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TRANSLATIONAL PHYSIOLOGY
ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
Exercise Metabolism Research Group, McMaster University, Hamilton, Ontario, Canada
Submitted 4 September 2007 ; accepted in final form 9 November 2007
ABSTRACT
Ten healthy young men (21.0 ± 1.5 yr, 1.79 ± 0.1 m, 82.7 ± 14.7 kg, means ± SD) participated in 8 wk of intense unilateral resistance training (knee extension exercise) such that one leg was trained (T) and the other acted as an untrained (UT) control. After the 8 wk of unilateral training, infusions of L-[ring-d5]phenylalanine, L-[ring-13C6]phenylalanine, and d3-
-ketoisocaproic acid were used to measure mixed muscle protein synthesis in the T and UT legs by the direct incorporation method [fractional synthetic rate (FSR)]. Protein synthesis was determined at rest as well as 4 h and 28 h after an acute bout of resistance exercise performed at the same intensity relative to the gain in single repetition maximum before and after training. Training increased mean muscle fiber cross-sectional area only in the T leg (type I: 16 ± 10%; type II: 20 ± 19%, P < 0.05). Acute resistance exercise increased muscle protein FSR in both legs at 4 h (T: 162 ± 76%; UT: 108 ± 62%, P < 0.01 vs. rest) with the increase in the T leg being significantly higher than in the UT leg at this time (P < 0.01). At 28 h postexercise, FSR in the T leg had returned to resting levels; however, the rate of protein synthesis in the UT leg remained elevated above resting (70 ± 49%, P < 0.01). We conclude that resistance training attenuates the protein synthetic response to acute resistance exercise, despite higher initial increases in FSR, by shortening the duration for which protein synthesis is elevated.
hypertrophy; feeding; weight lifting
Training can affect the acute response of MPS to a bout of resistance exercise. Phillips et al. (18) noted increases in the rates of MPS that persisted for up to 48 h in untrained individuals. In contrast, MacDougall et al. (13) reported that the resistance exercise-induced increase in MPS in trained individuals returned to baseline by 36 h. MPS has also been shown to be reduced following resistance training at the same absolute workload (17), which might be expected since the same absolute workload would result in a lower relative stress posttraining. When taken together, these studies suggest that training alters the time course of the acute response of MPS to resistance exercise; however, to date no longitudinal study has directly confirmed this thesis nor examined it in the fed state. Indeed, feeding to provide amino acids augments the postexercise response of MPS, which would maximize the anabolic effect of any exercise.
The purpose of this study, therefore, was to examine resistance training-induced changes in MPS after a bout of resistance exercise in the fed state. We were interested in determining the extent and time course of changes in MPS after an acute bout of resistance exercise performed at the same intensity relative to the increase in maximal strength following training in the fed state. We hypothesized that resistance training would alter resting MPS as well as the time course of the increase in MPS after an acute bout of resistance exercise.
METHODS
Subjects. Ten healthy men volunteered to participate in the study (21.0 ± 1.5 yr, 1.79 ± 0.08 m, 82.7 ± 14.7 kg, means ± SD). All subjects were recreationally active (i.e., exercised < 1–2 d/wk) and had not been regularly engaging in resistance exercise for at least 8 mo. All subjects were informed of the purpose of the study, experimental procedures to be used, and potential risks. Written consent was obtained from all subjects prior to commencing the study. This study was conducted in accordance with the standards set by the Declaration of Helsinki and was approved by the local McMaster University and Hamilton Health Sciences Research Ethics Boards.
General experimental protocol. This study involved an 8-wk unilateral resistance training program followed by three stable isotope infusion trials to determine changes in MPS following training. The unilateral resistance training program consisted of knee extension exercise only. One leg was assigned to perform the exercise such that it would become trained (T) while the other leg would serve as a nonexercised, untrained control (UT). All subjects participated in a familiarization session with the dynamic knee extension exercise machine and an isokinetic dynamometer (Biodex System 3; Biodex Medical Systems, Shirley, NY) at least 2 wk prior to any initial testing. Strength testing was performed on each leg separately and included a voluntary dynamic one repetition maximum (1 RM) test and isometric strength tests performed on the isokinetic dynamometer. Strength was assessed before and after completion of the training program.
Prior to the start of training, a muscle biopsy was obtained for histochemical analysis from the vastus lateralis of the leg to be trained of each subject; the biopsy was performed under local anesthesia (2% xylocaine) using our custom suction-modified 5-mm Bergstrom needle. The leg to be trained was selected randomly, counterbalanced for dominance based on strength, such that equal numbers of subjects trained their dominant and nondominant legs. Training was performed 3 d/wk (Monday to Friday) initially (weeks 1–4) and then only 2 d/wk at the latter stages of the training (weeks 5–8); at least 1 day of rest was provided between each training session. For weeks 1–2, training began with three sets of knee extension exercise performed at a workload equivalent to each subject's 10–12 RM. For weeks 3–4, participants performed four sets at their 8–10 RM. The number of sets was increased to five for weeks 5–6. Finally, for weeks 7–8, participants performed six sets at a workload equivalent to their 6–8 RM. Each subject performed all 20 training sessions, which were supervised by a study investigator.
Stable isotope infusion protocol.
L-[ring-d5]phenylalanine (98% enriched), L-[ring-13C6]phenylalanine (99% enriched), and d3--ketoisocaproic acid (d3--KIC; sodium salt, 98% enriched) were purchased from Cambridge Isotope Laboratories (Andover, MA). All isotopes were dissolved in 0.9% saline, passed through a 0.2-µm filter, and stored in evacuated bottles prior to infusion. Isotopes were passed through an in-line 0.2-µm filter during the infusion by using a calibrated syringe pump (74900 Series; Cole Palmer Instrument, Niles, IL).
Stable isotope infusions were used to measure MPS in the T and UT legs on three occasions: at rest, 4 h following an acute bout of resistance exercise, and 28 h thereafter. The experimental infusion protocols are shown schematically in Fig. 1. Four days following their last training session, subjects participated in the infusion trial to measure MPS at rest. Following the resting infusion trial, subjects then performed two additional training sessions according to their last recorded training volume and workload. A second infusion trial was performed 1 wk following the resting infusion trial to examine the response of MPS 4 h after an acute bout of resistance exercise (6 sets of 8–10 repetitions at 
80% 1-RM knee extension at the same relative intensity for the T and UT leg) in the fed state. Subjects then returned to the lab 24 h following the bout of resistance exercise for the third and final infusion trial. Dietary intake was controlled 48 h prior to the infusions as well as between the infusion sessions by providing subjects with prepackaged diets. The diets contained standard food items as well as at least 30% of the subjects' dietary energy in the form of a liquid meal replacement drink (Boost; Novartis, Mississauga, ON, Canada) to provide vitamins and minerals at the recommended levels. Diets were formulated based on the subjects' estimated energy requirements according to the Harris-Benedict equation using a moderate activity level. Protein was held constant in each diet package at 1.2 g·kg–1·d–1. Compliance with the diets was checked by having subjects report to the lab on each day they were consuming the diet and providing dietary checklists as well as close monitoring of the subjects weight during this time.
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0700–0800 after an overnight fast (10 h). Subjects had a 20-gauge catheter inserted in an antecubital vein from which blood samples were drawn throughout the infusion period (t = 0, 60, 90, 120, 180, 240, and 270 min; see Fig. 1). The catheter was kept patent by periodic flushing with 1–2 ml of 0.9% saline. Following the baseline blood sample, a second catheter was inserted in the contralateral arm in which participants received a primed constant infusion of L-[ring-d5]phenylalanine, L-[ring-13C6]phenylalanine, or d3--KIC, in a randomized order. We chose to use three different tracers to prevent carryover in isotope enrichment in the bound muscle protein from one trial to the next, which can result in increased variability in the measure of MPS (M. J. Rennie, personal communication). The priming dose was 3 µmol/kg for the phenylalanine tracers and 10 µmol/kg for the d3--KIC tracer. A continuous infusion was initiated following the priming dose at a rate of 0.08 µmol·kg–1·min–1 for the phenylalanine tracers and 0.15 µmol·kg–1·min–1 for d3--KIC. Subjects received 35% and 50% of their recommended daily caloric intake, as predicted by the Harris-Benedict equation, through a liquid meal supplement (Boost: 68% carbohydrate, 17% protein, 15% fat) during the rested and postexercise (both 4 h and 28 h) infusions, respectively. Participants ingested the meal supplement in equally divided aliquots every 30 min (0.1 g protein·kg–1·h–1). The supplement aliquots where enriched with whichever isotope was being infused to maintain isotopic steady state following consumption. Muscle biopsies were obtained from both legs within 10 min of each other at t = 90 and 270 min during the first and third infusion trials and at t = 60 and 240 min during the exercise infusion. A portion of a resting biopsy obtained from each leg was processed for histochemical analysis (see below) upon excision, while all other tissue samples were immediately frozen in liquid nitrogen and stored at –80°C until further analysis. Blood analyses. All blood samples were collected into heparinized tubes (Vacutainer) and processed as previously described (14, 30). Blood amino acid concentrations were determined by HPLC from plasma samples as previously described (14, 30). Plasma glucose was measured by an enzymatic assay adapted for fluorometry (15), while insulin concentrations were analyzed by RIA (Coat-A-Count; Diagnostic Products, Los Angeles, CA).
For determination of blood amino acid enrichment, the perchloric acid extracted whole blood was transferred into a threaded Pyrex tube and lyophilized in a Speed-Vac Plus rotary evaporator (Savant Instruments, Farmingdale, NY). To determine the isotopic enrichment in the blood, the tert-butyl dimethylsilyl derivative of phenylalanine and leucine was prepared. To the dried sample 50 µl of acetonitrile (HPLC grade) and 50 µl N-methyl-N-(tert-butyl dimethylsilyl) trifluoro-acetamide ± 1% tert-butyl dimethylchlorosilane (MTBSTFA ± 1% TBDMCS; Pierce Chemical, Rockford, IL) were added, and then the samples were heated for 1 h at 90°C. The tert-butyl dimethylsilyl derivatives of phenylalanine and leucine were analyzed by splitless injection using electron-impact ionization capillary gas chromatography-mass spectrometry (models 6890 GC and 5973 MS; Hewlett-Packard, Palo Alto, CA) with selected ion monitoring of mass-to-charge ratios of 234 and 239 for L-[ring-d5]phenylalanine (m+0 and m+5), 234 and 240 for L-[ring-13C6]phenylalanine (m+0 and m+6), and 200 and 203 for d3-leucine (m+0 and m+3), where m is mass.
Muscle fiber analyses.
Frozen tissue samples embedded in optimal cutting temperature compound (Sakura Finetechnial, Tokyo, Japan) were sectioned and stained for myofibrillar ATPase activity and analyzed as previously described (12, 24, 26, 29). We have only included data for subjects for whom we could obtain a complete data set (Pre, Post-UT, and Post-T) with each sample providing 100 fibers. We were unable to obtain fiber data from two Post-UT samples, while one Post-T sample had only 15 usable fibers. Thus, only data from 7 of 10 subjects were included in the fiber analyses.
Gas chromatography-mass spectrometry analyses.
For mixed-muscle protein-bound and free intracellular enrichment 20 mg of wet muscle tissues were analyzed. Muscle samples were lyophilized to dryness overnight, while being incubated on dry ice. Samples were then manually powdered, and 500 µl of 0.6 M PCA was added. After gentle shaking, the samples sat on ice for 10 min and were then centrifuged at 2,000 g for 2 min. The supernatant (i.e., intracellular free amino acids) was collected and processed in the same manner as the whole blood samples. The remaining pellet from the muscle sample was washed twice with 1 ml of distilled deionized water and then lyophilized to dryness overnight. The dried pellet was placed in 2 ml of 6 N HCl and hydrolyzed for 24 h at 100°C. The acid hydrolysate was passed over acid-washed cation exchange column (Dowex Analytical Grade 50W-X8, 100–200 mesh hydrogen form; Bio-Rad Labs, Hercules, CA) for amino acid isolation/purification to determine the protein-bound enrichments. The samples were dried by using a rotary evaporator, and the dried pellet was then derivatized in the same manner as the blood samples. Intracellular enrichments were determined in the same manner as the blood, while bound muscle enrichments were determined using the standard curve approach (16).
Calculations. Fractional synthetic rate (FSR) was calculated from the determination of the rate of tracer incorporation into muscle protein and using the muscle intracellular free phenylalanine or leucine enrichment as a precursor, according to the following equation: FSR (%/h) = (Et1 – Et0)/[Ep * (t1 – t0)] * 100%, where Et0 is the enrichment of the protein-bound isotope tracer from the first biopsy, Et1 is the enrichment of the protein-bound isotope tracer from the second biopsy, Ep is the mean intracellular tracer enrichment during the time period for determination of protein incorporation, and (t1 – t0) is the incorporation time.
Statistics.
Strength, amino acid, blood glucose, and insulin data were analyzed using a two-factor repeated-measures ANOVA. Fiber cross-sectional area data were analyzed using a paired t-test for two sample means (pre and post) for each fiber type. Rates of mixed MPS were analyzed using a two-factor ANVOA with training status (T and UT) and condition (rest, 4 h postexercise, and 28 h postexercise) as within-subject factors. Where ANOVA revealed significance, a Tukey's post hoc procedure was used to identify pairwise differences. Residuals and comparisons of FSR determined by the phenylalanine and -KIC tracers were made using GraphPad Prism (version 5.00, GraphPad Software) Significance was accepted at P < 0.05. All data are expressed as means ± SD.
RESULTS
Strength measurements. Prior to training there was no difference between legs in unilateral knee extension 1-RM strength (Fig. 2). Unilateral 1-RM strength increased in both legs with training (T = 62.3 ± 27%;, UT = 19.7 ± 13%, P < 0.01; Fig. 2). However, by the completion of the training protocol, the increase in the T leg was threefold greater than of that seen in the UT leg (P < 0.01; Fig. 2). There was no pretraining difference between legs in isometric strength (T = 284.5 ± 43.5; UT = 297.4 ± 46.8 N·m, P = 0.64). After training, isometric strength increased in the T leg (17.7 ± 12%, P < 0.01), with no change in the UT leg (P = 0.86).
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We report here that 8 wk of unilateral resistance training (20 total training sessions) results in hypertrophy and alters the response of fed state-mixed MPS to an acute bout of resistance exercise performed at the same relative intensity (i.e., greater work performed by the T leg). In the present study the subjects were fed a meal supplement to maximize anabolic potential by creating a hyperinsulinemic and hyperaminoacidemic environment (3–6, 10, 21, 28). Despite the fact that mixed MPS was higher in the immediate postexercise period in the T vs. UT leg, the protein synthetic response was reduced in the T leg over 28 h.
The limited number of studies that have aimed to define the time course of increased MPS following a bout of resistance exercise suggests that protein synthesis rises rapidly (within 3–4 h) postexercise and remains elevated for at least 24 h (7, 13, 18). Combined cross-sectional data from Chesley et al. (7) and MacDougall et al. (13) suggests that MPS rises at 4 h postexercise, peaks at 24 h, and subsequently returns to resting levels by 36 h postexercise in trained subjects. Conversely, Phillips et al. (18) demonstrated that protein synthesis peaks at 3 h postexercise, and remains elevated above resting levels, albeit declining progressively both at 24 h and 48 h postexercise after an acute bout of resistance exercise. It is difficult to reconcile these previous results, both to each other and to the present study, as there are considerable differences in the training status of the participants and the study designs employed (i.e., cross-sectional vs. longitudinal). Indeed, Chesley et al. (7) and MacDougall et al. (13) utilized different groups of trained individuals for each time point studied, while Phillips et al. studied the same subjects at each time in a longitudinal design. Moreover, Chesley et al. (7) and MacDougall et al. (13) studied subjects in the fed state while the participants in the study by Phillips et al. (18) were fasted. To our knowledge, the present study is the first study to longitudinally examine the time course of changes in MPS in the fed state following an acute bout of resistance exercise after training. We found an increase in mixed MPS in the immediate period after exercise (4 h) in both the T and UT legs by 162% and 108%, respectively. However, while MPS remained elevated by 70% at 28 h postexercise in the UT leg, rates of MPS had returned to resting levels in the T leg. Thus, our data suggests that in the T state the response of MPS to the same relative intensity of resistance exercise is accelerated such that it reaches a greater peak initially but returns to baseline more rapidly compared with the UT state.
It has previously been reported that resistance training attenuates the protein synthetic response to an acute bout of resistance exercise (17). However, in the study by Phillips et al. (17), resistance exercise was performed at the same absolute intensity before and after training. Thus, the reduced rates of MPS in trained individuals could be expected as the stimulus would be relatively lower in the trained state due to the training-induced increase in muscle strength. In the present study, we compared MPS in T and UT muscle in the same individual following a bout of resistance exercise as the same relative intensity. We observed a greater immediate increase in MPS after exercise in the T compared with the UT leg (T = 162%; UT = 108%), but one which returned to baseline by 28 h postexercise compared with the UT leg. These results are in agreement with the general concept of adaptation to stress, manifest here as a reduction in the duration of the postexercise stimulation of protein synthesis. While there has been some evidence to suggest that rate of protein synthesis is dependent on the tracer used (25, 27), a residual plot (Fig. 5) of our FSR values determined by either phenylalanine or -KIC tracer infusion indicates no systematic bias. Moreover, when we compare the slopes between the residuals for each tracer there is no difference (P = 0.43), indicating that the selected tracers, with all of the inherent assumptions of each tracer (including the reciprocal pool model used with the -KIC tracer) did not unduly influence our data in magnitude or overall pattern of response.
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We report here that 8 wk of progressive unilateral leg training reduces the mixed muscle protein synthetic response over 28 h to an acute bout of resistance exercise performed at the same relative intensity before and after training. Despite the reduced anabolic response, MPS was higher initially in the T leg (4 h postexercise) but returned to baseline by 28 h postexercise at which time rates of MPS in the UT leg remained elevated. We conclude, therefore, that resistance training alters the time course of the increase in mixed MPS in the fed state.
Perspectives and Significance
As the protein synthetic response becomes attenuated with training, our results suggest that it likely becomes increasingly important for individuals to consume a source of amino acids in the immediate postexercise period during training to maximize MPS and thus protein accretion. While these results may also suggest that the frequency or intensity of lifts being performed during training sessions needs to be increased as individuals progress through their training, consideration must be made for adequate recovery time between training sessions and the risk of overtraining. Finally, additional research is required to determine the mechanisms that underlie the training-induced changes in the protein synthetic response to exercise.
GRANTS
This work was supported by a Natural Science and Engineering Research Council of Canada (NSERC) grant (to S. M. Phillips). S. M. Phillips is a Canadian Institutes for Health Research (CIHR) New Investigator Award recipient, J. E. Tang is a CIHR Doctoral Scholarship Award recipient, J. G. Perco was supported by an NSERC PGS Master's Scholarship, D. R. Moore is a CIHR Canada Graduate Scholarship (CGS) Scholarship Award recipient, S. B. Wilkinson is a CIHR CGS scholarship award recipient, and all authors acknowledge those sources of support during the conduct of this research.
ACKNOWLEDGMENTS
We thank the subjects for their time and effort and Mr. E. T. Prior for his technical assistance.
FOOTNOTES
Address for reprint requests and other correspondence: S. M. Phillips, Exercise Metabolism research Group, Dept. of Kinesiology, McMaster Univ., 1280 Main St. West, Hamilton, ON L8S 4K1 (e-mail: phillis{at}mcmaster.ca)
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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  R. Koopman and L. J. C. van Loon Aging, exercise, and muscle protein metabolism J Appl Physiol, June 1, 2009; 106(6): 2040 - 2048. [Abstract] [Full Text] [PDF]
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V. Kumar, P. Atherton, K. Smith, and M. J. Rennie Human muscle protein synthesis and breakdown during and after exercise J Appl Physiol, June 1, 2009; 106(6): 2026 - 2039. [Abstract] [Full Text] [PDF]
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N. A. Burd, J. E. Tang, D. R. Moore, and S. M. Phillips Exercise training and protein metabolism: influences of contraction, protein intake, and sex-based differences J Appl Physiol, May 1, 2009; 106(5): 1692 - 1701. [Abstract] [Full Text] [PDF]
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 E. I. Glover, B. R. Oates, J. E. Tang, D. R. Moore, M. A. Tarnopolsky, and S. M. Phillips Resistance exercise decreases eIF2B{varepsilon} phosphorylation and potentiates the feeding-induced stimulation of p70S6K1 and rpS6 in young men Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R604 - R610. [Abstract] [Full Text] [PDF]
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 S. B. Wilkinson, S. M. Phillips, P. J. Atherton, R. Patel, K. E. Yarasheski, M. A. Tarnopolsky, and M. J. Rennie Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle J. Physiol., August 1, 2008; 586(15): 3701 - 3717. [Abstract] [Full Text] [PDF]
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) is taken from Kim et al., (