Contraction-induced adaptations in the skeletal muscle are largely determined by the mode, volume, and intensity of exercise (10). Endurance training leads to multiple adaptations in the skeletal muscle including, but not limited to, mitochondrial biogenesis (21) and increases in the maximal activities of oxidative enzymes (21). Resistance training generally promotes a phenotype of myofibrillar protein accretion and increased cross-sectional area of Type II fibers (13,33). Exercise–nutrient interactions may also be important in mediating skeletal muscle adaptation and may have the capacity to modulate the specificity of training response (19).
The cellular mechanisms regulating the specificity of training adaptations with concurrent resistance and endurance exercise are undoubtedly complex, given the capacity for single-mode endurance and resistance training to generate divergent phenotypes (13,39) and the potential confounding factors of exercise order and recovery between bouts. For example, Wilson et al. (40) have reported that endurance exercise inhibits hypertrophy/strength with increasing training volume and frequency in a concurrent training paradigm. We have previously reported various cell signaling responses related to translation initiation and messenger RNA (mRNA) expression of mitochondrial/metabolic and myogenic adaptation when endurance or sprint exercise was performed directly before/after resistance exercise in the fasted state (11,12). Although the molecular profile generated by an acute bout of concurrent training has yet to be clearly established, the possibility exists that successive resistance and endurance exercise may have the capacity to promote myofibrillar and mitochondrial protein synthesis.
Consumption of high-quality protein in close temporal proximity to resistance exercise enhances translation initiation signaling, maximally stimulates rates of muscle protein synthesis (25), and augments hypertrophy and strength gains (8). Likewise, protein feeding after endurance exercise can increase the transcriptional profile of mitochondrion-related genes and increase rates of myofibrillar protein synthesis (5,34). To date, no study has determined the effect of protein ingestion after a bout of concurrent training on the acute myofibrillar and mitochondrial protein synthesis rates in the skeletal muscle. Accordingly, the primary aim of the present investigation was to examine the acute effects of protein ingestion on rates of myofibrillar and mitochondrial protein synthesis in association with selected cellular/molecular responses after a bout of consecutive resistance exercise and cycling. We hypothesized that, compared with placebo, protein ingestion would enhance anabolic and metabolic signaling and subsequent protein synthesis during the early (1–4 h) recovery period after exercise.
Eight healthy male subjects (age, 19.1 ± 1.4 yr; body mass, 78.1 ± 15.6 kg; peak oxygen uptake (V˙O2peak), 46.7 ± 4.4 mL·kg−1·min−1; leg extension one-repetition maximum (1RM), 130 ± 14 kg; values are mean ± SD) who had been participating in regular concurrent resistance and endurance training (approximately three times per week, for >1 yr) volunteered to participate in this study. Our subject inclusion criteria were based on subjects with concurrent training history to quantify the habitual rather than novel adaptation response in the skeletal muscle. The experimental procedures and possible risks associated with the study were explained to all subjects, who gave a written informed consent before participation. The study was approved by the human research ethics committee of the Royal Melbourne Institute of Technology University.
The study used a randomized, counterbalanced, double-blind, crossover design in which each subject completed two bouts of concurrent resistance exercise and cycling with either postexercise placebo (PLA) or protein (PRO) ingestion separated by a 3-wk recovery period, during which time, subjects maintained their habitual physical activity pattern. Given the limited data on molecular responses to concurrent training, we chose to undertake resistance exercise before endurance exercise on the basis of our previous finding that endurance exercise may attenuate the acute anabolic response if performed before resistance exercise (12). We chose to quantify the fractional synthetic rates in skeletal muscle during the 1- to 4-h recovery period because of potential complications generated by limb hyperemia with strenuous exercise and because protein digestion and absorption are reduced during the acute postexercise period (37).
V˙O2peak was determined during an incremental test to volitional fatigue on a Lode cycle ergometer (Groningen, The Netherlands). The protocol has been described in detail previously (7). In brief, subjects commenced cycling at a workload equivalent to 2 W·kg−1 for 150 s. Thereafter, the workload was increased by 25 W every 150 s until volitional fatigue, defined as the inability to maintain a cadence >70 rpm. Throughout the test, subjects breathed through a mouthpiece attached to a metabolic cart (Parvomedics) to determine oxygen consumption.
Quadriceps strength was determined during a series of single repetitions on a plate-loaded leg extension machine until the maximum load lifted was established (1RM). Repetitions were separated by 3-min recovery and were used to establish the maximum load/weight that could be moved through the full range of motion once but not for a second time. Exercise range of motion was 85°, with leg extension end point set at −5° from full extension.
Before an experimental trial (described subsequently), subjects were instructed to refrain from exercise training and vigorous physical activity and alcohol and caffeine consumption for a minimum of 48 h. Subjects were provided with standardized prepacked meals that consisted of 3 g CHO·kg−1 body mass, 0.5 g protein·kg−1 body mass, and 0.3 g fat·kg−1 body mass consumed as the final caloric intake the evening before reporting for an experimental trial.
Experimental Testing Session
On the morning of an experimental trial, subjects reported to the laboratory after an approximately 10-h overnight fast. After resting in the supine position for approximately 15 min, catheters were inserted into the antecubital vein of each arm and a baseline blood sample (approximately 3 mL) was taken (Fig. 1). A primed constant intravenous infusion (prime, 2 μmol·kg−1; infusion, 0.05 μmol·kg−1·min−1) of l-[ring-13C6] phenylalanine (Cambridge Isotopes Laboratories) was then administered. Under local anesthesia (2–3 mL of 1% Xylocaine), a resting biopsy was obtained 3 h after commencement of the tracer infusion from the vastus lateralis using a 5-mm Bergström needle modified with suction. Subjects then completed the exercise intervention (described subsequently). Immediately after the cessation of exercise, subjects ingested 500 mL of either PLA (water and artificial sweetener) or PRO (25 g whey protein). The PRO beverage was enriched to 5% L-[ring-13C6] phenylalanine to prevent dilution of the steady-state isotope enrichment implemented by constant infusion. Subjects rested throughout a 240-min recovery period, during which, additional muscle biopsies were taken 60 min after exercise to investigate early cell signaling and mRNA responses and 240 min after exercise to determine rates of muscle protein synthesis. Each muscle biopsy was taken from a separate site 2–3 cm distal from the right leg for the first trial and left leg for the second trial, with all samples stored at −80°C until subsequent analysis. Blood samples were collected into EDTA tubes at regular intervals during the postexercise recovery period.
After a standardized warm-up (2 × 5 repetitions at approximately 50% and approximately 60% 1RM, respectively), subjects performed eight sets of five repetitions at approximately 80% 1RM. Each set was separated by a 3-min recovery period, during which time, the subject remained seated on the leg extension machine. Contractions were performed at a set metronome cadence approximately equal to 30° per second, and strong verbal encouragement was provided during each set. Subjects then rested for 15 min before beginning the cycling protocol.
Subjects performed 30 min of continuous cycling at a power output that elicited approximately 70% of individual V˙O2peak. Subjects were fan-cooled and allowed ad libitum access to water throughout the ride. Visual feedback for pedal frequency, power output, and elapsed time was provided to subjects.
Blood glucose and plasma insulin concentration
Whole blood samples (5 mL) were immediately analyzed for glucose concentration using an automated glucose analyzer (YSI 2300; YSI Life Sciences, Yellow Springs, OH). Blood samples were then centrifuged at 1000g at 4°C for 15 min, with aliquots of plasma frozen in liquid N2 and stored at −80°C. Plasma insulin concentration was then measured using a radioimmunoassay kit according to the manufacturer’s protocol (Linco Research, Inc., St. Charles, MO).
Plasma amino acids and enrichment
Plasma amino acid concentrations were determined by high-performance liquid chromatography from a modified protocol (31). Briefly, 100 μL of plasma was mixed with 500 μL of ice-cold 0.6-M perchloric acid and neutralized with 250 μL of 1.25-M potassium bicarbonate (KHCO3). Samples were then subsequently derivatized for high-performance liquid chromatography analysis. Plasma [ring-13C6] phenylalanine enrichments were determined as previously described (31).
Mitochondrial and Myofibrillar Protein Synthesis
A piece of frozen wet muscle (approximately 100 mg) was homogenized with a Dounce glass homogenizer on ice in an ice-cold homogenizing buffer (1-M sucrose, 1-M Tris/HCl, 1-M KCl, 0.5-M EDTA) supplemented with a protease inhibitor and phosphatase cocktail tablet (PhosSTOP; Roche Applied Science, Mannhein, Germany) per 10 mL of buffer. The homogenate was transferred to an Eppendorf tube and centrifuged (700g, 15 min, 4°C) to pellet a fraction enriched with myofibrillar proteins and collagen that was stored at −80°C for subsequent extraction of the myofibrillar fraction (to be described later). The supernatant was transferred to another Eppendorf tube and centrifuged (12,000g, 20 min, 4°C) to pellet the mitochondrion-enriched protein fraction. The supernatant was placed in a separate Eppendorf and stored at −80°C for Western blot analysis (to be described later). The mitochondria-enriched pellet was then washed and lyophilized, and amino acids were liberated by adding 1.5 mL of 6-M HCl and heating to 110°C overnight. Rates of mitochondrial protein synthesis were unable to be determined for two subjects because of limited muscle tissue obtained from the biopsy samples.
The myofibrillar pellet stored at −80°C was washed twice with the homogenization buffer and centrifuged (700g, 10 min, 4°C), and supernatant was discarded. Myofibrillar proteins were solubilized in 0.3-M sodium hydroxide and precipitated with 1-M perchloric acid. Amino acids were then liberated from the myofibril-enriched precipitate by adding 2.0 mL of 6-M HCl and heating to 110°C overnight.
Free amino acids from myofibril- and mitochondrion-enriched fractions were purified using cation-exchange chromatography (Dowex 50WX8-200 resin; Sigma-Aldrich Ltd.) and converted to their N-acetyl-n-propyl ester derivatives for analysis by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) (Hewlett Packard 6890, IRMS model Delta Plus XP; Thermo Finnagan, Waltham, MA).
Intracellular amino acids (IC) were extracted from a separate piece of wet muscle (approximately 20 mg) with ice-cold 0.6-M perchloric acid for determination of intracellular phenylalanine enrichment. Muscle was homogenized, and the free amino acids in the supernatant were purified by cation-exchange chromatography and converted to their heptafluorobutyric derivatives before analysis by GC-MS (models 6890 GC and 5973 MS; Hewlett-Packard, Palo Alto, CA), as previously described (31).
The fractional synthetic rate of mitochondrial and myofibrillar protein synthesis was calculated using the standard precursor–product method: fractional synthetic rate (% per hour) = [(E2b − E1b)/(EIC × t)] × 100 where E2b − E1b represents the change in the bound protein enrichment between two biopsy samples, EIC is the average enrichment of intracellular phenylalanine between the two biopsy samples, and t is the time between two sequential biopsies. The inclusion of “tracer-naive” subjects permitted use of the preinfusion blood sample (i.e., single biopsy method) as the baseline enrichment (E1b) for the calculation of resting muscle protein synthesis. The single biopsy method for quantifying baseline tracer enrichment in tracer-naive subjects has been clearly shown to be a reliable and valid experimental technique in previous studies (15).
The supernatant frozen at −80°C from the previous mitochondrion-enriched fraction extraction was used to determine protein concentration using a BCA protein assay (Pierce, Rockford, IL). The supernatant was subsequently resuspended in a Laemmlli sample buffer and separated by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes and incubated with primary antibody (1:1000) overnight at 4°C and secondary antibody (1:2000) and proteins detected via chemiluminescence (Amersham Biosciences, Buckinghamshire, United Kingdom; Pierce Biotechnology, Rockford, IL) and quantified by densitometry (ChemiDoc; Bio-Rad, Gladesville, Australia). All sample (40 μg) time points for each subject were run on the same gel. Polyclonal anti-phospho-AktSer473 (number 9271), -mTORSer2448 (number 2971), -eukaryotic elongation factor 2Thr56 (eEF2) (number 2331), and monoclonal anti-p70S6KThr389 (number 9234) were from Cell Signaling Technology (Danvers, MA). Data are expressed relative to α-tubulin (number 3873; Cell Signaling Technology, Danvers, MA) in arbitrary units.
RNA Extraction and Quantification
Briefly, approximately 20 mg of skeletal muscle was homogenized in TRIzol and chloroform added to form an aqueous RNA phase. This RNA phase was then precipitated by mixing with isopropanol alcohol, and the resulting pellet was washed and resuspended in 50 μL of ribonuclease-free water. Extracted RNA was quantified using a QUANT-iT analyzer kit (catalog number Q32852; Invitrogen, Melbourne, Australia) and on a NanoDrop 1000 spectrophotometer (Nanodrop Technologies, Wilmington, NC) by measuring absorbance at 260 and 280 nm, with a 260/280 ratio of approximately 1.88 recorded for all samples.
Reverse Transcription and Real-Time Polymerase Chain Reaction
First-strand complementary DNA synthesis was performed using commercially available TaqMan reverse transcription reagents (Invitrogen, Melbourne, Australia) in a final reaction volume of 20 μL. All RNA and negative control samples were reverse-transcribed to complementary DNA in a single run from the same reverse transcription master mix. Serial dilutions of a template RNA (catalog number AM7982; Ambion) were included to ensure efficiency of reverse transcription and for calculation of a standard curve for real-time quantitative polymerase chain reaction (PCR). Quantification (in duplicate) was performed using a Rotor-Gene 3000 Centrifugal Real-Time Cycler (Corbett Research, Mortlake, Australia). Taqman-FAM-labeled primer/probes for muscle RING finger 1 (MuRF1) (catalog number Hs00261590), atrogin (catalog number Hs01041408), myostatin (catalog number Hs00976237), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (catalog number Hs01016719), hexokinase (catalog number Hs00175976), and vascular endothelial growth factor (VEGF) (catalog number Hs00900055) were used in a final reaction volume of 20 μL. PCR treatments were 2 min at 50°C for UNG activation, 10 min at 95°C then 40 cycles of 95°C for 15 s and 60°C for 60 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number Hs Hs99999905) was used as a housekeeping gene, and expression was not different at any time point (data not shown). The relative amounts of mRNA were calculated using the relative quantification (ΔΔCT) method (27).
Blood, cell signaling, and mRNA data were analyzed by two-way ANOVA (two factor, time × treatment) with repeated measures, and myofibrillar and mitochondrial protein syntheses were analyzed by one-way ANOVA with Student–Newman–Keuls post hoc analysis when P < 0.05 (SigmaStat for windows Version 3.11). All data are expressed as mean ± SD. Magnitude-based inferences and Cohen effect sizes (ES) were used to identify physiologically meaningful differences in rates of myofibrillar and mitochondrial protein synthesis (22). The precision of the effect was determined using 90% confidence limits, making the same assumptions about sampling distributions that statistical packages use to derive P values. Differences between PRO and PLA conditions were interpreted using inferences on the basis of the magnitude of effect, as described previously (2). Results are determined using ES similar to the Cohen conventional threshold values of 0.2 as the smallest effect, 0.5 as a moderate effect, and 0.8 as a large ES (22).
Plasma Insulin, Amino Acids, and Blood Glucose
There were main effects for plasma insulin and total amino acid concentration with PRO but not with PLA (P < 0.001) (Fig. 2A and B). Peak plasma insulin (approximately 535%) and amino acid (approximately 70%) concentrations occurred 40 min after exercise (P < 0.001). The same effect was evident for branch chain amino concentration (approximately 180%, P < 0.001) (Fig. 2C). Blood glucose was not different at any time in either treatment (data not shown).
Intracellular and Plasma Tracer Enrichments
Intracellular free phenylalanine enrichments showed a stable precursor pool at rest and at 60 and 240 min after exercise for PRO (0.0455, 0.0502, and 0.0488 tracer-to-tracee ratio (t·T−1)) and PLA (0.0468, 0.0468 and 0.0473 t·T−1). Linear regression analysis indicated that the slopes of the plasma L-[ring-13C6] phenylalanine enrichments were not significantly different from zero or between treatments, showing isotopic plateau/steady state.
AktSer473 phosphorylation increased above rest with PRO (approximately 175%, P < 0.05) but not with PLA 1 h after exercise (Fig. 3A). This disparity in AktSer473 resulted in a significant difference between treatments at 1 h (P < 0.05). Phosphorylation in PRO then returned to resting levels 4 h after recovery from exercise (P < 0.05).
mTOR phosphorylation increased after PRO (approximately 400%, P < 0.001) and PLA (approximately 100%, P < 0.05) ingestion at 1 h, and this increase was markedly higher with PRO (approximately 300%, P < 0.001) (Fig. 3B). mTORSer2448 phosphorylation remained elevated above rest 4 h after exercise with PLA only (approximately 130%, P < 0.05), resulting in significant disparity between treatments (P < 0.05).
p70S6KThr389 phosphorylation increased above rest with PRO (approximately 3000%, P < 0.001) but not PLA 1 h after exercise (Fig. 3C). This disparity in p70S6KThr389 resulted in significant difference between treatments at 1 h (P < 0.05). Phosphorylation of p70S6K after PRO returned to resting levels after 4 h of recovery from exercise (P < 0.001).
There were main effects for eEF2Thr56 phosphorylation for time in both treatments (P < 0.05) (Fig. 3D). One hour after exercise, phosphorylation of eEF2 decreased approximately 60% (P < 0.05) with PLA and approximately 75% (P < 0.05) with PRO and remained at this level for the duration of the recovery (4 h).
MuRF1 increased significantly above resting levels at 1 h (approximately 315% vs approximately 230%, P < 0.001) and 4 h (approximately 250% vs approximately 140%, P < 0.05) after exercise after both PLA and PRO, respectively (Fig. 4A). MuRF1 was higher in PLA compared with that in PRO at both postexercise time points (1 h, 78%; 4 h, 105%; P < 0.05).
Atrogin-1 mRNA expression increased above rest only with PLA 1 h after exercise (approximately 50%, P < 0.05) (Fig. 4B). The disparity in atrogin-1 mRNA at 1 h resulted in a significant difference between treatments (P < 0.05).
There was a main effect of time for myostatin mRNA abundance (P < 0.05) (Fig. 4C). Myostatin decreased from rest at 1 h (approximately 40% vs approximately 55%, P < 0.05) and 4 h (approximately 70% vs approximately 80%, P < 0.001) after both PLA and PRO, respectively. Myostatin mRNA at 1 h was different from that at 4 h after PLA (approximately 120%, P < 0.05).
There were main effects for PGC-1α mRNA abundance for time (P < 0.05) (Fig. 5A). PGC-1α expression increased above resting and 1 h levels after 4-h postexercise recovery in PLA (approximately 730%, P < 0.001) and PRO (approximately 620%, P < 0.001).
Hexokinase increased above rest at 4 h in PLA only (approximately 120%, P < 0.05), whereas in PRO, there were no changes. This disparity resulted in a significant difference between treatments at 4 h (P < 0.05) (Fig. 5B).
VEGF mRNA expression increased above rest at both 1 h (approximately 200%, P < 0.001) and 4 h (approximately 210%, P < 0.001) with PLA (Fig. 5C). Likewise, VEGF also increased with PRO at 1 h (approximately 170%, P < 0.05) and 4 h (approximately 180; P < 0.05). There were no differences between treatments at any postexercise time point.
Rates of Muscle Protein Synthesis
Rates of myofibrillar protein synthesis increased above rest between 1 and 4 h after exercise after both PLA (approximately 75%, P < 0.05) and PRO (approximately 145%, P < 0.001) (Fig. 6A). This postexercise increase in the rate of myofibrillar synthesis was greater with PRO compared with that in PLA (P < 0.05). Magnitude-based inferences revealed the chances that the true value of the statistic is mechanistically or physiologically positive for PRO compared with PLA was 91%, and Cohen ES demonstrated a large effect (ES, >1.0). Rates of mitochondrial protein synthesis (n = 6) were unchanged during the acute postexercise period, and there were no differences in postexercise fractional synthesis rates between treatments (Fig. 6B).
Athletes from a variety of sports undertake resistance and endurance training concurrently as part of their training. It has been reported that hypertrophy/strength adaptations to concurrent resistance and endurance exercise are “compromised” when compared with training for either exercise mode alone (10,40). Our results show that in moderately trained individuals, the combined effects of resistance and endurance exercise resulted in elevated rates of myofibrillar but not mitochondrial protein synthesis in the early (1–4 h) postexercise recovery period. In addition, we provide new information to demonstrate that postexercise PRO ingestion attenuates mRNA expression of markers of muscle catabolism after a concurrent training session.
Early studies examining the specificity of adaptation to concurrent training indicate an “interference” in adaptation for hypertrophy and strength relative to resistance training (17,20). In contrast, there are also reports of little or no decrements in strength gain with combined resistance and endurance training (1,30). However, the vast majority of studies of concurrent training have concluded that adaptation is compromised compared with each exercise mode undertaken in isolation (26,40). Our findings indicate that resistance exercise seems to generate a sufficient signal to stimulate myofibrillar protein synthesis despite a subsequent bout of endurance exercise (Fig. 6A). This increase in myofibrillar protein synthesis is similar to previous maximal levels observed when PRO is ingested after resistance exercise (31). What is not known, however, is whether the myofibrillar synthetic response we observed is exclusively the result of the resistance exercise or of some interaction with endurance exercise (24). Regardless, such an acute response may have potential to promote hypertrophy with repeated training bouts in a chronic concurrent training program. Importantly, we show that postexercise PRO ingestion was beneficial for promoting myofibrillar protein synthesis with concurrent training and may have the capacity to reduce the potential interference effect of endurance exercise on skeletal muscle hypertrophy.
Interestingly, we observed substantial variation in the individual response of myofibrillar protein synthetic rates with PLA and PRO ingestion after a bout of concurrent training. The percentage of “low” responders who did not increase rates of myofibrillar protein synthesis with PLA in the present study (25%) is similar to that reported after resistance or endurance training in isolation (32,36). After a 21-wk training study, Karavirta et al. (23) have also shown within a concurrent training paradigm that high “responders” for aerobic adaptation were not also high “responders” to resistance training (and vice versa) and that simultaneous endurance and resistance training-induced adaptation occurred in only 50% of subjects. Consequently, undertaking concurrent training increases the complexity of the genotype–exercise interaction in promoting skeletal muscle adaptation, with individual nutrient responses further complicating this response. In this regard, a limitation of the present study is the modest subject numbers, and understandably, further studies are needed to corroborate our findings.
We have previously demonstrated a similar selective increase in myofibrillar compared with mitochondrial protein synthesis rates in response to protein–CHO coingestion after a high-intensity repeated-sprint protocol (9). Given the high load (0.75 N·m·kg−1) and subsequent mechanical force required to complete maximal sprint cycling repetitions, the loading stimulus in our previous study was somewhat resistance-like and may have promoted a modest hypertrophy response with PRO ingestion (9). However, Breen et al. (5) have also recently reported increases in rates of myofibrillar, but not mitochondrial, protein fractional synthetic rates when CHO–protein was coingested compared with CHO feeding alone after 90 min of steady-state cycling at approximately 75% V˙O2max. Our present study shows variable rates of mitochondrial protein synthesis that failed to increase after the concurrent training bout with either treatment (Fig. 6B). Few studies have investigated rates of mitochondrial protein synthesis after exercise, with, to the best of our knowledge, only one previous study reporting mitochondrial fractional synthetic rates after a bout of concurrent training (15). Specifically, Donges et al. (15) showed comparable increases in mitochondrial protein synthesis after concurrent training compared with endurance exercise alone. The exercise intervention was undertaken in an untrained, middle-age cohort, and the results may reflect an adaptive response to unaccustomed contractile stimuli. Consequently, we suggest that the training status of the subjects in the present study may have required a greater overload stimulus to generate an acute increase in mitochondrial protein synthesis (14). However, Scalzo et al. (35) have recently shown accretion of new mitochondrial protein concomitant with increased PGC-1α protein and mitochondrial enzyme content after 3 wk of chronic training. Rowlands et al. (34) have also reported an enhanced mitochondrial transcriptome associated with protein ingestion after endurance exercise, an effect that was only evident late (48 h) but not early (3 h) in the postexercise period. Therefore, we cannot rule out that quantification of mitochondrial protein synthesis later in recovery (e.g., 24 h) or after an extended training period may have revealed changes in phenotype that reflect a different adaptation response to exercise and protein ingestion (14).
The enhanced myofibrillar protein synthesis was associated with increases in the phosphorylation status of signaling proteins that regulate translation initiation and elongation. We have previously demonstrated a similar time course for Akt-mTOR-S6K phosphorylation during the early recovery period after single bouts of resistance exercise and cycling (6). Others have also previously shown that endurance and resistance exercise in isolation activate the insulin/IGF signaling pathway (3,16). Collectively, these findings indicate that specific translational processes in skeletal muscle are not an important factor in determining the specificity of training adaptation. More recently, a concurrent training bout has been shown to enhance Akt/mTOR-mediated signaling responses (29,38). The results of the present study extend these findings by demonstrating that PRO ingestion augments Akt-mTOR-S6K phosphorylation after concurrent training (Fig. 3). Consequently, we contend that Akt-mTOR-S6K signaling may be indicative of nutrient sensitivity and/or muscle overload but fails to discriminate between divergent contraction stimuli. Exercise also generated a decrease in phosphorylation (activation) of the peptide chain elongation factor eEF2, although there were no differences between treatments indicating that it may be unresponsive to PRO ingestion (Fig. 3D). Thus, the nutrient-mediated increases in muscle protein synthesis after exercise are likely partly due to enhanced translation initiation rather than elongation.
Expressions of MuRF1 and atrogin-1 mRNA were elevated above rest after the concurrent training bout; however, this increase was attenuated with PRO ingestion (Fig. 4A and B). Harber et al. (18) have previously shown a similar effect on MuRF1 mRNA abundance with ingestion of a protein/CHO supplement after 60 min of cycling, and Borgenvik et al. (4) demonstrated that an amino acid-enriched beverage decreased MuRF1 protein levels at rest and after a resistance exercise bout. Therefore, coordinated attenuation in MuRF1 and Atrogin-1 expression with provision of exogenous amino acids in the present study may have provided substrate for muscle remodeling/hypertrophy that might otherwise be achieved through muscle proteolysis after exercise in the fasted state. However, without direct measures of proteasome activity or muscle protein breakdown, the effect of the altered atrogene mRNA expression remains unclear. In contrast, there were no differences between treatments in myostatin mRNA expression during the acute recovery period (Fig. 4C). Reduced myostatin expression has been demonstrated after an acute bout of endurance (28) and resistance (7,28) exercise, and it seems that myostatin mRNA expression is responsive to contraction per se rather than a specificity of training response and/or nutrient availability. There were also comparable increases in mRNA abundance of metabolic/mitochondrial proteins after the consecutive resistance and endurance exercise bouts, but PRO ingestion failed to induce any noteworthy increase in PGC-1α, hexokinase, or VEGF mRNA levels (Fig. 5).
In conclusion, the results of the present study demonstrate that PRO ingestion after consecutive resistance and endurance exercise selectively increased rates of myofibrillar, but not mitochondrial, protein synthesis in the early (1–4 h) recovery period. PRO ingestion also attenuated postexercise increases in genetic markers associated with muscle proteolysis. Given that endurance exercise undertaken in close temporal proximity to resistance exercise may interfere in hypertrophy/strength adaptation responses with concurrent training, our findings suggest that protein intake can be beneficial after successive resistance and endurance exercise by promoting myofibrillar protein synthesis and decreasing ubiquitin ligase expression. Accordingly, we suggest that postexercise PRO ingestion may have potential to ameliorate “interference” of endurance exercise on muscle hypertrophy and represents an important nutritional strategy for concurrent training.
The authors wish to thank Dr. José Areta, Dr. Daria Camera, and Mr. Stephen Lane for their assistance with the experimental trials. We are grateful to Todd Prior for technical expertise.
This study was funded by Nestec Ltd., Nestlé Nutrition, Vevey, Switzerland. D. M. C. is supported by a National Health and Medical Research Council postgraduate scholarship. The authors also acknowledge the support received from the Royal Melbourne Institute of Technology Higher Degrees Research Publication Grant.
The authors declare no conflicts of interest. The results of the present study do not constitute endorsement by American College of Sports Medicine.
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