postresistance exercise elevations of protein synthesis has been obtained from work using resistance exercise in rats (3, 17). Bolster et al. (3) recently suggested that early steps, within 1 h, in the postexercise response following a similar type of resistance exercise included increased activity of mTOR and p70S6K in gastrocnemius muscle. However, rates of protein synthesis were not measured in that study (3), and it is unlikely that the increased activity of these kinases resulted in elevations of muscle protein synthesis at those early time points. This speculation is based on the observation of Hernandez et al. (17), using a similar resistance exercise paradigm in rats, that elevations of muscle protein synthesis did not occur until ~12 h after exercise. However, it is possible that the activation of the mTOR pathway may lead to expression of specific proteins in the translational apparatus. For example, Kubica and colleagues (21, 22) demonstrated that mRNA content of eIF2B-{epsilon}, an important factor involved in peptide chain initiation, is elevated after only 4 days of resistance exercise and that this increase of eIF2B-{epsilon} is blocked by a preexercise administration of rapamycin and is effective for abolishing the postexercise increases of muscle protein synthesis.

Although the work from Kubica and colleagues was conducted in gastrocnemius, our present work in soleus muscles demonstrated that eIF2B-{epsilon} is also overexpressed in response to 2 days of resistance exercise, and although activation of the mTOR pathway cannot completely explain the later postexercise elevation of muscle protein synthesis reported here, we cannot discount that early postexercise activation of mTOR, perhaps leading to an overexpression of eIF2B-{epsilon}, is necessary for this anabolic process regardless of muscle. This conclusion is consistent with the observation that rates of protein synthesis after resistance exercise in the presence of insulin and rapamycin were similar to rates of synthesis in control rats and did not achieve the elevations of muscle protein synthesis compared with insulin alone after exercise. Thus, at present, we may only conclude that a rapamycin-sensitive pathway is not solely responsible for the elevation of muscle protein synthesis after resistance exercise in soleus muscle. It is interesting to note, however, that the effect of the ERK inhibitor alone on muscle protein synthesis after resistance exercise was similar to the presence of both inhibitors. Thus the present data strongly suggest that the transient increase of muscle protein synthesis in response to insulin after resistance exercise is accompanied by a concomitant shift of insulin signaling through pathways involving ERK kinases.

In summary, resistance exercise facilitates an elevation of muscle protein synthesis, of which insulin is a necessary component (11, 12). We found that the MAP kinase pathway is at least in part responsible for the elevation in protein synthesis after two sessions of resistance exercise in addition to the previously implicated mTOR pathway. Future studies should be directed toward determining the underlying mechanisms related to this shift of insulin signaling after resistance exercise. Although not fully resolved at this time, results presented here have important implications toward our understanding of general muscle protein homeostasis as well as how muscle adapts under hypertrophying conditions, such as in response to intermittent muscle overload.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-47577 to C. A. Peterson.


   ACKNOWLEDGMENTS
 
We thank Patrick Bennett for expert technical assistance on this project, and the Department of Laboratory Animal Medicine for their expert care in handling of the animals used in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Fluckey, Muscle Biology Laboratory, Dept. of Health and Kinesiology, Mail Stop 4243, Texas A&M University, College Station, TX 77845 (e-mail: jfluckey{at}hlkn.tamu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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