The anabolic potential of dietary protein intake on skeletal muscle is prolonged by prior light-load exercise

Clinical Nutrition 32 (2013) 236e244

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Original article

The anabolic potential of dietary protein intake on skeletal muscle is prolonged by prior light-load exercise Rasmus Bechshoeft a, Kasper J. Dideriksen a, Søren Reitelseder a, Thomas Scheike b, Michael Kjaer a, Lars Holm a, * a

Institute of Sports Medicine, Dept. of Orthopedic Surgery M81, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Dept. of Biostatistics, Institute of Public Health, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

b

a r t i c l e i n f o

s u m m a r y

Article history: Received 25 April 2012 Accepted 23 June 2012

Background & aims: Hyperaminoacidemia stimulates myofibrillar fractional synthesis rate (myoFSR) transiently in resting skeletal muscle. We investigated whether light-load resistance exercise can extent this responsiveness. Methods: Ten healthy males exercised one leg with a light-load resistance-like exercise at 16% of 1 repetition maximum and received oral protein boluses every hour for a 10-h period. Their myoFSR was determined by [1-13C]-leucine incorporation. Muscle biopsies were obtained from the resting (REST) and exercised (EXC) muscles every 2.5-h in the protein-fed period. Results: Protein feeding significantly elevated plasma leucine and essential amino acids by an average of 39
9% (mean
SEM) and 20
4%, respectively, compared to the basal concentrations: 197
12 mmol L 1 and 854
35 mmol L1, respectively. The myoFSR was similar in EXC and REST muscles in the first 8 h (all time intervals p > 0.05). After 8 h the myoFSR dropped in the REST muscle to 0.041
0.005%$h1, which was 65
5% of the rate in EXC leg at the same time point (0.062
0.004%$h1) and 80
14% of the level in REST leg from 0.5 to 8 h (0.056
0.005%$h1) (interaction p < 0.05). Conclusions: Compared to rest, light-load exercise prolonged the stimulatory effect of dietary protein on muscle biosynthesis providing perspectives for a muscle restorative effect in clinical settings where strenuous activity is intolerable. Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Caseinate Muscle protein synthesis rate Protein feeding

1. Introduction Maintenance of the muscle protein synthesis rate is markedly dependent on amino acid availability and abundance. Reductions in circulating amino acid concentrations by hemodialysis decrease muscle protein synthesis rate1 and nutritional energy provision without elevations in amino acid availability fails to improve the synthesis rate.2 Correspondingly, it has been shown that the circulating abundance of amino acids is decisive for regulating the muscle protein synthesis rate.3 Hence, the availability of amino acids stimulates the biosynthetic processes in skeletal muscle in a doseAbbreviations: BMI, body mass index; EAA, essential amino acids; FSR, fractional synthesis rate; KIC, ketoisocaproic acid; LBM, lean body mass; NEAA, non-essential amino acids; NIDDM, non-insulin dependent diabetes mellitus; RM, repetition maximum. * Corresponding author. Institute of Sports Medicine, Building 8, 1st floor, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Tel.: þ45 3531 6662; fax: þ45 3531 2733. E-mail address: [email protected] (L. Holm).

dependent manner,3,4 with the potency explored at low (2.5 g) intakes and peaking at approximately 10 g essential amino acids ingested in one bolus.4 Amino acids transiently become excessively available after ingestion of a protein bolus and the temporal abundance is closely followed by a transient improvement in muscle protein synthesis rate.5,6 Not even repeated whole-nutrient feeding7 or prolonged continuous amino acid infusion8 can maintain the resting muscle protein synthetic apparatus responsive to amino acids for a longer period. Such transient and self-limiting stimulatory effect of amino acids on the skeletal muscle protein synthesis regulation is very likely the reason why muscle deterioration during periods of bed rest cannot fully be counteracted by daily extra intake of protein9 or highly potent amino acid supplementations.10,11 In contrary, a single bout of heavy-loading muscle activity exerts a prolonged stimulatory effect on the muscle protein synthesis rate lasting up to 48 h.12 Similarly it has been shown that as long as 24 h after heavy contractile activity, the sensitivity towards hyperaminoacidemia following protein intake is markedly enhanced.13

0261-5614/$ e see front matter Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. http://dx.doi.org/10.1016/j.clnu.2012.06.015

R. Bechshoeft et al. / Clinical Nutrition 32 (2013) 236e244

We have shown that 12 weeks of very light load resistance exercise followed by intake of a protein-containing nutrient supplement resulted in a 3% hypertrophy of the quadriceps muscle in young males.14 However, we were unable to demonstrate that a single bout of exercise at the same low intensity could increase the myofibrillar protein synthesis rate, neither in the overnight fasted nor in the fed condition.15 Interestingly, we showed that during repeated whole-nutrient feeding, completion of light load resistance exercise after 4 h of rest maintained the muscle protein synthesis rate elevated for additional 5.5 h.15 Since the purpose was related to contraction intensity in that study, we had no resting control-muscle to directly verify this potential. As the anabolic potential of the light contractile activity per se was negligible, we speculated that the hypertrophy was accomplished by making the tissue more sensitive to the potent amino acids. The present study was purposely designed to investigate the potential of lightload contractions to make the muscle more responsive to hyperaminoacidemia. We therefore hypothesized that completion of light-load resistive exercise would improve the sensitivity of the muscle tissue towards a sub-maximal abundance of amino acids. The measure of the enhanced sensitivity and hence the primary outcome, the amount of incorporated tracer into myofibrillar proteins as well as their fractional synthesis rate in the exercised muscle is compared to that in the resting muscle. 2. Materials and methods 2.1. Subjects Through a web advertisement we recruited ten sedentary male subjects (mean age 22.7
3.1 y (mean
SD)) who appeared healthy, determined by interviews and evaluation of blood levels of standard health parameters. Following exclusion criteria were used: BMI>28, parents/siblings with NIDDM, smoking, alcohol consumption more than 21 units per week (1 unit equals 12 g of alcohol), daily or frequent intake of medication, and regular (>1 per week) participation in strenuous sport. Subjects were individually informed about the trial protocol and their rights as voluntary subjects, in accordance with the Declaration of Helsinki II, before giving their informed written consent. The study was approved by the local ethical committee of the Capital Region of Denmark, journal H-42010-008. Subject characteristics are presented in Table 1. 2.2. Pretesting and standardisation At least 7 days prior the experimental day, subjects met in at the Institute of Sports Medicine where they had their lean body mass (LBM) determined by a DEXA scan (Lunar Corp. Model DPX-IQ, Madison, WI, USA) at medium speed level (24 mSv). Subsequently subjects were familiarised to the knee-extension device (Technogym, Superexecutive Line, Gambottola, Italy), which was individually adjusted. The knee extension range of motion was set to 20 e100 (0 equals full leg extension). After a brief warm up on a cycle ergometer and on the knee-extension device at low loads, Table 1 Subject characteristics. Parameter

Mean
SD

Included males: Age (yrs): BMI (kg/m2) LBM (kg) 1RM (kg) Exercise load (% of 1RM)

10 22.7 22.3 59.0 77 16







3.1 1.5 6.0 8

237

the 1 repetition maximum (1RM) was determined on one leg, randomly chosen between dominant and non-dominant legs, as the exercise leg. Results of pre-testing and calculated exercise loads are shown in Table 1. Based on a general food-consumption interview, a meal-plan was made to support subjects in adhering to their regular and sufficiently nourished diet for the last three days prior the trial ensuring adequate energy and protein intake. Subjects were asked not to consume alcohol and caffeine-containing foods for seven days and one day, respectively prior to the experiment and to refrain from exercise for the last three days before the experimental day. Restrictions were monitored by food and activity diaries and indicated acceptable compliance for the included subjects. 2.3. Experimental protocol On the morning of the trial day, the overnight fasted subjects arrived by taxi at the Institute of Sports Medicine, Bispebjerg Hospital where they were placed supine and remained rested. A catheter was inserted into an antecubital vein of each forearm; one used for tracer infusion and one used for collection of blood samples throughout the study. The trial design and sampling protocol is shown in Fig. 1. After obtaining a background blood sample, the L-[1-13C]-leucine tracer (sterile and pyrogen free, 99% enriched; Cambridge Isotopes Laboratories, Andover, MA, USA), which was sterilely prepared; weighted, mixed in sterile saline, and filtered through 0.2 mm sterile disposable filter (Sartorius, Hannover, Germany), was administered as a primed (13 mmol kg LBM1), continuous (13 mmol kg LBM1 h1) infusion aiming at a venous plasma enrichment of around 10%. Shortly after, the subjects were transported to the exercise equipment and the one-legged light load knee extension exercise consisting of 10 sets (3 min each, separated by a 30 s break) of 36 unilateral leg extensions at 16% of 1RM was completed in 35 min (EXC). The contra-lateral leg remained rested (REST). Subjects remained seated in the device between sets and the whole exercise intervention was guided and assisted by instructors. Hereafter the subjects returned to supine rest for the remaining time (10.5 h). Blood samples were obtained at time points 65, 0, 30, 60, 120, 180, 240, 300, 360, 480, and 600 min (with time zero being immediately after completion of exercise e Fig. 1) for determination of plasma glucose-, insulin-, and amino acid concentrations as well as 13C-leucine- and 13C-ketoisocaproic acid (KIC) enrichments. Bilateral muscle biopsies were obtained at time points 30, 180, 330, 480 and 630 min providing four postprandial (REST vs. EXC) intervals for fractional synthesis rate (FSR) measures; 30-180, 180-330, 330-480, and 480e630 min. 2.4. Protein ingestion Protein drinks were administered repeatedly throughout the post exercise period (for specific contents in drinks see Table 2) and consisted of whey and calcium caseinate protein (for amino acid contents of the two milk protein fractions, as reported by the manufacturer, see Table 3). The milk proteins were intrinsically labelled with 10.0% [1-13C]-leucine meaning that we avoided fluctuations in the tracer steady state enrichments, which else wise likely would have been the case if standard unlabelled proteins were ingested and unlabelled leucine absorbed. Further, the intrinsically labelled proteins allowed us to maintain the tracer enrichment as high as possible and there by maximise the incorporation of tracer into muscle proteins, hence, improving the analytical sensitivity. The labelled protein was produced in collaboration with Danish Dairy Board, Danish Dairy Research Foundation and Arla Foods (all Sønderhøj, Viby J, Denmark), which is explained

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Fig. 1. Trial design. All subjects had a primed, continuous infusion of tracer (1-13C-leucine) started at 6 am. Shortly after they conducted the one-legged knee extensor exercise session (10 sets of 36 reps at 16% 1RM) while the other leg remained in the resting condition. Hereafter, at time zero (0), a whey protein bolus was ingested and at 30 min and 60 min a calcium caseinate protein supplement was administered as half-boluses. Every hour from 120 min, whole boluses of caseinate were given until 600 min. The total amount of protein ingested corresponded to 0.1 g kg LBM1 h1. Muscle biopsies were obtained bilaterally at 30, 180, 330, 480, and 630 min and blood samples were obtained frequently throughout the trial. \: Muscle biopsy, B: Whey drink, ◖: ½ dose of calcium caseinate drink, C: whole bolus of calcium caseinate drink, [: Blood sample.

in detail elsewhere.16 The total administered protein amount corresponded to 0.1 g protein$kg LBM1 h1 and the absolute amounts are reported in Table 2. With the purpose quickly to obtain an elevated concentration of circulating amino acid, we gave the first bolus as the more quickly digested and absorbed whey protein.17 Subsequently, calcium caseinate protein was administered with the purpose to keep a continuous release of amino acids to the circulation and there by maintain a constant elevated amino acid concentration in the blood. To initiate the digestion of the caseinate protein early after the whey intake, we provided half doses at 30 and 60 min. Hereafter, full doses of caseinate was ingested each hour, the last at 10 h (outlined in Fig. 1). 2.5. Plasma and blood samples and analysing procedures Venous blood samples were drawn into 9 mL K3EDTAvaccuettes (Greiner-Bio one, Kremsmünster, Austria), cooled for 10 min, centrifuged (3060g at 4  C for 10 min), and the plasma phase was stored at 80  C until analysis of insulin- and amino acid concentrations as well as ketoisocaproic acid and leucine 13Cenrichments. Blood glucose concentrations were measured immediately from venous whole blood droplets on Accu-Chek Inform II System (F. Hoffmann-La Roche AG, Basel, Switzerland). Plasma insulin concentration was measured using a standard insulin ELISA kit, Code K6219 (Dako Denmark A/S, Glostrup, Denmark). The plasma amino acid concentration was measured using High-Performance Liquid Chromatography (HPLC) coupled with a UV detector [HPLC: SpectraSystem P4000, Thermo Separation Products (FinniganMat, Paris, France); column: Nova-Pak C18 60Å 4 mm, 3.9  300 mm (Waters, Milford, MA, USA); UV detector: Table 2 Contents of administered protein. Protein

Total (g)

Whey, one bolus and total Caseinate, one bolus Caseinate, total Protein, total

6.0 5.9 59.0 64.9







0.2 0.2 1.9 2.1

Leucine (g) 0.9 0.6 6.2 7.1







0.03 0.02 0.2 0.2

EAA (g) 3.5 2.9 28.5 32.0







0.1 0.1 0.9 1.0

UV6000LP PDA FINN 50 mm Cell UV photo array detector, Thermo Separation Products (FinniganMat)] using a buffer gradient program from 100% buffer A (0.05 mol L1 ammonium acetate) to 100% buffer B (0.1 mol L1 ammonium acetate in 44:10:46 acetonitrile-methanol-water). Both buffer A and B were adjusted to pH 6.8 with acetic acid and filtered and degassed before use. To 100 mL plasma sample 100 mL internal standard (0.5 mmol L1 norleucine) was added into 1.5 mL Nanosep tubes (Pall Corporation, Port Washington, NY, USA). Samples were centrifuged at 20820 g for 45 min, after which 100 mL of the filtrate was transferred to glass tubes. The solvent was evaporated under a stream of N2 and the pellet re-suspended in 20 mL coupling buffer (2:2:1 methanolwater-triethylamine) and vortexed. Again the solvent was evaporated using N2, and the amino acids were derivatised with phenylisothiocyanate (20 mL of 1:1:1:7 triethylamine-waterphenylisothiocyanate-methanol) and vortexed, and after 30 min at room temperature the solvent was again evaporated using N2.

Table 3 Amino acid contents of milk protein fractions. Protein

Alanine Arginine Asparagine Cysteine Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Total

Whey

Caseinate

4.66 2.65 11.25 2.65 16.58 1.78 2.25 5.72 11.77 9.68 2.10 3.54 4.62 4.78 5.05 2.04 3.59 5.29 100

2.84 3.31 6.71 0.35 20.61 1.73 2.87 5.03 8.77 7.44 2.71 4.80 10.10 5.73 3.97 1.16 5.38 6.46 100

Single amino acids in grams per 100 g protein, dry matter.

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239

The derivatised amino acids were finally reconstituted in 200 mL buffer A and transferred to analysis vials. To determine the plasma 13C-ketoisocaproic acid (KIC) and 13Cleucine enrichments the samples were prepared prior to analysis with mass spectrometry. For KIC analysis, protein from 140 mL plasma was precipitated with 1 mL ethanol (99.8%). The supernatant was dried under N2 at 50  C. With 200 mL acidified 2% w/v ophenylenediamine in 200 mL Millipore water the substrate was prepared for the subsequent derivatisation using pyridine as a solvent and BSTFA þ 1% TMCS (#38831, Pierce, Bie & Berntsen & VWR International, Rødovre, Denmark) as the derivatising agent. For 13C-leucine enrichment analysis, 200 mL plasma was added 1 mL of 50% acetic acid, vortexed, and poured over an acetified hydrogenform cation exchanger (Dowex AG 50W-X8 resin 100e200 mesh; Bio-Rad, Copenhagen, Denmark) collected in disposable Slow Flow open tip pin-holed columns (Image Molding, Denver, CO, USA). The amino acids were eluted with 2 mL 2 mol L1 NH4OH, which was evaporated under a stream of N2, azeotroped with 0.5 mL methylene chloride and derivatised using MTBSTFA þ 1% TBDMCS (Regis Technologies, Morton Grove, Il, USA) and acetonitrile as solvent. The KIC and leucine 13C-enrichments were determined as the tracer-to-tracee ratio ([Mþ1]/M ratio) with m/z being M ¼ 232 and M ¼ 302 for KIC and leucine, respectively, with the purpose to evaluate the precursor enrichments for protein synthesis.18 One mL sample was injected in the PTV (programmed-temperature vaporisation) injection mode and carried by a constant helium flow (1.8 mL,min1) into the gas chromatograph (GC, Trace GC 2000 series, Thermo Quest Finnigan, Paris, France) and separated by a capillary column (CP-Sil 8 CB low bleed 30 m  0.32 mm, coating 0.25 mm, ChromPack, Varian, Palo Alto, CA). The analytes were fragmented by electron ionization (EI) and the mass spectrometer (MS, Automass Multi, Thermo Quest Finnigan, Paris, France) was operated in the SIM mode.

gently for 6 h and left overnight at 4  C. The day after, the samples were centrifuged (515 g, 20 min, 4  C). The supernatant containing the myofibrillar proteins was added 4.6 mL icecold ethanol (99%), vortexed, left for 2 h at 4  C, and hereafter centrifuged (515 g, 20 min, 4  C) and the supernatant discarded. The pellet was hydrolysed at 110  C in 6 mol L1 HCl for 18 h. The constituent amino acids were purified over Dowex resin columns using 2 mol L1 NH4OH for elution. Hereafter the amino acids were derivatised as the N-acetyl-propyl (NAP) derivative. Briefly, the dried amino acids were mixed with 200 mL propyl acetate and 100 mL BF3:propanol (14%) followed by heating at 110  C for 30 min and thereafter cooled down. The solvent was evaporated under N2 at 70  C. Another 100 mL BF3:propanol (14%) was added and then evaporated by N2. To the dried pellet, 50 mL of acetonitril and 26 mL 1,4-dioxan were added and the solution mixed and subsequently 38 mL triethylamine was added and mixed, and 24 mL acetic anhydride added and mixed. Samples were then heated to 55  C for 15 min, cooled, and added 50 mL chloroform. Further 3  50 mL of 1 mmol L1 NaHCO3 was added and the samples were allowed to settle. The water-phase was carefully removed and the sample dehydrated overnight using a Molecular Sieve UOP Type 3A, pore diam 3 Å (Fluka, Switzerland) in the tube. The myofibrillar protein bound 13C-leucine abundance was determined on a GCcombustion-isotope ratio MS (Delta Plus XL 6890, Thermo Finnigan, Bremen, Germany) by converting the obtained d (delta) value (sample ratio against a reference CO2 gas internal standard ratio in &) to an actual enrichment by referring the reference gas to the PDB limestone standard (13C-enrichment: 1.12372%). Prior to combustion, the leucine was isolated in the GC on a capillary column (CP-Sil 19 CB 60 m  0.25 mm, coating 1.5 mm, ChromPack, Varian, Palo Alto, CA, USA).

2.6. Muscle biopsy sampling and analysis procedure

The myofibrillar protein 13C-leucine enrichment found by GC-CIRMS were used to calculate the myofibrillar fractional synthesis rate (myoFSR) reported in percent per hour using the standard 1 precursor-product model: FSR ¼ DEproduct,E1 precursor,Dtime ,atom dilution factor,100%, where DEproduct is the change in tracer enrichment in two tissue samples taken with a time interval of Dtime hours during which period the precursor pool enrichment was constant and equal to Eprecursor. We used the muscle free 13Cleucine enrichments as estimates for the precursor pool to calculate the myoFSR. To convert the actual 13C-enrichments to leucine atomic percent excess (ape) enrichments we multiplied with an atom dilution factor of 11 (given by the ratio between the number of unlabelled carbons divided by number of labelled carbons in the NAP-derivatised leucine).

Five muscle biopsies from each vastus lateralis muscle were obtained through separate incisions after prior local anesthetisation (1% Lidocaine) and disinfection (0.5% chlorhexidine) using a 5 mm Bergström needle with suction. Two muscle specimens were freed from visible fat and connective tissue, washed and wiped clean from blood in ice-cold saline, weighted, and stored at 80  C until further analysis. One piece of muscle (20e30 mg wet weight) was homogenised (FastPrep 120A-230; Thermo Savant, Holbrook, NY, USA) for 4  15 s in 1.0 mL 2% perchloric acid (PCA), centrifuged at 3060 g for 20 min at 4  C, and the supernatant saved. Two times more, the pellet was added 1.0 mL 2% PCA, homogenised once, spun, and the supernatants saved. Hereafter, the muscle free amino acids present in the pooled supernatants were purified over Dowex resin columns using 2 mol L1 NH4OH for elution. The amino acids were derivatised as the TBDMS-derivatives and intramuscular free leucine 13C-enrichment was determined on GCMS as described for plasma leucine above. To isolate the myofibrillar proteins, separate muscle specimens of 20e40 mg wet weight were homogenised (FastPrep 120A-230; Thermo Savant, Holbrook, NY, USA) for 4  15 s in 1.0 mL buffer (NaCl 0.15 mol L1, Triton X-100 0.05%, EDTA 2 mmol L1, Tris 0.02 mol L1, pH 7.4), left overnight at 4  C, and centrifuged at 130 g for 20 min at 4  C. The pellet containing mainly structural proteins was homogenised once in 1 mL buffer (sucrose 0.25 mol L1, NaCl 0.15 mol L1, Triton X-100 0.5%, EDTA 2 mmol L1, Tris 0.02 mol L1, pH 7.4), allowed to settle for 1 h, and centrifuged 130 g for 20 min at 4  C. The supernatant was discarded and the pellet added 1.8 mL buffer (KCl 0.7 mol L1 and pyrophosphate 0.1 mol L1), shaken

2.7. Calculations

2.8. Statistical analysis Plasma insulin, glucose and amino acid concentrations as well as the KIC/Leucine 13C-enrichment ratio was evaluated using a one way ANOVA. In case of a significant factorial outcome, Holm-Sidak post hoc tests were used to locate specific temporal differences. Paired t-tests were used to compare the weighted mean concentrations over time with the basal level for glucose, insulin, and amino acids. Two-way ANOVA with repeated measures were used to evaluate venous plasma a-13C-KIC and 13C-leucine enrichments as well as muscle free 13C-leucine enrichments, and myofibrillar protein derived leucine 13C-abundances. In case of significant factorial outcomes, Holm-Sidak post hoc tests were made to locate specific differences. The Sigma Plot 12 (Systat Software Inc., San Jose, CA, USA) software was used to perform the above-mentioned statistical tests.

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The FSR results were analysed using cluster corrected ANOVA to compare the rest and exercise interventions, but we got similar results with random effects models. We stress that the default repeated measures analyses under compound symmetry does not lead to correct conclusion, due to an incorrect assumption about the correlation structure. Post-hoc tests were based on multiple pairwise t-tests and the p-values were corrected by Bonferonicorrections. We compared the rest/exercise rate at the four timepoints, by paired t-tests. In addition, we also looked at the rest and exercise groups to see if there was a time-change within these groups, thus comparing the starting value with the subsequent rates, again by paired t-tests (3 tests within each group). We got similar results by the non-parametric Wilcoxon rank test in all these post-hoc tests. These statistical analyses were performed in the R software (R Development Core Team (2012), Vienna, Austria. ISBN 3-900051-07-0 (URL http://www.R-project.org/), R 2.15-0) and in SAS (Copyright (c) 2002e2008 by SAS Institute Inc., Cary, NC, USA. NOTE: SAS (r) Proprietary Software 9.2 (TS2M3)). Unless otherwise stated, all data are expressed as means
SEM and significance level was set at p < 0.05. 3. Results 3.1. Protein and amino acid ingestion The contents of the whey and calcium caseinate protein boluses and the total doses are shown in Table 2. We based the total protein dose on the lean body mass of the subjects, providing a total of 0.1 g protein$kg LBM1 h1. In the whey protein bolus, the subjects got a total of 6.0
0.2 g protein of which 3.5
0.1 g was essential amino acids (EAA) including 0.9
0.03 g leucine (Table 2). With in the first hour after start of protein intake, additional 5.9
0.2 g of protein, 2.9
0.1 g of EAA, and 0.6
0.02 g leucine in the form of calcium caseinate was ingested. In total over the 10 h, subjects consumed 64.9
2.1 g protein, 32.0
1.0 g EAA, and 7.1
0.2 g leucine. 3.2. Plasma glucose and insulin concentrations Plasma glucose concentration is shown in Fig. 2A. Due to technical problems with the Accu-Chek Inform II System on a trial-day, glucose measures only exist for 8 subjects. A one-way ANOVA on

glucose-values showed an effect of time shown as a slightly lowered level compared to basal from 240 min and throughout the study (p < 0.05). When basal fasting values were compared to a weighted mean of glucose concentrations from 0 to 600 min with a paired t-test (Fig. 2A), no difference was found (5.1
0.2 vs. 4.9
0.2 mmol L1, p > 0.05). Plasma insulin concentration is shown in Fig. 2B. The concentration varied with time (one-way ANOVA, p < 0.001), presumably due to a peak-elevation of 57
31% (p < 0.05) above baseline at 30 min. The plasma insulin concentration returned to values not statistically different from basal fasting level from 120 min and throughout the study. When basal fasting insulin concentrations were compared to a weighted mean from 0 to 600 min (Fig. 2B) no difference was found (33.8
3.7 vs. 34.4
4.7 pmol L1, p > 0.05). 3.3. Plasma amino acid concentrations Venous plasma leucine and essential amino acids (EAA) concentrations during the trial are shown in Fig. 3A and AB. An effect of time appeared for leucine and EAA concentrations, which rose at 30 min by 43
8 and 22
4% (both p < 0.05), respectively, compared to basal fasting values. Leucine concentrations were maintained significantly elevated throughout the feeding period and the EAA were elevated at 30 and 60 min and from 180 min. Averaging the concentration measures from 30 min and throughout the trial and comparing that weighted average with basal fasting concentration with a paired t-test revealed an average elevation in concentrations of leucine and EAA by 39
9 and 20
4% (both p < 0.05), respectively (Fig. 3). 3.4. Precursor and product enrichments Venous plasma leucine and KIC 13C-enrichments were compared by a two-way ANOVA with repeated measures, revealing a higher 13C-enrichment in leucine than in KIC (Fig. 4A, left y-axis, interaction: p < 0.001). Leucine 13C-enrichments were steady from 60 to 600 min, except from a small peak at 360 min. KIC 13 C-enrichments were steady from 240 to 600 min with a slow and steady rise in the prior time points. The ratio of KIC/Leucine 13 C-enrichments was calculated at each time point (Fig. 4A, circles and right y-axis) revealing a drop at 60 min compared to 30 min and the 480 and 600 min time points (p < 0.05), and the ratio at the

Fig. 2. Plasma glucose and insulin concentrations. A: Glucose concentrations at individual time points throughout the trial and in the upper right hand corner the weighted mean of post-exercise glucose concentration compared to the basal, overnight fasted level. All values are means
SEM, N ¼ 8. Time effect: p < 0.05, *: p < 0.05 vs. basal value. B: Insulin concentrations at individual time points throughout the trial and in the upper right hand corner the weighted mean of post-exercise insulin concentration compared to the basal, overnight fasted level. All values are means
SEM, N ¼ 10. Time effect: p < 0.05, *: p < 0.05 vs. basal value.

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241

Fig. 3. Plasma leucine and essential amino acids concentrations. A: Leucine concentrations at individual time points throughout the trial and in the upper right hand corner the weighted mean of post-exercise leucine concentration compared to the basal, overnight fasted level. All values are means
SEM, N ¼ 10. Time effect: p < 0.001, *: p < 0.05 and y: p ¼ 0.001 vs. basal value. B: Essential amino acids concentrations at individual time points throughout the trial and in the upper right hand corner the weighted mean of postexercise total essential amino acid (EAA) concentration compared to the basal, overnight fasted level. All values are means
SEM, N ¼ 10. Time effect: p < 0.001, *: p < 0.05 and y: p ¼ 0.001 vs. resting value.

latter time point was higher than 120 and 240 min as well (p < 0.05). The muscle free 13C-leucine enrichments were also measured demonstrating a steady level from 30 min through 630 min with an average value of 9.00
0.20% (Fig. 4B, right y-axis). The myofibrillar proteins from REST and EXC muscles started out with an even enrichment at 30 min e shortly after end of exercise in one leg and start of protein ingestion (see Figs. 1 and 4B). Hereafter, the myofibrillar protein-bound leucine derived 13Cabundances increased in subsequent biopsies in both the resting and exercised muscle, but the exercised muscle got more tracer incorporated than the resting muscle from the 180 min time point and succeeding (two way ANOVA with repeated measures, interaction: p < 0.001, Fig. 4B, left y-axis) despite even precursor (muscle free 13C-leucine) enrichments. 3.5. Myofibrillar fractional synthesis rate The myoFSR revealed no difference between REST and EXC muscles up to 480 min, with a grand mean of 0.057
0.003%$h1

(from 30 to 480 min for pooled REST and EXC muscles). In the last time-period (480e630 min) the resting myofibrillar FSR dropped to 0.041
0.005%$h1 but was maintained in the previously exercised muscle 0.062
0.004%$h1 (Fig. 5, interaction p < 0.05). 4. Discussion In the present study we verified our hypothesis, namely that completion of light-load resistance-like exercise compared to resting state prolonged the responsiveness of muscle contractile protein synthesis rate to sub-maximal elevations in circulating amino acids obtained by repeated dietary protein intake. It was recently demonstrated that as late as 24 h after completion of heavy-load resistance exercise or low intensity resistance exercise performed till exhaustion the muscle protein synthesis rate after protein intake was higher than in a non-exercised muscle.13,19 No comparable data exist on more brief, light-loading exercise, but conduction of endurance exercise had no effect on 24-h whole body protein turnover.20,21 Thus, the herein demonstration of a prolonged (as long as 10 h) responsiveness to feeding

Fig. 4. Plasma surrogate precursor and product enrichments. A: Venous plasma leucine (:) and KIC (;) 13C-enrichments (N ¼ 10). A two-way ANOVA comparison with repeated measures revealed an interaction (p < 0.001). For leucine and KIC 13C-enrichments letters a, b, and c denote different from 30, 60, and 120 min, respectively within the same precursor. Further, there was significant difference between venous plasma leucine and KIC 13C-enrichments at all time points (p < 0.001, not shown on figure). The plasma KIC/ Leucine 13C-enrichment ratio was calculated at each time point (circles at right y-axis) and the ratio fluctuated with time (p < 0.001). For the KIC/Leucine 13C-enrichment-ratio, letters b, c, and d denote different from 60, 120, and 240 min, respectively. All values are means
SEM. B: Left y-axis: Myofibrillar protein-bound leucine-derived 13C-abundance in atomic excess. : represents means of EXC and ; means of REST (N ¼ 10). A two-way ANOVA with repeated measures revealed interaction: p < 0.001. A significant increase in enrichment was seen over time irrespective of intervention and the enrichment was significantly higher in the EXC leg compared to the REST leg at 180, 330, 480, and 630 min (denoted: #: p < 0.05, ##: p < 0.01, and ###: p < 0.001). Right y-axis: Muscle free 13C-leucine tracer-to-tracee ratio enrichment, calculated as: Muscle sample 13C-leucine abundance subtracted the plasma free background 13C-leucine abundance. 6 represents means of EXC and 7 means of REST (N ¼ 10). A two-way ANOVA with repeated measures revealed no significant differences in tracer enrichments. Values are means and error bars SEM.

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Fig. 5. Myofibrillar protein fractional synthesis rate. In four time intervals of 150 min, the contractile protein FSR was calculated using the muscle free 13C-leucine enrichment as precursor pool. Using the cluster corrected ANOVA with repeated measures a significant interaction (p < 0.05) appeared. The post hoc test found that the FSR in the resting muscle at 480e630 min dropped and was lower than the EXC at the same time point and than REST at the first (80e180 min) time point (both p < 0.05), both significant differences are denoted by #. All values are means
SEM, N ¼ 10.

with dietary protein of the myofibrillar protein synthesis rate after completion of the light-load exercise supports a mechanism by which this type of exercise may exert an anabolic potential.14 4.1. Resting muscle fractional synthesis rate (FSR) in response to hyperaminoacidemia When amino acids are abundant in the circulation, the muscle protein synthesis rate is immediately elevated over the level in the fasting state.22e24 This response is ubiquitous in various functionally different protein pools in the skeletal muscle.6,8 When providing the protein as a single bolus, the increment in muscle protein FSR fades out along with the drop in circulating amino acids to basal concentrations.5,6 This pattern, which probably is coordinated via a direct stimulus of the amino acids on the myocyte signalling,24e27 are interrupted by prior completion of heavy-load resistance exercise6,28 as the synthesis rate are maintained elevated beyond the peak in amino acid concentrations. In the resting muscle, the myofibrillar protein synthesis rate is only temporarily elevated when hyperaminoacidemia is obtained via constant intra-venous infusion of amino acids.8 Similarly in other animal species, which, like humans, also have an intermittent eating behaviour, the stimulatory effect of protein meals also disappeared despite excessive abundance of amino acids.29 In the present experimental setting, we verified that this self-limiting synthetic response in resting skeletal muscle also exists during oral protein feeding. 4.2. Muscle-full concept and sensitivity to amino acids In our set up, the resting muscle FSR remained unchanged for the first 480 min (8 h) of excess amino acid delivery (Fig. 5), where after it dropped to a rate of 80
14% of the 8-h grand mean value. With in the first hour of the present study, we provided the subjects with a total of 12 g protein (containing 6.3 g EAA of which 1.5 g was leucine). Although some delay in the appearance of protein-derived amino acids into the circulation is obvious, comparable quantities of amino acids have earlier been shown adequate to significantly stimulate the muscle protein synthetic

processes.30,31 From studies with intravenous amino acid infusion the doseeresponse relationship between hyperaminoacidemia and muscle protein synthesis rate is well-described.3,4 Intake as low as 2.5 g of EAA stimulate myofibrillar FSR in the resting young muscle and the response reaches a maximal effect after intake of 10 g EAA,4 or 10e20 g whole protein.32 Thus, the size of the stimulatory effect determined in the present study confers with that seen after the small protein bolus intake. Throughout the entire trial subjects were provided with an average of 65 g protein, corresponding to a sufficient daily intake (0.87
0.02 g kg body weight1). This amount contained 7.1 g or 54 mmol of leucine, which is approximately 100 times the abundance in the circulation (though without accounting for the intracellular pool). Despite not all ingested caseinate protein was digested and thus absorbed by the end of the experiment, the provided amount exceeds what is reflected by the increase in concentration (Fig. 3). By the use of intrinsically labelled proteins, Pennings et al. recently demonstrated, that the absorption rate from the gut exceeded what was reflected by the changes in plasma concentration,33 thereby demonstrating the quantitative discrepancy between absorbed amounts and changes in circulating amino acid levels. For leucine a higher rate of clearance from the circulation34 as well as an elevated turnover rate35 are probably the causes for that discrepancy, which is probably also the case for other amino acids when supplied. Similar inverse relationship between the amounts of available amino acids and plasma concentrations has also been demonstrated during periods with different dietary protein ingestion.36 The prolonged 8 h long elevated resting muscle protein synthesis rate seen in the present study, compared to the 2e3 h long stimulation in the iv-infusion study by Bohe et al., 2001,8 could partly be the result of the oral/ enteral feeding route, as this has been shown superior to stimulate whole body protein turnover when compared to parenteral provision.37 Alternatively and more likely, the discrepancy is also in concordance with the muscle-full concept,5 which comprises a quantitative limitation in the amino acid storage capacity in the resting skeletal muscle. Once the absolute capacity to extend the protein pool is reached, the sensitivity to increase the storage of amino acids (enhanced synthesis rate) shuts down and the muscle protein balance (probably) returns to net zero. In accordance, the storage rate (synthesis rate) of the muscle proteins is elevated with increased offer of amino acids (plasma concentration) in a dosedependent manner, until a maximal rate is reached.3,4,30 Therefore, the larger increase in circulating amino acids, the more will the synthesis rate increase, the faster the storage will be filled, and the sooner the synthetic processes will return to basal level. Being the link between amino acid abundance, muscle protein synthesis and storage capacity, this could explain why the w100% increase in plasma essential amino acid concentrations in the Bohe study stimulated the resting muscle FSR by 180% for 2e3 h8 while our 20
4% increased EAA plasma concentration increased the muscle FSR by w50% for 8 h. Being rather experimental in the protein/ amino acid administration protocols, these studies mainly demonstrate a nutritive-physiological phenomenon. In terms of its relevance during normal large bolus eating behaviour, the contractile work would be expected to prolong the nutrientinduced elevation in muscle protein anabolism beyond that seen in a resting muscle, demonstrating a pattern similar to that seen as a result of heavy resistance exercise by Moore et al.6 Irrespective of the mechanism, we showed that brief light-load resistive exercise is sufficient to overrule or recruit new intrinsic pathways that allow more amino acids to be incorporated into and stored as contractile proteins (Figs. 4B and 5). This ability, of the muscle to find new storage space for contractile proteins just by completion of brief contractile efforts, underlines the necessity of contractile work per se for maintenance of skeletal muscle and

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leaves hope for the light-load intensity to inhere muscle-restoring properties. 4.3. Protocol limitations The repeated caseinate protein feeding protocol was not designed to reflect a normal meal pattern and hence not as much to mimic an applied set up. Instead we aimed at providing a continuous absorption of amino acids from an orally provided protein source resulting in a prolonged elevated amino acid concentration in the circulation. This was accomplished, although the hyperaminoacidemia was not as marked as we had expected from the high amount of protein provided. However, with reference to the literature as described above, ingestion of protein doses as low as 5 g significantly stimulates and elevates the muscle protein synthesis rate,4 and thus we are confident that the protein feeding protocol stimulated the muscle protein synthetic rate above the basal level. This we support by the fact that we observed the decline in FSR in the resting muscle after 8 h (beyond 3 pm in the afternoon). To our knowledge no data have previously reported or even discussed the phenomenon that the basal muscle protein turnover rate would be likely to deteriorate during the day after an overnight fast. Therefore, we believe that the protein intake actually enhanced the muscle FSR and that it dropped to basal level in the resting leg after 8 h. However, admittedly it would have been nice if the basal, overnight fasted muscle FSR had been measured to verify the protein-stimulated increase, which we for ethical reasons refrained from and instead prioritised to extend the postprandial period. 4.4. Conclusion and perspectives The regulation of skeletal muscle contractile protein synthesis rate at rest demonstrated a self-limiting response to excess amino acid availability obtained by oral protein intake. Completion of non-exhaustive light-load contractions prior to protein intake prolonged the elevation in muscle protein synthesis rate in response to hyperaminoacidemia. Although it should be acknowledged that heavy load exercise is the most potent exercise-stimulus to increase muscle protein turnover13,15,28,38e40 and muscle accretion,14 the herein demonstrated phenomenon that resistance exercise-type light-load contractions is sufficient to improve the muscle sensitivity to protein feeding, may lend support for a muscle mass preserving effect. In perspective, the results could be relevant for hospitalized or partly immobilized patients, who could be hypothesised to attenuate muscle atrophy by completing brief light-load contractile activity combined with protein-supplemented diets. Further, the physiological phenomenon should be verified in elderly and applied in a clinical setting to investigate its relevance for population groups with special needs. Statement of authorships Each author has contributed to the following: RB and LH carried out the studies and data analyses and drafted the manuscript. LH, SR, and KJD carried out the samples analyses. RB, LH and TS participated in the design of the study and performed the statistical analysis. LH and MK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Conflict of interest No author had any conflict of interest.

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Acknowledgements Source of funding: Danish Medical Research Council, Lundbeck Foundation, Novo Foundation, and Nordea Foundation (Center for Healthy Aging, Health Faculty, University of Copenhagen). All subjects are thanked for their efforts and patience during the experimental protocol. Lab technician Ann-Marie Sedstroem is thanked for preparation of samples. Prof. Gerrit van Hall and engineer Flemming Jessen at Clinical Metabolomics Core Facility at Rigshospitalet, Copenhagen, Denmark are thanked for kindly access to their lab and mass spec equipment. Chief physicians Lene Roerdam and Jens Bülow are thanked for kindly access to the DEXA scanner. References 1. Kobayashi H, Borsheim E, Anthony TG, Traber DL, Badalamenti J, Kimball SR, et al. Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B. Am J Physiol Endocrinol Metab 2003;284:E488e98. 2. Bell JA, Fujita S, Volpi E, Cadenas JG, Rasmussen BB. Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases. Am J Physiol Endocrinol Metab 2005;289:E999e1006. 3. Bohe J, Low A, Wolfe RR, Rennie MJ. Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a doseresponse study. J Physiol 2003;552:315e24. 4. Cuthbertson DJ, Smith K, Babraj J, Leese G, Waddell T, Atherton P, et al. Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. FASEB J 2005;19:422e4. 5. Atherton PJ, Etheridge T, Watt PW, Wilkinson D, Selby A, Rankin D, et al. Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling. Am J Clin Nutr 2010;92:1080e8. 6. Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA, Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. J Physiol 2009;587:897e904. 7. McNurlan MA, Essen P, Milne E, vinnars e, Garlick PJ, Wernerman J. Temporal responses of protein synthesis in human skeletal muscle to feeding. Br J Nutr 1993;69:117e26. 8. Bohe J, Low JF, Wolfe RR, Rennie MJ. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J Physiol 2001;532:575e9. 9. Lemoine JK, Lee JD, Trappe TA. Impact of sex and chronic resistance training on human patellar tendon dry mass, collagen content, and collagen cross-linking. Am J Physiol Regul Integr Comp Physiol 2009;296:R119e24. 10. Ferrando AA, Paddon-Jones D, Hays NP, Kortebein P, Ronsen O, Williams RH, et al. EAA supplementation to increase nitrogen intake improves muscle function during bed rest in the elderly. Clin Nutr 2010;29:18e23. 11. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Sanford AP, Aarsland A, Wolfe RR, et al. Essential amino acid and carbohydrate supplementation ameliorates muscle protein loss in humans during 28 days bedrest. J Clin Endocrinol Metab 2004;89:4351e8. 12. Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol 1997;273:E99e107. 13. Burd NA, West DW, Moore DR, Atherton PJ, Staples AW, Prior T, et al. Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men. J Nutr 2011;141:568e73. 14. Holm L, Reitelseder S, Pedersen TG, Doessing S, Petersen SG, Flyvbjerg A, et al. Changes in muscle size and MHC composition in response to resistance exercise with heavy and light loading intensity. J Appl Physiol 2008;105:1454e61. 15. Holm L, van Hall G, Rose AJ, Miller BF, Doessing S, Richter EA, et al. Contraction intensity and feeding affect collagen and myofibrillar protein synthesis rates differently in human skeletal muscle. Am J Physiol Endocrinol Metab 2010;298:E257e69. 16. Reitelseder S, Agergaard J, Doessing S, Helmark IC, Lund P, Kristensen NB, et al. Whey and casein labeled with L-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion. Am J Physiol Endocrinol Metab 2011;300:E231e42. 17. Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci U S A 1997;94:14930e5. 18. Matthews DE, Schwarz HP, Yang RD, Motil KJ, Young VR, Bier DM. Relationship of plasma leucine and alpha-ketoisocaproate during a L-[1-13C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment. Metabolism 1982;31:1105e12. 19. Burd NA, Holwerda AM, Selby KC, West DW, Staples AW, Cain NE, et al. Resistance exercise volume affects myofibrillar protein synthesis and anabolic

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