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Differential effects of resistance and endurance exercise in the fed state on signaling molecule phosphorylation and protein synthesis in human muscle

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Resistance (RE) and endurance (EE) exercise stimulate mixed skeletal muscle protein synthesis. The phenotypes induced by RE (myofibrillar protein accretion) and EE (mitochondrial expansion) training must result from differential stimulation of myofibrillar and mitochondrial protein synthesis. We measured the synthetic rates of myofibrillar and mitochondrial proteins and the activation of signalling proteins (Akt-mTOR-p70S6K) at rest and after an acute bout of RE or EE in the untrained state and after 10 weeks of RE or EE training in young healthy men. While untrained, RE stimulated both myofibrillar and mitochondrial protein synthesis, 67% and 69% (P < 0.02), respectively. After training, only myofibrillar protein synthesis increased with RE (36%, P = 0.05). EE stimulated mitochondrial protein synthesis in both the untrained, 154%, and trained, 105% (both P < 0.05), but not myofibrillar protein synthesis. Acute RE and EE increased the phosphorylation of proteins in the Akt-mTOR-p70S6K pathway with comparatively minor differences between two exercise stimuli. Phosphorylation of Akt-mTOR-p70S6K proteins was increased after 10 weeks of RE training but not by EE training. Chronic RE or EE training modifies the protein synthetic response of functional protein fractions, with a shift toward exercise phenotype-specific responses, without an obvious explanatory change in the phosphorylation of regulatory signalling pathway proteins.
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J Physiol 586.15 (2008) pp 3701–3717 3701
Differential effects of resistance and endurance exercise
in the fed state on signalling molecule phosphorylation
and protein synthesis in human muscle
SarahB.Wilkinson
1,StuartM.Phillips
1,PhilipJ.Atherton
2,RekhaPatel
2, Kevin E. Yarasheski3,
Mark A. Tarnopolsky4and Michael J. Rennie2
1Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada
2School of Biomedical Science, Graduate Entry Medical School, University of Nottingham, Derby, UK
3Department of Internal Medicine, Division of Metabolism, Endocrinology and Lipid Research, Washington University School of Medicine,
St Louis, MO, USA
4Departments of Pediatrics and Neurology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
Resistance (RE) and endurance (EE) exercise stimulate mixed skeletal muscle protein synthesis.
The phenotypes induced by RE (myofibrillar protein accretion) and EE (mitochondrial
expansion) training must result from differential stimulation of myofibrillar and mitochondrial
protein synthesis. We measured the synthetic rates of myofibrillar and mitochondrial proteins
and the activation of signalling proteins (Akt–mTOR–p70S6K) at rest and after an acute
bout of RE or EE in the untrained state and after 10 weeks of RE or EE training in young
healthy men. While untrained, RE stimulated both myofibrillar and mitochondrial protein
synthesis, 67% and 69% (P<0.02), respectively. After training, only myofibrillar protein
synthesis increased with RE (36%, P=0.05). EE stimulated mitochondrial protein synthesis
in both the untrained, 154%, and trained, 105% (both P<0.05), but not myofibrillar protein
synthesis. Acute RE and EE increased the phosphorylation of proteins in the Akt–mTOR–p70S6K
pathway with comparatively minor differences between two exercise stimuli. Phosphorylation
of Akt–mTOR–p70S6K proteins was increased after 10 weeks of RE training but not by EE
training. Chronic RE or EE training modifies the protein synthetic response of functional
protein fractions, with a shift toward exercise phenotype-specific responses, without an obvious
explanatory change in the phosphorylation of regulatory signalling pathway proteins.
(Received 11 March 2008; accepted after revision 11 June 2008; first published online 12 June 2008)
Corresponding author S. M. Phillips: Exercise Metabolism Research Group, Department of Kinesiology, McMaster
University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1. Email: phillis@mcmaster.ca
Resistance exercise (RE) training results in increases in
strength and muscle fibre cross-sectional area (CSA)
(Hasten et al. 2000; Balagopal et al. 2001) and, particularly,
of myofibrillar protein myosin and actin. Endurance
exercise (EE) training is characterized by fatigue resistance
due in part to increased oxidative capacity secondary to
increased mitochondrial density and thus mitochondrial
protein (Morgan et al. 1971; Gollnick et al. 1972; Hoppeler
et al. 1973; Fink et al. 1977). Acutely, the rate of mixed
muscle protein synthesis (MPS), an average measure of all
muscle proteins, is stimulated by resistance (Chesley et al.
1992; Yarasheski et al. 1993; Biolo et al. 1995; MacDougall
et al. 1995; Phillips et al. 1997) and endurance (Carraro
et al. 1990; Sheffield-Moore et al. 2004) exercise, but
the nature of responses of classes or individual proteins
within this mixed protein response is unknown. Resistance
and endurance exercise must induce protein synthesis
in differing fractions of muscle protein, most obviously
myofibrillar versus mitochondrial proteins. Chronic
repeated performance of resistance exerciseinduces a more
rapid but less long lived rise in mixed muscle protein
synthesis after acute resistance exercise than occurs in
untrained muscle (MacDougall et al. 1995; Phillips et al.
1997; Phillips et al. 1999, 2002; Rasmussen & Phillips,
2003; Tang et al. 2008). Furthermore, endurance exercise
training (Short et al. 2004; Pikosky et al. 2006) as well as
resistance training (Yarasheski et al. 1993, 1999; Phillips
et al. 2002; Kim et al. 2005) have been shown to increase
mixed muscle protein synthesis for up to 2 and 4 days,
respectively, after the last exercise session
Feeding (Cuthbertson et al. 2005; Fujita et al. 2007b)and
resistance exercise (Coffey et al. 2006; Dreyer et al. 2006;
Eliasson et al. 2006; Fujita et al. 2007a) can independently
activate translation initiation via multiple signalling
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2008 The Authors. Journal compilation C
2008 The Physiological Society DOI: 10.1113/jphysiol.2008.153916
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3702 S. B. Wilkinson and others J Physiol 586.15
proteins (Kimball & Jefferson, 2006), including the
protein kinase B (Akt)–mammalian target of rapamycin
(mTOR)–70 kDa S6 protein kinase (p70S6K) signal trans-
duction pathway. Several recent investigations have found
that the activation of proteins in the Akt–mTOR–p70S6K
pathway are enhanced following resistance exercise if
amino acids or protein is ingested (Karlsson et al. 2004;
Koopman et al. 2007; Dreyer et al. 2008; Drummond
et al. 2008) versus resistance exercise alone. These
observations corroborate the evidence that provision of
amino acids/protein with concurrent resistance exercise
synergistically enhances muscle protein synthesis (Biolo
et al. 1997; Tipton et al. 1999; Rasmussen et al. 2000;
Borsheim et al. 2002, 2004; Miller et al. 2003; Tipton
et al. 2004). Therefore, in an effort to contextualize the
results of our planned measurements of protein synthesis
we also sought to measure phosphorylation of signalling
proteins of the Akt–mTOR–p70S6K (Nautilus, Vancouver,
USA) pathway shown by others to be responsive to
resistance exercise (Karlsson et al. 2004; Coffey et al. 2006;
Cuthbertson et al. 2006; Dreyer et al. 2006; Eliasson et al.
2006; Fujita et al. 2007a;Terziset al. 2007), and endurance
exercise (Coffey et al. 2006; Mascher et al. 2007).
Exercise mode-specific responses have been observed in
terms of AMP-activated protein kinase (AMPK) activation
(Nielsen et al. 2003; Frosig et al. 2004; Coffey et al. 2006;
Dreyer et al. 2006; Koopman et al. 2006; Drummond
et al. 2008) and inhibition of mTOR in rodents has been
speculated to be the underlying reason why resistance
exercise is anabolic whereas endurance exercise is not
(Atherton et al. 2005). Phosphorylation and enzyme
activity of AMPK, which is increased during and briefly
after endurance exercise (Nielsen et al. 2003; Frosig et al.
2004; Coffey et al. 2006) as well as resistance exercise
(Dreyer et al. 2006; Koopman et al. 2006), has been
associated with inhibition of mTOR, p70S6K and 4EBP-1
phosphorylation in rat skeletal muscle (Bolster et al. 2002;
Atherton et al. 2005; Thomson et al. 2008); whether a
similar mechanism is at play in human muscle is unknown.
Membrane-associated integrins respond to mechanical
stimuli by transducing the signal into a cellular response.
Focal adhesion kinase (FAK) has been suggested as a
possible integrator of load-activated stimuli and integrin
signalling in hypertrophying skeletal muscle because
stretch has been shown to activate FAK in muscle (Fluck
et al. 1999). Moreover, FAK phosphorylation is reduced
in unloaded human skeletal muscle (de Boer et al. 2007)
suggesting that it is a reasonable candidate for sensing
mechanical loads.
We hypothesized that in an untrained state an acute
bout of resistance or endurance exercise would elicit
a non-exercise-specific rise in the synthetic rate of all
protein fractions. As more and more acute bouts are
carried out (i.e. comprising a training programme),
increasingly exercise-mode and protein-specific protein
synthetic responses must occur. Thus, the primary
aim of this study was to measure myofibrillar and
mitochondrial protein synthesis after an acute bout of
either resistance or endurance exercise and repeat this
after 10 weeks of resistance or endurance exercise training.
The second aim of this study was to examine the effects
of bouts of different exercise modes carried out in
the fed state, on the activation of signalling proteins
regulating protein synthesis (in the Akt–mTOR–p70S6K
pathway), to see how these responses were affected by
endurance or resistance training. We hypothesized that
the phosphorylation of all anabolic signalling molecules
would be changed appropriately (increases or decreases
of phosphorylation) to promote muscle protein synthesis
after acute resistance exercise but possibly suppressed
after endurance exercise, at least in the untrained state
for the time required for AMP/ATP ratios to normalize.
We considered that only endurance exercise would be
affected by this mechanism, despite evidence that AMPK
is activated with resistance exercise (Dreyer et al. 2006;
Koopman et al. 2006; Drummond et al. 2008), since
ATP turnover was more than 80-fold greater than that
during resistance exercise. After training we expected a
different response in as much as training would attenuate
the rise in protein synthesis (MacDougall et al. 1995;
Phillips et al. 1997, 1999, 2002; Rasmussen & Phillips,
2003; Tang et al. 2008) and that this would be accompanied
by a reduced extent of change of the phosphorylation
of the regulatory anabolic proteins. We also undertook
to measure the responses of molecules likely to regulate
activities of members of the Akt–mTOR–p70S6K pathway,
namely AMPK and FAK.
Methods
Subjects
Ten healthy men (mean ±S.E.M.: age, 20.5 ±0.6 years;
mass, 89.4 ±4.8 kg; height, 179.6 ±2.2 cm, ˙
VO2,peak:
43.9 ±2.1mlkg
1min1) were recruited for the study.
Subjects were not actively participating in any weightlifting
activities or any programmed endurance activity
(<1dayweek
1) for >8 months prior to the study. Each
participant was advised of the purposes of the study and
associated risks. Participants were required to complete a
health questionnaire and based on responses were deemed
healthy. All subjects were non-smokers and were not
taking any medication. All subjects gave their written
and verbal informed consent prior to participation. The
Hamilton Health Sciences Research Ethics Board approved
the project, which complies with all standards set by the
Declaration of Helsinki on the use of human research
subjects.
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3703
Experimental protocol
The subjects underwent two metabolic investigations
(described later) separated by 10 week of unilateral leg
resistance or endurance exercise training. Participants
served as their own controls, one leg being assigned
to the resistance exercise and one leg to the endurance
exercise condition. The choice of legs for training mode
was randomized in a counter-balanced manner. We chose
to use a unilateral model of exercise for within-subject
comparisons of adaptations, because this had substantial
advantage in not having to recruit and train two groups of
individuals in two different exercise modes and also being
able to directly compare the metabolic responses in the
same person (thereby increasing statistical power) and also
saving on resources by using only a single isotopic tracer
infusion per acute study. Thus, while we acknowledge that
our design does not reflect typical training behaviour,
it was not designed to do this. Rather we wished to
elucidate aspects of the muscle specific changes that under-
pin changes in muscle phenotype with various forms
of exercise training. For these purposes the design was
efficient and practical.
Exercise testing. At least 2 weeks before the first infusion
trial, subjects reported to the Exercise Metabolism
Research Laboratory for familiarization and explanation
of all procedures. Afterwards, the baseline physiological
characteristics of the legs were measured, in a randomized
order. Single leg knee extension strength measurements
were determined for both legs over several days of testing
before the first and last metabolic investigation. Strength
measures included isotonic single repetition maximum
(1RM) (Nautilus, Vancouver, WA, USA), isometric 1RM
and isokinetic (0.52 rad s1) 1RM (Biodex-System 3,
Biodex Medical Systems, Shirley, NY, USA). Participants
completed three ˙
VO2,peak testsonseparatedaysover
the 2 week period prior to the first infusion metabolic
investigation: a two leg ˙
VO2,peak test and a single leg ˙
VO2,peak
test on each leg.
To estimate isotonic 1RM for the knee extension, sub-
jects were seated with their hips at 90 deg and their back
against a backrest inclined at 30 deg from horizontal. The
fulcrum of the machine was aligned with the lateral aspect
of the midline of the subject’s knee. The leg pad of the
machine was positioned 5cm abovethesubjectsankle
and the rotation arm was positioned so that the subject’s
knee was bent to 90 deg. The subject’s ‘settings’ for pin
placements for seating in the machine were recorded and
kept constant throughout the study. A full repetition was
when the subject was able to move the weight through
an arc of 80 deg (from 90 deg to 170 deg). Subjects
warmed up with 8–10 repetitions using a light weight.
Subjects then performed a single best effort at a weight
estimated to be the subject’s 1RM based on body weight
and height by an experienced trainer. The weight was
increased or decreased depending on whether the sub-
ject could just manage to perform the task. This estimated
1RM was checked on two subsequent occasions by simply
having the subject report to the lab and asking them to
once more perform a 1RM at the previously determined
weight. For a ‘true’ 1RM to be determined, the coefficient
of variation (CV) between two attempts had to be less than
5%. For no subject did it require more than three attempts
to determine this 1RM.
Isometric and isokinetic (concentric at an angular
velocity of 0.52 rad s1) knee extensor peak torques were
determined using the dynamometer after having a prior
familiarization on the dynamometer. The order of testing
for each mode was randomized. Subjects had their
shoulders strapped to the chair and the chair was adjusted
so that the lateral aspect of the midline of the subject’s
knee lined up with the dynamometer fulcrum. Chair
settings were recorded and set to the same settings for
subsequent dynamometer testing. The participant’s knee
was maintained at an angle of 70 deg during three
isometric repetitions (5 s) with 90 s of rest between
repetitions. For the isokinetic contractions, subjects
carried out 10 repetitions throughout the complete 65 deg
range of motion. The subjects were given more than
2 min of recovery time between each exercise mode. All
subjects were verbally encouraged to voluntarily produce
their maximal force and given visual feedback of their force
production. The highest peak torque value was considered
as the maximal value.
˙
VO2,peak values for both legs together and separately
were determined using an incremental exercise test to
exhaustion (usually 7–12 min) on a Lode cycle ergometer
(Groningen, Netherlands) with oxygen uptake measured
continuously (AEI Technologies, Pittsburgh, PA, USA).
Exhaustion was defined at a respiratory exchange ratio
>1.2, a heart rate within 5 beats min1of the subject’s
age-predicted maximal heart rate (for the two leg test), and
the inability to maintain 60 r.p.m. on the cycle ergometer
at the set workload. The participant’s foot was secured to
the pedal to enable transmission of force when pushing
and pulling the pedals. Prior to training, participants’
heart rates during the single leg test were 95 ±1% of
the maximum heart rate achieved in the two leg test.
Following training the EE leg achieved 98 ±3% and the
RE leg achieved 95 ±4% of the two leg maximum heart
rate. From the single leg test on the leg assigned to end-
urance exercise activity, a workload designed to elicit a
˙
VO2equivalent to 75% of the subject’s single leg ˙
VO2,peak
was selected and confirmed using a 15 min test ride 1 week
before the metabolic investigation.
Muscle metabolic investigation. Subjects participated in
two investigations before and after training (see Fig. 1).
They were asked to refrain from any strenuous exercise
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3704 S. B. Wilkinson and others J Physiol 586.15
for 2 days beforehand. Therefore, the post-training
investigations were carried out at least 2 days after the
last training session (9/10 subjects performed the study
4 days post-exercise and only one participant performed
the testing 2 days post-exercise due to scheduling).
Participants were asked to record what they ate in the after-
noon and evening before the first investigation and were
asked to consume the same meals again before the second
investigation. Subjects were instructed not to consume
any food after 20.00 h on the night beforehand and not
to consume beverages containing caffeine for 24 h. On
each study trial day, subjects consumed a defined formula
beverage (2000 kJ; 82 g carbohydrate, 20 g protein and 8 g
fat, Boost R, Novartis Nutrition Corporation, Mississauga,
ON, Canada) in the morning (05.00 h) after an overnight
fast. Two and a half hours later (effectively postabsorptive),
subjects reported to the exercise metabolism laboratory.
A 20-g polyethylene catheter was then inserted into an
antecubital vein from which a baseline blood sample was
taken to determine background amino acid enrichment.
After the baseline blood sample was drawn, a primed
constant infusion of D3-α-ketoisocaproic acid (D3-α-KIC
prime: 10 μmol kg1; infusion rate: 9 μmol kg1h1)was
initiated. All isotopes were purchased from Cambridge
Isotopes (Andover, MA, USA), dissolved in 0.9% saline,
filtered through a 0.2 μm filter and infused using a
calibrated syringe pump (KD Scientific, Holliston, MA,
USA). The infusion protocol was designed so that
steady state was achieved within 1 h in both the intra-
muscular and plasma pools (Moore et al. 2005; Tang
et al. 2008). Immediately after the beginning of the
infusion the participant ingested an aliquot of Boost R
(75% carbohydrate, 18% protein and 7% fat), which
they ingested every 30 min (22 times) for the duration
of the metabolic study. The total energy content of all
the drinks provided 75% of the daily energy requirements
based on the Harris–Benedict equation (Roza & Shizgal,
1984), using a physical activity level of 1.5 (providing
1.1 g of protein per kg body mass). On the basis of the
Muscle Biopsy
(leg 1)
D3- -ketoisocaproic acid
Exercise
leg 1
Muscle Biopsy
(leg 2)
Blood
Samples
0
Rest 0h 4 h
Exercise
leg 2
Time (h) 145 9610
0h
Rest 4 h
Drink
ingestion
Figure 1. Infusion trial protocol
leucine content in protein and the food composition
we added sufficient tracer to the drink to maintain
expected blood D3-α-KIC labelling of 7–8%. After base-
line sampling, subjects rested for 1 h, during which time
another polyethylene catheter was inserted into the contra-
lateral arm to sample blood for the remainder of the
protocol. After 1 h, a blood sample and a percutaneous
quadriceps muscle biopsy were obtained using a 5 mm
Bergstr¨
om biopsy needle modified for manual suction
under local anaesthesia (1% xylocaine). The muscle was
dissected free of any visible fat and connective tissue and
was immediately frozen in liquid nitrogen and stored
at 80C prior to analysis. In the untrained situation
a single biopsy was taken, but after training, biopsies
were taken from each leg. After resting for another 3 h,
another blood sample and two more muscle biopsies
were taken (one from each leg) via separate incisions.
Half the participants were randomized to perform the
resistance exercise first and the others performed the end-
urance first. After 10 week of training, the order of exercise
was reversed for each participant so that participants
who performed the exercised with resistance exercise first
when untrained, underwent endurance exercise first in
the untrained situation, and endurance first after training.
The acute resistance exercise consisted of five sets of 8–10
repetitions at 80% 1RM of single leg knee extension and
the endurance exercise consisted of single leg cycling for
45 min at 75% ˙
VO2,peak. The same relative intensities for
the knee extension and single leg cycling were used in
the second trial after training and re-testing of strength
and ˙
VO2,peak. After the first exercise bout, in whatever
mode, a blood sample was taken and then a percutaneous
quadriceps muscle biopsy was taken from the leg that had
exercised; the second acute bout of exercise in a different
mode was then performed followed by blood sampling and
a percutaneous muscle biopsy taken from the exercised
leg. The participants then rested and blood samples and
biopsies were taken 4 h after each respective mode of
exercise.
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2008 The Authors. Journal compilation C
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3705
Training protocol. After the aforementioned baseline
testing was complete, subjects underwent a 10 week
training programme in which one leg carried out
a resistance-training programme, consisting of knee
extension exercise, while the contralateral leg carried
out an endurance training programme consisting of
one-legged cycling on a cycle ergometer specially
designed for this study. The participant’s foot was
secured to the pedal to enable transmission of
force when pushing and pulling the pedals. Subjects
alternated training between resistance and endurance
training each day so that the first week they
performed endurance training three days and resistance
training twice. The following week, resistance training
was carried out three times and endurance exercise
twice.
The training for the resistance-trained leg began with
each session consisting of three sets with 10–12 repetitions
persetat80%oftheir1RM.Onthethirdweek,the
volume of training was increased to four sets with 8–10
repetitions per set at 80% 1RM with the last set performed
to failure (unable to lift the weight). If the participant
could lift more than 10 repetitions on the last set, the
training weight was increased on the subsequent session.
This mode of adjusting training weight was carried
on for the remainder of the protocol. On the fourth
and fifth weeks, subjects performed five sets of 8–10
repetitions. At weeks six and seven, the training weight
was increased so that participants’ goal was to lift eight
repetitions for five sets. During weeks 8 to 10, participants
completed five sets of six to eight repetitions. After the
completion of the training protocol, subjects were tested
again to determine their dynamic, isometric and isokinetic
strength.
The training for the endurance-trained leg began
with exercise for 30 min at 75% of predetermined
single leg ˙
VO2,peak for 2 weeks. Participants’ heart rates
were monitored throughout each training session. As
a participant adapted to the training, workload was
increased to elicit a heart rate equivalent to the heart
rate observe at 75% single leg ˙
VO2,peak in the untrained
state. The duration of the exercise was increased in week
3 to 45 min. During the midweek session of weeks 4,
6, 8 and 10, participants completed interval sessions.
In brief, participants alternated between two workloads
designed to elicit heart rates of 140–150 and 160–170
for the duration of the training session. During week
5, training workload was adjusted so that participants
were training at a heart rate of 160. For participants with
a below or above average maximal heart rate, workload was
adjusted on an individual basis. In week 6, training time
was increased to 1 h. After the completion of the training
protocol subjects’ two leg and single leg ˙
VO2,peak was tested
again on separate days.
Analytic methods
Blood sample analysis. Blood samples were collected into
heparinized evacuated containers. Whole blood (100 μl)
was added to ice-cold perchloric acid (PCA; 0.6 M,
500 μl), mixed and allowed to sit on ice for 10 min to
precipitate all proteins. This mixture was then centrifuged
at 20 000 gfor 2 min at 4C. The PCA was neutralized
with 250 μl of 1.25 MKHCO3and the reaction was
allowed to proceed on ice for 10 min. Samples were then
centrifuged at 4000 gfor 2 min at 4C. The supernatant
was stored at 20C for further analysis of blood amino
acid concentrations (Wilkinson et al. 2007) and leucine
enrichments. In the fed state there is a rapid equilibration
between leucine and α-KIC such that plasma and muscle
intracellular enrichments converge (Chinkes et al. 1996).
The PCA blood sample was derivatized by adding 50 μl
of N-methyl-N-t-butyl-dimethylsilyl-trifluoroacetamide
+1% t-butyl-dimethylchlorosilane (MTBSTFA +1%
TBDMCS, Regis Chemical). 2H3-Leu enrichment was
quantified using capillary gas chromatography–electron
impact ionization–quadrupole mass spectrometry
(GCMS; GC Hewlett Packard 6890, Palo Alto, CA,
USA; MSD Agilent 5973, Palo Alto, CA, USA) by
monitoring ions at m/z200 and 203. Plasma was
obtained by centrifuging the evacuated tube at 4C
for 10 min at 4000 g. Plasma was stored at 20C for
quantifying insulin and glucose concentrations. Insulin
levels were determined using a commercially available
radioimmunoassay kit from Diagnostic Products Corp.
(Los Angeles, CA, USA). Glucose levels were determined
on neutralized blood PCA extracts using a standard
enzymatic method (Passoneau & Lowry, 1993).
Muscle biopsy sample analysis. Needle biopsies from the
vastus lateralis were obtained under local anaesthesia (1%
xylocaine). A 5 mm Bergstr¨
om biopsy needle modified
for manual suction was used to obtain 100–200 mg of
muscle tissue from each biopsy. Biopsies were obtained
from separate incisions from the same leg during each
session. The muscle was dissected free of any visible fat
and connective tissue and was immediately frozen in liquid
nitrogen and stored at 80C prior to analysis.
One piece of frozen wet muscle (10–15 mg) was
homogenized using the method described by Henriksson
et al. (1986) to a 50 times dilution. The homogenate
was subsequently analysed to determine the maximal
activity of CS on a spectrophotometer (Ultrospec 3000
pro UV/Vis) using a method described by Carter et al.
(2001) and corrected for protein content using a Bradford
assay (Passoneau & Lowry, 1993). A second portion
of wet muscle (10–20 mg) was saved for Western
blotting; 10 mg wet muscle was added to 150 μlof
homogenizing buffer (1 mMNa3VO4,50mMNaF, 40 mM
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3706 S. B. Wilkinson and others J Physiol 586.15
β-glycerolphosphate, 20 mMsodium pyrphosphate, 0.5%
Triton X-100, complete mini protease inhibitor tabs
(Roche, Indianapolis, IN, USA) in Tris buffer pH 7.2).
Afterthoroughhomogenizationonice,homogenateswere
spun in a 4C centrifuge at 4500 gfor 10 min. The super-
natant was removed and stored at –80C until Western
blot analysis. A small portion was saved for Bradford assay
for protein (Passoneau & Lowry, 1993).
Aliquots from the homogenates were boiled at 100C
for 5 min in 4 ×sample buffer (300 mMTr i s-H C l,
pH 6.8, 50% glycerol, 8% SDS, 20% β-mercaptoethanol,
0.02% bromophenol blue). Fifty micrograms of sample
were added per lane and separated by electrophoresis
in running buffer (0.1% SDS, 192 mMglycine, 25 mM
Tris base) on 7.5–15% SDS-PAGE gels at 100 V until
dye marker had passed through the stacking layer and
then at 200 V until the dye marker reached the gel
bottom. After electrophoresis, proteins were transferred to
polyvinylidene difluoride (PVDF) membranes (Bio-Rad,
Hercules, CA, USA) at 100 V for 2 h at 4C in transfer
buffer (192 mMglycine, 25 mMTris base, 10% methanol).
After transfer, the PVDF membranes were placed in
blocking buffer: 5% BSA in Tris-buffered saline (50 mM
Tris, 150 mMNaCl) and 0.1% Tween-20 (TBST) for
1 h. Blots were incubated in primary antibody in 5%
BSA in TBST overnight at 4C with constant agitation.
Thenextmorning,blotswerewashedinTBSTthree
times for 5 min and then incubated with secondary anti-
body (Amersham Biosciences, Little Chalfont, UK) in
5% BSA in TBST for 1 h at room temperature, with
continual agitation. Blots were washed in TBST three
times for 5 min and then incubated for 5 min with Chemi
GlowTM chemiluminescence reagent (Alpha Innotech,
San Leandro, CA). Optical density measurements were
obtained with a CCD camera, mounted in a Fluorchem
SP imaging system (Alpha Innotech). Once the image
was captured, densiometric analysis was performed using
AlphaEaseFC software (Alpha Innotech). After detection
of the phosphorylated antibody, the antibodies were
stripped off the PVDF membrane by incubating the
blot in stripping buffer (25 mMglycine-HCl, pH 2.0, 1%
SDS) for 1 h. We confirmed this stripping buffer was
effective by reincubating the blot with secondary anti-
body then Chemi GlowTM chemiluminescence reagent and
determining that no chemiluminescence was observable
after incubation with stripping buffer. The blot was
then washed in TBST three times for 10 min each,
blocked and exposed to the total primary antibody over-
night. All data are expressed as the ratio between the
phosphorylated protein to the total protein. Primary anti-
bodies were purchased from Cell Signalling (Beverly,
MA, USA) as follows: phospho-Akt (Ser473; 1 : 1000),
total-Akt (1 : 1000), phospho-AMPKα(Thr172; 1 : 1000),
total-AMPKα(1 : 1000), phospho-GSK3β(Ser9; 1 : 1000),
total-GSK3β(1 : 1000) phospho-mTOR (Ser2448; 1 : 1000),
total-mTOR (1 : 1000), phospho-S6 ribosomal protein
(Ser235/236; 1 : 1000), total-S6 ribosomal protein (1 : 1000);
and from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA, USA): phospho-eIF4E (Ser209; 1 : 500), total-eIF4E
(1 : 500), phospho-FAK (Tyr576/577, 1 : 1000), total-FAK
(1 : 1000), phopho-p70 S6K (Thr389; 1 : 1000), total-
p70S6K (1 : 1000).
The procedure for the isolation of the mitochondria was
adapted from Bezaire et al. (2004) and myofibrillar protein
from Bohe et al. (2001). Briefly, the remaining portion of
muscle was homogenized with a Dounce homogenizer in
ice-cold homogenizing buffer (0.1 mMKCl, 50 mMTr is ,
5m
MMgCl21mMEDTA, 10 mMβ-glycerophosphate,
50 mMNaF, 1.5% BSA pH 7.5). The homogenate was
transferred to a 2 ml Eppendorf tube and spun at 650 g
for 10 min at 4C. The supernatant was transferred
to another Eppendorf tube and spun at 10 000 gfor
10 min at 4C to pellet the sarcoplasmic mitochondria
(SMs). The supernatant was removed and discarded. The
pellet that remained from the original 650 gspin was
washed twice with homogenization buffer. A glass pestle
was utilized to forcefully homogenize the pellet in ice
cold homogenization buffer to liberate intermyofibrillar
mitochondria (IMs). The resulting mixture of myofibrils
and IMs was spun at 650 gfor 10 min at 4Ctopelletout
the myofibrils. The supernatant was removed and spun
at 10 000 gfor 10 min at 4C to pellet the IMs The myo-
fibrils and SM and IM pellets were washed three times
with homogenizing buffer containing no BSA. A BSA-free
homogenate was confirmed by electrophoresis. The myo-
fibrils were separated from any collagen by dissolving them
in 0.3 MNaCl, removing the supernatant and precipitating
them with 1.0 MPCA. All samples were washed once with
95% ethanol and then lyophilized to dryness (Savant,
Rockville, MD, USA).
We confirmed qualitatively using electron micro-
scopy that the mitochondrial fractions were enriched for
mitochondria and that the myofibrillar fraction contained
exclusively myofibrils. Furthermore, we measured citrate
synthase activity in the mixed muscle homogenate (homo-
genate prior to differential centrifugation) and each of
the mitochondrial fractions. We found that the citrate
synthase activities of the mitochondrial fractions were
3-fold higher than those of the mixed muscle homo-
genates.
It was determined the SM and IM protein pellets should
be combined in order to quantify 2H3-Leu enrichment.
The mitochondria- and myofibrillar-enriched proteins
were hydrolysed in 6 MHCl at 100C for 24 h. Hydro-
lysates were applied to a cation exchange resin (Dowex
AG-50W X8, 100–200 mesh H+form) and washed with
0.01 MHCl. The amino acids were eluted with 6 MNH4OH,
the heptafluorobutyric propyl esters were prepared (Reeds
et al. 2006), and 2H3-Leu enrichment in the myofibrillar-
and mitochondria-enriched protein fractions was
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Table 1. Strength measurements
Endurance exercise Resistance exercise
Untrained Trained % change Untrained Trained % change
1RM (kg) 48 ±362±330 ±647±379±3∗+ 75 ±10
Isometric peak torque (N) 299 ±15 285 ±16 4±4 293 ±16 346 ±17∗+ 20 ±7
Isokinetic peak torque (N m) 0.52 rad s1241 ±10 243 ±10 1 ±2 233 ±11 290 ±10∗+ 25 ±4
Values are means ±S.E.M.significantly different from those in the untrained state (same leg);+significantly different from endurance
exercise (same training state), P<0.05.
Table 2. Measures of endurance capacity
Endurance exercise Resistance exercise
Untrained Trained % change Untrained Trained % change
One leg ˙
VO2,peak (l min1)2.9±0.2 3.5 ±0.2∗+ 17 ±43.1±0.2 3.1 ±0.2 2±2
One leg ˙
VO2,peak as a percentage
of two 2 leg ˙
VO2,peak 77 ±290±2∗+ 19 ±381±281±30±3
CS activity (mol kg1protein h1)9.6±0.3 11.7 ±0.5∗+ 22 ±89.6±0.3 9.0 ±0.2 4±5
Values are means ±S.E.M.significantly different from untrained (same leg),+significantly different from resistance exercise (same
training state), P<0.05. Two leg ˙
VO2,peak (l min1): untrained, 3.9 ±0.2; trained, 3.8 ±0.2.
determined using gas chromatography–negative chemical
ionization–quadrupole mass spectrometry (GC Hewlett
Packard 6890; MSD Agilent 5973) by monitoring ions at
m/z349 and 352. Unfortunately, during processing, the
mitochondria-enriched samples from 3 participants were
lost, so data represent n=7. No myofibrillar samples were
lost (n=10).
Calculations. The fractional synthetic rate (FSR) of
myofibrillar and mitochondrial proteins was calculated
as the rate of tracer incorporation into the appropriate
muscle proteins using plasma 2H3-Leu to reflect the
precursor pool enrichment, according to the previously
published equation (Phillips et al. 1997).
Statistics. Sample size estimates were based on the ability
to detect a 25% difference between groups in mixed muscle
fractional synthetic rate using αat 0.05 and βat 0.2
(2-sided), with an estimated variance in the measure based
on past studies from our lab and from literature values. To
protect power we added two subjects to the final calculated
sample size estimate. Data were analysed using Statistica
(v 6.0, Statsoft, Tulsa, OK, USA) using a repeated measures
analysis of variance with planned comparisons. Where a
significant Fratio was observed, post hoc analysis using
Tukey’s test was utilized to identify individual differences.
Significance was set at P<0.05. Data are presented as
means ±S.E.M. All relevant comparisons were made for
time (within a trial), leg (comparison between EE and
RE at each time point within a given trial) and training
(comparison of the same leg at the same time point
between trials).
Results
Training and functional capacities
Nine out of 10 participants completed 100% of their
allocated training sessions. One subject completed 80%
of his training sessions, but his results did not differ sub-
stantially from those of the other participants so his data
are included. All participants maintained stable weight
throughout the study (data not shown). There were
no significant differences in any strength measurements
between legs before training (Table 1). As expected,
voluntary 1RM, isokinetic and isometric knee extension
strengths increased by 75, 25 and 20%, respectively, after
resistance exercise, with all increases being greater or
occurring only in the resistance trained leg. We have
previously shown that the same programme of resistance
training also elicits increases in muscle fibre and whole
muscle cross-sectional area with no changes in the contra-
lateral leg (Wilkinson et al. 2006; Tang et al. 2008).
Measurements of functional adaptations considered to
be characteristic of endurance training are presented in
Table 2. There were no significant differences in single
leg ˙
VO2,peak between legs before training. Two-leg (i.e.
whole-body) ˙
VO2,peak did not change significantly after
10 weeks of training, but there was a simultaneous 17%
increase in the endurance trained single leg ˙
VO2,peak
which was significant, with no change observed in the
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3708 S. B. Wilkinson and others J Physiol 586.15
resistance exercise leg. Before training, one leg ˙
VO2,peak
represented 77 ±2% of two leg ˙
VO2,peak capacity. This
capacity increased to 90 ±2% afterwards (P<0.05;
Table 2) only in the endurance trained leg and did not
change after resistance exercise training. Citrate synthase
activity, a marker of muscle mitochondrial content (Leek
et al. 2001), increased 22 ±8% (P<0.05) after endurance
training but no change was observed after resistance
exercise training.
Feeding during the protocol resulted in the expected
hyperaminoacidaemia, hyperinsulinaemia, and hyper-
glycaemia (Fig. 2). Isotopic equilibrium was attained in
plasma after 1 h of infusion and maintained throughout
the tracer infusion (data not shown).
Protein synthetic responses
Resting myofibrillar fractional synthetic rate (FSR) was
not different between legs prior to training (Fig. 3A). After
training, resting myofibrillar FSR was significantly greater
0
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a
b
bb
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[ TAA] (m M)
A
B
Figure 2
A, plasma glucose (mM) and insulin (IU ml1) concentrations. B,
whole-blood venous total amino acid concentration (mM). Values are
means ±S.E.M.Significantly different from values in untrained, letters
depict a significant effect of time, P<0.05.
in the resistance trained leg than in the endurance trained
leg. Prior to training, resistance exercise resulted in a 67%
increase in myofibrillar FSR. After 10 weeks of unilateral
resistance exercise training, the same relative intensity
of acute resistance exercise resulted in a 37% increase
above resting myofibrillar FSR. Single leg cycling had no
statistically significant effect on myofibrillar FSR in the
postexercise period compared to rest prior to (P=0.96)
and after 10 weeks (P=0.12) of endurance training.
Resting mitochondrial FSR was not different between
legs before training (Fig. 3B). Both single-leg knee
extension resistance exercise and cycling endurance
exercise increased in mitochondrial FSR in the untrained
state (Fig. 3). However, this postexercise increase in
mitochondrial FSR was significantly more pronounced
after endurance exercise than after resistance exercise.
After training, there were no significant differences
between legs or changes due to training at rest. However,
the postexercise increase in mitochondrial FSR above
resting values was only observed in the endurance trained
leg.
Signalling protein phosphorylation
Figure 4 shows representative blots for all signalling
proteins measured, in all conditions, in the study. All
bands for a particular antibody were obtained from a single
Western blot (for details see Methods).
Akt. In the untrained state trial, Akt phosphorylation
was increased by 1.5-fold above rest immediately and 4 h
after acute exercise in both the endurance and resistance
modes (Fig. 5A). After training in both modes, resting Akt
phosphorylation was 1.3-fold greater than in the untrained
state. The postexercise phosphorylation response to
endurance exercise vanished after endurance training.
After resistance exercise training, resistance exercise
increased phosphorylation of Akt by 1.5-fold above rest
immediately postexercise, but this difference did not
persist 4 h later.
GSK-3β.GSK-3β(Fig. 5B) phosphorylation increased
immediately after and returned to resting phosphorylation
values by 4 h after acute exercise in the endurance
and resistance modes before (endurance 1.7-, resistance
1.8-fold increases above rest, respectively) and after
training (endurance 1.5-, resistance 1.4-fold increases
above rest, respectively). After each type of training, resting
GSK-3βphosphorylation was 1.3-fold greater than before
training.
mTOR. mTOR (Fig. 5C) phosphorylation increased
immediately after and returned to resting phosphorylation
values by 4 h after acute endurance (untrained 1.7-, trained
1.4-fold increase above rest, respectively) and resistance
(untrained 1.6-, trained 1.5-fold increase above rest,
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3709
respectively) exercise irrespective of training state. The
phosphorylation of mTOR was 1.1-fold greater 4 h after
acute resistance exercise after training than after endurance
exercise.
p70S6K. Acute exercise before training in both modes of
exercise increased p70S6K phosphorylation above resting
values immediately after exercise (Fig. 5D, endurance
1.9-fold, resistance 1.7-fold). p70S6K phosphorylation
remained 1.4-fold elevated above rest 4 h after resistance
exercise, whereas after endurance exercise it had
returned to baseline. After training acute endurance and
resistance exercise caused a similar pattern of immediate
postexercise stimulation of p70S6K phosphorylation
(endurance 2.2-fold, resistance 2.7-fold), but by 4 h,
p70S6K phosphorylation was no different from that at
baseline.
rpS6. rpS6 phosphorylation was significantly increased
by 1.8-fold 4 h after acute resistance exercise before
training (Fig. 6A). Endurance exercise had no
Rest Post exercise
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(% h-1
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Rest Post exercise
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Figure 3. Myofibrillar (A) and mitochondrial enriched (B) FSR at rest and for the 4 h period after
single-leg cycling (endurance) or single leg knee extension exercise (resistance) in the fed state
Values are means ±S.E.M. as percentage per hour. Training effect: significantly different from untrained values
with same leg at same time; +leg effect: significantly different from endurance values, same training state and
at same time; #time effect: significantly different from values at rest with same leg at same time and from those
with same training state, P<0.05.
effect on rpS6 phosphorylation. After training rpS6
phosphorylation was 50% lower at rest than in the
untrained state. Endurance exercise increased rpS6
phosphorylation by 1.6-fold immediately and 4 h after
exercise but resistance exercise had no significant effect
after training.
eIF4E. In the untrained state, eIF4E phosphorylation did
not change immediately after acute exercise in either
mode (Fig. 6B). However, 4 h after each type of exercise,
eIF4E phosphorylation was significantly increased above
resting values (endurance 1.5-fold, resistance 1.8-fold).
At rest, after resistance training, eIF4E phosphorylation
was 1.5-fold greater than in the resting condition in the
untrained trial. Increased eIF4E phosphorylation was seen
immediately (1.5-fold above trained rest) and persisted
until 4 h after both modes of exercise in the TR trial. At 4 h
post-exercise, the resistance trained leg had significantly
greater (1.8-fold greater than rest) eIF4E phosphorylation
than its endurance trained (1.5-fold greater than rest)
counterpart.
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3710 S. B. Wilkinson and others J Physiol 586.15
AMPK. AMPK phosphorylation increased by 6-fold
immediately after acute exercise of both modes in both
the untrained and trained states, but returned to baseline
values by 4 h after exercise (Fig. 7A). There was no effect
of mode of exercise or training.
FAK. In the untrained state, immediately after acute
exercise of each mode, FAK phosphorylation increased
(endurance 2.6-, resistance 2.5-fold) and remained
significantly elevated above baseline for 4 h (Fig. 6B, end-
urance 1.2- and resistance 1.3-fold), but phosphorylation
was significantly greater at this time after resistance
exercise. After both modes of training resting FAK
phosphorylation was significantly greater than before
training (endurance 1.4-, resistance 1.5-fold). After both
modes of training, FAK phosphorylation was elevated
above rest immediately after exercise (endurance 1.8-,
Figure 4. Examples of analysis of protein
Western blots
Representative blots for all signalling proteins
measured, in all conditions, in this study. All
bands for a particular antibody were obtained
from a single Western blot.
resistance 1.7-fold), but returned to baseline at 4 h after
exercise.
Discussion
We have characterized the human muscle protein synthetic
response and intracellular phosphoprotein signalling
alterations (Akt–mTOR–p70S6K) that occur after both
endurance and resistance exercise in the fed state and
examined how it is altered with exercise training. We
observed that in untrained muscle, resistance exercise
stimulated both myofibrillar and muscle mitochondrial
protein synthesis. We contend that this is the first study to
report an increase of human muscle mitochondrial protein
synthesis after acute exercise.
After 10 weeks of unilateral resistance training the
protein synthetic response was more specific, in that only
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3711
Figure 5. Ratio of phosphorylated to total Akt (Ser473 )(A), GSK3β(Ser9)(B), mTOR (Ser2448)(C),
p70S6Kinase (Thr389)(D) at rest, immediately after (0 h) and 4 h (4 h) after single-leg knee extension
(resistance) or cycling (endurance) in the fed state prior to and after 10 weeks of unilateral resistance
or endurance training
Values are means ±S.E.M. in arbitrary units. Training effect: significantly different from untrained values with same
leg at same time; +leg effect: significantly different from endurance values, same training state and at same time;
time effect: letters depict a significant effect of time (lower case represent untrained and upper case represent
trained), P<0.05.
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3712 S. B. Wilkinson and others J Physiol 586.15
myofibrillar, and not mitochondrial, protein synthesis
increased after an acute bout of resistance exercise.
Furthermore, resistance training resulted in a greater
resting rate of myofibrillar protein synthesis. Before and
after training single-leg endurance exercise, performed at
the same relative intensity, stimulated only mitochondrial
protein synthesis. Single leg endurance exercise did not
acutely stimulate myofibrillar protein synthesis regardless
of training state.
In the untrained trial, both forms of exercise increased
Akt and mTOR phosphorylation. Only resistance exercise
increased rpS6 phosphorylation. Furthermore, increased
p70S6K phosphorylation seen immediately after both
forms of exercise remained elevated at 4 h after resistance
exercise only. Our observation mirror those of pre-
viousworkerswhoshowedincreasedAktandmTOR
phosphorylation with no concurrent change in p70S6K
or rpS6 phosphorylation after endurance exercise (Coffey
et al. 2006; Mascher et al. 2007). A different response was
observed after 10 weeks of training; we observed higher
resting Akt, GSK3-β, eIF4E and FAK phosphorylation.
Similarly, Leger et al. (2006) observed increased Akt,
Figure 6. Ratio of phosphorylated to total rpS6 (Ser235/236 )(A) and eIF4E (Ser209)(B) at rest, immediately
after (0 h) and 4 h after single-leg knee extension (resistance) or cycling (endurance) in the fed state
before and after 10 weeks of unilateral resistance or endurance training
Values are means ±S.E.M. in arbitrary units. Training effect: significantly different from untrained values with same
leg at same time; +leg effect: significantly different from endurance values, same training state and at same time;
time effect: letters depict a significant effect of time (lower case represent untrained and upper case represent
trained), P<0.05.
GSK3-βand mTOR phosphorylation at rest after 8 weeks
of resistance exercise training. This increased resting
phosphorylation status could explain the observed higher
resting protein synthesis after resistance training or it
may allow for translation initiation to be activated more
rapidly after exercise in the trained state. Phosphorylation
of several of the key proteins (Akt, p70S6K, GSK3-β)which
had remained elevated 4 h after exercise in the untrained
state returned to baseline at this stage in the trained state.
We propose that exercise training shifts the activation
state of key anabolic signalling molecules to a heightened
state of ‘responsiveness’ so that they are activated and
deactivated more rapidly than in the untrained state.
This training enhanced increase in ‘signalling efficiency’ is
congruent with a more rapid but much shorter response
in the trained versus the untrained state, which we have
recently reported (Tang et al. 2008).
Resistance exercise training stimulates mixed MPS at
rest and after an acute bout of resistance exercise (Phillips
et al. 1999, 2002; Kim et al. 2005). Those studies have
also shown that resistance training attenuates the acute
postexercise increase of mixed MPS to the same relative
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3713
exercise intensity (Phillips et al. 1999, 2002; Kim et al.
2005). We did not find an attenuation of myofibrillar
protein synthesis after 10 weeks of resistance exercise
training. This observation is similar to that reported by
Kim et al. (2005). The differing response of mixed and
myofibrillar protein synthesis could be due to dampening
of the synthetic response of non-myofibrillar proteins
while maintaining the synthesis of myofibrillar proteins
after training. In support of this we observed that the
increase in mitochondrial protein synthesis seen after
resistance exercise in the untrained state was absent after
10 weeks of resistance training. This response does not
appear to be mediated by changes in the phosphorylation
status of known regulatory signalling proteins; however,
it is possible that increased specific gene transcripts for
myofibrillar proteins remain elevated after resistance
training, promoting production of myofibrillar proteins,
whereas those for mitochondrial/sarcoplasmic proteins
arenot.Alternatively,feedinghasbeenshowntohave
a marked effect on signalling proteins (Karlsson et al.
2004; Dreyer et al. 2008); because our participants were fed
throughout the infusion trial and we did not take a biopsy
Figure 7. Ratio of phosphorylated to total AMPKα(Thr172)(A)andFAK(Tyr
576/577)(B)atrest,
immediately after (0 h) and 4 h after single-leg knee extension (resistance) or cycling (endurance) in the
fed state prior to and after 10 week of unilateral resistance or endurance training
Values are means ±S.E.M. in arbitrary units. Training effect: significantly different from untrained with same leg
at same time; +leg effect: significantly different from endurance same training state and at same time; time
effect: letters depict a significant effect of time (lower case represent untrained and upper case represent trained),
P<0.05.
in the fasted state, we may have missed observing a smaller
effect of exercise on the phosphorylation of signalling
proteins.
Several workers have reported an increase in mixed
MPS after endurance or dynamic exercise of various
kinds (Carraro et al. 1990; Sheffield-Moore et al. 2004).
However, to our knowledge, no other study has examined
the relative rates of myofibrillar or mitochondrial protein
synthesis after endurance exercise. Miller et al. (2005) used
dynamic leg extensor exercise and reported an elevation of
myofibrillar and sarcoplasmic (which would include the
subsarcolemmal mitochondria that are not adherent to
the myofibrils) MPS for 48 h after a 1 h bout. Based on
this work (Miller et al. 2005) and the results of others
(Carraro et al. 1990; Sheffield-Moore et al. 2004) we
proposed that we would observe increases, albeit one that
may have been delayed due to AMPK activation (Bolster
et al. 2002; Atherton et al. 2005; Thomson et al. 2008),
in both mitochondrial and myofibrillar protein synthesis
following endurance exercise, although we did not see
such a phenomenon. The single leg cycling model we
used and the single leg kicking model (Miller et al. 2005)
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3714 S. B. Wilkinson and others J Physiol 586.15
both involve endurance work, since both elicit increases in
mitochondrial protein. However, the intensity of muscular
work (W per kg active muscle) of the vastus lateralis in the
single leg kicking exercise would be greater. This difference
in work intensity could explain why our response was
confined to mitochondrial proteins, but Miller et al. (2005)
reported increases in myofibrillar protein synthesis. Those
authors cited the cellular tensegrity hypothesis (Ingber,
2006) as a reason for the robust stimulation of protein
synthesis. As a test of this hypothesis we quantified
FAK phosphorylation as a reasonable loading responsive
protein complex (Gordon et al. 2001; de Boer et al.
2007) but found it to be phosphorylated equally in all
conditions, a result which did not illuminate our protein
synthetic data. We did, however, observe an elevation of
mitochondrial protein synthesis after an acute bout of
one-leg cycling before and after training, consistent with
the observation of an increased mitochondrial content
with endurance training (Morgan et al. 1971; Gollnick
et al. 1972; Hoppeler et al. 1973; Fink et al. 1977).
Scheper & Proud (2002) speculate that eIF4E
phosphorylation at Ser209 subsequent to formation of the
initiation complex may allow for the eIF4F complex to
detach from the 5-cap during elongation, allowing for
the rapid recruitment of the next initiation complex or
rendering the cap binding factors available for trans-
lation of different proteins. Before training, both forms of
exercise resulted in an increased eIF4E phosphorylation
at Ser209 4 h after exercise. After resistance exercise
training, resting eIF4E phosphorylation was elevated.
Furthermore, both modes of exercise increased eIF4E
phosphorylation immediately and it remained elevated for
4 h. In the resistance trained leg, eIF4E phosphorylation
was significantly greater than that observed in the
endurance trained leg at 4 h post-exercise. Limited data
exist on the activation of eIF4E in skeletal muscle.
Williamson et al. (2003) found that in untrained
participants, resistance exercise acutely stimulated Mnk1,
the upstream kinase of eIF4E, but this was not associated
with an increase in eIF4E phosphorylation immediately
after exercise. In consonance with the observations made
by Williamson et al. (2003), we did not observe an increase
in eIF4E phosphorylation immediately after exercise in our
untrained participants.
Atherton et al. (2005) used electrical stimulation in iso-
lated rat muscle to mimic endurance exercise or resistance
exercise. AMPK was activated immediately and 3 h after
low frequency stimulation mimicking endurance exercise,
but was not altered after high-frequency stimulation
mimicking resistance exercise. Coffey et al. (2006) found
that when trained cyclists and weightlifters performed
exercise in their familiar disciplines, no activation of
muscle AMPK was observed. However, when they under-
took a novel form of exercise (cycling for weightlifters
and weightlifting for cyclists) an increase in muscle
AMPK phosphorylation was observed immediately after
exercise and this returned to baseline within 3 h after
exercise. AMPK is also activated following resistance
exercise in humans (Dreyer et al. 2006, 2008; Koopman
et al. 2006, 2007; Drummond et al. 2008); however, one
would suspect that such an elevation would be relatively
transient and possibly has less of an effect on mTOR
signalling compared to aerobic exercise which has a much
greater ATP turnover. Our results are similar in that
AMPK phosphorylation was elevated immediately after
exercise, but returned to baseline within 4 h post-exercise.
In contrast to previous reports (Coffey et al. 2006),
we found that 10 weeks of training was insufficient to
blunt the exercise stimulation of AMPK activity seen in
athletes who had trained in their respective disciplines
for years. In rat skeletal muscle, evidence exists that an
increase in AMPK phosphorylation is associated with a
reduced activation of mTOR, p70S6K and 4EBP-1 (Bolster
et al. 2002; Atherton et al. 2005; Thomson et al. 2008).
Nevertheless, we observed a simultaneous increase in
mTOR and p70S6K phosphorylation above resting values
coincident with the increased AMPK phosphorylation.
Although unclear, in the current study it is possible that
AMPK, mTOR, and p70S6K were concomitantly increased
by the feeding-induced hyperaminoacidaemia and hyper-
insulinaemia during exercise, and that these factors may
override the inhibitory effect that active AMPK has on
mTOR (Dreyer et al. 2006; Morrison et al. 2008). Recently,
Morrison et al. (2008) reported that 3 h of swimming did
not alter the phosphorylation of mTOR, p70S6K, rpS6 or
4EBP-1 compared to rest when rats were fasted. However,
when fed carbohydrate and protein, phosphorylation of
these signalling proteins increased. While, no measure of
AMPK activation was included, this study lends evidence
that feeding with exercise may serve to override the
inhibition that AMPK has on mTOR (Morrison et al.
2008). Evidence suggests that a similar feeding-induced
phenomenon may be occurring in humans (Beelen et al.
2008).
These findings provide evidence that the human skeletal
muscle protein synthetic response to different modes
of exercise is, as hypothesized, specific for proteins
needed for structural and metabolic adaptations to the
particular exercise stimulus, and that this specificity of
response is altered with training to be more specific.
Furthermore, our findings indicate that several factors
involved in translation initiation explained little of this
differential protein synthetic response. The only notable
changes were that resistance exercise resulted in a more
prolonged (p70S6K) or later (rpS6 and eIF4E) activation
of signalling components than with endurance exercise.
It is possible that due to the timing of muscle samples,
we missed changes in phosphorylation status of the
target phosphoproteins measured, or feeding throughout
the protocol may have masked any exercise-specific
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2008 The Authors. Journal compilation C
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J Physiol 586.15 Protein synthesis, resistance and endurance exercise 3715
changes in phosphorylation, or perhaps there are other
phosphorylation sites (that were not quantified) or
phosphoproteins that are involved in exercise-induced
adaptations to muscle protein synthesis. In contrast,
we propose that, as opposed to marked changes in
signalling protein activation, at least of those proteins that
would appear to be most obviously affected, that exercise
and training-specific changes in gene transcription may
be involved in determining the specific nature of the
protein synthetic response that we observed and the
changes in phenotype seen with training. What controls
these upstream transcriptional changes remains to be
answered.
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Acknowledgements
This study was sponsored by the National Science and
Engineering Research Council of Canada and the Canadian
Institutes for Health Researchto S.M.P.There were contributions
from the UK BBSRC (BB/X510697/1 and BB/C516779/1)
and EC EXGENESIS to M.J.R., and K.E.Y. was supported
by NIH RR00954, DK20579, DK56341, DK49393, DK74345
and DK59531. Jennifer (Xianghong) Chen provided mass
spectrometry analytical support. No authors have any financial
or other conflicts of interest to declare.
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Age-related loss of muscle mass, strength, and performance, commonly referred to as sarcopenia, has wide-ranging detrimental effects on human health, the ramifications of which can have serious implications for both morbidity and mortality. Various interventional strategies have been proposed to counteract sarcopenia, with a particular emphasis on those employing a combination of exercise and nutrition. However, the efficacy of these interventions can be confounded by an age-related blunting of the muscle protein synthesis response to a given dose of protein/amino acids, which has been termed “anabolic resistance.” While the pathophysiology of sarcopenia is undoubtedly complex, anabolic resistance is implicated in the progression of age-related muscle loss and its underlying complications. Several mechanisms have been proposed as underlying age-related impairments in the anabolic response to protein consumption. These include decreased anabolic molecular signaling activity, reduced insulin-mediated capillary recruitment (thus, reduced amino acid delivery), and increased splanchnic retention of amino acids (thus, reduced availability for muscular uptake). Obesity and sedentarism can exacerbate, or at least facilitate, anabolic resistance, mediated in part by insulin resistance and systemic inflammation. This narrative review addresses the key factors and contextual elements involved in reduction of the acute muscle protein synthesis response associated with aging and its varied consequences. Practical interventions focused on dietary protein manipulation are proposed to prevent the onset of anabolic resistance and mitigate its progression.
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The effect of resistance training with higher- and lower-loads on muscle mass and strength has been extensively studied while changes in muscle endurance have received less attention. This trial aimed to assess the effect of training load on absolute (AME) and relative muscle endurance (RME). Sixteen untrained women (22.7±3.3 yr: mean ± SD) had one arm and leg randomly assigned to train with higher-loads (HL; 80-90% 1RM), and the contralateral limbs trained with lower-loads (LL; 30-50% 1RM) thrice weekly to volitional fatigue for 10 weeks. Heavy and light load AME and RME, strength, and muscle mass were assessed pre- and post-training. Strength increased more in the HL compared to LL leg (P = <0.01), but similar increases in strength were observed between upper body conditions (P = 0.46). Lower body heavy and light load AME improved in both conditions, but HL training induced a larger improvement in heavy load AME (HL, 9.3±4.3, vs. LL, 7.5±7.1 repetitions, Time × Limb P < 0.01) and LL training induced a larger improvement in light load AME (LL, 24.7±22.2, vs. HL, 15.2±16.7 repetitions, Time × Limb P = 0.04). In the upper body, HL and LL training induced similar increases in both heavy (Time × Limb P = 0.99), and light load (Time × Limb P = 0.16) AME. Dual-energy x-ray absorptiometry showed no change in leg fat-and-bone-free mass (FBFM) for either condition, and an increase in only LL arm FBFM. AME improved in a manner specific to the training loads used. ClinicalTrials.gov (NCT04547972).
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Cardiovascular diseases are the most common cause of death in the world. One of the major causes of cardiac death is excessive apoptosis. However, multiple pathways through moderate exercise can reduce myocardial apoptosis. After moderate exercise, the expression of anti-apoptotic proteins such as IGF-1, IGF-1R, p-PI3K, p-Akt, ERK-1/2, SIRT3, PGC-1α, and Bcl-2 increases in the heart. While apoptotic proteins such as PTEN, PHLPP-1, GSK-3, JNK, P38MAPK, and FOXO are reduced in the heart. Exercise-induced mechanical stress activates the β and α5 integrins and subsequently, focal adhesion kinase phosphorylation activates the Akt/mTORC1 and ERK-1/2 pathways, leading to an anti-apoptotic response. One of the reasons for the decrease in exercise-induced apoptosis is the decrease in Fas-ligand protein, Fas-death receptor, TNF-α receptor, Fas-associated death domain (FADD), caspase-8, and caspase-3. In addition, after exercise mitochondrial-dependent apoptotic factors such as Bid, t-Bid, Bad, p-Bad, Bak, cytochrome c, and caspase-9 are reduced. These changes lead to a reduction in oxidative damage, a reduction in infarct size, a reduction in cardiac apoptosis, and an increase in myocardial function. After exercising in the heart, the levels of RhoA, ROCK1, Rac1, and ROCK2 decrease, while the levels of PKCε, PKCδ, and PKCɑ are activated to regulate calcium and prevent mPTP perforation. Exercise has an anti-apoptotic effect on heart failure by increasing the PKA-Akt-eNOS and FSTL1-USP10-Notch1 pathways, reducing the negative effects of CaMKIIδ, and increasing the calcineurin/NFAT pathway. Exercise plays a protective role in the heart by increasing HSP20, HSP27, HSP40, HSP70, HSP72, and HSP90 along with increasing JAK2 and STAT3 phosphorylation. However, research on exercise and factors such as Pim-1, Notch, and FAK in cardiac apoptosis is scarce, so further research is needed. Future research is recommended to discover more anti-apoptotic pathways. It is also recommended to study the synergistic effect of exercise with gene therapy, dietary supplements, and cell therapy for future research.
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Background: Duchenne muscular dystrophy (DMD) is a severe form of rare genetic disorder. It was noted that it affects 0.8 million Indian children. Coronaviruses disease 19 is a highly contagious pandemic that causes severe respiratory symptoms. This COVID 19 may cause devastation to the lives of DMD children. Respiratory muscles are already weaker in DMD children, and because of this, they are more prone to lung infections. No interventions are advised in the early part of Covid 19 for the DMD children. This study was conducted to identify the effect of low-intensity aerobic exercises in children with DMD. Materials and Methods: This longitudinal study was conducted with eleven DMD children who are also wheelchair-dependent. The children are already under the proper medical, physiotherapeutic, and nursing care. Since the WHO declared a pandemic in 2020, these children have been helpless in managing themselves. The pandemic was announced in India in March 2020. Since then, the physiotherapist created a protocol for these children and advised them to follow it. All the selected children and their whole families were given a clear picture of Covid 19 and its severity on lungs. Researchers created a set of quarantine exercises, which involved limb exercises, breathing exercises, trunk mobility training, and neck exercises. A low-intensity aerobic exercise which included cycling to the upper limb for 15 minutes, and upper limb movements with a frequent rest period, was given. Covid 19 preventions like social distancing, frequent handwashing, and wearing the mask are compulsory for these children. A logbook was given to all the children and advised them to note the exercise time and duration. Frequent Video calls, and WhatsApp videos, were used to monitor them regularly. Result: The online mode continuously monitored all the children; the face-to-face visit was done by the researcher in June 2020 (i.e., after two months). All the exercises were reviewed, few exercises were added during the personal visit. The researcher also did a subsequent visit in August 2020, October 2020, December 2020, February 2021, and May 2021. All the children noted Flu infection but recovered within ten days without hospitalization. The parents monitored their SPo2 and temperature and updated them in the logbook. A lung function test was done using a handheld incentive spirometer during the personnel visit by the therapist and found satisfactory. Conclusion: The study concluded that low-intensity aerobic exercises with quarantine protocols significantly improve lung functions and prevent Covid 19 in children with DMD.
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Background: Resistance exercise leads to net muscle protein accretion through a synergistic interaction of exercise and feeding. Proteins from different sources may differ in their ability to support muscle protein accretion because of different patterns of postprandial hyperaminoacidemia. Objective: We examined the effect of consuming isonitrogenous, isoenergetic, and macronutrient-matched soy or milk beverages (18 g protein, 750 kJ) on protein kinetics and net muscle protein balance after resistance exercise in healthy young men. Our hypothesis was that soy ingestion would result in larger but transient hyperaminoacidemia compared with milk and that milk would promote a greater net balance because of lower but prolonged hyperaminoacidemia. Design: Arterial-venous amino acid balance and muscle fractional synthesis rates were measured in young men who consumed fluid milk or a soy-protein beverage in a crossover design after a bout of resistance exercise. Results: Ingestion of both soy and milk resulted in a positive net protein balance. Analysis of area under the net balance curves indicated an overall greater net balance after milk ingestion (P < 0.05). The fractional synthesis rate in muscle was also greater after milk consumption (0.10 ± 0.01%/h) than after soy consumption (0.07 ± 0.01%/h; P = 0.05). Conclusions: Milk-based proteins promote muscle protein accretion to a greater extent than do soy-based proteins when consumed after resistance exercise. The consumption of either milk or soy protein with resistance training promotes muscle mass maintenance and gains, but chronic consumption of milk proteins after resistance exercise likely supports a more rapid lean mass accrual.
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Enzymatic Analysis: A Practical Guide is a multipurpose manual of laboratory methods. It offers a systematic scheme for the analysis of biological materials from the level of the whole organ down to the single cell and beyond. It is intended as a guide to the development of new methods, to the refinement of old ones, and to the adaptation in general of methods to almost any scale of sensitivity. As some may realize, the book is a sequel to A Flexible System of Enzymatic Analysis, originally published in 1972. The major changes, other than an appropriate interchange of authors, consist of a wholly new chapter of methods and protocols for measuring enzymes, the addition of 13 new entries in the metabolite chapter, and a much superior chapter on enzymatic cycling. With considerable nostalgia, we have switched from DPN and TPN to NAD and NADP nomenclature, which no doubt will make Otto Warburg turn over in his grave. The incentives for the methodology in this book came from the rigorous demands of quantitative histochemistry and cytochemistry. These demands are specificity, simplicity, flexibility, and, of course, sensitivity—all likewise desirable attributes of methods for other purposes. The specificity is provided by the use of enzyme methods. Simplicity is achieved by leading all reactions to a final pyridine nucleotide step.
Chapter
In this study we have examined muscle adaptations to long term exercise in man. Studies of Holloszy (1), Kraus (2), Gollnick (3) and their coworkers leave little doubt that exercise training stimulates muscle mitochondrial changes. We will present further evidence that exercise training in man stimulates mitochondrial growth, oxidative capacity, and capacity for syntheses of glycogen and lipid.
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• The aim of this study was to describe the time course of the response of human muscle protein synthesis (MPS) to a square wave increase in availability of amino acids (AAs) in plasma. We investigated the responses of quadriceps MPS to a ≈1.7-fold increase in plasma AA concentrations using an intravenous infusion of 162 mg (kg body weight)−1 h−1 of mixed AAs. MPS was estimated from D3-leucine labelling in protein after a primed, constant intravenous infusion of D3-ketoisocaproate, increased appropriately during AA infusion. • Muscle was separated into myofibrillar, sarcoplasmic and mitochondrial fractions. MPS, both of mixed muscle and of fractions, was estimated during a basal period (2.5 h) and at 0.5-4 h intervals for 6 h of AA infusion. • Rates of mixed MPS were not significantly different from basal (0.076 ± 0.008 % h−1) in the first 0.5 h of AA infusion but then rose rapidly to a peak after 2 h of ≈2.8 times the basal value. Thereafter, rates declined rapidly to the basal value. All muscle fractions showed a similar pattern. • The results suggest that MPS responds rapidly to increased availability of AAs but is then inhibited, despite continued AA availability. These results suggest that the fed state accretion of muscle protein may be limited by a metabolic mechanism whenever the requirement for substrate for protein synthesis is exceeded.
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Muscles and tendons are highly adaptive to changes in chronic loading, though little is known about the adaptative time course. We tested the hypothesis that, in response to unilateral lower limb suspension (ULLS), the magnitude of tendon mechanical adaptations would match or exceed those of skeletal muscle. Seventeen men (1.79 ± 0.05 m, 76.6 ± 10.3 kg, 22.3 ± 3.8 years) underwent ULLS for 23 days (n = 9) or acted as controls (n = 8). Knee extensor (KE) torque, voluntary activation (VA), cross-sectional area (CSA) (by magnetic resonance imaging), vastus lateralis fascicle length (L f) and pennation angle (θ), patellar tendon stiffness and Young's modulus (by ultrasonography) were measured before, during and at the end of ULLS. After 14 and 23 days (i) KE torque decreased by 14.8 ± 5.5% (P < 0.001) and 21.0 ± 7.1% (P < 0.001), respectively; (ii) VA did not change; (iii) KE CSA decreased by 5.2 ± 0.7% (P < 0.001) and 10.0 ± 2.0% (P < 0.001), respectively; L f decreased by 5.9% (n.s.) and 7.7% (P < 0.05), respectively, and θ by 3.2% (P < 0.05) and 7.6% (P < 0.01); (iv) tendon stiffness decreased by 9.8 ± 8.2% (P < 0.05) and 29.3 ± 11.5% (P < 0.005), respectively, and Young's modulus by 9.2 ± 8.2% (P < 0.05) and 30.1 ± 11.9% (P < 0.01), respectively, with no changes in the controls. Hence, ULLS induces rapid losses of KE muscle size, architecture and function, but not in neural drive. Significant deterioration in tendon mechanical properties also occurs within 2 weeks, exacerbating in the third week of ULLS. Rehabilitation to limit muscle and tendon deterioration should probably start within 2 weeks of unloading.
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Twenty-one enzymes of different metabolic systems were measured in the rabbit fast-twitch tibialis anterior (TA) muscle after electrical stimulation (10 Hz, 24 h/day) for 1 day to 10 wk. Nine analytical methods are either new, (3-oxoacid CoA - transferase, branched-chain-amino-acid aminotransferase, carnitine acetyltransferase, thiolase), improved (glutamate dehydrogenase, glycogen synthase, adenylic acid deaminase), or specially adapted (hexokinase, phosphoglucomutase). The activities (based on protein) of 12 mitochondrial or partly mitochondrial enzymes were lower in control TA than in control (slow) soleus (30-84% of soleus level). After 2 wk, 11 of these had surpassed the control soleus level. Maximal increases (3- to 14-fold) occurred after 2-5 wk, and thereafter six of the enzymes declined, whereas the other five maintained or increased their levels. Five glycolytic and two high-energy phosphate transfer enzymes, originally much higher in control TA than in control soleus, decreased gradually to levels at 8-10 wk only 27-123% higher than in soleus. Noncollagen protein concentration dropped 46%, explained largely by a sixfold increase in extracellular (chloride) space and a modest increase in collagen. The data constitute strong evidence for coordinate regulation of (mainly cytosolic) enzymes of glycolysis, glycogenolysis, gluconeogenesis, and high-energy phosphate transfer. Changes in the (mainly mitochondrial) enzymes of oxidative metabolism were more divergent, partly because of a hitherto undescribed secondary phase in the metabolic response. This phase may reflect a lower energy consumption in muscles adapted to continuous activity.
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In contrast to the impact of nutritional intervention on post-exercise muscle protein synthesis, little is known about the potential to modulate protein synthesis during exercise. This study investigates the impact of protein co-ingestion with carbohydrate on muscle protein synthesis during resistance type exercise. Ten healthy males were studied in the evening after consuming a standardized diet throughout the day. Subjects participated in 2 experiments, in which they ingested either carbohydrate or carbohydrate with protein during a 2 h resistance exercise session. Subjects received a bolus of test drink prior to and every 15 min during exercise, providing 0.15 g·kg-1 ·h-1 carbohydrate with (CHO+PRO) or without (CHO) 0.15 g·kg-1 ·h-1 protein hydrolysate. Continuous intravenous infusions with L-[ring-13 C 6 ]phenylalanine and L-[ring-2 H 2 ] tyrosine were applied, and blood and muscle biopsies were collected to assess whole-body and muscle protein synthesis rates during exercise. Protein co-ingestion lowered whole-body protein breakdown rates by 8.4±3.6% (P=0.066), compared to the ingestion of carbohydrate only, and augmented protein oxidation and synthesis rates by 77±17 and 33±3%, respectively (P<0.01). As a consequence, whole-body net protein balance was negative in CHO, whereas a positive net balance was achieved following the CHO+PRO treatment (-4.4±0.3 vs 16.3±0.4 µmol phe·kg-1 ·h-1 , respectively; P<0.01). In accordance, mixed muscle protein fractional synthetic rate (FSR) was 49±22% higher following protein co-ingestion (0.088±0.012 and 0.060±0.004 %·h-1 in CHO+PRO vs CHO treatment, respectively; P<0.05). We conclude that, even in a fed state, protein co-ingestion stimulates whole-body and muscle protein synthesis rates during resistance type exercise.
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Muscle biopsies were taken from the middle part of the vastus lateralis muscle of 9 men, who were not regularly involved in endurance training (M, average [(V)\dot]\textO\text2 \dot V_{{\text{O}}_{\text{2}} } max=61.3 ml/minkg), 3 sedentary women (W, [(V)\dot]\textO\text2 \dot V_{{\text{O}}_{\text{2}} } max=43.7 ml/minkg) and 5 well trained orienteers (TO, [(V)\dot]\textO\text2 \dot V_{{\text{O}}_{\text{2}} } max=76.1 ml/minkg). Morphometric analysis of 60 electron micrographs per biopsy gave the following significant differneces: 1. The volume density of central mitochondria was 1.47-fold higher in TO than in M, and 1.44-fold higher in M than in W. 2. The volume density of peripheric mitochondria was 3.22 times higher in TO compared to M. 3. The ratio of the central mitochondrial volume to the volume of myofibrils was 1.54-fold higher in TO compared to M, while the respective ratio was 1.49 for M compared to W. 4. The surface of the central mitochondria was 1.28-fold higher in TO than in M and 1.35-fold higher in M than in M. 5. The surface of mitochondrial cristae was higher by a factor of 1.62 in TO compared to M and 1.35 in M compared to W. 6. The central mitochondria were larger in TO compared to M by a factor of 1.12. 7. The volume density of intracellular lipid (triglyceride droplets), was 2.5-fold higher in TO than in M. There were highly significant correlations between [(V)\dot]\textO\text2 \dot V_{{\text{O}}_{\text{2}} } max and volume density of central mitochondria (r=0.82), surface of mitochondrial cristae (r=0.80) and the ratio of mitochondrial volume to myofibrillar volume (r=0.78). No quantitative changes could be observed in mitochondrial fine structure. Neither volume density of sarcoplasma nor volume and surface density of the tubular system showed any difference as a function of training and sex. It is postulated that a) an individual's maximum oxygen intake is limited not only by the capacity of the oxygen transport system but also by the oxidative capacity of mitochondria in the skeletal muscles, and b) the skeletal muscle of trained athletes contains a much higher quantity of intracellular lipids (triglyceride droplets) as a substrate directly available for energy production.