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Departments of 2 Movement Sciences and 3 Human Biology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht 6200 MD, The Netherlands
* To whom correspondence should be addressed. E-mail: r.koopman{at}hb.unimaas.nl.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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We recently reported that a single bout of resistance type exercise increases the phosphorylation status of S6K1 at T421/S424 in human skeletal muscle in a fiber type-dependent manner, with the most pronounced phosphorylation ocurring in the type II muscle fibers (8). In addition, we found that AMPK was temporarily phosphorylated after cessation of exercise. The latter has been reported to induce a reduction in 4E-BP1 phosphorylation and implies that translation initiation is suppressed immediately after cessation of exercise (8–10). Furthermore, the phosphorylation of S6K1 at T421/S424 after exercise is not accompanied by a substantial phosphorylation of S6K1 at T389 and/or S6, indicating that merely a single bout of resistance type exercise is not sufficient to further increase signaling through S6K1 (8,9). The latter suggests that other stimuli are warranted to allow S6K1 phosphorylation at T389 and to initiate the mRNA translational machinery (8,11).
Amino acid and insulin availability are factors that play a permissive role in translation initiation [as reviewed by Kimball et al. (5)]. The administration of essential amino acids after an overnight fast increases the phosphorylation status of 4E-BP1, mTOR, and S6K1 (12). In accordance, continuous administration of specific amino acids during and after resistance type exercise can substantially elevate S6K1 phosphorylation status (11). Increased amino acid availability under conditions of elevated plasma insulin levels has been shown to further augment phosphatidylinositol-3 kinase/mTOR signaling and increase protein synthesis rates in a rat model (5). It is generally advised to ingest both carbohydrate and protein following exercise to enhance postexercise muscle recovery and to promote net muscle protein accretion. However, it has not yet been established whether the availability of dietary protein further augments postexercise S6K1 phosphorylation in vivo in humans. In addition, information on muscle fiber-type specific changes in the phosphorylation status of S6 following exercise and/or nutritional intervention are not yet available.
Therefore, the purpose of this study was to assess the impact of carbohydrate and/or protein ingestion before and after resistance type exercise on S6K1 and S6 phosphorylation status in humans. Besides measuring the phosphorylation state of S6K1, 4E-BP1, and S6 in mixed muscle tissue, we also aimed to investigate whether changes in S6 phosphorylation are specific for muscle fiber type and/or subcellular localization.
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Methods |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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Seven healthy male volunteers with no history of participating in any regular exercise program were recruited to participate in this study. Their characteristics are provided in Table 1. All subjects were informed on the nature and possible risks of the experimental procedures before their written informed consent was obtained. This study was approved by the Medical Ethical Committee of the Academic Hospital Maastricht.
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Body composition was assessed using the hydrostatic weighing method in the morning after an overnight fast, as previously described (8). Thereafter, maximum strength for leg press and leg extension was estimated using the multiple repetitions testing procedure (13). In an additional exercise session, at least 1 wk before the first experimental treatment, the subjects' 1-repetition maximum (RM) was determined (14). The mean 1-RM for the leg press and leg extension averaged 198 ± 7 and 105 ± 3 kg, respectively.
Diet and activity prior to testing
All subjects consumed a standardized meal (63 ± 2 kJ·kg–1 body weight (BW), consisting of 65 energy percent (En%) carbohydrate, 15 En% protein, and 20 En% fat) the evening prior to the experiments. All volunteers were instructed to refrain from any sort of heavy physical exercise and to keep their diet as constant as possible for 3 d before the experiments. In addition, subjects were asked to record their food intake for 48 h before the start of the first experiment and to consume the same diet 48 h prior to the second test.
Protocol
After an overnight fast, subjects arrived at 08:00 at the laboratory by car or public transportation. A Teflon catheter was inserted in a heated dorsal hand vein of the contra-lateral arm and placed in a hot-box (60°C) for arterialized blood sampling. Subjects rested for 30 min in a supine position, after which a basal blood sample was collected and a muscle biopsy was taken from the vastus lateralis muscle. Before the start of the exercise session, subjects received the initial bolus of a given test drink (5 mL·kg–1). After 5 min of warm-up on a cycle ergometer (
75 W), subjects completed a session of 3 upper-body resistance exercises, which featured 3 sets of 10 repetitions for each of the exercises. This was performed with weight set at 40% of their BW for the chest press and shoulder press, and at 50% BW for the front pull-down (Jimsa Benelux BV), with 1 min resting intervals between sets. These exercises were included to provide a whole-body warm-up and, as such, to reduce the risk of musculoskeletal injuries. This was followed by a blood draw (t = –30 min) and a session of lower-limb exercise, consisting of 8 sets of 10 repetitions on the leg press and leg extension machines (Technogym BV), both performed at 75% of the individual 1-RM, with 2 min rest intervals between sets. All subjects were verbally encouraged during exercise and the entire exercise protocol required 1 h to complete. Within 5 min after cessation of exercise, a second muscle biopsy sample and an additional blood sample was collected, after which subjects received the second bolus of the test drink and rested supine for 4 h. Following 60 min of postexercise recovery, an additional muscle biopsy was taken and the third beverage was provided. Arterialized blood samples were collected during recovery at t = 30, 60, 90, 120, 150, 180, 210, and 240 min with a fourth muscle biopsy taken at t = 240 min.
Beverages
Subjects received a beverage volume of 5 mL·kg–1 before, immediately after, and 1 h after exercise to ensure a given dose of 0.3 g carbohydrate·kg–1 (50% glucose and 50% maltodextrin) with or without the addition of 0.3 g·kg–1 of a protein hydrolysate. The total amount of protein (3 x 0.3 g·kg–1) provided in the carbohydrate+protein (CHO+PRO) treatment was chosen to exceed the calculated amount of protein that was estimated to provide ample amino acids to sustain maximal protein synthesis rates for at least 5 h (15). Glucose and maltodextrin were obtained from AVEBE. Casein protein hydrolysate was prepared by DSM Food Specialties. To make the taste comparable in all treatments, beverages were uniformly flavored as previously described (16,17). Treatments were performed in a randomized order, with test-drinks provided in a double-blind fashion.
Muscle biopsies
The first 2 biopsies were taken from the same incision in one leg (18); the other 2 biopsies were taken from the same incision in the contra-lateral leg under aseptic conditions and local anesthesia (1% Lidocaine+). When biopsy samples were taken from the same incision, the first sample was taken from a different region (distal of the incision, with the needle pointing inwards) than the second (proximal of the incision, with the needle pointing outwards). Muscle samples were freed from any visible nonmuscle material and rapidly frozen in liquid nitrogen. Muscle samples (50 mg) for Western blotting analyses were treated and homogenized using a previously described buffer containing several protease inhibitors (10). Another part of each muscle sample (20 mg) was frozen in liquid nitrogen-cooled isopentane and embedded in Tissue-Tek for immunohistochemical analyses (Sakura Finetek).
Plasma sample analyses
Blood samples were collected in EDTA-containing tubes and centrifuged at 1000 x g and 4°C for 5 min. Aliquots of plasma were frozen in liquid nitrogen and stored at –80°C. Plasma glucose (Uni Kit III, 07367204, Roche) and lactate (19) concentrations were analyzed with the COBAS-FARA semiautomatic analyzer (Roche). Insulin was analyzed by radio immunoassay (Insulin RIA kit, LINCO Research).
Muscle sample analyses
Antibodies. Primary phospho-specific antibodies [anti-phospho S6K1 (T421/S424), anti-phospho S6K1 (T389), anti-phospho S6 (S235/236), anti-phospho 4E-BP1 (T37)], and anti-S6K1, anti-S6, and anti-4E-BP1 were purchased from Cell Signaling Technologies. Anti-caveolin-3 was purchased from BD Biosciences and the monoclonal antibody raised against adult human slow myosin heavy chain or A4.840 was from Developmental Studies Hybridoma Bank, developed by Dr. H. Blau (Stanford University). Appropriate secondary conjugated antibodies (GARIgGAlexa555 and GAMIgG1Alexa488) were purchased from Molecular Probes.
Western blotting.
Quantification of phosphorylation status of S6K1, S6, and 4E-BP1 was performed using western blotting with phospho-specific and a-specific antibodies as previously described (8). 
-Actin was used to standardize for the amount of protein loaded. Phosphorylation of 4E-BP1, S6K1, and S6 was expressed relative to the total amount of each protein. Data were analyzed for each subject as the % change in phosphorylation state from preexercise values.
Immunohistochemical analyses. Multiple serial sections (5 µm) from muscle biopsy samples collected before, immediately after, 1 h, and 4 h after exercise were thaw-mounted together on uncoated, precleaned glass slides for each subject, carefully aligned for cross-sectional analyses. Sections were fixed for 5 min in methanol, followed by 1 min in acetone. Slides were then incubated overnight at 4°C with anti-caveolin-3, anti-MHCI (A4.840) (20), and anti-phospho S6 antibodies (1:200, 1:50, and 1:50 in PBS, respectively). Slides were rinsed for 35 min with PBS and then incubated for 45 min with appropriate secondary antibodies diluted together with 5 mg/L 4',6-diamidino-2-phenylindole (DAPI; to visualize nuclei) in PBS. After several washes with PBS, stained sections were embedded in Mowiol and covered with a coverslip. All muscle cross-sections were stained and prepared within a single batch using the same antibody-preparation to minimize variability in staining efficiency (8). The phospho-S6 specific fluorescence signal was quantified for each muscle fiber, resulting in a total of 180 ± 14 muscle fibers analyzed for each muscle cross-section (108 ± 8 type I and 71 ± 6 type II muscle fibers). Intracellular phospho-S6 content was expressed as mean staining intensity.
Statistics
All data are expressed as means ± SEM. The plasma insulin and glucose responses were calculated as area under the curve above baseline (AUC) values. Two-way ANOVA for repeated measures were applied to determine differences in plasma insulin and glucose concentrations, as well as in phosphorylation status in the protein of interest over time between treatments. In case of significant interaction between time and treatment effects, paired t tests were applied to locate differences between treatments, and Scheffe post-hoc tests were applied to locate differences over time. Statistical significance was set at P < 0.05.
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Results |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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Plasma insulin and glucose levels increased in both treatments after the intake of the beverages (Fig. 1) and returned to baseline values within 1 h after ingestion of the last beverage. The plasma insulin response, expressed as AUC values (t = –60 min, AUC), was greater in CHO+PRO (47 ± 6 µmol·5 h·L–1) than in CHO (24 ± 8 µmol·5 h·L–1, P < 0.01). The concomitant plasma glucose response (AUC) was significantly lower in CHO+PRO (10 ± 23 mmol·5 h·L–1) than in CHO (124 ± 40 mmol·5 h·L–1, P < 0.01). Plasma lactate concentrations increased from 0.8 ± 0.1 mmol·L–1 at rest to 7.2 ± 0.6 mmol·L–1 (estimated marginal means, P < 0.0001) immediately following exercise, and decreased during recovery in both treatments. Plasma lactate concentrations over time did not differ between treatments.
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Exercise. Following resistance exercise, 4E-BP1 phosphorylation status was lower in CHO (–48 ± 7%) vs. CHO+PRO (–15 ± 14%; P < 0.01, Fig. 2A). S6K1 phosphorylation at T421/S424 increased to the same extent following exercise in both treatments (P < 0.05) (Fig. 2B). In contrast to the CHO treatment (–4 ± 2%), S6K1 phosphorylation at T389 was higher following exercise in the CHO+PRO treatment (+78 ± 2%, P < 0.01, Fig. 2C). S6 phosphorylation was substantially higher following exercise in CHO+PRO (169 ± 35% of preexercise values) than in CHO (45 ± 7% of preexercise values, P < 0.01, Fig. 2D).
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Exercise. Staining for phospho-specific S6 in the pre- and postexercise muscle biopsy samples showed a predominant localization of phospho-S6 in the cytosol (Fig. 4). Following exercise, S6 phosphorylation status in type I increased to a greater extent in CHO+PRO than CHO (Table 2, P < 0.05). In type II muscle fibers, S6 phosphorylation increased following resistance exercise. S6 phosphorylation status in the type II muscle fibers did not differ between treatments (Table 2).
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Discussion |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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Changes in protein synthesis rates occur before changes in mRNA accumulation (2). Therefore, it is assumed that post-transcriptional mechanisms play a key regulatory role in the activation of muscle protein synthesis (3). The initiation of mRNA translation is thought to be rate-limiting in the overall control of muscle protein synthesis. The initial binding of the tRNA to the ribosome is partly regulated by eIF2B (21), whereas the binding of the mRNA to the ribosome is regulated by the phosphatidylisositol 3-kinase signaling pathway (5,6) The activation of mTOR and subsequent phosphorylation of S6K1 and 4E-BP1 are thought to represent key events in the activation of translation initiation (5), as 4E-BP1 (22) and S6 (23) may facilitate the binding of the mRNA to the ribosome. In rodents, the phosphorylation status of S6K1 following resistance type exercise has been reported to form an excellent marker for the long-term increase in skeletal muscle mass (24).
S6K1 and S6 phosphorylation increase following maximal lengthening contractions in rat (24–27) and human (28) muscle tissue. However, the phosphorylation status of S6K at T389 (11,28) and/or S6 (8,28) does not seem to be substantially increased in humans following conventional resistance type exercise in the fasted state. The latter suggests that the response of signaling through S6K1 is dependent of the mode and intensity of muscle contraction, and that merely a single bout of resistance type exercise is not sufficient to further increase S6K1 signaling. In line with data provided by Karlsson et al. (11), we observed in this study a strong increase in the phosphorylation status of S6K1 at T421/S424 following exercise (Fig. 2B), with no differences between treatments. Clearly T421/S424 phosphorylation status of S6K1 immediately following exercise does not seem to be greatly affected by the differences in circulating plasma insulin (Fig. 1A) or amino acid concentrations. Phosphorylation of S6K1 at T421/S424 results in a conformational change in S6K1, allowing the phosphorylation at T389, which subsequently activates the kinase (29,30).
Phosphorylation of S6K1 at T421/S424 is not necessarily associated with increased activity of S6K1. The latter is confirmed by the absence of increased phosphorylation status of S6K1 at T389 and S6 at S235/236 in the CHO treatment immediately following exercise, even though T421/S424 phosphorylation had increased severalfold (Fig. 2). The administration of protein during postexercise recovery substantially increased the phosphorylation status of S6K1 (T389) and S6 during postexercise recovery (Fig. 3), when compared with the ingestion of carbohydrate only. These observations are in line with Karlsson et al. (11), who showed enhanced S6K1(T389) and S6 phosphorylation during postexercise recovery after administration of the branched chain amino acids. We extend on these data by showing the phosphorylation of some of the key events in translation initiation following the ingestion of a single bolus of carbohydrate and protein, before and after resistance type exercise. We found that the combined ingestion of protein with carbohydrate further increases 4E-BP1 phosphorylation compared with the ingestion of only carbohydrate. These observations seem to be in line with rat data showing no effect of carbohydrate intake on 4E-BP1 phosphorylation (22). The greater increase in phosphorylation status of S6K1, S6, and 4E-BP1 suggests that elevated insulin concentrations and/or greater amino acid availability are instrumental to allow enhanced translation initiation process and, as such, to promote subsequent muscle protein anabolism. In accordance, numerous previous studies have shown that protein intake following exercise accelerates muscle protein synthesis [reviewed in (31)].
S6 is one of the main substrates of S6K1. As a result, the phosphorylation status of S6 can be used as an indirect measure of S6K1 activity (23). Using an immunohistochemical approach, we assessed potential differences in muscle fiber-type specific activation of S6 after resistance type exercise following CHO or CHO+PRO ingestion. We observed increased S6 phosphorylation in both type I and II fibers in both treatments immediately following exercise. Interestingly, phosphorylation of S6 in type I fibers immediately postexercise was substantially higher in CHO+PRO than in CHO treatment, whereas no differences were observed between treatments in type II fibers. This difference in response may in part be attributed to difference in metabolic demand of the exercise between fiber-types (8). In accordance, during postexercise recovery, S6 phosphorylation was more pronounced in the CHO+PRO vs. CHO treatment in both fiber-types. Again these finding confirmed the western blotting analyses of the mixed muscle tissue samples.
In conclusion, the combined ingestion of protein and carbohydrate further elevates the phosphorylation status of 4E-BP1, S6K1, and S6 during recovery from resistance type exercise. These findings provide a mechanistic background for the observation that protein ingestion is instrumental to stimulate postexercise muscle protein accretion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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4 Abbreviations used: 4E-BP1, eIF4E binding protein; AUC, area under the curve above baseline; BW, body weight; CHO+PRO carbohydrate+protein; En%, energy percent; mTOR, mammalian target of rapamycin; RM, repetition maximum, S6K1, ribosomal protein S6 kinase.
Manuscript received 21 March 2007. Initial review completed 9 April 2007. Revision accepted 12 May 2007.
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LITERATURE CITED |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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