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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Coingestion of Carbohydrate and Protein Hydrolysate Stimulates Muscle Protein Synthesis during Exercise in Young Men, with No Further Increase during Subsequent Overnight Recovery^1–3,

⁴ Department of Movement Sciences and ⁵ Department of Human Biology, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, 6200 MD Maastricht, The Netherlands; ⁶ Stable Isotope Research Center, Academic Hospital Maastricht, 6229 HX Maastricht, The Netherlands; and ⁷ DSM Food Specialties, 2600 MA Delft, The Netherlands

^* To whom correspondence should be addressed. E-mail: milou.beelen{at}bw.unimaas.nl.

	ABSTRACT

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
We investigated the effect of carbohydrate and protein hydrolysate ingestion on whole-body and muscle protein synthesis during a combined endurance and resistance exercise session and subsequent overnight recovery. Twenty healthy men were studied in the evening after consuming a standardized diet throughout the day. Subjects participated in a 2-h exercise session during which beverages containing both carbohydrate (0.15 g·kg^–1·h^–1) and a protein hydrolysate (0.15 g·kg^–1·h^–1) (C+P, n = 10) or water only (W, n = 10) were ingested. Participants consumed 2 additional beverages during early recovery and remained overnight at the hospital. Continuous i.v. infusions with L-[ring-¹³C₆]-phenylalanine and L-[ring-²H₂]-tyrosine were applied and blood and muscle samples were collected to assess whole-body and muscle protein synthesis rates. During exercise, whole-body and muscle protein synthesis rates increased by 29 and 48% with protein and carbohydrate coingestion (P < 0.05). Fractional synthetic rates during exercise were 0.083 ± 0.011%/h in the C+P group and 0.056 ± 0.003%/h in the W group, (P < 0.05). During subsequent overnight recovery, whole-body protein synthesis was 19% greater in the C+P group than in the W group (P < 0.05). However, mean muscle protein synthesis rates during 9 h of overnight recovery did not differ between groups and were 0.056 ± 0.004%/h in the C+P group and 0.057 ± 0.004%/h in the W group (P = 0.89). We conclude that, even in a fed state, protein and carbohydrate supplementation stimulates muscle protein synthesis during exercise. Ingestion of protein with carbohydrate during and immediately after exercise improves whole-body protein synthesis but does not further augment muscle protein synthesis rates during 9 h of subsequent overnight recovery.

	Introduction

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

In general, previous studies have assessed the impact of food intake on the muscle protein synthetic response to exercise in the overnight food-deprived state. Under these conditions, it seems reasonable to assume that the limited endogenous availability of amino acids from the gut and/or the intramuscular free amino acid pool limits an increase in postexercise muscle protein synthesis rates. Such postabsorptive conditions differ from normal everyday practice, in which recreational sports activities are often performed in the evening after dinner. Therefore, it might be speculated that the impact of protein and carbohydrate supplementation during and/or after exercise does not further elevate muscle protein synthesis rates in the fed state. No data are available regarding the impact of protein and/or carbohydrate supplementation on muscle protein synthesis after exercise performed in the evening following the consumption of a normal, standardized diet throughout the day.

Moreover, in daily practice where recreational exercise is often performed in the evening, postexercise recovery predominantly occurs during subsequent overnight sleep. Studies in rodents suggest that protein synthesis is stimulated in various tissues during the sleeping state (16). In vivo measurements of muscle protein synthesis rates during sleep in humans are scarce. Tipton et al. (17) reported a 30% greater muscle protein synthesis rate during the night following exercise compared with the nonexercised condition. However, studies investigating the impact of dietary modulation on muscle protein synthesis during overnight recovery are entirely lacking. Therefore, the aim of the present study was to assess the impact of carbohydrate and protein hydrolysate coingestion during and after exercise on whole-body and muscle protein synthesis rates when exercise is performed in the evening following the consumption of a standardized diet throughout the day. We hypothesized that coingestion of carbohydrate and protein stimulates muscle protein synthesis during exercise performed in the fed state as well as during subsequent overnight recovery.

	Methods

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Participants. Twenty healthy, male volunteers participated in this study. All participants were recreationally active, which was defined as participating in sports <2 times a week, and not on a regular and/or competitive basis. Participants were randomly assigned to either the carbohydrate and protein supplement (C+P⁸; n = 10) or placebo (water only) group (W; n = 10). All participants were fully informed on the nature and possible risks of the experimental procedures before their written informed consent was obtained. The study was approved by the Medical Ethical Committee of the Academic Hospital Maastricht, The Netherlands, and is part of a greater project on the impact of nutrition on postexercise recovery.

    Pretesting. All participants participated in 2 screening sessions separated by at least 5 d. In the morning following an overnight fast, body composition was determined by the hydrostatic weighing method. Body fat percentage was calculated using Siri's equation (18) and leg volume was measured by anthropometry (19). Thereafter, participants were familiarized with the exercise equipment and exercise procedures. Proper lifting technique was demonstrated and practiced for each of the upper-body exercises (chest press, shoulder press, and lat pulldown) and for the 2 lower-limb exercises (leg press and leg extension). Thereafter, maximum strength for the 2 leg exercises was estimated using the multiple repetition testing procedure (20).

In the 2nd screening session, participants' one repetition maximum (1RM) was determined for the 2 leg exercises (21). In addition, participants performed an incremental exhaustive exercise test on an electronically braked cycle ergometer (Lode Excalibur) to measure their maximal oxygen uptake and workload capacity (W_max) (22).

    Design. During the experimental days, all participants received the same standardized diet (breakfast, lunch, dinner, and snacks). Apart from the standardized diet, participants participated in their normal daily activities and reported to the hospital in the evening. Subsequently, participants performed a 2-h endurance and resistance exercise session during which either carbohydrate and a protein hydrolysate (C+P) or a placebo (W) was ingested. Participants received 2 additional boluses of the test drink during early recovery and remained overnight at the hospital. Plasma samples were collected every 15 min during exercise, every 30 min during the first 2 h of postexercise recovery, and every hour during overnight sleep. Muscle biopsies from the vastus lateralis muscle were taken before and immediately after exercise and in the morning after 9 h of postexercise recovery (at 0700). Tests were designed to simultaneously assess whole-body amino acid kinetics and mixed muscle protein fractional synthetic rate (FSR) by the incorporation of L-[ring-¹³C₆]-phenylalanine in the mixed muscle protein pool of the collected tissue samples.

    Diet and activity prior to and during the experiments. All participants received a standardized diet the evening prior to the experimental day (3.7 MJ, consisting of 62 energy% (En%) carbohydrate, 16 En% protein, and 22 En% fat) and during the entire experimental day (0.16 ± 0.01 MJ·kg body weight^–1·d^–1, consisting of 62 ± 0.4 En% carbohydrate, 12 ± 0.2 En% protein, and 26 ± 0.4 En% fat). Participants' energy requirements were calculated with the Harris and Benedict equation (23), with a physical activity index of 1.7 (24). The investigator provided the participants with measured amounts of all food products and participants were instructed to take all meals/snacks at predetermined time intervals. During the test day, participants ingested 1.1 ± 0.1 g/kg body weight protein via the standardized diet, with an additional 0.6 g/kg body weight supplemented in the C+P group. All volunteers were instructed to refrain from any sort of exhaustive physical labor and to keep their diet as constant as possible 2 d before the experimental day.

    Beverages. Participants received either a carbohydrate and protein hydrolysate-containing beverage (C+P) or water only (W) at a volume of 1.5 mL/kg every 15 min during exercise and 4 mL/kg at 30 and 90 min after cessation of exercise. The C+P beverages provided 0.15 g·kg^–1·h^–1 carbohydrate (50% glucose and 50% maltodextrin) and 0.15 g·kg^–1·h^–1 protein hydrolysate during the first 4 h following the onset of exercise. The first bolus was provided in a volume of 4.5 mL/kg to stimulate gastric emptying and, as such, to allow a more continuous supply of glucose and amino acids from the gut during exercise. Glucose and maltodextrin were obtained from AVEBE. The casein protein hydrolysate (PeptoPro; 85.3% protein) was prepared by DSM Food Specialties and involved the enzymatic hydrolysis of intact casein protein by specific endopeptidases and proline-specific endoprotease, which resulted in a di- and tripeptide content of 70–80%. To make the taste comparable, all solutions were flavored by adding 0.05 g/L sodium saccharinate, 0.9 g/L citric acid, and 5.0 g/L cream vanilla flavor (Quest International). Treatments were performed in a randomized, double-blind fashion.

    Experimental protocol. At 1830, participants reported to the laboratory, where a Teflon catheter was inserted into an antecubital vein for the primed, continuous infusion of isotopically labeled phenylalanine and tyrosine (priming dose: 2 µmol/kg L-[ring-¹³C₆]-phenylalanine, 0.775 µmol/kg L-[ring-²H₂]-tyrosine; infusion rate: 0.05 µmol·kg^–1·min^–1 L-[ring-¹³C₆]-phenylalanine, 0.02 µmol·kg^–1·min^–1 L-[ring-²H₂]-tyrosine). Another Teflon catheter was inserted into a contralateral hand vein, which was placed in a hotbox for arterialized blood sampling. After a background blood sample was collected (t = –180 min), continuous tracer infusion was started and participants rested in a supine position for 1 h. Before engaging in the exercise protocol (t = –120), the first muscle biopsy was collected, after which participants ingested the first bolus of test drink (4.5 mL/kg). During exercise, participants received subsequent boluses (1.5 mL/kg) of the test drink every 15 min. The exercise protocol consisted of an interval-cycling program followed by (whole-body) resistance exercise. This exercise protocol was designed to mimic a practical fitness training session. At 2200, immediately after the end of the exercise protocol (t = 0), an arterialized blood sample from the heated hand vein and a 2nd muscle biopsy from the vastus lateralis muscle were obtained. Participants rested supine during the remainder of the evening and were provided with 2 beverages (4 mL/kg) after 30 and 90 min of postexercise recovery. This was followed by 7 h of sleep, after which participants were awoken in the morning at 0700 for a 3rd muscle biopsy. The total postexercise recovery time was 9 h. Blood samples (8 mL) were taken from the arterialized hand vein at t = –180, –120, –105, –90, –75, –60, –45, –30, –15, 0, 30, 60, 90, and 120 min, and t = 3, 4, 5, 6, 7, 8, and 9 h during sleep. Blood samples at t = 3, 4, 5, 6, 7, 8, and 9 h during sleep were not arterialized, as sleeping turned out to be impossible with the hand in a hotbox. Muscle biopsies were taken before and immediately after exercise and after 9 h of postexercise overnight recovery (t = –2, 0, and 9 h).

    Exercise protocol. After 10 min of warming-up on a cycle ergometer (50% W_max), participants cycled 4 x 5 min at 65% W_max, alternated by 4 x 2.5 min at 45% W_max. After a 5-min resting period, participants started with the resistance exercise protocol, consisting of an upper- and a lower-body workout. The upper-body workout was performed with a workload set at 40% of the total body weight in which participants completed 5 sets of 10 repetitions on 3 upper-body machines (chest press, shoulder press, and lat pulldown). A resting period of 1 min between sets was allowed. This was followed by a lower-limb workout, which consisted of 9 sets of 10 repetitions on the horizontal leg press machine (Technogym BV) and 9 sets of 10 repetitions on the leg extension machine (Technogym). On both machines, 3 sets were completed at 55% of participants' 1RM, 3 at 65% 1RM, and 3 at 75% 1RM, with 2-min rest periods between sets. Finally, participants performed 2 sets of 30 abdominal crunches. All participants were verbally encouraged during the exercise regimen to complete the entire protocol within 120 min.

    Tracer. The stable isotope tracers, L-[ring-¹³C₆]-phenylalanine and L-[ring-²H₂]-tyrosine, were purchased from Cambridge Isotopes and dissolved in 0.9% saline before infusion. Continuous i.v. infusion (over a period of 12 h, 0.05 µmol·kg^–1·min^–1 L-[ring-¹³C₆]-phenylalanine, 0.02 µmol·kg^–1·min^–1 L-[ring-²H₂]-tyrosine) was performed using a calibrated IVAC 560 pump. Both the phenylalanine and tyrosine pool were primed (2 µmol/kg L-[ring-¹³C₆]-phenylalanine, 0.775 µmol/kg L-[ring-²H₂]-tyrosine) to enable the calculation of whole-body phenylalanine kinetics using established tracer models (25–27).

    Muscle biopsies. Muscle biopsies were obtained from the middle region of the vastus lateralis muscle (15 cm above the patella) and 2 cm below the entry through the fascia by means of the percutaneous needle biopsy technique described by Bergström et al. (28). The pre- and postexercise muscle biopsies were taken through the same incision, with the needle pointed in distal and proximal directions, respectively. The biopsy at 9 h postexercise was taken from the contralateral leg. All samples were carefully freed from any visible fat and blood, rapidly frozen in liquid nitrogen, and stored at –80°C until subsequent analysis.

    Plasma analysis. Blood samples (8 mL) were collected in EDTA-containing tubes and centrifuged at 1000 x g; 10 min at 4°C. Aliquots of plasma were frozen in liquid nitrogen and stored at –80°C until analysis. Plasma glucose concentrations were analyzed using the COBAS-FARA semiautomatic analyzer (Uni Kit III, 07367204, La Roche). Insulin was analyzed using RIA (Linco, Human Insulin RIA kit, LINCO Research). Plasma (500 µL) for amino acid analyses was deproteinized on ice with 100 µL of 24% (wt:v) 5-sulphosalicylic acid, mixed, and the clear supernatant was collected after centrifugation. Plasma amino acid concentrations were analyzed on an automated dedicated amino acid analyzer (LC-A10, Shimadzu Benelux) using an automated precolumn derivatization procedure and a ternary solvent system. For plasma phenylalanine and tyrosine enrichment measurements, plasma phenylalanine and tyrosine were derivatized to their t-butyldimethylsilyl derivatives and their ¹³C and/or ²H enrichments were determined by electron ionization GC-MS (Agilent 6890N GC/5973N MSD) using selected ion monitoring of masses 336 and 342 for unlabeled and labeled phenylalanine, respectively and masses 466, 468, and 472 for unlabeled and [ring-²H₂] and [ring-¹³C₆] labeled tyrosine, respectively.

    Muscle analyses. For measurement of L-[ring-¹³C₆]-phenylalanine enrichment in the free amino acid pool and mixed muscle protein, 55 mg of wet muscle was freeze-dried. Collagen, blood, and other nonmuscle fiber material were removed from the muscle fibers under a light microscope. The isolated muscle fiber mass (2–3 mg) was weighed and 8 volumes (8x dry weight of isolated muscle fibers x wet:dry ratio) of ice-cold 2% perchloric acid was added. The tissue was then homogenized and centrifuged. The supernatant was collected and processed in the same manner as the plasma samples, such that intracellular free L-[ring-¹³C₆]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[ring-¹³C₆]-tyrosine enrichments could be measured using their t-butyldimethylsilyl derivatives on a GC-MS. The free amino acid concentration in the supernatant was measured using an HPLC technique after precolumn derivatization with o-phthaldialdehyde (29). The protein pellet was washed with 3 additional 1.5-mL washes of 2% perchloric acid, dried, and the proteins were hydrolyzed in 6 mol/L HCl at 120°C for 15–18 h. The hydrolyzed protein fraction was dried under a nitrogen stream while heated to 120°C, dissolved in a 50% acetic acid solution, and passed over a Dowex exchange resin (AG 50W-X8, 100–200 mesh hydrogen form, Bio-Rad) using 2 mmol/L NH₄OH. Thereafter, the eluate was dried and the purified amino acid fraction was derivatized into the ethoxycarbonyl-ethylesters to determine the ¹³C-enrichment of protein-bound phenylalanine using GC-isotope ratio MS (Finnigan, MAT 252).

    Calculations. Infusion of L-[ring-¹³C₆]-phenylalanine and L-[ring-²H₂]-tyrosine with muscle and arterialized blood sampling was used to simultaneously assess whole-body amino acid kinetics and FSR of mixed muscle protein. Whole-body rates of appearance (Ra) and disappearance (Rd) of phenylalanine were calculated using the nonsteady–state Steele equations adapted for stable isotope methodology (25–27). FSR of mixed muscle protein was calculated by dividing the increment in enrichment in the product, i.e. protein-bound L-[ring-¹³C₆]-phenylalanine, by the enrichment of the precursor (plasma L-[ring-¹³C₆]-phenylalanine enrichment) as described previously (10).

    Statistics. Values are expressed as means ± SEM. The plasma insulin, glucose, and amino acid responses were calculated as area under the curve (AUC). We used a 2-factor repeated-measures ANOVA with time and treatment as factors to compare differences between groups over time. In the case of a significant F-ratio, Scheffé post hoc tests were applied to locate the differences. For nontime–dependent variables, an unpaired Students' t test was used to compare differences between groups. Significance was set at P < 0.05. All calculations were performed using StatView 5.0 (SAS Institute).

	Results

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
Participants

At baseline, the groups did not differ in age (21 ± 1 y), BMI (21.7 ± 0.4 kg/m²), body fat (13.8 ± 1.0%), leg volume (8.3 ± 0.3 L), 1RM leg press (211 ± 8 kg), 1RM leg extension (122 ± 3 kg), W_max (292 ± 7 W), or maximal oxygen uptake (3.4 ± 0.1 L/min).

Plasma analyses

    Insulin. Although there was a treatment x time interaction (P < 0.01), groups did not differ at specific time points. Total plasma insulin responses, measured as AUC, were 566 ± 60 pmol·L^–1·11 h^–1 in the W group and 1037 ± 270 pmol·L^–1·11 h^–1 in the C+P group (P = 0.12).

    Glucose. Although there was a treatment x time interaction (P < 0.01), groups did not differ at specific time points. Total plasma glucose responses, measured as AUC, were 57.4 ± 1.3 and 59.2 ± 0.8 mmol·L^–1·11 h in the W and C+P group, respectively (P = 0.25).

    Amino acids. Plasma phenylalanine and tyrosine concentrations were higher during the exercise period in the C+P than in the W group (P < 0.05). Plasma phenylalanine concentrations increased during the first 3 h of recovery in the C+P group, after which they returned to baseline values. The plasma tyrosine concentration was elevated throughout the entire recovery period in the C+P group, whereas it remained at baseline values in the W group. Total plasma amino acid responses were higher in the C+P compared with the W group for all amino acids (P < 0.05), except for glutamic acid, glutamine, -aminobutyrate, glycine, and taurine during exercise and recovery, and alanine, histidine, and serine during recovery only (Table 1).

View larger version (10K): [in this window] [in a new window]   FIGURE 1  Plasma L-[ring-13C6]-phenylalanine (A), L-[ring-2H2]-tyrosine (B), and L-[ring-13C6]-tyrosine (C) enrichment in healthy young men during and after W (n = 10) and C+P (n = 10) ingestion. MPE, mole percent excess. Values are means ± SEM. Data were analyzed with ANOVA repeated measures (treatment x time). Plasma L-[ring-13C6]-phenylalanine enrichment: treatment effect, P < 0.01; time effect, P < 0.01; interaction of treatment and time, P < 0.01. Plasma L-[ring-2H2]-tyrosine enrichment: treatment effect, P < 0.01; time effect, P < 0.01; interaction of treatment and time, P < 0.01. Plasma L-[ring-13C6]-tyrosine enrichment: treatment effect, P < 0.01; time effect, P < 0.01; interaction of treatment and time, P < 0.01. *Different from W, P < 0.05.

View larger version (10K): [in this window] [in a new window]   FIGURE 2  Plasma phenylalanine Ra and Rd in healthy young men during and after W (n = 10) and C+P (n = 10) ingestion. Values are means ± SEM. Data were analyzed with ANOVA repeated measures (treatment x time). Plasma phenylalanine Ra: treatment effect, P < 0.01; time effect, P < 0.01; interaction of treatment and time, P < 0.01. Plasma phenylalanine Rd: treatment effect, P < 0.01; time effect, P < 0.01; interaction of treatment and time, P < 0.01. *Different from W, P < 0.05.

	Discussion

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
The present study examined the impact of carbohydrate and protein hydrolysate supplementation on muscle protein synthesis rate during exercise performed in the evening and subsequent overnight recovery. Our data show that, even in the fed state, supplementation with carbohydrate and a protein hydrolysate elevates muscle protein synthesis during exercise, but does not seem to further augment muscle protein synthesis over the subsequent overnight recovery period.

Studies on the impact of carbohydrate and protein ingestion on muscle protein synthesis have generally focused on nutrient intake during acute postexercise recovery (4–7,9–12,15). It has been proposed that carbohydrate and protein coingestion prior to and/or during exercise might be more effective to augment muscle protein synthesis rates (13), because protein synthesis rates were higher when protein and carbohydrate were administered before exercise than immediately after cessation of exercise. The latter has been attributed to a more rapid provision of amino acids to the muscle following the cessation of exercise. However, it has also been speculated that protein coingestion prior to and/or during exercise stimulates muscle protein synthesis during exercise activities. So far, only 2 studies have addressed the potential impact of nutrition on protein synthesis during exercise (13,30). Their results show that the combined ingestion of carbohydrate and protein stimulates whole-body protein synthesis and improves net protein balance during both endurance (30) and resistance (13) exercise activities. Because whole-body protein kinetics do not necessarily reflect skeletal muscle protein turnover (31), it still remains to be established how protein and carbohydrate supplementation modulates skeletal muscle protein synthesis during exercise. The present study shows that ingestion of carbohydrate and protein hydrolysate during a combined endurance and resistance exercise session stimulates both whole-body and skeletal muscle protein synthesis rates during exercise conditions. The latter clearly shows that, even in the fed state, skeletal muscle protein synthesis can be further augmented by nutrient intake during exercise activities. This may be attributed to the intermittent character of the exercise regimen that we applied, allowing muscle protein synthesis rates to be accelerated during the rest intervals between sets. Therefore, our findings indicate that it might be preferable to ingest carbohydrate and protein already during resistance exercise to further augment the skeletal muscle adaptive response to exercise training (32). Whether this is also relevant to athletes participating in more continuous endurance exercise activities remains to be established. In addition to carbohydrate and protein provided during exercise, participants ingested another bolus of test drink 30 and 90 min after the cessation of exercise. To evaluate the impact of carbohydrate and protein hydrolysate supplementation on whole-body protein turnover during the early stages of postexercise recovery, we assessed whole-body protein turnover rates by measuring plasma phenylalanine kinetics. Carbohydrate and protein ingestion increased whole-body protein synthesis by 40% during the first 3 h of postexercise recovery compared with the control treatment (47.8 ± 2.4 vs. 34.1 ± 2.0 µmol phenylalanine·kg^–1·h^–1, respectively; P < 0.05). These findings are consistent with previous studies that investigated the impact of carbohydrate and protein ingestion during acute postexercise recovery (5,13,15). However, we extend on these previous findings as we also assessed the impact of carbohydrate and protein supplementation on muscle protein synthesis during subsequent overnight recovery. Whole-body protein synthesis rates during the 9-h recovery period were 19% higher in the C+P group than in the W group. However, muscle protein synthesis rates did not differ between groups during this period. These results show that carbohydrate and protein hydrolysate supplementation during and immediately after exercise do not seem to further enhance muscle protein synthesis over the subsequent overnight recovery period. Whole-body protein turnover does not necessarily reflect protein synthesis on a skeletal muscle tissue level. As a consequence, the greater whole-body protein synthesis rates observed during overnight recovery might be attributed to tissues other than skeletal muscle. It could be speculated that gut proteolysis during exercise (33) is compensated for by greater protein synthesis in the gut during subsequent overnight recovery. Furthermore, it has been suggested that brain tissue contributes largely to overall protein synthesis rates during the night (16,34). Research on the diurnal variation of protein synthesis rates in different tissues is warranted.

Only 1 previous study has measured overnight muscle protein synthesis rates in vivo in humans. Tipton et al. (17) studied the combined effect of resistance exercise and amino acid supplementation muscle protein synthesis and protein balance over a 24 period. They reported a (nonsignificant) 29% greater muscle protein synthesis rate during the night following exercise (performed in the morning) when compared with the nonexercised condition. Our findings extend on Tipton et al. (17), and show that protein and carbohydrate supplementation during and immediately after exercise does not further stimulate muscle protein synthesis during subsequent overnight recovery when performed in a fed state.

For obvious methodological limitations, we were unable to assess muscle protein FSR during different stages of overnight recovery. The fact that the mean muscle protein synthesis rate assessed over the entire 9-h overnight recovery period did not differ between groups might be attributed to the inability of the dietary regimen to elevate overnight plasma amino acid availability and/or increase circulating plasma insulin concentrations. Increased amino acid availability (8,35–38) can elevate muscle protein synthesis but only under conditions where circulating insulin concentrations are higher than 69.5 pmol/L (35,37,39). In the present study, plasma insulin concentrations were 55.4 ± 8 pmol/L in the C+P group and 48.5 ± 3 pmol/L in the W group during the last 6 h of overnight recovery. Increasing plasma insulin and/or amino acid availability during the night, either by i.v. infusion of amino acids and/or the ingestion of more slowly digestible protein sources may represent interesting strategies to augment muscle protein accretion during the night. Studies addressing potential diurnal variation in muscle protein synthesis rates are needed to define more effective dietary strategies that can stimulate muscle protein accretion in both sports and clinical settings.

In conclusion, the combined ingestion of carbohydrate and protein during and immediately after exercise stimulates muscle protein synthesis during exercise conditions but does not further augment net muscle protein accretion during subsequent overnight recovery.

	ACKNOWLEDGMENTS

 
We thank Joan Senden and Jos Stegen for expert technical assistance.

	FOOTNOTES

 
¹ Supported by DSM Food Specialties, Delft, The Netherlands

² Author disclosures: M. Beelen, M. Tieland, A. Gijsen, H. Vandereyt, A. Kies, H. Kuipers, W. Saris, R. Koopman, and L. van Loon, no conflicts of interest.

³ A supplemental description of amino acid tracer calculations is available with the online posting of this article at jn.nutrition.org.

⁸ Abbreviations used: AUC, area under the curve; C+P, carbohydrate and protein group; EN%, energy percent; FSR, fractional synthetic rate; Ra, rate of appearance; Rd, rate of disappearance; 1RM, one repetition maximum; W, water group; W_max, maximal workload capacity.

Manuscript received 14 May 2008. Initial review completed 5 June 2008. Revision accepted 24 August 2008.

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