The influence of carbohydrate protein co ingestion following endurance exercise on

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The influence of carbohydrate-protein co-ingestion following endurance exercise on 
myofibrillar and mitochondrial protein synthesis. 
 
 
Leigh Breen1, 5, Andrew Philp2, Oliver. C. Witard1, 4, Sarah. R. Jackman1, Anna Selby3, Ken 
Smith3, Keith Baar2, Kevin. D. Tipton1, 4 
 
1School of Sport and Exercise Sciences, University of Birmingham, Birmingham, UK. 
2Functional Molecular Biology Lab, Neurobiology, Physiology and Behaviour, University of 
California, Davis, US .3School of Graduate Entry Medicine & Health, University of Nottingham, 
Derby, UK. 4Department of Sports Studies, University of Stirling, Stirling, UK. 5Department of 
Kinesiology, McMaster University, Ontario, Canada. 
Running title: Carbohydrate-protein consumption for endurance athletes 
Word count: 7320 (exclusive of references and legends); Figures: 6; Tables: 2 
TOC Category: Skeletal muscle and exercise 
Address for Correspondence: 
Leigh Breen, Ph.D. 
McMaster University 
Department of Kinesiology 
1280 Main St West, Hamilton, ON. 
L8P 2P9 
Canada 
Email: breenl@mcmaster.ca
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Non-technical summary 1 
A single bout of exercise stimulates the production of new muscle proteins. Furthermore, 2 
ingesting protein in close proximity to exercise enhances the metabolic response. Long-term 3 
exercise training promotes muscle adaptation, and the mode of exercise performed determines 4 
the type of proteins that are made. To date, the types of proteins that are made when protein is 5 
ingested after endurance exercise are not known. We report that when well-trained male cyclists 6 
ingest protein with a carbohydrate drink after a high-intensity ride, production of proteins 7 
responsible for muscle contraction is increased. Proteins responsible for aerobic energy 8 
production are not responsive to protein feeding. Furthermore, specific signals within the muscle 9 
that control protein synthesis are responsive to protein ingestion, providing a potential 10 
mechanism to underpin our primary findings. These results suggest that protein feeding after 11 
intense endurance exercise may be important in maintaining the structural quality and power 12 
generating capacity of the muscle. 13 
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Abstract 24 
Purpose: The aim of the present study was to determine mitochondrial and myofibrillar 25 
muscle protein synthesis (MPS) when carbohydrate (CHO) or carbohydrate plus protein (C+P) 26 
beverages were ingested following prolonged cycling exercise. The intracellular mechanisms 27 
thought to regulate MPS were also investigated. Method: In a single-blind, cross-over study, 10 28 
trained cyclists (age 29 ± 6 yr, V&O2max 66.5 ± 5.1 mL·kg¯1·min¯1) completed two trials in a 29 
randomized order. Subjects cycled for 90 min at 77 ± 1% V&O2max before ingesting a CHO (25 g 30 
of carbohydrate) or C+P (25 g carbohydrate + 10 g whey protein) beverage immediately and 30 31 
min post-exercise. A primed constant infusion of L-[ring-13C6] phenylalanine began 1.5 h prior 32 
to exercise and continued until 4 h post-exercise. Muscle biopsy samples were obtained to 33 
determine myofibrillar and mitochondrial MPS and the phosphorylation of intracellular 34 
signalling proteins. Arterialized blood samples were obtained throughout the protocol. Results: 35 
Plasma amino acid and urea concentrations increased following ingestion of C+P only. Serum 36 
insulin concentration increased more for C+P than CHO. Myofibrillar MPS was ~35% greater 37 
for C+P compared with CHO (0.087 ± 0.007 and 0.057 ± 0.006 %h-1, respectively; P = 0.025). 38 
Mitochondrial MPS rates were similar for C+P and CHO (0.082 ± 0.011 and 0.086 ± 0.018 %h-1, 39 
respectively; P = 0.025). mTORSer2448 phosphorylation was greater for C+P compared with CHO 40 
at 4 h post-exercise (P < 0.05). p70S6KThr389 phosphorylation increased at 4 h post-exercise for 41 
C+P (P < 0.05), whilst eEF2Thr56 phosphorylation increased by ~40% at 4 h post-exercise for 42 
CHO only (P < 0.01). Conclusions: The present study demonstrates that the ingestion of protein 43 
in addition to carbohydrate stimulates an increase in myofibrillar, but not mitochondrial, MPS 44 
following prolonged cycling. These data indicate that the increase in myofibrillar MPS for C+P 45 
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could, potentially, be mediated through p70S6K, downstream of mTOR, which in turn may 46 
suppress the rise in eEF2 on translation elongation. 47 
Key words: cycling, skeletal muscle, protein turnover, training adaptation. 48 
 49 
Abbreviations: EE, endurance exercise; RE, resistance exercise; MPS, muscle protein synthesis; 50 
C+P, carbohydrate plus protein; CHO, carbohydrate-only; V&O2, oxygen uptake; mTOR, 51 
mammalian target of rapamycin; p70S6K, 70kDa S6 protein kinase; Akt, protein kinase B; 4E-52 
BP1, eukaryotic initiation factor 4E binding protein 1; eEF2, eukaryotic elongation factor 2; 53 
PRAS40, proline-rich Akt substrate 40 kDa; p38 MAPK, p38 mitogen-activated protein kinase; 54 
AMPK, AMP-activated protein kinase. 55 
 56 
Introduction 57 
Endurance (EE) and resistance exercise (RE) training regimens result in divergent 58 
phenotypic adaptations. Whereas RE promotes muscle hypertrophy and an increase in contractile 59 
force output (Jones & Rutherford, 1987; Hartman et al., 2007), EE training is characterized by an 60 
expansion of oxidative capacity, brought about through an increase in the size and density of 61 
mitochondria and accumulation of myosin heavy chain I (Holloszy, 1967; Harber et al., 2002; 62 
Tarnopolsky et al., 2007). At the metabolic level, the adaptation to exercise is determined by 63 
summing the acute transcriptional (Pilegaard et al., 2000; Hildebrandt et al., 2003) and 64 
translational responses (Wilkinson et al., 2008) to each exercise stimulus and the subsequent 65 
increase in synthesis of muscle proteins (Mahoney et al., 2005; Hawley et al., 2006; Tipton, 66 
2008). Recently, Wilkinson and colleagues (2008) showed the response of myofibrillar and 67 
mitochondrial muscle protein synthesis (MPS) were dependent on the type of exercise 68 
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performed. Furthermore, the interaction of RE and protein nutrition varies between different 69 
protein fractions (Moore et al., 2009). Thus, it is important to examine the response of different 70 
protein fractions in order to determine whether a particular nutritional intervention elicits the 71 
desired effect following varied types of exercise. 72 
 73 
Whereas the response of mixed MPS to different types of exercise has been investigated 74 
(Tipton et al., 1996; Phillips et al., 1997; Harber et al., 2010), there is less information on the 75 
response of various proteins to exercise and nutrition. It is widely recognized that ingesting 76 
protein potentiates the anabolic effect of RE (Tang et al., 2007), seemingly due to the essential 77 
amino acid content (Tipton et al., 1999). Recently, Moore et al. (2009) showed that myofibrillar 78 
and sarcoplasmic proteins respond in a similar manner to protein feeding, however, rates of 79 
synthesis of only myofibrillar proteins were further increased when RE was combined with 80 
protein ingestion. Similarly, myofibrillar, but not mitochondrial protein synthesis increased in 81 
response to nutrient, including protein, ingestion following repeated sprints (Coffey et al., 2010). 82 
To date, no study has investigatedthe interactive effect of EE and protein ingestion on different 83 
muscle protein fractions. 84 
 85 
There are many studies demonstrating the response of MPS to protein ingestion following 86 
RE (Phillips et al., 1997; Moore et al., 2009; Burd et al., 2010) but very few studies have sought 87 
to determine the effect of protein ingestion on MPS following EE. Levenhagen et al. (2002) 88 
reported that adding protein to a carbohydrate treatment increased post-EE leg and whole-body 89 
protein synthesis. This increase in synthesis was associated with increased net muscle protein 90 
balance. However, it was unclear whether the benefits observed were due to the protein per se, or 91 
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an increase in total energy intake. To address this possibility, Howarth and colleagues (2009) 92 
showed that the addition of protein to carbohydrate increased mixed MPS compared with CHO 93 
treatments matched for total energy and carbohydrate content. Thus, it seems clear that the rate 94 
of mixed MPS responds to protein ingestion following EE, as well as RE. However, none of 95 
these studies (Levenhagen et al., 2002; Howarth et al., 2009) attempted to determine the specific 96 
protein fractions that contribute to changes in mixed MPS in response to protein ingestion or the 97 
mechanisms accounting for the response. Recent evidence from Coffey et al. (Coffey et al., 98 
2010) suggests that nutrient provision prior to a high-intensity, repeat-sprint bout of cycling 99 
increases the rate of myofibrillar MPS in the post-exercise period. However, this finding may 100 
have been influenced by the unique overload stimulus of repeated sprint exercise, characterized 101 
by a greater rate of force production and large disturbances to ion homeostasis (Coffey et al., 102 
2009). Thus, our primary aim was to investigate the impact of ingesting protein in addition to 103 
carbohydrate on the response of myofibrillar and mitochondrial protein synthesis rates following 104 
prolonged EE. 105 
 106 
Acute changes in transcription and translation that occur following exercise regulate 107 
skeletal muscle protein turnover through a number of intracellular signalling proteins. The 108 
mammalian target of rapamycin (mTOR) is a key regulator of translational control, integrating 109 
environmental signals from nutrients and exercise to control cell growth (Fingar et al., 2004). 110 
The activation of mTOR signalling leads to the phosphorylation of downstream targets involved 111 
in mRNA translation initiation and elongation (Bolster et al., 2003), e.g. p70 ribosomal protein 112 
S6 kinase-1 (p70S6K). In concert with an increase in mitochondrial MPS following EE, 113 
Wilkinson et al. (2008) showed an increase in the phosphorylation of signalling proteins in the 114 
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mTOR–p70S6K pathway. Furthermore, human (Ivy et al., 2008) and rat studies (Morrison et al., 115 
2008) indicate that post-EE C+P ingestion increases the phosphorylation of intermediates in the 116 
mTOR-p70S6K pathway. To date, no study has characterized the response of signalling proteins 117 
to changes in myofibrillar and mitochondrial MPS following post-EE protein ingestion. Hence, 118 
the secondary aim of the study was to elucidate the potential intracellular signalling mechanisms 119 
(in the mTOR-p70S6K pathway) regulating the response of myofibrillar and mitochondrial MPS 120 
following post-EE protein ingestion. 121 
 122 
Methods 123 
Participants 124 
 Ten well-trained, male cyclists were recruited from local cycling clubs through 125 
advertisements. Their mean (±SD) age was 29 ± 6 yr, body mass was 77.2 ± 6.5 kg, maximal 126 
oxygen uptake was 66.5 ± 5.1 mL·kg¯1·min¯1 and maximal power output was 383 ± 25 W. Only 127 
cyclists who undertook 2 or more training sessions per week of 1-5 h duration were eligible to 128 
participate. Participants had 7.5 ± 3.0 yr of competitive cycling experience. All tests were 129 
completed within a 4-week period with both treatment trials separated by 14 to 21-days, with the 130 
exception of one participant who completed the second trial 32-days after the first. The purpose 131 
and methodology of the study were clearly explained to the participants. All participants gave 132 
their informed consent prior to taking part in the study and were deemed healthy based on their 133 
response to a general health questionnaire. The experimental protocol was approved by the Black 134 
Country Research Ethics Committee (Rec No: 08/H1202/130). 135 
 136 
Study Design 137 
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 Participants reported to the laboratory on three separate occasions. During the first visit 138 
maximal aerobic fitness was determined. Approximately 2-weeks later participants performed 139 
the first blinded trial in which they consumed a carbohydrate (CHO) or carbohydrate-protein 140 
beverage (C+P; Lucozade Sport Recovery Powder, GlaxoSmithKline, Brentford, UK). During 141 
each trial participants performed a 90 min high intensity, steady-state cycle before consuming 142 
CHO or C+P immediately and 30 min following the exercise bout. Mitochondrial and 143 
myofibrillar MPS were measured by combining isotopic tracer infusion and muscle biopsy 144 
techniques. Participants returned to the laboratory for the second blinded trial 14 to 21-days after 145 
the first trial, thereby serving as their own control. Trial order was randomized in a counter-146 
balanced fashion. 147 
 148 
Preliminary Testing 149 
 Body Mass: Body mass was determined to the nearest 0.1 kg. Each participant was 150 
weighed in their cycling clothing without shoes on. Measurement of body mass was repeated 151 
prior to each of the two testing visits to ensure body mass remained constant throughout the 152 
study. 153 
 154 
 Maximal Cycling Test: Maximum oxygen uptake (V&O2max) and maximal power output 155 
(Wmax) were determined using an incremental cycle test-to-exhaustion on an electrically braked 156 
cycle ergometer (LODE Excalibur Sport V 2.0 Groningen, Netherlands). The test consisted of a 157 
3 min warm-up cycling at a self-selected cadence at 95 W followed by an increase of 35 W every 158 
3 min until volitional exhaustion. Breath-by-breath measurements were taken throughout 159 
exercise using an OxyCon Pro automated gas analysis system (Jaeger, Wuerzeburg, Germany). 160 
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The gas analyzers were calibrated using a 4.99% CO2 – 15.01% O2 gas mixture (BOC Gases, 161 
Surrey, UK) and the volume transducer was calibrated with a 3L calibration syringe. Heart rate 162 
(HR) was measured continuously via telemetry using a HR monitor (Polar S625X; Polar Electro 163 
Oy, Kempele, Finland). V&O2 was considered maximal if 2 of the 4 following conditions were 164 
met: 1) a plateau in V&O2 with further increasing workloads (an increase of < 2 mL·min¯1·kg¯1); 2) 165 
a HR within 10 beats/min of the age predicted maximum (220 bpm - age); 3) a respiratory 166 
exchange ratio (RER) of >1.05; and 4) a rate of perceived exertion (RPE) greater than 17. The 167 
seat position, handlebar height and orientation used during baseline testing were recorded and 168 
replicated on subsequent visits to the laboratory. All exercise bouts were conducted in thermo-169 
neutral conditions (21°C, 40% relative humidity). 170 
 171 
 Dietary Analysis & Control: Participant diet was standardized for 48 h prior to each 172 
treatment. During the preliminary testing phase, participants completed a 3-day food diary, 173representative of their average week (2 weekdays and 1 weekend day). A questionnaire of food 174 
preferences was also completed by participants. Using an on-line diet planner (Weight Loss 175 
Resources), each of the 3-days was logged and energy and macronutrient intake was estimated. 176 
In our study cohort the average total daily energy intake was 2787 ± 164 kcal (5.3 ± 0.4 g·kg-1 177 
BM carbohydrate; 1.2 ± 0.1 g·kg-1 BM fat; 1.5 ± 0.2 g·kg-1 BM protein). The food parcels given 178 
to each participant matched their habitual energy and macronutrient intake. Participants were 179 
instructed to refrain from caffeine and alcohol and to consume only the food provided for them 180 
over the two-days prior to arriving for each trial. Participants also were asked to consume their 181 
final meal no later than 2200 h to ensure a 10 h fast prior to measuring myofibrillar and 182 
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mitochondrial protein synthesis rate. An identical two-day food parcel was provided to each 183 
participant prior to the second trial. 184 
 185 
 Physical activity control: Participants were instructed to maintain their normal training 186 
volume and intensity throughout the course of the study but to refrain from training for 48 h prior 187 
to each treatment trial. To monitor physical activity between trials, participants were asked to 188 
record all training 7-days prior to each trial. No differences were noted in training volume in the 189 
7-days prior to each trial. 190 
 191 
Experimental trial protocol 192 
 Each participant was instructed to arrive at the Human Performance Laboratory at ~0630 193 
h after an overnight fast, where standard measures of height and weight were taken. A cannula 194 
was placed into the forearm vein of one arm and a wrist vein of the other. The forearm cannula 195 
was used to infuse a stable isotopic tracer whilst the hand vein was heated for frequent 196 
arterialized blood sampling (Abumarad et al., 1981). After a resting blood sample had been 197 
obtained, participants then received a primed constant infusion of L-[ring-13C6] phenylalanine 198 
(prime: 2 μmol·kg-1; infusion: 0.05 μmol·kg-1·min-1; Cambridge Isotope Laboratories, MA, USA) 199 
to determine MPS (described below). Approximately 90 min after the start of the infusion, 200 
having rested in a supine position, participants were asked to complete a 90 min cycling exercise 201 
bout on a Lode Cycle Ergometer at a self-selected cadence ≥ 60 revolutions per minute (rpm). 202 
The exercise bout consisted of a warm-up cycle at 50% Wmax (189 ± 4 W) for 10 min and then 203 
80 min, initially at 75% Wmax (283 ± 6 W). V&O2, RER, HR and RPE were recorded over 25-30 204 
min, 55-60 min and 85-90 min of the exercise bout (as described above). If participants indicated 205 
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during exercise that the workload was too difficult and they were unlikely to complete the full 80 206 
min, workload was lowered in 5% decrements to no less than 65% Wmax. Air conditioning and 207 
a fan were used when requested by participants. The exact settings of the air conditioning/fan 208 
and the time of any change in workload were recorded and replicated during the second trial. 209 
Throughout each trial, participants were allowed to drink water ad libitum. The amount of water 210 
consumed throughout the course of the each trial was found to be similar (1556 ± 249 mL for 211 
CHO and 1674 ± 233 mL for C+P). 212 
 213 
 Muscle biopsy and blood sampling Using a 5-mm Bergstrom biopsy needle, two muscle 214 
biopsies (~100-150 mg of muscle tissue per biopsy) were obtained from the same leg during 215 
each trial. The order of biopsied leg was randomized and counterbalanced for each trial. Prior to 216 
the exercise bout (~15min) under local anaesthetic (1% Lidocaine), the lateral portion of one 217 
thigh was prepared for the extraction of a needle biopsy sample from the vastus lateralis muscle. 218 
Biopsy incisions were made prior to exercise to allow the sample to be obtained as quickly as 219 
possible after exercise (5 ± 1 min post-exercise). Immediately after the post-exercise muscle 220 
biopsy was obtained, participants were asked to consume one of two treatment beverages 221 
described below. Four hours after consuming the treatment beverage, the second muscle biopsy 222 
was obtained (Figure 1). The second biopsy was taken ~2 cm proximal to the first biopsy. Biopsy 223 
samples were quickly rinsed in saline, blotted and divided into two to three aliquots, before being 224 
frozen in liquid nitrogen and stored at -80°C until later analysis. Arterialized blood samples from 225 
a heated wrist vein were collected at rest, immediately post-exercise and every 15 min following 226 
beverage consumption for 2 h. Thereafter, blood samples were obtained at regular intervals for 227 
remainder of the infusion. Blood was collected in ethylenediaminetetraacetic-containing, lithium 228 
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heparin-containing and serum separator tubes and spun at 3500 rpm for 15 min at 4°C. Aliquots 229 
of plasma and serum were the frozen at -80°C for subsequent analysis. 230 
 231 
 Treatment beverages Immediately after the first muscle biopsy sample was obtained, 232 
subjects ingested either 25.2 g of carbohydrate (CHO) or 25.4 g of carbohydrate plus 10.2 g of 233 
whey protein isolate (C+P) dissolved in 250 mL of cold water (~11 ± < 1 min post-exercise). A 234 
second identical beverage was consumed 30 min after the first beverage was finished. This dose 235 
regime provided a total carbohydrate and protein intake of 50.8 g and 20.4 g, respectively, in 236 
C+P and a total carbohydrate intake of 50.4 g in CHO. Participants were encouraged to consume 237 
the beverages within 2 min. Both CHO and C+P treatment beverages were matched for flavour 238 
(orange and passion fruit) and appearance. Beverages were administered to participants in a 239 
single-blinded manner, the order of which was randomized. Eight out of the ten participants 240 
correctly identified the order of the treatments. The amino acid content of the whey protein was 241 
(in percent content, wt:wt): Ala, 5.2; Arg, 2.2; Asp, 11.4; Cys, 2.3; Gln, 18.8; Gly, 1.5; His, 1.8; 242 
Ile, 6.7; Leu, 11; Lys, 10; Met, 2.3; Phe, 3.1; Pro, 5.7; Ser, 4.8; Thr, 7; Trp, 1.5; Tyr, 2.7; and 243 
Val, 6.1. A small amount of L-[ring-13C6] phenylalanine tracer was added to the C+P drink (6% 244 
of phenylalanine content) in order to minimize changes in blood phenylalanine enrichment after 245 
drink ingestion. The need to add isotopic tracer to the C+P beverage dictated that the 246 
investigators were not blinded to the treatment beverage order. 247 
 248 
Analyses 249 
 Blood analyses: Plasma glucose, lactate and urea concentrations were analyzed using an 250 
ILAB automated analyzer (Instrumentation Laboratory, Cheshire, UK). Serum insulin 251 
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concentrations were analyzed using a commercially available ELISA kit (IBL International, 252 
Hamburg, Germany), following the manufacturer’s instructions. L-[ring-13C6] phenylalanine 253 
tracer-to-tracee (t/T) enrichment was determined by gas chromatography, mass spectrometry 254 
(GCMS) (model 5973; Hewlett Packard, Palo Alto, CA). Upon thawing, plasma samples were 255 
combined with diluted acetic acid and purified on cation-exchange columns (Dowex 50W-X8-256 
200, Sigma-Aldrich Poole, UK). The amino acids were then converted to their N-tert-257 
butyldimethyl-silyl-N-methyltrifluoracetamide (MTBSTFA)derivative. Plasma 13C6 258 
phenylalanine enrichment was determined by ion monitoring at masses 234/240. Appropriate 259 
corrections were made for overlapping spectra contributing to the t/T ratio. Phenylalanine, 260 
leucine and threonine concentrations were determined using an internal standard method (Tipton 261 
et al., 1999; Tipton et al., 2001), based on the known volume of blood and internal standard 262 
added. The internal standards used were U-[13C9-15N] phenylalanine (50 µmol/L), U-[13C6] 263 
leucine (120 μmol/L) and U-[13C9-15N] threonine (182 μmol/L) added in a ratio of 100 µL/mL of 264 
blood. Leucine, threonine and phenylalanine concentrations were determined by monitoring at 265 
ions 302/308, 404/409 and 336/346, respectively. 266 
 267 
Muscle tissue analyses: Muscle samples were analyzed for enrichment of L-[ring-13C6] 268 
phenylalanine in the intracellular pool and bound myofibrillar and mitochondrial protein 269 
fractions. Intracellular amino acids were liberated from ~20 mg of muscle. The tissue was 270 
powdered under liquid nitrogen using a mortar and pestle and 500 µL of 0.2M perchloric acid 271 
(PCA) was added. The mixture was centrifuged at 10,000 g for 10 min. The pH of the 272 
supernatant was then adjusted to 5–7 with 2M KOH and 0.2M PCA and treated with 20 µL of 273 
urease for removal of urea. The free amino acids from the intracellular pool were purified on 274 
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cation-exchange columns (described above). Intracellular amino acids were converted to their 275 
MTBSTFA derivative and 13C6 phenylalanine enrichment determined by monitoring at ions 276 
234/240 (as described above) using GCMS. 277 
 278 
 Mitochondrial and myofibrillar protein isolation was achieved using a protocol adapted 279 
from Wilkinson et al. (2008). Approximately 70-100 mg of muscle tissue was homogenized in a 280 
2mL Eppendorf with a Teflon pestle in 10 µL·mg-1 of ice-cold homogenizing buffer (0.1mM 281 
KCl, 50mM Tris, 5mM MgCl, 1mM EDTA, 10mM β-glycerophosphate, 50mM NaF, 1.5% 282 
BSA, pH 7.5). The homogenate was spun at 1,000 g for 10 min at 4ºC. The supernatant was 283 
transferred to another Eppendorf and spun at 10,000 g for 10 min at 4ºC to pellet the 284 
sarcoplasmic mitochondria (SM). The supernatant was then removed and discarded. The pellet 285 
that remained from the original 1,000 g spin was washed twice with homogenization buffer. A 286 
glass Dounce homogenizer and tight fitting glass pestle were used to forcefully homogenize the 287 
pellet in homogenization buffer to liberate intermyofibrillar mitochondria (IM). The resulting 288 
mixture of myofibrillar proteins (MYO) and IM was spun at 1,000 g for 10 min at 4ºC to pellet 289 
out the MYO. The supernatant was removed and spun at 10,000 g for 10 min at 4ºC to pellet the 290 
IM. The MYO, SM and IM pellets were washed twice with homogenizing buffer containing no 291 
BSA. The MYO fraction was separated from any collagen by dissolving in 0.3M NaCl, 292 
removing the supernatant and precipitating the proteins with 1M PCA. All samples were washed 293 
once with 95% ethanol. In order to determine 13C6 phenylalanine enrichment in the 294 
mitochondrial protein fraction, IM and SM fractions were combined as per Wilkinson et al. 295 
(Wilkinson et al., 2008). Mitochondrial and myofibrillar fractions were then hydrolysed 296 
overnight at 110ºC in 0.1M HCl/Dowex 50WX8-200 (Sigma Ltd, Poole, UK) and the constituent 297 
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amino acids purified on cation-exchange columns (Dowex 50W-X8-200, Sigma-Aldrich Poole, 298 
UK). The amino acids were then converted to their N-acetyl-n-propyl ester derivative. 299 
Phenylalanine labelling was determined by gas-chromatography-combustion-isotope ratio mass 300 
spectrometry (GC-C-IRMS, Delta-plus XL, Thermofinnigan, Hemel Hempstead, UK) by 301 
monitoring at ions 44/45 for labelled and unlabelled CO2. Unfortunately, during processing two 302 
mitochondrial fraction samples were lost, therefore these data represent an n = 8. 303 
 304 
Western Blots: The remaining muscle tissue (25-40 mg) was powdered on dry ice under 305 
liquid nitrogen using a mortar and pestle. Approximately 20 mg of powdered muscle was 306 
homegenized in 10uL of lysis buffer per mg of powdered muscle (50mM Tris pH 7.5; 250mM 307 
Sucrose; 1mM EDTA; 1mM EGTA; 1% Triton X-100; 1mM NaVO4; 50mM NaF; 0.50% PIC), 308 
using a hand-held homogenizer (PRO200, UK). Samples were shaken at 4ºC for 30 min (12,000 309 
rpm), centrifuged for 5 min at 6,000 g and the supernatant removed for protein determination. 310 
Protein concentration was determined using the DC protein assay (Bio Rad, Hertfordshire, UK). 311 
Equal aliquots of protein were boiled in Laemmli sample buffer (250mM Tris-HCl, pH 6.8; 2% 312 
SDS; 10% glycerol; 0.01% bromophenol blue; 5% β-mercaptoethanol) and separated on SDS 313 
polyacrylamide gels (10 – 12.5%) for 1 h at 58 mA. Following electrophoresis; proteins were 314 
transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) at 100 V for 1 h. 315 
The membranes were incubated overnight at 4°C with the appropriate primary antibody. The 316 
primary antibodies used were AMPKThr172 (Millipore 15-115), mTORSer2448 (Cell signalling 317 
2976), S6KThr389 (Cell signalling 9234), AktThr308 (Cell signalling 4056), 4E-BP1Thr37 (Santa Cruz 318 
SC6025), eEF2Thr56 (Cell signalling 2332), PRAS40Thr246 (Cell Signalling 2610) and p38 319 
MAPKThr180 (Cell Signalling 9212). The following morning the membrane was rinsed in wash 320 
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buffer (TBS with 0.1% Tween-20) three times for 5 min. The membrane was then incubated for 321 
1 h at room temperature within wash buffer containing the appropriate secondary antibody, either 322 
horseradish (HRP)-linked anti-mouse IgG (New England Biolabs, 7072; 1:1,000) or anti-rabbit 323 
IgG (New England Biolabs, 7074; 1:1,000). The membrane was then cleared in wash buffer 324 
three times for 5 min. Antibody binding was detected using enhanced chemiluminescence 325 
(Millipore, Billerica, MA). Imaging and band quantification were carried out using a Chemi 326 
Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK). Intracellular signalling targets 327 
were determined with n = 8 for CHO and C+P trials. Protein phosphorylation was expressed 328 
relative to the total protein. 329 
 330 
Calculations 331 
The fractional synthetic rate (FSR) of mitochondrial and myofibrillar proteins were 332 
calculated using the standard precursor-product method: 333 
 334 
FSR (%·h-1) = ΔEb ⁄ Ep × 1 ⁄ t × 100 (1) 335 
 336 
Where ΔEb is the change in bound 13C6 phenylalanine enrichment between two biopsy 337 
samples, Ep is the precursor enrichment and t is the time between muscle biopsies. 338 
 339 
The true precursor enrichment would be the labelled phenylalanine-tRNA (Baumann et al., 340 
1994). However, measurement of this enrichment requires large amounts of tissue, which cannot 341 
typically be obtained from human volunteers. Thus, the intracellular (IC) free phenylalanine 342 
enrichment is commonly used in studies (Tipton et al., 1999; Howarth et al., 2009; Moore et al., 343 
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2009), primarily because it often is considered to be the best available correlate of muscle tRNA 344 
(Ljungqvist et al., 1997). Unfortunately, due to technical difficulties, the IC enrichment was 345 
available for only 5 participants. Thus, we chose to use the plasma precursor to estimate the IC 346enrichment. A comparison of arterialized plasma and IC enrichments from the samples available 347 
(n =5) revealed the IC enrichment to be 70 ± 2% of the plasma (range 67-77%). Furthermore, a 348 
comprehensive examination of studies, e.g. (Koopman et al., 2006; Beelen et al., 2008; Tang et 349 
al., 2009), in which phenylalanine tracers were used to determine fed-state FSR revealed the IC 350 
phenylalanine enrichment to be ~70% of the plasma enrichment. We chose to use the IC 351 
enrichment estimated from the plasma enrichment as the precursor for ease of comparison of the 352 
results to other previously published studies, e.g. (Tipton et al., 1996; Howarth et al., 2009). 353 
However, FSRs also were calculated with the unadjusted plasma precursor. 354 
 355 
Statistics 356 
A within-subject repeated measures design was utilized for the current study. Exercise 357 
variables, blood analytes and Western blot data were analyzed using a two-way ANOVA with 358 
repeated measures (treatment x time) to determine differences between each treatment beverage 359 
across time. When a significant main effect or interaction was identified, data were subsequently 360 
analyzed using a Bonferroni post hoc test. Myofibrillar and mitochondrial FSR data were 361 
analyzed using one-factor (treatment) repeated measures ANOVA. All statistical tests were 362 
analyzed using statistical package for social sciences (SPSS) version 18.0 (Illinois, Chicago, 363 
U.S). Significance for all analyses was set at P < 0.05. All values are presented as means ± 364 
standard error of the mean (SEM). 365 
 366 
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Results 367 
Exercise variables: There was no between-trial difference in heart rate, cadence, V&O2 and 368 
RER measured at 25-30, 55-60 and 85-90 min of the steady state cycle (Table 1). Average HR 369 
over the 90 min cycle was 172 ± 3 and 172 ± 4 bpm for CHO and C+P, respectively. Average 370 
V&O2 over 90 min (51.3 ± 1.6 mL·kg-1·min-1 for CHO and 51.6 ± 1.2 mL·kg-1·min-1 for C+P) and 371 
RER over 90 min (0.88 ± 0.01 for CHO and C+P) were not different between treatments. 372 
Metabolic data indicated participants were cycling at 77 ± 1% of V&O2 during CHO and C+P 373 
trials. Average RPE over 90 min was similar for CHO and C+P (16 ± 4 for CHO and C+P). 374 
 375 
Blood analytes: Fasted blood glucose was 5.1 ± 0.3 mmol·L and 5.3 ± 0.3 mmol·L for 376 
CHO and C+P, respectively and remained similar immediately post-exercise. Approximately 30 377 
min after consuming the first treatment beverage, plasma glucose concentration increased by 378 
~32% and ~20% for CHO and C+P, respectively, with no significant difference between 379 
treatments. Plasma glucose concentration returned to basal values by 1.5 h post-exercise for 380 
CHO and C+P and was constant for the remainder of the trial. Fasted serum insulin concentration 381 
was 6.0 ± 0.8 and 5.5 ± 0.5 μU·mL-1 for CHO and C+P, respectively (Figure 2A). Following 382 
drink ingestion, serum insulin concentration increased for both CHO and C+P, peaking at 30 min 383 
post-exercise (P < 0.001). Serum insulin increased to a greater extent for C+P (285 ± 32%) 384 
compared with CHO (60 ± 8%; P < 0.001). Serum insulin returned to basal values by 1.5 h post-385 
exercise for CHO and C+P and remained constant until the end of the trial. Following exercise, 386 
plasma lactate concentration increased by 150 ± 16% and 175 ± 19% compared with resting 387 
values for CHO and C+P, respectively (P < 0.001), with no difference between treatments. 388 
Lactate concentration returned to basal values by 3 h post-exercise for CHO and C+P. Resting 389 
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plasma urea concentration was similar for CHO and C+P and was stable immediately post-390 
exercise (Figure 2B). Following ingestion of C+P, plasma urea concentration increased by 20 ± 391 
2% compared with resting values and remained elevated at 4 h post-exercise (P < 0.05). Plasma 392 
urea concentration remained unchanged for CHO compared with resting values. From 15 min to 393 
4 h post-exercise, plasma urea concentration for C+P was significantly greater than CHO (P < 394 
0.05). 395 
 396 
Plasma amino acid concentrations: Prior to and immediately post-exercise, plasma amino 397 
acid concentrations of phenylalanine, leucine and threonine were similar for CHO and C+P 398 
(Figure 3). Following ingestion of C+P, plasma concentrations of phenylalanine, leucine and 399 
threonine increased by 37, 130 and 58%, respectively (P < 0.001) and peaked at 1 h post-400 
exercise (30 min after the second drink was ingested), after which, amino acid concentrations 401 
returned to basal levels such that there were no differences between CHO and C+P at 4 h post-402 
exercise. Following CHO ingestion, plasma phenylalanine, leucine and threonine concentrations 403 
were reduced at 30 min compared with immediate post-exercise values (P < 0.05). Phenylalanine 404 
and threonine concentrations remained lower for the remainder of the infusion. In contrast, 405 
plasma leucine concentration returned to pre-exercise values by 150 min post-exercise. 406 
 407 
Plasma and intracellular 13C6 phenylalanine enrichment: Plasma 13C6 phenylalanine 408 
enrichment increased from immediately pre- to post-exercise (P < 0.05) but was stable across the 409 
time of tracer incorporation, immediately post-exercise to 4 h post-exercise for CHO and C+P 410 
(7.20 ± 0.03% t/T for both; Figure 4). These data demonstrate that the additional tracer added to 411 
the C+P treatment beverage did not appear in the circulation more rapidly than the amino acids 412 
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from the protein and that all measurements were made at isotopic equilibrium. Intracellular 13C6 413 
phenylalanine enrichment for an available sub-set of participants (n = 5) was stable across the 414 
time of tracer incorporation for CHO and C+P (4.8 ± 0.5 and 4.5 ± 0.7% t/T, respectively; P > 415 
0.05). However, due to the small sub-set of available intracellular samples (n = 5) our 416 
calculations revealed intracellular phenylalanine enrichments for 8 participants would have been 417 
required to achieve sufficient statistical power (≥ 0.8). Based on a close correlation between 418 
available intracellular (n = 5) and plasma enrichments (r = 0.63), the mean predicted intracellular 419 
enrichment for the complete study cohort (n = 10) was 5.0 ± 0.1 and 5.1 ± 0.2% t/T for C+P and 420 
CHO, respectively. Trial order did not influence the tracer enrichment in plasma (7.0 ± 0.3 and 421 
7.4 ± 0.2% t/T for trials 1 and 2, respectively; P = 0.2) or in the available intracellular samples 422 
(4.5 ± 0.4 and 4.8 ± 0.3% t/T for trials 1 and 2, respectively; P = 0.2). 423 
 424 
Post-exercise protein phosphorylation: Immediately post-exercise, mTORSer2448 425 
phosphorylation was similar for CHO and C+P. At 4 h post-exercise mTOR phosphorylation 426 
tended to increase for C+P (P = 0.1), whereas mTOR phosphorylation tended to decrease for 427 
CHO (P = 0.08). A group effect at 4 h post-exercise revealed mTOR phosphorylation was 428 
greater for C+P compared with CHO (P = 0.02; Figure 5A). However, there was no group by 429 
time interaction for mTOR phosphorylation. Immediately post-exercise, eEF2Thr56 430 
phosphorylation was similar for CHO and C+P. At 4 h post-exercise phosphorylation increased 431 
1.4-fold for CHO compared with immediately post-exercise (P = 0.02). Furthermore, eEF2 432 
phosphorylation for CHO was greater than C+P at 4 h post-exercise (P = 0.04; Figure 5B). 433 
Immediately post-exercise, p38MAPKThr180 phosphorylation was similar for CHO and C+P. At 434 
4 h post-exercise p38 MAPK phosphorylation was unchanged for CHO and C+P (Table 2). 435 
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Immediately post-exercise, p70S6KThr389 phosphorylation was similar for CHO and C+P. A time 436 
effect revealed p70S6K phosphorylation was increased at 4 h for C+P compared with 437 
immediately post-exercise (P = 0.05), however, p70S6K phosphorylation was not significantly 438 
different from CHO at 4 h (Figure 5C). Immediately post-exercise, 4E-BP1Thr37 phosphorylation 439 
was similar for CHO and C+P. At 4 h post-exercise 4E-BP1 phosphorylation showed a tendency 440 
to increase for CHO and C+P (P = 0.09) with no difference between treatments (Table 2). There 441 
was no difference in the phosphorylation of AMPKThr172, AktThr308 and PRAS40Ser246 442 
immediately post-exercise between CHO and C+P. Furthermore, the phosphorylation of AMPK, 443 
Akt and PRAS40 remained unchanged from immediately post-exercise at 4 h post-exercise for 444 
CHO and C+P (Table 2). 445 
 446 
Mitochondrial and myofibrillar FSR: Mitochondrial protein synthesis rates were similar 447 
for CHO and C+P (95% confidence interval [CI]: 0.06 - 0.12 and 0.06 - 0.10 %·h-1, respectively; 448 
Figure 6). Myofibrillar protein synthesis rates were ~35% higher for C+P compared with CHO 449 
(CI: 0.07 - 0.11 and 0.05 - 0.07 %·h-1, respectively; P = 0.025). Rates of mitochondrial and 450 
myofibrillar protein synthesis were 30.2 ± 0.9% lower when the unadjusted plasma precursor 451 
was used in the calculation of FSR for C+P and CHO (0.061 ± 0.005 and 0.040 ± 0.004 %·h-1, 452 
for myofibrillar FSR respectively). The difference in myofibrillar FSR between groups remained 453 
significant with the unadjusted precursor (P = 0.03). 454 
 455 
Discussion 456 
This is the first study to investigate the response of various muscle protein fractions to 457 
protein ingestion after endurance exercise. The present study findings expand on those of 458 
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previous investigations (Levenhagen et al., 2002; Howarth et al., 2009) to show that the addition 459 
of protein to carbohydrate (C+P) ingestion following 90 min of intense cycling by well-trained 460 
individuals stimulates an increase in rates of myofibrillar muscle protein synthesis (MPS) 461 
compared with carbohydrate alone (CHO). Interestingly, C+P did not increase mitochondrial 462 
MPS rates compared with CHO. The mechanism facilitating the adaptive response of 463 
myofibrillar MPS could potentially be due to enhanced mRNA translation as evidenced by 464 
differences (albeit marginal) in the phosphorylation of cell signalling intermediates with C+P 465 
compared with CHO. 466 
The response of myofibrillar MPS to hyperaminoacidemia with protein ingestion is not 467 
unique to post-exercise recovery from EE. Following RE, Holm et al. (Holm et al., 2010) 468 
recently demonstrated that myofibrillar MPS increased in response to protein ingestion. No 469 
measurement of mitochondrial protein synthesis was made in that study. Additionally, an amino 470 
acid infusion increased myofibrillar MPS in resting muscle (Bohe et al., 2001; Bohe et al., 471 
2003). In addition, Moore et al (Moore et al., 2009) demonstrated that protein ingestion 472 
increased myofibrillar MPS at rest and after RE. Finally, increased myofibrillar protein synthesis 473 
was noted with protein ingestion following repeated sprints (Coffey et al., 2010). Taken together 474 
with our results, it seems clear that myofibrillar protein synthesis rates are particularly sensitive 475 
to hyperaminoacidemia from ingested protein. 476 
Mitochondrial protein synthesis rates are increased by EE with little response of 477 
myofibrillar proteins (Wilkinson et al., 2008). Thus, it may seem logical that others (Levenhagen 478 
et al., 2002; Howarth et al., 2009) have assumed that an increase in mixed MPS reported with 479 
post-EE protein ingestion would be due, primarily, to mitochondrial proteins. However, this 480 
notion is not supported by our data or others (Coffey et al., 2010). Instead, it seems that the 481 
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increase in mixed muscle protein synthesis with protein ingestion immediately following EE 482 
(Levenhagen et al., 2002; Howarth et al., 2009) is attributed more aptly to the increase in the 483 
myofibrillar protein synthesis rates. However, a contribution of mitochondrial synthesis to the 484 
mixed muscle protein response cannot be completely dismissed. Whereas we studied 485 
mitochondrial protein synthesis in well-trained cyclists, previous investigations demonstrated 486 
increased mixed muscle protein synthesis in untrained subjects (Levenhagen et al., 2002; 487 
Howarth et al., 2009). It has been shown previously (Wilkinson et al., 2008) that training reduces 488 
the response of mitochondrial protein synthesis rates to exercise. Thus, it is possible that the 489 
exercise bout maximized the acute response in these well-trained cyclists causing a ‘ceiling’ 490 
effect of mitochondrial MPS. Therefore, although mitochondrial synthesis rates do not respond 491 
to protein ingestion in our trained subjects, it is possible that the increase in mixed muscle 492 
protein synthesis rates reported previously (Levenhagen et al., 2002; Howarth et al., 2009) may 493 
have included a mitochondrial component. Accordingly, whereas our data provide more 494 
information regarding the impact of protein nutrition on the acute stimulation of mitochondrial 495 
protein synthesis, clearly there is much more to be learned. 496 
The reason for this lack of response of mitochondrial MPS to protein ingestion, in our 497 
hands, is not clear. Moore et al. (2009) demonstrated that the synthesis of the sarcoplasmic 498 
protein pool, composed mostly of mitochondrial proteins, was increased by ~70% with protein 499 
ingestion, both at rest and following RE. Thus, these results suggest that rates of mitochondrial 500 
MPS increase in response to protein ingestion after RE. A response of mitochondrial protein 501 
synthesis to protein ingestion following RE, but not EE, seems somewhat counterintuitive, 502 
particularly given that mitochondrial protein synthesis responds to elevated amino acid levels at 503 
rest (Bohe et al., 2001; Bohe et al., 2003). 504 
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Despite the evident responsiveness of mitochondrial protein synthesis rates to protein 505 
ingestion in other situations (Bohe et al., 2001; Bohe et al., 2003; Moore et al., 2009), we and 506 
others (Coffey et al., 2010) were not able to detect an increase in response to protein following 507 
intense cycling. However, we cannot dismiss the possibility that the magnitude and/or duration 508 
of the response may have been smaller than that which is detectable with our current methods. In 509 
line with our data, a recent study by Coffey et al. (Coffey et al., 2010) showed that when a mixed 510 
macronutrient meal was ingested prior to a short bout of high-intensity, repeat-sprint cycling, 511 
myofibrillar proteins were preferentially synthesized in the post-exercise period, with no effect 512 
on mitochondrial proteins. The precise mechanism to explain the apparent synthesis of only 513 
myofibrillar proteins after nutrient ingestion in combination with exercise should be investigated 514 
further. 515 
Finally, it is possible that the lack of response may be related to the temporal pattern of the 516 
response of mitochondrial proteins to the exercise. Thissupposition is supported by recent 517 
preliminary data suggesting that mitochondrial protein synthesis is much greater at 24 h than 6 h 518 
post-exercise (Burd, Phillips et al. personal communication). Further support comes from 519 
evidence that transcription of mitochondrial protein mRNA does not produce elevated mRNA 520 
levels for several hours after exercise - at least for some proteins (Yang et al., 2005). Therefore, 521 
the stimulation of mitochondrial MPS detected 24 h after exercise (Burd, Phillips et al. personal 522 
communication) may be due primarily to translation of the new mRNA. Thus, the stimulation of 523 
mitochondrial protein synthesis may manifest itself far later than when we made our 524 
measurements. However, it should be noted that the 24 h response was following RE and, to our 525 
knowledge, there are no data investigating the temporal pattern of mitochondrial protein 526 
synthesis following EE. Nevertheless, the acute increase in mixed muscle protein synthesis 527 
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following post-EE protein ingestion noted in previous studies (Levenhagen et al., 2002; Howarth 528 
et al., 2009) may not be explained by an acute increase in mitochondrial FSR. 529 
Since training adaptations result, ultimately, from the summation of the acute responses to 530 
exercise and nutrition (Hawley et al., 2006), protein consumption may potentiate this response. It 531 
is unclear from our results what impact protein ingestion following cycling may have on training 532 
adaptations. Increased myofibrillar MPS is normally associated with increased muscle mass and 533 
strength in the context of resistance training (Tipton, 2008; Burd et al., 2009). Thus, our data 534 
could be interpreted to suggest that protein ingestion following EE would lead to increased 535 
muscle mass (and overall body weight) with training, potentially decreasing the power-to-mass 536 
ratio. Alternatively, it is possible that muscle hypertrophy may increase power output, thus 537 
offsetting the increase in overall body weight. In support of this notion, Hickson et al. (1980) 538 
showed that gains in thigh mass after strength training, extended short-term endurance capacity. 539 
Alternatively, EE training increases myosin heavy chain I content, a key protein in type I fibres 540 
leading to more fatigue resistant fibres (Harber et al., 2004; Kohn et al., 2007). Thus, increased 541 
rates of myofibrillar protein synthesis with protein intake after EE may be a contributing factor 542 
to potentiate the adaptive response to EE training. Additionally, elevated rates of myofibrillar 543 
protein synthesis may simply reflect increased turnover due to increased degradation of 544 
myofibrillar proteins. Moderate intensity EE has been shown to stimulate muscle protein 545 
breakdown during (Blomstrand & Saltin, 1999; Van Hall et al., 1999) and immediately following 546 
exercise (Sheffield-Moore et al., 2004), this is thought to be due, at least in part, to increased 547 
breakdown of myofibrillar proteins (Carraro et al., 1990). Therefore, the increase in myofibrillar 548 
synthesis may be contributing to repair and remodelling of proteins damaged during the bout. 549 
Thus, enhanced myofibrillar protein synthesis with post-exercise ingestion of protein may impact 550 
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training adaptations by one or more of several mechanisms. Clearly, these mechanisms require 551 
more study. 552 
The increase in myofibrillar MPS with post-EE protein ingestion coincided with 553 
significant, albeit relatively modest, alterations in the phosphorylation of factors previously 554 
associated with increased mRNA translation (Fingar et al., 2004). These findings were surprising 555 
given that we were only able to determine signalling phosphorylation at 0 and 4 h post-exercise 556 
and the phosphorylation of signalling intermediates with anabolic stimuli is relatively transient 557 
(Atherton et al., 2010; Camera et al., 2010). EE is known to activate proteins that regulate 558 
translation initiation (Mascher et al., 2007; Wilkinson et al., 2008; Camera et al., 2010) (i.e. 559 
mTOR) and elongation (Mascher et al., 2007) (i.e. eEF2) following EE. In addition, ingestion of 560 
protein increases rates of MPS via mTOR-p70S6K-mediated mechanism (Fujita et al., 2007). 561 
Previously, Ivy et al. (2008) demonstrated that mTOR and rpS6 phosphorylation was greater 562 
when C+P is ingested after EE as compared with a non-energetic placebo. However, due to the 563 
study design, it was unclear whether the effect of C+P was due to greater amino acid availability, 564 
elevated plasma insulin or a combination of the two. In contrast, an earlier experiment, in which 565 
rats were fed immediately after an exhaustive swim (Morrison et al., 2008) showed that the 566 
phosphorylation of translational proteins was sustained with C+P ingestion (Morrison et al., 567 
2008). In line with these data, our results show that protein ingestion after EE modified the 568 
translational signalling response in the mTOR-p70S6K pathway and, potentially, contributed 569 
towards prolonging the effect of EE on protein synthesis. We also show that eEF2 570 
phosphorylation increased by ~40% at 4 h post-EE in the CHO group, whereas there was no 571 
change with protein co-ingestion. Reduced phosphorylation of eEF2, due to eEF2 kinase 572 
inhibition, results in a more efficient translocation of the ribosome along the mRNA, thereby 573 
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contributing to faster elongation and greater MPS (Carlberg et al., 1990). Our findings are 574 
further supported by prior human (Fujita et al., 2007) and cell culture (Wang et al., 2001) studies 575 
showing that mTOR regulation of p70S6K is inversely related to eEF2 phosphorylation. 576 
Together with an increase in initiation due to prolonged mTOR phosphorylation, the suppression 577 
of eEF2 phosphorylation (and improved elongation) may have contributed to the rise in 578 
myofibrillar MPS for C+P. Finally, we acknowledge that from our findings it is unclear whether 579 
alterations in translational signalling with C+P are causative, or merely associative with the 580 
increase in myofibrillar MPS. 581 
In conclusion, we have shown that when protein is co-ingested with carbohydrates after 582 
cycling exercise myofibrillar, but not mitochondrial, protein synthesis is increased. It is possible 583 
that frequent post-endurance exercise protein ingestion may promote muscle hypertrophy over 584 
time. Thus, the implications of larger, more powerful muscles for cyclists should be carefully 585 
considered prior to making nutritional recommendations. On the other hand, increased 586 
myofibrillar MPS may enhance the synthesis of proteins associated with fatigue resistance or 587 
may serve to counteract a fasted-state rise in myofibrillar protein breakdown during and 588 
immediately following endurance exercise. Thus, the maintenance and structural integrity of 589 
contractile proteins may be enhanced. Thus, we posit that post-endurance exercise protein 590 
nutrition could have important implications for the adaptive response to endurance exercise and, 591 
potentially, the recovery of muscle function. Finally, the synthetic response of different protein 592 
fractions to endurance exercise and protein ingestion may be dependent on the individual 593 
training status, intensity and duration of the exercise bout, as well as the timing and quantity of 594 
post-EE nutrient strategies. Future studies should seek to address these issues in greater depth. 595596 
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Author contributions 597 
LB, OCW, SRJ, KB and KDT contributed to the conception and design of the experiment. 598 
LB, AP, OCW, SRJ, KB and KDT contributed to the collection of the data. LB, AP, OCW, SRJ, 599 
AS, KS, KB and KDT contributed to the analysis and interpretation of the data. LB, AP, OCW, 600 
SRJ, KS, KB and KDT contributed to drafting or revising the content of the manuscript. 601 
 602 
The authors declare no conflicts of interest. 603 
 604 
Acknowledgements 605 
We would like to thank Daniel Moore, Debbie Rankin and Robert Bagabo, for cooperation 606 
during method development and data analyses and Ms Lorna Webb for assistance during data 607 
collection. We would also like to extend our appreciation to the participants for their time and 608 
effort. This study was funded by a research grant from GlaxoSmithKline Nutritional Healthcare, 609 
Brentford, UK to KDT. 610 
 611 
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 33
 
 
 
 
 
CHO C+P 
Ex time (min) 
 
25-30 55-60 85-90 25-30 55-60 85-90 
HR (bpm) 
 
172 ± 5 172 ± 3 172 ± 1 174 ± 4 172 ± 1 171 ± 2 
Cadence (rpm) 
 
*84 ± 5 79 ± 5 80 ± 3 *84 ± 4 80 ± 3 79 ± 5 
V&O2 (mL·kg¯1·min¯1) 
 
53.2 ± 2.0 50.7 ± 2.0 50.9 ± 2.0 52.6 ± 1.9 50.5 ± 1.9 50.9 ± 2.6 
%V&O2max 
 
80 ± 3 77 ± 2 76 ± 5 79 ± 2 76 ± 2 77 ± 3 
RER 
 
0.89 ± 0.01 0.87 ± 0.02 0.87 ± 0.02 0.91 ± 0.01 0.87 ± 0.02 0.87 ± 0.01 
RPE 16 ± 1 17 ± 1 17 ± 1 16 ± 1 17 ± 1 18 ± 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 1. Exercise variables 
Values are the average recording over each 5 min phase and are presented as mean ± SEM. * indicates significant 
difference at 25-30 min compared with other time phases (P < 0.05). 
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Treatment CHO CHO C+P C+P CHO C+P 
 
Time post-exercise (h) 0 4 0 4 Fold-change 
0 – 4 
Fold-change 
0 – 4 
 
AMPKThr172 
 
1.40 ± 0.04 
 
1.36 ± 0.06 
 
1.45 ± 0.06 
 
1.33 ± 0.05 
 
0.95 ± 0.03 
 
0.89 ± 0.08 
 
AktThr308 
 
 
0.80 ± 0.02 
 
0.73 ± 0.02 
 
0.74 ± 0.03 
 
0.77 ± 0.05 
 
0.92 ± 0.04 
 
1.04 ± 0.04 
4E-BP1Thr37 
 
1.71 ± 0.07 1.77 ± 0.09 1.66 ± 0.08 1.77 ±0.07 1.03 ± 0.02 1.04 ± 0.02 
eEF2Thr56 0.65 ± 0.11 0.91 ±0.11* 0.71 ± 0.08 0.75 ± 0.07† 1.40 ± 0.11*† 1.05 ± 0.12 
 
 
mTORSer2448 
 
1.53 ± 0.21 1.40 ± 0.07 1.49 ± 0.12 1.58 ± 0.13† 0.91 ± 0.09 1.05 ± 0.05† 
P38 MAPKThr180 
 
0.65 ± 0.07 0.62 ± 0.10 0.61 ± 0.14 0.64 ± 0.08 1.15 ± 0.21 1.28 ± 0.08 
p70S6KThr389 
 
1.13 ± 0.06 1.20 ± 0.04 1.17 ± 0.03 1.32 ± 0.06* 1.06 ± 0.04 1.13 ± 0.05* 
PRAS40Ser246 
 
0.83 ± 0.12 0.80 ± 0.13 0.70 ± 0.08 0.72 ± 0.06 0.97 ± 0.08 1.03 ± 0.08 
 
 
 
 
 
 
Table 2. Post-exercise protein phosphorylation 
Values are means ± SEM protein phosphorylation at 0 and 4 hours post-exercise (arbitrary units) and fold-change over 4 h post-exercise. 
Protein phosphorylation is expressed as a ratio of phospho to total protein. * indicates significant increase in phosphorylation at 4 h 
compared with 0 h post-exercise (P < 0.05). † indicates significant difference between CHO and C+P at 4 h post-exercise (P < 0.05); n = 8. 
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 35
Figure1. Schematic diagram of the experimental protocol. 
 
Figure 2. (A) Serum insulin and (B) Plasma urea concentrations in CHO and C+P trials. Means 
within each trial with different subscripts are significantly different from each other (P < 0.05). 
*: significant difference between CHO and C+P at each respective time point. Values are means 
± SEM; n = 10. 
 
Figure 3. Plasma concentrations of (A) phenylalanine, (B) leucine and (C) threonine. Means 
within each trial with different subscripts are significantly different from each other (P < 0.05). *: 
significant difference between CHO and C+P (P < 0.05). Values are means ± SEM; n = 10. 
 
Figure 4. Enrichment of 13C6 phenylalanine in plasma. % t/T: percentage of tracer-to-tracee ratio. 
Values are means ± SEM; n = 10. 
 
Figure 5. Post-exercise protein phosphorylation of (A) mTORSer2448, (B) eEF2Thr56 and (C) 
p70S6KThr389 *: significant time effect for protein phosphorylation at 4 h compared with 0 h post-
exercise (P < 0.05) †: significant group effect for protein phosphorylation at 4 h post-exercise 
between CHO and C+P (P < 0.05). Values are means ± SEM; n = 8. 
 
Figure 6. Myofibrillar (n = 10) and mitochondrial (n = 8) fractional synthetic rate. *: significant 
difference between CHO and C+P (P < 0.05). Values are means ±SEM. 
 
 
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-180 2400 60 120 180-120 -60
* * * * ** * * ** * * *
Exercise
Time (min)
Biopsy
Blood
Primed-continuous L-[ring-¹³C6] phenylalanine infusion
Beverage
Figure 1
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Figure 2
-90 -60 -30 0 30 60 90 120 150 180 210 240
Se
ru
m
 
in
su
lin
 
co
n
ce
n
tra
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n
 
(µU
/m
L)
0
5
10
15
20
25
30
35
40 C+P 
CHO 
a
a
b
c
a
a
a
*
*
b
 Exercise
 
b
a
a a
aa
A
Time (min)
-90 -60 -30 0 30 60 90 120 150 180 210 240
Pl
a
sm
a
 
u
re
a
 
co
n
ce
n
tra
tio
n
 
(m
m
o
l/L
)
0
3
4
5
6
7
CHO 
C+P 
b b b b
b b
 Exercise
b
a
a
a a a a
a a a
a
a
 
 
*
B
 Time (min)
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Figure 3
Pl
as
m
a
 
le
u
ci
n
e 
co
n
ce
n
tra
tio
n
 
(µ m
ol
/L
)
200
250
300
350 CHO 
C+P 
b
b
c
c
c
b
b
ab
 
*B
 Time (min)
-180 -120 -60 0 60 120 180 240
Pl
a
sm
a
 
ph
e
n
yla
la
n
in
e
 
co
n
ce
n
tra
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(µm
o
l/L
)
0
40
50
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80
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100
CHO 
C+P 
a
b
b
bc
c c c
b
b
a
a ab
ab
a
a c
c
ac
ab
a a
 
 Exercise
 
A
B
 Time (min)
-180 -120 -60 0 60 120 180 240
Pl
a
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a
 
le
u
ci
n
e
 
co
n
ce
n
tra
tio
n
 
(
0
50
100
150
a
a
a
ab
ab ab
a
b b b b
c c c
bc c
b
b b
 Exercise
 
 Time (min)
-180 -120 -60 0 60 120 180 240
Pl
a
sm
a 
th
re
on
in
e
 
co
n
ce
n
tra
tio
n
 
(µm
o
l/L
)
0
50
100
150
200
250
300
350
CHO
C+P
 
a
ab ab
ab
b b b b b b b
b b
a
a
a a
a
b
b
b b
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a
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*
 Exercise
 
C
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Figure 4
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1
3
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6
]
 
p
h
e
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a
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a
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e
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m
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(
%
 
t
/
T
)
7
8
9
10 CHO 
C+P
Time post-exercise (min)
-90 -60 -30 0 30 60 90 120 150 180 210 240
P
l
a
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m
a
 
L
-
[
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 Exercise
 
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Figure 5
C+P CHO
p 
-
 
m
TO
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Se
r2
44
8 
(A
rb
itr
ar
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u
n
its
)
0.0
0.8
1.0
1.2
1.4
1.6
1.8
2.0 0h Post-ex 
4h Post-ex
 
† 
 
(A
rb
itr
a
ry
 
u
n
its
)
0.8
1.0
1.2
0h Post-ex 
4h Post-ex *
 
† 
A
B
C+P CHO
p 
-
 
eE
F2
 
Th
r5
6 
(A
rb
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ry
 
u
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0.0
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0.8
C+P CHO
p 
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p7
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S6
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89
 
(A
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itr
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ry
 
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0.6
0.8
1.0
1.2
1.4
1.6
0h Post-ex
4h Post-ex 
*
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Figure 6
M
u
s
c
l
e
 
p
r
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i
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F
S
R
 
(
%
·
h
-
1
)
0.06
0.08
0.10
0.12
CHO 
C+P
*
Myofibrillar Mitochondrial
M
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0.02
0.04) by guest on April 19, 2014
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