A v h a n d l i n g s s e r i e f ö r
G y m n a s t i k - o c h i d r o t t s h ö g s k o l a n
Nr 07
EFFECTS OF EXERCISE AND AMINO ACID INTAKE ON MECHANISMS REGULATING PROTEIN SYNTHESIS AND
BREAKDOWN IN HUMAN MUSCLE
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Effects of exercise and amino acid intake on
mechanisms regulating protein synthesis and
breakdown in human muscle
Marcus Moberg
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© Marcus Moberg Gymnastik- och idrottshögskolan 2016 ISBN 978-91-980862-6-3 Tryckeri: Universitetsservice US-AB, Stockholm 2016 Distributör: Gymnastik- och idrottshögskolan
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To Madeleine, Jacqueline and
baby boy Moberg, without you
these words have no meaning.
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ABSTRACT
Skeletal muscle is a highly malleable tissue that responds effectively to stimuli such as
nutrition and different modes of exercise. Resistance training stimulates the growth of
contractile proteins in the muscle, increasing muscle mass and strength, while endur-
ance training stimulates mitochondrial proliferation and angiogenesis leading to an
improved oxidative capacity. These adaptations are the accumulated result of changes in
signaling pathways that occur in the acute phase following an exercise bout. The
mTORC1 signaling pathway is in control of the rate of protein synthesis in the muscle
cell and is in a healthy muscle mainly responsive to resistance type exercise, amino
acids and insulin. For a muscle cell to grow, the rate of protein synthesis must be greater
than the rate of protein breakdown, and this can be achieved by the coordinated stimula-
tion by resistance exercise and amino acid ingestion.
The PGC-1α pathway is defined to in part control the adaptations to endurance exer-
cise, being in master control of mitochondrial biogenesis. The energy sensing protein
AMPK that is activated during strenuous exercise is described to activate PGC-1α sig-
naling. Interestingly, activated AMPK has in cell and animal models been shown to
inhibit mTORC1 signaling and consequently decrease the rate of protein synthesis. It is
therefore speculated that endurance exercise is incompatible with resistance exercise
with regard to stimulation of protein synthesis and muscle growth. However, such a
mechanism has not been established in human skeletal muscle.
With regard to amino acids and the stimulation of protein synthesis, it is believed
that the group of essential amino acids (EAA) is responsible for this effect. Special
attention has been attributed to leucine, which has been shown to possess quite unique
anabolic properties. The particular role of leucine among the EAA, and the involvement
of the other branched-chain amino acids (BCAA) in the anabolic stimulus by amino
acids is however unknown.
The mechanisms in control of protein breakdown in human skeletal muscle are
largely unidentified, but much attention has been given the proteins MuRF-1 and
MAFbx. These proteins are described to play a pivotal role in the degradative ubiquitin-
proteasome pathway and have been shown to be up-regulated in a number of models of
atrophy. Interestingly, the expression of these proteins is also influenced by exercise,
but there is a lack of knowledge concerning the regulation and the function of this al-
tered expression.
The aim of thesis was to examine how different modes of exercise and amino acids
affect mTORC1 signaling, protein synthesis and markers of protein breakdown. Particu-
lar emphasis is on the effects of concurrent exercise in relation to resistance exercise
only, examining both the effects in the same muscle group and potential systemic influ-
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ences. Furthermore, the role of leucine and the BCAA in the anabolic response the EAA
was also a primary objective of this thesis.
In study I, the immediate influence of high intensity endurance exercise on subsequent
resistance exercised induced mTORC1 signaling was examined. Despite robust activa-
tion of AMPK by the endurance exercise there was no inhibition of mTORC1 signaling
or protein synthesis during the three hour recovery from resistance exercise. The con-
current exercise did however substantially induce the mRNA expression of MuRF-1 as
well as that of PGC-1α. Study II had a similar set up as study I, but with the difference
that resistance exercise was performed with the arms. The cycling exercise reduced the
resistance exercise stimulated mTORC1 signaling in the triceps immediately after the
exercise, but during the recovery period mTORC1 signaling and protein synthesis was
similar between trials. Although the exercise modes were separated between legs and
arms, concurrent exercise induced the mRNA expression of MuRF-1 and PGC-1α in the
triceps muscle. In study III, the effect of an EAA supplement in the stimulation of
mTORC1 signaling in connection with resistance exercise was compared to an EAA
supplement without leucine included. Ingestion of EAA induced a robust stimulation of
mTORC1 signaling after exercise, but this was only minor when leucine was excluded
from the supplement. In study IV, subjects were orally supplied with leucine, BCAA,
EAA or placebo in a randomized fashion in connection with four sessions of resistance
exercise. Leucine alone stimulated mTORC1 signaling after the exercise, but both the
amplitude and extent of stimulation was substantially greater with EAA, an effect that
was largely mediated by the BCAA as a group.
In conclusion, high intensity cycling intervals prior to a bout of resistance exercise us-
ing the leg- or arm muscle does not affect mTORC1 signaling or protein synthesis dur-
ing the three hour recovery period from exercise, accordingly not supporting the theory
of incompatibility between resistance- and endurance exercise signaling. Concurrent
exercise increases the expression of the proteolytic marker MuRF-1 compared to re-
sistance exercise only, even when the exercise modes is separated between legs and
arms. The precise function of MuRF-1 induction is unclear, and could indicate both and
increased demand of cellular adaptive remodeling to the training or a more direct detri-
mental proteolytic effect.
Leucine is crucial among the EAA in the stimulation of mTORC1 signaling after re-
sistance exercise. The effect of leucine is readily potentiated by intake of the remaining
BCAA, isoleucine and valine, but the greatest response on mTORC1 signaling is at-
tained with a mixture of EAA. As a supplement in connection with exercise, a mixture
of EAA must be regarded preferable, although the effect on signaling is largely attribut-
ed to the BCAA.
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LIST OF SCIENTIFIC PAPERS I. William Apró, Marcus Moberg, D Lee Hamilton, Björn Ekblom, Gerrit van
Hall, Hans-Christer Holmberg and Eva Blomstrand. Resistance exercise-
induced S6K1 kinase activity is not inhibited in human skeletal muscle de-
spite prior activation of AMPK by high-intensity interval cycling. Am J
Physiol Endocrinol Metab 308: E470–E481, 2015.
II. Marcus Moberg, William Apró, Björn Ekblom, Gerrit van Hall, Hans-
Christer Holmberg and Eva Blomstrand. Lower body endurance exercise
acutely affects resistance exercise induced transcriptional and translational
signalling in the tricpes brachii muscle. In manuscript.
III. Marcus Moberg, William Apró, Inger Ohlsson, Marjan Pontén, Antonio
Villanueva, Björn Ekblom and Eva Blomstrand. Absence of leucine in an es-
sential amino acid supplement reduces activation of mTORC1 signaling fol-
lowing resistance exercise in young females. Appl. Physiol. Nutr. Metab. 39:
183–194, 2014
IV. Marcus Moberg, William Apró, Björn Ekblom, Gerrit van Hall, Hans-
Christer Holmberg and Eva Blomstrand. Activation of mTORC1 by leucine is
potentiated by branched chain amino acids and even more so by essential
amino acids after resistance exercise in human muscle. Submitted.
Study I is reprinted with permission from The American Physiological Society.
Study III © 2008 Canadian Science Publishing or its licensors. Reproduced with permission from
the NRC Research Press provided by Copyright Clearance Center.
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CONTENTS 1 INTRODUCTION ............................................................................................... 15
1.1 Nutrition and exercise affects protein turnover .......................................................................... 16 1.1.1 Methods to study protein turnover .................................................................................... 17
1.2 Protein synthesis – effects of exercise ........................................................................................ 18 1.3 Protein synthesis – effects of amino acids .................................................................................. 20 1.4 Molecular regulation of protein synthesis .................................................................................. 21
1.4.1 Downstream of mTORC1 ................................................................................................. 22 1.4.2 Upstream of mTORC1 ...................................................................................................... 23 1.4.3 mTORC1 – importance for muscle protein accretion ........................................................ 26
1.5 Protein breakdown – effects of exercise and nutrition ................................................................ 27 1.6 Regulation of endurance exercise adaptations ............................................................................ 28 1.7 Crosstalk between divergent modes of exercise ......................................................................... 28
2 AIMS .......................................................................................................................... 31
3 METHODS ................................................................................................................. 32 3.1 Subjects ...................................................................................................................................... 32 3.2 Initial exercise tests .................................................................................................................... 33
3.2.1 Oxygen uptake .................................................................................................................. 33 3.2.2 Maximal strength .............................................................................................................. 33 3.2.3 Familiarization sessions .................................................................................................... 33
3.3 Intervention protocols ................................................................................................................ 34 3.3.1 Study I ............................................................................................................................... 34 3.3.2 Study II ............................................................................................................................. 35 3.3.3 Study III ............................................................................................................................ 36 3.3.4 Study IV ............................................................................................................................ 37
3.4 Muscle biopsy sampling ............................................................................................................. 38 3.5 Plasma analysis .......................................................................................................................... 38
3.5.1 Sample preparation ............................................................................................................ 38 3.5.2 Glucose, lactate, insulin and cortisol ................................................................................. 39 3.5.3 Amino acids ...................................................................................................................... 39 3.5.4 Stable isotope enrichment (Study I, II, IV) ........................................................................ 39
3.6 Muscle analysis .......................................................................................................................... 39 3.6.1 Sample preparation ............................................................................................................ 39 3.6.2 Muscle glycogen (Study I, II, IV)...................................................................................... 40 3.6.3 Amino acids (Study II, III, IV) .......................................................................................... 40 3.6.4 Immunoblotting ................................................................................................................. 40 3.6.5 Immunoprecipitation (Study I, II, IV) ............................................................................... 41 3.6.6 Kinase assays (Study I, II, IV)........................................................................................... 42 3.6.7 mRNA expression (Study I, II, III).................................................................................... 43 3.6.8 Stable isotope enrichment (Study I, II, IV) ........................................................................ 44 3.6.9 Statistical analysis ............................................................................................................. 44
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4 RESULTS ................................................................................................................... 46 4.1 Study I ........................................................................................................................................ 46 4.2 Study II ...................................................................................................................................... 49 4.3 Study III ..................................................................................................................................... 54 4.4 Study IV ..................................................................................................................................... 55
5 METHODOLOGICAL CONSIDERATIONS ............................................................ 59 5.1 Variability of immunoblotting.................................................................................................... 59 5.2 Muscle sample homogenization ................................................................................................. 60 5.3 Myofibrillar protein extraction ................................................................................................... 62 5.4 Fractional synthetic rate ............................................................................................................. 63
6 DISCUSSION ............................................................................................................. 68 6.1 The effects of concurrent exercise on mTORC1 signaling ......................................................... 68 6.2 Concurrent exercise increases the expression of PGC-1α .......................................................... 72 6.3 The effects of amino acids on mTORC1 signaling ..................................................................... 73 6.4 The role of insulin in amino acid induced stimulation of mTORC1 signaling ........................... 74 6.5 Details in exercise and amino acid regulation of mTORC1 signaling ........................................ 75 6.6 The effect of exercise and amino acids on markers of protein breakdown ................................. 77 6.7 Differences between the vastus lateralis and triceps brachii muscles ......................................... 80 6.8 The relationship between mTORC1 signaling and protein synthesis ......................................... 80 6.9 Gender differences ..................................................................................................................... 82 6.10 Representativeness and implications ........................................................................................ 83 6.11 Limitations ............................................................................................................................... 84 6.12 Future directions ...................................................................................................................... 85 6.13 Conclusions .............................................................................................................................. 86
7 SAMMANFATTNING............................................................................................... 87
8 ACKNOWLEDGEMENTS ........................................................................................ 90
9 REFERENCES ........................................................................................................... 93
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ABBREVIATIONS
4E-BP1
ANOVA
AMP
AMPK
4E binding protein 1
Analysis of variance
Adenosine monophosphate
AMP-activated protein kinase
ATP
AV
BCAA
Adenosine triphosphate
Arterio-venous
Branched-chain amino acids
CAMK Ca2+
/calmodulin-dependent protein kinase
cDNA Complementary deoxyribonucleic acid
CHAPS
COX IV
3-[(3-Cholamidopropyl) dimethylammonio]-1-
propanesulfonate
Cytochrome c oxidase IV
CT
DTT
Cycle threshold
Dithiothreitol
EAA
EDTA
eEF2
Essential amino acids
Ethylenediaminetetraacetic acid
Eukaryotic elongation factor 2
EGTA
ELISA
eIF4x
FOXO
FSR
Ethylene glycol tetraacetic acid
Enzyme-linked immunosorbent assay
Eukaryotic initiation factor 4x
Forkhead box O
Muscle protein fractional synthetic rate
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GC-C-IRMS Gas chromatography combustion isotope ratio mass spec-
trometry
GC-MS/MS Gas chromatography-tandem mass spectrometry
GDP Guanosine diphosphate
GTP
HEPES
HPLC
KOH
LC-MS/MS
LRS
Guanosine triphosphate
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
Reversed-phase high-performance liquid chromatography
Potassium chloride
Ultra-performance liquid chromatography - tandem mass
spectrometry
Leucyl-tRNA synthetase
MAFbx Muscle atrophy F-box
MgCl2
mRNA
mTOR
MTBSTFA
Magnesium chloride
Messenger ribonucleic acid
Mechanistic target of rapamycin
N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide
MuRF-1 Muscle Ring-finger protein 1
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NaCl
NaF
Sodium chloride
Sodium fluoride
NaOH Sodium hydroxide
Na3VO4
NF-κB
Sodium orthovanadate
Nuclear factor kappa-light-chain-enhancer of activated B
cells
p38 MAPK
PA
PCR
PDCD4
PDK-1
PGC-1α
PRAS40
Raptor
RER
Rheb
p38 mitogen-activated protein kinase
Phosphatidic acid
Polymerase chain reaction
Programmed cell death protein 4
3-phosphoinositide dependent protein kinase-1
Peroxisome proliferator-activated receptor gamma co-
activator 1α
Proline-rich Akt substrate of 40 kDa
Regulatory-associated protein of mTOR
Respiratory exchange ratio
Ras homologous enriched in brain
RM
RNA
RPE
rpS6
S6K1
SDS-Page
Repetition maximum
Ribonucleic acid
Rate of perceived exertion
Ribosomal protein S6
70 kDa ribosomal protein S6 kinase
Sodium dodecyl sulfate – polyacrylamide gel electropho-
resis
Ser
TBC1D7
Serine
Tre2-Bub2-Cdc16 1 domain family, member 7
Thr Threonine
Tris Base
tRNA
TSC1/2
Tris(hydroxymethyl)aminomethane
Transfer ribonucleic acid
Tuberous sclerosis 1/2
VO2max Maximal oxygen uptake
Wmax Maximal watt production
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1 INTRODUCTION
Skeletal muscle mass is the human body’s largest constituent, ranging between 30 to
40% depending on gender and age (1). The skeletal muscle has an imperative role in our
locomotion and metabolism and thus being of great importance for athletic performance
as well as in states of disease. Skeletal muscle force development capacity is closely
related to its size (2), which is a major determinant of strength, alongside with the ca-
pacity of neuromuscular activation (3, 4). A loss of muscle (disuse atrophy, sarcopenia
or cachexia) results in a loss of strength and physical performance and is associated with
immobility, falls/fractures, increased mortality and low quality of life (5, 6). In a anoth-
er health aspect, the total mass of skeletal muscle and its protein turnover has the largest
potential to influence the resting metabolic rate, and through contractions/exercise be
the largest contributor to total energy expenditure (7).
Skeletal muscle exhibits a remarkable plasticity and is able to change its phenotype
depending on external stimuli such as exercise and nutrition. For athletic performance it
is important that an optimal amount of muscle mass is developed while the appropriate
metabolic characteristics are attained for the specific sport. Different types of exercise
stimuli generate different adaptations at the muscular level. Repeated bouts of resistance
exercise results in increased muscle mass (hypertrophy), while repeated sessions of
endurance exercise collectively leads to an enhanced capacity to sustain muscle contrac-
tions at a given intensity.
The changes that occur in the skeletal muscle following a period of strength training
is predominantly a growth of contractile proteins, improved glycolytic capacity and an
enhanced capacity to generate force (8, 9). During the first hours of recovery from re-
sistance exercise there is an increased rate of muscle protein synthesis (10), and this
increase can be sustained for up to 48 hours (11). Muscle protein accretion as a result of
repeated bouts of exercise can be detected as early as after 20 days of structured re-
sistance training (12). The muscular adaptations to endurance training are characterized
by an increased mitochondrial content, vascularization and increased levels of oxidative
enzymes that collectively enhance the capacity to utilize fat as a fuel source (13, 14). In
similarity to resistance exercise these changes start to occur during early stages of re-
covery from exercise through activation of genes that regulate mitochondrial biogenesis
and vascular growth, along with an increased rate of mitochondrial protein synthesis
(15, 16).
The term protein turnover involves the processes of synthesizing new tissue proteins
and the breakdown of protein, which liberates amino acids as substrates for protein
synthesis, for gluconeogenesis, as precursors for amino acid derived compounds and for
oxidation to release energy. The turnover rate of muscle protein is relatively slow com-
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pared to other tissues such as the liver, intestine and kidneys, however, 1-2% of the
muscle proteins are broken down and re-synthesized each day (17), meaning that a
skeletal muscle is completely remodeled within two months. In order for the skeletal
muscle mass to increase, the rate of protein synthesis must exceed the rate of breakdown
i.e. an overall positive net balance must be attained. Two major factors that are able to
influence the processes of protein turnover are nutrition and exercise. Increasing our
knowledge of their influence on protein turnover and the underlying mechanisms will
have major implications in maintaining health and preventing disease.
1.1 Nutrition and exercise affects protein turnover
The proteins of skeletal muscle are constantly and simultaneously synthesized and de-
graded and nutrition and exercise have the greatest impact on these events in the healthy
individual. With regard to nutrition, its impact on protein turnover is mainly mediated
by the dietary derived amino acids with a lesser contribution by carbohydrates through
stimulation of insulin secretion. When we are in the fasted state, the exogenous provi-
sion of amino acids to the relatively small free amino acid pool is absent. Therefore, the
rate of protein synthesis is downregulated due to substrate limitation, while breakdown
is elevated to remove decrepit protein and regulatory proteins as well as to maintain the
free amino acid pool, and accordingly there is a negative net balance of muscle protein
(11). Ingestion of a protein containing meal results in an enlargement of the free amino
acid pool, which drives the stimulation of protein synthesis and to a minor degree re-
duces protein breakdown (18, 19). This enables a restoration of protein losses during
fasting and accordingly there is a net protein accretion. Importantly, an elevated rate of
synthesis cannot be withheld for longer than 2-3 hours despite preservation of an elevat-
ed pool of free amino acids achieved by infusion (20). There is evidently a need for a
refractory period where a functional demand for new proteins is attained in order for the
rate of synthesis to be increased again. All in all, during the course of the day as the
feeding-fasting periods replace one another the fluctuations in protein synthesis and
breakdown balance each other out and muscle mass remains stable under normal condi-
tions.
In the aspect of exercise, resistance type of exercise in particular, stimulate the rate
of protein synthesis during the recovery period in order to generate new proteins to
functionally adapt to the demands the muscle is exposed to while contracting. Alongside
with the robust increase in the rate of protein synthesis there is also an increase, alt-
hough lesser, in protein breakdown. Accordingly, exercise in the fasted state improves
muscle protein net balance but it remains slightly of the negative side (11). Therefore,
the coordination of exercise and amino acid ingestion act together in an additive manner
on protein synthesis so that it exceeds protein breakdown and generates a positive net
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protein balance (19), thus enabling muscle protein accretion to occur after repeated
periods of a positive net balance (21, 22).
The observation that amino acids and resistance exercise robustly stimulates protein
synthesis while having no or only minor effects on protein breakdown (11, 23), together
with findings that acute changes in synthesis following an exercise/nutrition interven-
tion are in a qualitative manner reflected in long term changes in muscle mass (22, 24),
argue that protein synthesis is the major determinant for muscle growth (17). As a cave-
at it must be emphasized that the methodology to study protein breakdown is intricate
and seldom applied in studies on muscle protein turnover. Hence, a more complete
understanding of how various nutritional and exercise factors influence protein break-
down could bring some nuance to that statement.
1.1.1 Methods to study protein turnover
The introduction of stable amino acid isotopes in the 1970s and the major advancements
in the field of molecular biology in the 1990s have had a major impact on our current
understanding of the regulation of protein turnover, especially with regard to protein
synthesis. One fundamental and tracer-free method to study protein turnover is via as-
sessment of arteriovenous differences in amino acid concentrations across a muscle.
This approach offers an assessment of net muscle protein balance but cannot dissect out
if the changes are synthesis or breakdown related (25). A combination of this methodol-
ogy with continuous infusion of a stable amino acid isotope and muscle biopsies to
assess the free amino acid pool, termed three-compartment model (26), enables quanti-
fication of both synthesis and breakdown. However, the three-compartment model is
limited by the fact that is does not enable direct quantification of the synthesis of pro-
teins in the muscle. Introduction of the now commonly practiced measurements of frac-
tional synthetic rate (FSR) has enabled direct quantification of the synthesis rate of
proteins without the necessity of catheterization (27) FSR is determined as the rate of
amino acid tracer incorporated into a fraction of the muscle protein pool during a certain
period of time (usually 2-4 hours). An advantage with FSR is that it allows quantifica-
tion of the synthesis rate of proteins either in the entire muscle pool (mixed muscle), or
in its sub fractions (myofibrillar, sarcoplasmic or mitochondrial) or even in specific
proteins (28, 29). The latter is of great importance in understanding the adaptations on
the protein level that occur as a result of different types of exercise stimuli. In addition
to evaluating the direct effect on protein synthesis after various interventions, a major
increase in our understanding of the underlying mechanisms has recently evolved. A
nutrition or exercise stimuli results in altered mRNA expression, changes in protein-
protein interactions and changes in enzyme activity within the muscle cell which we are
able to asses using various refined molecular methods. Although an important ground-
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work in the knowledge of these changes has been made, a lot is still unknown concern-
ing the mechanistic control of these events, especially in humans.
The methodological assessment of protein breakdown can on the whole be described
as more intricate than the assessment of protein synthesis. As described above break-
down can be assessed using AV-differences across the muscle, preferably in combina-
tion with stable isotope infusion. As with synthesis this approach cannot directly quanti-
fy the breakdown of muscle protein or determine effects on individual proteins. An
alternative method to assess fractional protein breakdown uses the approach of tracer-
dilution, where a tracer is infused and its dilution from tracee in muscle is monitored
and used to calculate the rate of breakdown (30). However, application of this method
involves a short time frame for assessment (11) and cannot surely determine if the
breakdown is related to the proteins in muscle per se. In order to accurately do so, the
proteins in muscle must be pre-labeled with isotope (31), which has the disadvantage of
being time consuming. In line with this, the understanding of the molecular mechanisms
controlling protein breakdown is also limited and revolves much around two proteins in
a proteolytic pathway that is to be discussed further in section 1.5.
Figure 1. The compartments and events involved in methods to study protein turnover. Modified
from Biolo et al (26).
1.2 Protein synthesis – effects of exercise
Given that resistance training generally results in muscle hypertrophy while endurance
training predominantly increases mitochondrial density and vascularization, exercise
induced rates of protein synthesis has been mainly studied in connection with resistance
type of exercise. While it is indeed clear that resistance exercise generates a profound
Muscle free amino acid pool Muscle protein
Artery
Vein Molecular signaling
Dietary amino acids
Excretion - Oxidation
Synthesis
Breakdown
Molecular signaling
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stimulation of the rate of protein synthesis (32, 33), there are several studies showing an
enhanced rate of protein synthesis in the recovery from endurance exercise at varying
exercise intensities (34-39). Acute changes in protein synthesis following exercise is a
response that enables the muscle to specifically adapt to the demands perturbated by the
exercise bout, hence it is logical that exercise, independent of mode, evokes alterations
in protein synthesis if the bout is physiological challenging to the muscle. Although
both modes of exercise stimulates protein synthesis there are a number of modulating
factors such as protein sub fractions, training status and intensity. In consideration of
resistance training adaptations, it is no wonder that resistance exercise primarily stimu-
lates myofibrillar protein synthesis (16, 40-42), and although studies are sparse, it is
generally appreciated that endurance exercise stimulates the synthesis of mitochondrial
proteins (16, 39, 43, 44).
In the aspect of training status, it can be generally claimed that the protein synthetic
response gets more specific with increasing training experience. In a study by Wil-
kinson and colleagues (16) the authors showed that in the untrained state both endur-
ance and resistance exercise stimulated mitochondrial protein synthesis following an
exercise bout. Following 10 weeks of mode specific training mitochondrial protein
synthesis was specifically stimulated after endurance exercise while that of the
myofibrillar proteins only after resistance exercise. This highlights the notion that it is
the physiological demands exerted by the exercise bout for a given individual that pro-
motes the adaptation and not only the mode of exercise per se. Moreover with regard to
training status, while resistance exercise induced protein synthesis is elevated above rest
for at least 48 hours (11) in untrained subjects, it is normalized after 36 hours in more
trained subjects (45) and also lower compared to untrained at 24 hours post exercise
(46). In addition, although not an entirely consistent finding, also the amplitude of the
protein synthetic response to resistance exercise seems to be reduced in trained subjects
(40, 47, 48) along with higher basal rate of synthesis in these individuals.
Concerning the third major modulator of the protein synthetic, exercise intensity,
performing resistance exercise repetitions at 70-90% of 1RM induces a greater stimula-
tion than performing work matched repetitions at 15-40% of 1RM (49, 50). Similar
observations has been made following endurance exercise, showing that cycling exer-
cise for 30 min at 60% of Wmax stimulates mitochondrial protein synthesis while 60
min at 30% of Wmax does not (39). All these modulators are likely to interact with each
other depending on the individual situation, e.g. a lower intensity may be sufficient for
an untrained individual (35), and when adding other modulators to the equation such as
concurrent exercise, age and muscle group the response becomes more complex, less
predictable and is required to be studied in specific.
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1.3 Protein synthesis – effects of amino acids
From a physiological point of view it is logical that protein turnover is largely regulated
by the availability of amino acids where a lack of substrate limits the process of protein
synthesis and an abundant supply of substrate enables a high rate. Findings that amino
acids have a role in regulation of the rate of protein synthesis started to emerge in the
late 1960s. For example, Jefferson and Korner (51) showed that perfusion of rat livers in
situ with two- to ten-fold the normal plasma levels of amino acids, stimulated amino
acid incorporation into liver proteins. Morgan et al. (52) showed similar effects in heart
muscle when raising amino acid levels in plasma to five times the fasting levels. The
first indications that amino acids stimulate the rate of protein synthesis in humans came
from the work of Rennie and colleagues (18), who showed that ingestion of a mixed
meal increased the rate of protein synthesis in skeletal muscle by about two-fold. Sever-
al studies have then shown that these changes are mediated by the increased amino acid
availability achieved either by infusion (19, 53, 54) or oral ingestion (23).
Dietary protein is composed of 20 amino acids and 9 out of these are so called es-
sential amino acids (EAA), meaning that they cannot be de novo synthesized in human
organs and need to be dietary derived. Interestingly the group of EAA is thought to be
the primary mediators of amino acid stimulated protein synthesis (55-58). In the group
of EAA the branched chain amino acid (BCAA) leucine has received particular interest
as the prime mediator of the anabolic stimulus. As early as in the 1970s, Buse and Reid
(59) demonstrated that leucine alone was capable of stimulating protein synthesis in
isolated rat muscle. In human muscle it was much later shown that a low dose continu-
ous infusion of leucine improved net protein balance (60) and that a large bolus infusion
was able to stimulate protein synthesis (61). It was however not until just recently estab-
lished that oral ingestion of leucine is able to stimulate protein synthesis in human skel-
etal muscle (62). While it is clear that leucine is a potent stimulator of anabolism in
skeletal muscle it is not established how important leucine is per se in the regulation of
protein synthesis in comparison to the other EAA and if those can exert individu-
al/additional effects in the response to amino acid ingestion in human skeletal muscle.
With regard to the amount of protein/amino acids that needs to be ingested to ac-
quire a maximal response on protein synthesis it is considered that 20 g of high quality
protein (63, 64) or 10 g of EAA is sufficient (65), which can be individualized to about
0.25 g/kg body weight in terms of protein. Given that amino acid stimulation of protein
synthesis is maintained for about three hours (20, 41) the most effective strategy for
muscle protein accretion seems to be consumption of at least 20 g of protein every third
hour (66).
Oral intake of protein/amino acids, and even more so a mixed meal containing car-
bohydrate, increases insulin secretion from the pancreas. Hyperinsulemia has the ability
to stimulate muscle protein synthesis (67), but the interplay between amino acids and
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insulin in the regulation of protein balance is somewhat complex. It is nonetheless es-
tablished that for elevated insulin to stimulate protein synthesis amino acid availability
must be maintained or increased (68). Moreover, insulin has a permissive role for amino
acids to stimulate protein synthesis (69, 70), meaning that a certain level of circulating
insulin needs to be present in order for amino acids to exert their effect. What that cer-
tain level is and at which concentration of insulin the maximal response is acquired
under amino acid stimulation is difficult to answer. It has however been shown that
raising insulin levels step wise from slightly above fasting to the end of what is physio-
logical relevant under amino acid infusion has no additional effect on protein synthesis
(71). In addition, adding carbohydrate to a protein or EAA supplement does not aug-
ment amino acid stimulated protein synthesis (72-74).
1.4 Molecular regulation of protein synthesis
At the molecular level, protein synthesis refers to the process of translating mRNA into
protein, where amino acid charged tRNA is brought to the ribosomes which read the
mRNA sequence and synthesizes an amino acid chain accordingly. The process of pro-
tein synthesis is energy demanding and largely dependent on the availability of amino
acid substrates, thus mammalian cells have developed a regulatory system for protein
synthesis that respond to nutrient availability, energy status, growth factors and hor-
mones. An essential signaling pathway that is able to respond and integrate these vari-
ous signals revolves around the key serine/threonine kinase mechanistic target of
rapamycin (mTOR), which exists in two distinct multi-protein complexes where mTOR
complex 1 (mTORC1) is the one responsible for growth regulation (75). Besides mTOR
itself, mTORC1 constitutes of five proteins where the regulatory-associated protein of
mTOR (raptor) is the defining component of mTORC1 and acts as a scaffolding protein
that recruits mTOR substrates with a so called TOS- (TOR signaling) motif (76, 77). In
addition to raptor, PRAS40 (proline-rich Akt substrate of 40 kDa) is a unique compo-
nent of mTORC1 and functions as an inhibitor of mTOR kinase activity that is regulat-
ed by insulin (78). The three additional proteins are deptor (DEP domain containing
mTOR-interacting protein), mLST8 (mammalian lethal with sec-13 protein 8) and the
Tit1/Tel2 complex (79). A number of defined and relatively undefined so called up-
stream pathways mediate the growth promoting and inhibitory signals to mTORC1,
which integrate these signals and transduce the input to well characterized downstream
targets that regulate the process of translation. The following sections will outline the
downstream events of mTORC1 as well as summarizing the current knowledge of the
upstream signals.
22
1.4.1 Downstream of mTORC1
Translation of mRNA into protein is a multistep process that includes three major steps;
initiation, elongation and termination, of which initiation is the rate limiting step and
also under major control by mTORC1. All mRNA transcripts contain a so called cap
structure in the 5’ end to which initiation factors are recruited resulting in ribosome
assembly and sequentially elongation. The eukaryotic initiation factor 4E (eIF4E) is the
protein responsible for the recognition of the mRNA 5’ cap, which upon binding recruit
eIF4G and eIF4A to form the so called eIF4F complex (80). The eIF4A possess RNA
helicase activity, meaning that it resolves secondary structures proximal to the mRNA
5’ cap allowing ribosome assembly, a function that can be potentiated by binding of the
translation factor eIF4B to eIF4A (80).
The protein 4E-BP1 is a well characterized substrate of mTORC1 and in the basal
state 4E-BP1 binds to eIF4E and prevents the formation of the eIF4F complex. Upon
stimulation by amino acids and growth factors mTORC1 phosphorylates 4E-BP1 result-
ing in the release of eIF4E, thus allowing its association with eIF4G (81). Seven phos-
phorylation sites have been identified on 4E-BP1 and the ones considered most im-
portant for its function and also early reported to response to insulin are Thr37
, Thr46
,
Thr70
and Ser65
(82). These sites are phosphorylated in the mentioned hierarchical order
(83), where Thr37
and Thr46
phosphorylation appear to be permissive for the latter to
occur (84, 85). It is shown that mTOR directly phosphorylates 4E-BP1 at Thr37
and
Thr46
in vitro (86, 87) while it remains to be concluded whether mTOR itself phosphory-
lates Thr70
and Ser65
, although it is shown that the latter sites are clearly mTOR-
dependent (85, 88, 89). The mechanisms regulating 4E-BP1:eIF4E interaction seem
rather complex but it is suggested that all three Thr sites are more or less important for
resolving the eIF4E interaction while Ser65
is dispensable for this event (83, 90, 91), but
is suggested to play a role in preventing re-association (92). Interestingly also, besides
the role of 4E-BP1 in controlling translation initiation and thus global rates of protein
synthesis it is also suggested to be in control of the synthesis of specific regulatory pro-
teins. Many mRNA transcripts that code for regulatory proteins such as initiation factors
and ribosomal proteins contain complex structures in the 5’ end, making them less ac-
cessible for translation and accordingly under a higher degree of control. Stimulation of
4E-BP1 phosphorylation and sequential release of eIF4E enhances the ability to trans-
late these complex mRNAs, thus 4E-BP1 directly controls the rate of protein synthesis
and the capacity for protein synthesis in more long term aspect (81).
The other well characterized substrate of mTORC1 that also regulates translation is
the 70 kDa ribosomal protein S6 kinase (S6K1). The primary phosphorylation site of
S6K1 is at Thr389
which is readily phosphorylated by active mTORC1 (86) and mutation
of this site completely ablates S6K1 activity (93). Although additional phosphorylation
sites can influence S6K1 activity, especially Thr229
which is phosphorylated by PDK-1
23
(94), S6K1 activity has the nearest correlation with the phosphorylation status of Thr389
(95). Being a principal target of mTORC1 and essential for its activity, Thr389
phos-
phorylation of S6K1 is the most common and functional read outs of mTORC1 activity.
A number of downstream targets of S6K1 that are involved in the translation machinery
have been identified. Two of these targets function to enhance the RNA helicase activity
of eIF4a which, as mentioned above, enhances the efficiency of translation initiation by
resolving the inhibitory secondary structure of the mRNA transcript. The first target,
eIF4B, gets phosphorylated by S6K1 on the Ser422
residue resulting in enhanced re-
cruitment of eIF4A to the initiation complex thus promoting its activity (96). The other
target is the programmed cell death protein 4 (PDCD4) which regulate eIF4A in a
slightly different manner. In the basal state PDCD4 binds to eIF4A and prevents its
association with eIF4G, but upon Ser67
phosphorylation of PDCD4 by active S6K1 the
protein is degraded allowing eIF4A to fully interact with eIF4G (97, 98). Ribosomal
protein S6 (rpS6) is another target of S6K1 and the Ser235/236
phosphorylation of rpS6 is
the most commonly assessed biomarker for S6K1 activity although the exact function of
rpS6 is unknown and its role in regulating protein synthesis is questioned (80). Lastly,
S6K1 also targets a kinase that is involved in translation elongation. The eukaryotic
elongation factor 2 (eEF2) is highly involved in peptide chain elongation and phosphor-
ylation of its Thr56
residue inhibits its activity (99). The phosphorylation of eEF2 is
under control by the eEF2 kinase (eEF2k) which is inhibited by S6K1 phosphorylation
of its Ser366
residue (100). Accordingly, active S6K1 inhibits the function of eEF2k
which results in the increased activity of eEF2, thus promoting translation elongation.
As made apparent mTORC1 is in control of multiple mechanisms in translation initia-
tion, affecting both the efficiency and the capacity of initiation as well as contributing to
the regulation of translation elongation.
1.4.2 Upstream of mTORC1
There are several factors that provide signal input to mTORC1 but the most prominent
are amino acids, insulin, energy stress and mechanical stretch of the muscle. While
these factors signal with parallel mechanism they all in some extent involve the small
GTPase Rheb (ras homologous enriched in brain) which is a key activator of mTORC1.
The effect of Rheb is dependent on its nucleotide status, meaning that when Rheb is
bound to GTP it is able to bind to mTORC1 and activate its kinase activity, while Rheb
∙ GDP does not activate mTORC1 (78, 101). The upstream regulator of Rheb nucleotide
status is the GTPase activating protein TSC2 (tuberous sclerosis 2) which functions in a
complex with TSC1 and TBC1D7. The TSC-complex has a role as an mTORC1 inhibi-
tor by stimulating the conversion of Rheb ∙ GTP to Rheb ∙ GDP, thus inhibiting the
stimulatory function of Rheb (102).
24
The best characterized stimulatory input to mTORC1 comes from insulin/IGF-1,
that upon binding to their extracellular tyrosine kinase receptors promote the sequential
activation of the kinase called Akt, which then promote mTORC1 activity by two sepa-
rate mechanisms (103). First, activated Akt phosphorylates TSC2 on Ser939
and Thr1462
which suppresses the ability of the TSC-complex to promote Rheb ∙ GDP status, thus
enabling mTORC1 to be activated by Rheb ∙ GTP (104). Secondly, Akt has the ability
to directly phosphorylate the mTORC1 inhibitory component PRAS40 (78), and once
phosphorylated it relives its inhibitory binding with raptor, thus enabling mTORC1
substrate binding (105). The mechanistic influence of cellular energy status on
mTORC1 is also quite well described and functions in a similar manner to that of insu-
lin, albeit with an opposite outcome; mTORC1 inhibition to conserve cellular energy.
The AMP-activated protein kinase (AMPK) is a major sensor of cellular energy status
which responds to the ratio between AMP and ATP. Phosphorylation of the Thr172
resi-
due of the AMPKα subunit by upstream kinases, along with AMP binding to the γ-
subunit results in full activation of AMPK (106). Activated AMPK can phosphorylate
TSC2 at Ser1387
which promotes its activity and thereby increasing the amount of Rheb ∙
GDP, thus decreasing mTORC1 activity (107). In addition, active AMPK is able to
phosphorylate the defining mTORC1 component raptor, leading to its binding of 14-3-3
protein thereby inhibiting mTORC1 activity (108). Accordingly, both growth factors
and energy stress alter TSC2 activity and modulates mTORC1 components, being either
stimulatory or inhibitory in their outcome.
Amino acids are potent stimulators of mTORC1 and they signal in a parallel and
dominant manner to that of growth factors since they do not require TSC2 and since
insulin fails to activate mTORC1 in the absence of amino acids, while the opposite
situation has a modest impact on its activity (109-111). In contrast to growth factors and
energy stress, amino acid signaling towards mTORC1 is not that well-characterized and
seems to be rather complex. However, important progress in the understanding has been
made in recent years. An important breakthrough was made when identifying the four
components of the Rag family of small GTPases (RagA, RagB, RagC and RagD) as
crucial mediators of the amino acid signaling. The Rag proteins functions as heterodi-
mers where Rag A or B bind to Rag C or D, enabling four different combinations (112),
and the activity of the complexes are determined by their nucleotide state. Under amino
acid rich conditions RagA/B becomes loaded with GTP and RagC/D with GDP result-
ing in active Rag heterodimers (113), whereas RagA/B ∙ GDP and RagC/D ∙ GTP re-
sults in inactive complexes. The active Rag heterodimers bind to the raptor component
of mTORC1 and recruits the complex to the surface of the lysosome where it is pro-
posed to physically interact with active Rheb which stimulates mTOR kinase activity
(113, 114). This model would explain why insulin fails to activate mTORC1 in the
absence of amino acids, since insulin stimulated Rheb is not able to physically interact
25
with mTORC1 prior to amino acid promoted translocation of mTORC1 towards Rheb.
In contrast to Rheb, the Rags do not contain a lipid-motif enabling their binding to the
lysosomal membrane. Instead the Rags require binding to the pentameric Ragulator-
complex for lysosomal anchoring, a complex that also is able to regulate the GTP status
of Rag A/B (115). How the amino acids are sensed and how this is mediated to the
Rags, and maybe also independently of the Rags, is a question of particular current
interest. Many factors/mechanisms have been introduced but the best characterized so
far involves the v-ATPase, which is located at the lysosomal membrane and binds to
both Rags and the Ragulator complex (116). It is proposed that the v-ATPase senses an
increase in the levels of amino acids within lysosome and mediates this by activating
the Rags (116). How this relates to the levels of amino acids in the cytosol and if amino
acids are translocated into the lysosome is unknown. Another interesting amino acid
sensor in this context, that is present in the cytosol, is the leucyl-tRNA synthetase
(LRS). In response to increasing levels of leucine in the cell LRS is activated and is
translocated to the lysosome where it interacts and activates the Rags (117).
The fourth major factor in upstream control of mTORC1 is mechanical stretch,
which acts by mechanisms less defined than the amino acid input. The most attracting
mediator of the mechanical stretch signal is phosphatidic acid (PA) which is synthesized
by among others phospholipase D and diacylglycerol kinases in response to muscle
contractions. PA is suggested to bind directly to mTOR and act as a positive regulator
of its kinase activity (81). A number of studies in animal muscle have pointed to a par-
ticular role for PA in mediating muscle contraction induced activation of mTORC1
(81), and interestingly, recent data suggest that exogenous supplementation of PA can
stimulate mTORC1 (118). While the role of PA in this context is intriguing it is war-
ranted further investigation in human muscle. In addition to PA, recent intriguing data
in mouse skeletal muscle has shown that muscle contractions induce translocation of
TSC2 from the lysosomal membrane, thus promoting formation of active Rheb ∙ GTP
and subsequent mTORC1 activation (119).
To summarize, Rheb is believed to be the ultimate activator of mTOR kinase activi-
ty and is under major control by TSC2. Growth factors and energy stress signal both via
direct actions on mTORC1 and in a TSC2-dependent mechanism, resulting in a positive
and a negative outcome, respectively. Amino acids, which are sensed in an inadequately
defined manner, seem to stimulate mTORC1 by activating the Rag-heterodimers which
translocate mTORC1 to the lysosomal membrane where it can interact with Rheb. Mus-
cle contractions is suggested to disrupt TSC2 inhibition of Rheb as well as increase
intracellular levels of PA which bind directly to mTOR to promote its activity. It must
however be emphasized that the present models are predominantly based on work con-
ducted in cell cultures and animal muscle, and there is an overall lack of data to confirm
these mechanisms in vivo in human skeletal muscle.
26
1.4.3 mTORC1 – importance for muscle protein accretion
As made apparent, mTORC1 is a major player in the control of the rate of protein syn-
thesis and responds to a variety of growth inducing and inhibiting factors. But as men-
tioned, the majority of studies examining the role of mTORC1 are performed in cells
that are often of non-muscle origin or in animal muscle under an acute (min-hour) ana-
bolic stimulus. It is thus difficult to make out the physiological relevance of these ob-
servations i.e. do they relate to muscle growth? In relation to mechanical stimuli, or
resistance training, there are several lines of evidence to support a pivotal role of
mTORC1 in the stimulation of muscle protein accretion. The first evidence in this mat-
ter came from Baar and Esser (120) who showed that acute changes in the degree of
S6K1 phosphorylation correlated well with the increase in muscle mass following six
weeks with continuous bouts of high frequency electric stimulation of the leg muscles in
rats. Such correlative data also exist in human skeletal muscle, were S6K1 activation 30
min after exercise correlated well with gains of muscle mass after a 14-week period of
progressive resistance training (121). In further relation to human muscle, administra-
tion of the mTORC1 specific inhibitor rapamycin blocks resistance exercise induced
protein synthesis (122). Moreover, in a pioneering study by Bodine and co-workers
(123) the authors showed that mTORC1 inhibition by rapamycin in rats was able to
prevent mechanical overload induced muscle hypertrophy. While the effect of
rapamycin in the study of Bodine et al (123) was not muscle specific, two more recent
studies using muscle specific genetic knock out models clearly established the specific
role of mTORC1 in muscle contraction induced hypertrophy (124, 125). In a more gen-
eral aspect, there is also a significant degree of hypertrophy in genetically modified rats
where mTORC1 is consistently active (126).
Since amino acids do not induce muscle hypertrophy per se, but require a mechani-
cal overload stimulus and vice versa, it is difficult to assess the role of mTORC1 in the
sole contribution of amino acids in muscle growth. However, evidence to support a
fundamental role of mTORC1 in amino acid stimulation of protein synthesis comes in
part from our laboratory, where we showed that ingestion of BCAA potently stimulates
S6K1 phosphorylation (127) and that this effect is potentiated by resistance exercise
(128). Importantly, in vivo studies in both animal and human skeletal muscle have
shown that rapamycin administration blunts amino acid induced stimulation of protein
synthesis (129, 130).
Given that it is not feasible to pharmacologically block mTORC1 in human muscle
over a prolonged period of time it is difficult to ultimately establish its role in muscle
growth in humans, but taken altogether there are several lines of evidence, from a wide
range of experimental models and species, which defines mTORC1 as an essential
component in stimulation of muscle protein synthesis and muscle hypertrophy.
27
1.5 Protein breakdown – effects of exercise and nutrition
In comparison to protein synthesis, the current knowledge of the influence of exercise
and nutrition on protein breakdown is limited, in part due to the methodological diffi-
culties in assessing protein breakdown, as discussed in the methodology section 1.1.1.
However, according to the literature available both resistance exercise (10, 11) and
endurance exercise (34, 35) induce an increase in the rate of protein breakdown after
exercise. Although in the case of resistance exercise it is of a lesser magnitude than the
increase in protein synthesis. Given the limited data available, it is not known which
mode of exercise that has the greatest impact on breakdown.
At rest, infusion of mixed amino acids or branched chain amino acids has been
shown to have a small to modest capacity in reducing muscle protein breakdown (60,
71, 131). In contrast, several studies report that infusion or oral ingestion of amino acids
do not affect protein breakdown after resistance exercise (19, 55, 57, 73, 132, 133).
Food intake/amino acids also stimulate insulin secretion, and while the sole role of
insulin in stimulating protein synthesis seems to be relatively minor, the evidence for its
capacity in reducing protein breakdown is more substantial. In a recently published
meta-analysis 22 out of the 25 studies analyzed exhibited a positive effect of insulin
infusion in reducing protein breakdown, and in seemingly quite modest concentrations
(68).
At the molecular level, several mechanisms or pathways are responsible for the
breakdown of protein within skeletal muscle. For example, lysosomal degradation and
specific proteases play an important role but the main contributor of protein breakdown
in muscle is the ubiquitin-proteasome pathway (134). This pathway constitutes of sev-
eral proteins that activate, transfer and ligates ubiquitin onto proteins as a marker for
ultimate degradation by the 26S proteasome (134). Two key regulators of this pathway,
termed muscle RING finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), were
identified in 2001 (135) and functions as ubiquitin ligases. These genes have been
shown to be up-regulated in numerous atrophy models such as in several catabolic dis-
eases, fasting, ageing, glucocorticoid treatment and immobilization (136). In addition,
genetic knock out models of these genes are reported to be protected from atrophy under
certain catabolic stimuli, displaying their crucial role in the regulation of protein break-
down (136). Interestingly, several studies have reported that both endurance and re-
sistance exercise affects the expression of these genes in human skeletal muscle (37,
137-140), presumably to support the increased demand for muscle tissue remodeling
and exercise adaptations.
The regulation of MuRF-1 and MAFbx expression is described to be under major
control by the FOXO family of transcription factors that are regulated up-stream by
among others Akt (141). In addition, inflammatory signaling through p38 MAPK and
NF-κB has been demonstrated to be able to control the expression (141). Although
28
exercise has the potential to affect both Akt and p38 it is yet to be determined how exer-
cise mediates its effects on MAFbx and MuRF-1 expression and on protein breakdown
1.6 Regulation of endurance exercise adaptations
Endurance training induces adaptations that promote an increased oxygen uptake and a
greater capacity to utilize fat as a fuel source. In skeletal muscle these changes mainly
relate to what is termed mitochondrial biogenesis, seen as both increases in the activity
of mitochondrial enzymes (142) and the mitochondrial density (143). A major break-
through in the understanding of the molecular regulation of mitochondrial biogenesis
was made in 1998 by Puigserver and colleagues (144) who identified the key transcrip-
tion factor peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α).
They found that PGC-1α induced the expression of several key mitochondrial enzymes
and PGC-1α has later been identified to control the expression of a plethora of genes
involved in metabolism, vascularization and reactive oxygen species defense (145). In
human skeletal muscle a number of studies using different exercise protocols have
shown a robust induction of PGC-1α expression in the recovery from exercise (146-
148). A crucial role for PGC-1α in inducing mitochondrial biogenesis has been shown
in models with both overexpression and genetic knout of PGC-1α that induce or blunt
the expression of mitochondrial enzymes, respectively (149). Several mechanisms have
been implied to control the expression of PGC-1α, and these are among others increased
intracellular [Ca2+
], activation of cellular stress responsive proteins and a decrease in
energy status (ATP/AMP ratio) (150). All of these events are well characterized to oc-
cur during a bout of endurance exercise in human skeletal muscle (151-153). Although
other mechanisms are likely present, exercise induced expression of PGC-1α is current-
ly viewed as the main regulator of the peripheral adaptive response to endurance exer-
cise.
1.7 Crosstalk between divergent modes of exercise
As made apparent resistance exercise and endurance exercise induce specific and di-
verse muscular adaptations. However, most athletes and recreationally active people
perform a combination of both types of exercise to a varying degree in order to meet the
metabolic and strength requirements of their sport or in everyday fitness. This notion
evokes a major and important question, are these mode specific adaptations and their
underlying molecular signals compatible or not (154, 155)? In a seminal study by
Hickson (156) it was reported that high volume concurrent resistance and endurance
training resulted in reduced gains of strength compared to resistance training only. Since
then a number of studies have investigated the adaptations to concurrent training and
generally concluded that it hampers strength development but has no detrimental effect
29
on oxygen uptake compared to the isolated modes of exercise (157). There is however
inconsistencies as to whether concurrent training reduces muscle hypertrophy compared
to resistance training alone. While some studies report a reduced muscle fiber growth
(158-161), other studies show no interference (162-165) and some actually show greater
gains of muscle mass with concurrent training (166-168). Due to differences in endur-
ance exercise modality, exercise intensity, duration, number of session, nutritional sta-
tus and initial training status between studies, it is difficult to decisively conclude
whether concurrent training affects muscle hypertrophy.
The concept that endurance exercise inhibits resistance exercise induced protein
synthesis and ultimately muscle hypertrophy has got some experimental support from
cell and rodent models. These studies have shown that active AMPK inhibits mTORC1
signaling (169), protein synthesis (170, 171) and cell size (172). From a biological point
of view this is rational since protein synthesis is an energy demanding process and
would accordingly be inhibited when the cell is under energy stress. In a more applica-
ble model, Atherton and co-workers (173) showed that 3 hours of low-frequency elec-
tric stimulation in rats, to mimic endurance exercise, activated AMPK and inhibited
mTORC1 signaling. Interestingly however, in human muscle mTORC1 signaling has
been reported to increase after both resistance and endurance exercise despite simulta-
neous activation of AMPK (16, 33, 174). Only a few human studies has tried to address
the question whether endurance exercise inhibits resistance exercise induced signaling.
However, these studies either separated the sessions by 6 hours (175) or used an endur-
ance exercise protocol with an intensity to low to activate AMPK (43, 176) and there-
fore found no detrimental effects on mTORC1 signaling. Accordingly, it is not com-
pletely resolved whether activation of AMPK inhibits resistance exercise induced
mTORC1 signaling and if divergent signaling pathways interfere in human skeletal
muscle.
30
Figure 2. Simplified overview of the mTORC1 signaling pathway
AMP/ATP↑
mRNA mRNA
TRANSLATION INITIATION
AMINO ACIDS INSULIN ENDURANCE EXERCISE
TRANSLATION ELONGATION
RESISTANCE EXERCISE
PROTEIN
Rapamycin
4E-BP1
RHEB
TSC1TSC2
PDCD4
PRAS40
RagC-RagD
eIF4B eEF2
eEF2K
eIF4E
mTORRaptor
p70-S6K1
RagA-RagB
RagC-RagD
RagA-RagB
RHEB
PI3K
AKTAMPK
PA
RTK
GTP
GTPGDP
GDP
GDPGTP
rpS6
eIF3eIF4G
eIF4E
eIF4A
40 S
eIF4B
PLD
Positive regulator Negative regulator
Stimulation Inhibition
31
2 AIMS
The overall objective of this thesis was to examine the underlying molecular mecha-
nisms in control of protein synthesis and breakdown in response to different modes
of exercise and amino acid ingestion in human skeletal muscle. The specific aims of
this thesis were as follows;
To examine if endurance exercise induced activation of AMPK inhibits re-
sistance exercise induced mTORC1 signaling and protein synthesis in the
vastus lateralis muscle
To explore whether resistance exercise induced protein signaling, protein
synthesis and gene expression in the triceps brachii muscle is affected by
preceding high intensity lower body endurance exercise
To examine the importance of leucine among the essential amino acids in
their ability to stimulate mTORC1 signaling following resistance exercise
To determine the separate effects of leucine, branched-chain amino acids
and essential amino acids on mTORC1 signaling and protein synthesis in
combination with resistance exercise
To examine how genetic markers of protein breakdown are affected by dif-
ferent models of concurrent exercise and amino acid intake
32
3 METHODS
3.1 Subjects
Table 1. The characteristics of the subjects in all four studies
Gender Number Age (yr) Weight
(kg) Height (cm)
VO2 Peak
(ml ∙ kg-1)
Study I Male 8 26 ± 2 85 ± 2 183 ± 2 55 ± 2
Study II Male 8 31 ± 2 80 ± 2 182 ± 2 55 ± 2
Study III Female 8 27 ± 2 60 ± 3 167 ± 2 45 ± 1
Study IV Male 8 27 ± 2 84 ± 3 181 ± 3 N/A
Mean 8 28 ± 2 77 ± 2 178 ± 2 52 ± 2
In all four studies the subjects were young, non-obese and healthy adults, who all gave
their written consents to participate after being informed of the purpose, the procedures
and all the associated risks of the experiments. All study protocols were pre-approved
by the Regional Ethics Review Board in Stockholm and performed in accordance with
the principles outlined in the Declaration of Helsinki. In study I and II the participants
were considered to be trained individuals who had at least performed resistance and
endurance exercise three and two times per week, respectively, for a minimum of six
months prior to the trials. In study I subjects were required to have a maximal leg
strength corresponding to four times their body weight or more, and in study II a maxi-
mal arm strength of at least 1.25 times their body weight, in the respective designated
exercise model. In study III subjects were recreationally active and performed endur-
ance and (or) resistance exercise on a regular basis. The subjects in study IV were re-
sistance exercise accustomed individuals who had a training history of one year or
more, and all had a maximal leg strength corresponding to five times their body weight.
In all four studies subjects were instructed to refrain from any type of vigorous physical
activity during the two days prior to the experiments. During those two days, in study
III, subjects were instructed to follow a standardized diet containing 15 energy % pro-
tein, 30% fat and 55% carbohydrate with a caloric content corresponding to their indi-
vidual estimated energy expenditure. In the remaining three studies subjects were in-
structed to report their habitual food intake during the two days prior to the first trial
and to repeat that intake prior to the following trials. In all trials subjects reported to the
laboratory early in the morning, arriving by car, bus or train and not by active transpor-
tation. The subjects were in the fasted state since 9 PM the evening before and orally
declared that they had followed the preparatory instructions.
33
3.2 Initial exercise tests
3.2.1 Oxygen uptake
The oxygen uptake tests were performed on a cycle ergometer with VO2 and VCO2
being measured continuously utilizing an on-line system and heart rate being recorded
continuously. The tests consisted of a two stage protocol were the first part consisted of
4 min at 4-5 sub maximal intensities, designed to determine the relationship between
oxygen uptake and work load. Following 10 min of rest the subjects initiated an incre-
mental protocol were the work load was increased by 20 W every min until volitional
exhaustion, reached after 6-8 min. VO2peak was defined as the highest recorded oxygen
uptake during 30-40 seconds when the following criteria where met; RPE above 18,
RER above 1.1 and a plateau in VO2 despite increased work load.
3.2.2 Maximal strength
In study I, III and IV the one repetition maximum (1-RM) for each subject was deter-
mined on a leg press machine. Following a low intensity warm-up the 1 RM was deter-
mined by gradually increasing the load until the subject could not perform more than
one repetition (90 – 180° knee angle). Every effort was separated by five minutes of
rest, and subjects were allowed two additional attempts once they failed to perform one
repetition. The resistance exercise protocol in study II was designed to target the triceps
brachii muscle, therefore exercise was performed in a seated arm extension machine.
Here, maximal strength was determined using a 10-RM protocol which consisted of
three warm-up sets with 10 repetitions each at 0, 25 and 75% of the subjects’ body
weight, followed by a maximal effort at 125%, which was the criterion for enrolment. If
the maximal effort resulted in more than 10 repetitions, the load was gradually increased
during additional sets until the subject failed to perform 10 approved repetitions. All
repetitions were performed in a 75°–180° elbow angle and all maximal efforts were
separated by a 5 min rest.
3.2.3 Familiarization sessions
In order to minimize any training effects during the experiments the subjects conducted
two (study II and III) or three (study I and IV) familiarization sessions where they per-
formed the actual exercise protocols. During these sessions the intensity and load was
gradually adjusted so that the subjects could perform the designated protocol. In study I
the number of repetitions and load during resistance exercise in the last familiarization
session set the criteria for both experimental trials, since the endurance exercise had an
impact on resistance exercise performance in that study. Each familiarization session
was separated by one week and the last one performed 7-13 days prior to the first exper-
imental trial.
34
3.3 Intervention protocols
3.3.1 Study I
In this study subjects each performed one session of high intensity interval cycling
combined with directly succeeding resistance exercise in the leg press (ER) and one
session of resistance exercise only (R), in a randomized cross-over fashion, where each
trial was separated by 9 – 14 days. We employed a primed-continuous infusion of L-
[ring-13
C6]-phenylalanine during the trials in order to quantify muscle protein fractional
synthetic rate (FSR) at rest and during recovery. Upon arrival to the laboratory (~5.30
AM) the subjects took a supine position and had catheters placed in both antecubital
veins for tracer infusion and repeated blood sampling. Following initiation of tracer
infusion subjects rested for two hours after which a first muscle biopsy was taken. After
three additional hours of rest a second biopsy was taken, which marked the end of the
resting measurement and the beginning of the exercise period. Thereafter, the subjects
initiated the exercise protocol which in the ER-trial consisted of 15 min warm-up on the
cycle ergometer (50 W 5 min, 100 W 10 min), followed by 5 x 4 min at 85% of individ-
ual VO2peak, each separated by 3 min of cycling at 100 W. Within one minute after com-
pletion of the last interval a third muscle biopsy was taken, after which subjects recov-
ered by 10 min low intensity cycling and 5 min of complete rest. In the R-trial the cor-
responding period of time was replaced by rest in a supine position. Next, in both trials,
subjects were seated in the leg press machine and performed three warm-up sets of 10
repetitions at ~10, 30 and 60% of their 1 RM. Thereafter the heavy-resistance exercise
protocol was initiated and consisted of 4 sets of 8-10 repetitions at 80% of 1 RM, 4 sets
of 10-12 repetitions at 70% of 1RM and finally 2 sets to fatigue at 60% of 1 RM, were
all sets were separated by 3 min of rest while still seated in the leg press. Immediately
after completion of the final set a fourth biopsy was taken, thereafter the subjects rested
for three hours in a supine position with additional biopsies taken after 90 and 180 min
of recovery. Blood samples were taken every 30 min during the initial five hours of rest.
During exercise blood was sampled after cycling warm-up, after the third and fifth in-
terval, prior to leg press warm up and following the third, seventh and thirteenth set.
Additional blood was collected after 15 and 30 min of recovery and thereafter every 30
min until completion of the trials.
35
Figure 3. Schematic overview of the experimental protocol in study I
3.3.2 Study II
In study II the subjects performed two resistance exercise session using the arms, with
(ER-Arm) or without (R-Arm) a directly preceding bout of high intensity cycling in a
randomized cross-over fashion. The intervention protocol was identical to study I with
three exceptions, first all biopsies were taken from the triceps brachii muscles, secondly
there were no biopsy taken directly following cycling exercise and thirdly the resistance
exercise was performed in a seated arm extensions machine. The resistance exercise
was commenced by three warm-up sets of 10 repetitions at 25, 50 and 75% of 10RM.
Thereafter, the subjects performed 10 sets of heavy resistance exercise where the load
was initiated at their 10RM, and for the following sets the load was gradually decreased
so that the subjects could perform 9-12 repetitions to fatigue. In the last two sets, at
fatigue, the load was lowered with 10 kg after which the subjects immediately per-
formed 5 additional repetitions. The load with corresponding number of repetitions in
each set during the first trial was used in the second trial. All sets were separated with 3
min of rest in a seated position.
R - protocol
Biopsy samples
Rest RecoveryRest Rest R-Ex
120 min 180 min 90 min 90 min~60 min ~60 min
Blood samples
ER - protocol
Biopsy samples
Rest RecoveryRest E-Ex R-Ex
120 min 180 min 90 min 90 min~60 min ~60 min
Continuous infusion L -[ring-(13)C6]phenylalanine
Blood samples
0Time (min) 120 300 360 420 510 600
36
Figure 4. Schematic overview of the experimental protocol in study II
3.3.3 Study III
In this study subjects performed two sessions of leg press exercise while supplied with
an oral EAA supplement, with (EAA) or without leucine (EAA-Leu). Following arrival
to the laboratory on the day of each trial, subjects took a supine position, had a catheter
placed in the antecubital vein of one arm and rested for 30 min. After the rest a first
muscle biopsy was taken from one leg, after which subjects warmed up on a cycle er-
gometer for 10 min at 60 W and then positioned themselves in the leg press machine.
Resistance exercise consisted of one set of 10 repetitions at 40% of 1RM, three min of
rest and then four sets of 10 repetitions at 80% of 1RM separated by five min each.
After exercise muscle biopsies were taken after 60 and 180 min of recovery. Blood
samples were taken at rest, after warm up, after the second set, immediately after exer-
cise and following 15, 30, 60, 90, 120 and 180 min of recovery. The amino acid sup-
plements were given as seven 150 ml drinks prior to the cycle warm up, before leg press
warm up, after the third set and following 15, 30, 60 and 90 min of recovery. The EAA
supplement provided the subjects with 260 mg EAA ∙ kg-1
body weight and had the
following composition; 13.7% L-histidine, 9.4% L-isoleucine, 17.3% L-leucine, 18.0%
L-lysine, 2.9% L-methionine, 14.4% L-phenylalanine, 13.7% L-threonine and 10.7% L-
valine. In the EAA-Leu trial the amount of leucine was replaced with glycine to match
for nitrogen content. The order of the drinks was randomized and double-blinded, and
besides amino acids the drinks contained salts, artificial sweetener and were lemon
flavored. The two trials were separated by four weeks so that subjects were in the same
phase of their menstrual cycle.
R-Arm protocol
Biopsy samples
Rest RecoveryRest Rest R-Ex
120 min 180 min 90 min 90 min~60 min ~60 min
Blood samples
ER-Arm protocol
Biopsy samples
Rest RecoveryRest E-Ex R-Ex
120 min 180 min 90 min 90 min~60 min ~60 min
Continuous infusion L -[ring-(13)C6]phenylalanine
Blood samples
0Time (min) 120 300 360 420 510 600
37
Figure 5. Schematic overview of the experimental protocol in study III
3.3.4 Study IV
Study IV utilized a randomized, double-blind, cross over design in which subjects were
orally supplied with leucine, BCAA, EAA or placebo in combination with resistance
exercise. On the day of each trial, subjects reported to the laboratory at ~6.00 AM after
which a primed-continuous infusion of L-[ring-13
C6]-phenylalanine was started, fol-
lowed by two hours of rest and a sequential first muscle biopsy. Next, the subjects were
seated in the leg press and initiated the exercise protocol which consisted of three sets of
10 repetitions at ~10, 30 and 60% of 1RM, followed by 10 sets of heavy resistance
exercise to fatigue, starting at 85% of their 1 RM and gradually reducing the load so that
they could perform at least eight, but no more than 12 repetitions to fatigue. The load
with corresponding number of repetitions in each set during the first trial was used in
the following trials. Each set was separated by three min of rest, with subjects remaining
seated in the leg press. Immediately following exercise, a second biopsy was taken and
then subjects rested for three hours in a supine position with additional biopsies taken
after 90 and 180 min. In total, subjects ingested 1350 ml of the supplements, which was
administered as nine 150 ml drinks consumed immediately before the resistance exer-
cise, after the third, seventh and eleventh sets, and following 15, 30, 60, 90 and 120 min
of recovery. The supplements were given at the following doses; EAA 290 mg ∙ kg-1
and BCAA 110 mg ∙ kg-1
, with the following composition; (EAA) 13.6% L-histidine,
9.5% L-isoleucine, 17.1% L-leucine, 17.8% L-lysine, 2.9% L-methionine, 14.3% L-
phenylalanine, 13.6% L-threonine and 11.4% L-valine, and (BCAA) 25% L-isoleucine,
45% L-leucine and 30% L-valine. Leucine alone was given at a dose of 50 mg ∙ kg-1
, so
that the amount of leucine in all of the amino acid supplements was identical. In addi-
tion to amino acids, all drinks contained salts and artificial sweetener, and were lemon-
flavored. Blood samples of 4 ml were collected every 30 min during the first two hours
of rest and then immediately prior to beginning the exercise, after completion of the
third, seventh, tenth and thirteenth set, as well as 15, 30, 60, 90, 120, 150 and 180 min
after completion of exercise. All trials were separated by approximately one week.
0 min 60 min 120 min 180 min 240 min
Rest Recovery
Biopsy sample
Drink
Blood sample
WU RE
38
Figure 6. Schematic overview of the experimental protocol in study IV
3.4 Muscle biopsy sampling
In all trials, muscle biopsies were collected with a Weil-Blakesley conchotome under
local anesthesia following a 0.5-1 cm incision in the skin and the fascia, as described by
Henriksson (177). Immediately after collection, the muscle sample was blotted free
from visible blood, fat and connective tissue and frozen in liquid nitrogen within 15
seconds, and subsequently stored in -80° C until analysis. In the vastus lateralis muscle
the first biopsy was collected 12-15 cm from the mid-patella and in the triceps brachii
~12 cm from the olecranon. All biopsies were taken from a new incision, 2-4 cm prox-
imal to the previous one, and when three biopsies had been taken in a longitudinal series
the next was taken ~2 cm medial or lateral to the first. In study I the first biopsy was
taken in the right leg and then in alternating leg throughout the trial, while in the second
trial the first was taken in the left leg and sequentially alternated. Accordingly, starting
leg and order of trial was alternated in that study. In study II the first biopsy was taken
in the right arm and then followed the pattern as in study I. In study III the first biopsy
was taken in the right leg for four subjects and in the left leg for four subjects, thereafter
the remaining two biopsies in each trial were taken in the same leg as the first one. In
study IV the initial biopsy in each trial was taken from alternating legs (1st; right, 2
nd;
left, 3rd
; right, 4th
; left) and the sequential biopsies in each trial was continuously alter-
nated between legs.
3.5 Plasma analysis
3.5.1 Sample preparation
Blood samples were collected in EDTA-tubes (study I, II and IV) or in heparinised
tubes (study III), set on ice and within minutes transferred to Eppendorf-tubes and cen-
trifuged at 10 000 g for 3 min. The plasma thus obtained was transferred to new tubes
and stored at -80° C until analysis.
Biopsy samples
RecoveryRest
120 min 90 min 90 min50 min
Blood samples
- -
0Time (min) 120 170 230 290 350
Resistance exercise
Drinks
Continuous infusion of L-[ring(13)C6]phenylalanine
39
3.5.2 Glucose, lactate, insulin and cortisol
Plasma levels of lactate and glucose were analyzed spectrophotometrically as described
by Bergmeyer (178), except for study II and IV where levels of glucose were deter-
mined automatically with a Biosen C Line. Levels of insulin were determined using an
ELISA kit (study I, II and IV) or a radioimmunoassay kit (study III) in accordance with
the manufacturer’s instructions. In study II, plasma cortisol levels were determined
using an ELISA kit as described by the manufacturer.
3.5.3 Amino acids
In study III the plasma samples were deproteinized by addition of 5% trichloroacetic
acid (1:5), set on ice for 20 min and then centrifuged for 3 min at 10 000 g. The free
amino acids in the resulting supernatant were measured by reversed-phase high-
performance liquid chromatography (HPLC), utilizing ortho-phthalaldehyde as
derivatizing agent as described by Pfeifer et al. (179). In study IV the plasma samples
was combined with an internal standard with isotope labeled amino acids (1:1) and 50%
acetic acid (1:1.2) and then passed through a cation exchange resin column. The puri-
fied amino acids were dervatizied with phenylthiocarbamyl and quantified using ultra
performance liquid chromatography - tandem mass spectrometry (LC-MS/MS) as de-
scribed by Bornø and van Hall (180).
3.5.4 Stable isotope enrichment (Study I, II, IV)
In study I and II, plasma enrichment was analyzed in 200 µl of sample that was com-
bined with 100 µl of internal standard (L-[ring-13
C9] phenylalanine, 50 µmol ∙ l-1
) and
500 µl of acetic acid before being passed through a cation exchange resin column. The
eluted amino acids were then dervatizied with N-methyl-N-(tert-butyldimethylsilyl)
trifluoroacetamide (MTBSTFA) and the enrichment measured using gas chromatog-
raphy-tandem mass spectrometry (GC-MS/MS) with electron impact ionization and
selective ion monitoring for 336, 342, and 345 m/z. In study IV, plasma enrichment was
analyzed as described above using LC-MS/MS (180).
3.6 Muscle analysis
3.6.1 Sample preparation
All muscle samples were freeze dried and thoroughly dissected free from blood, fat and
connective tissue under a light microscope. The dissection resulted in very small intact
fiber bundles that were mixed extensively to obtain a highly homogenous sample. All
subsequent analysis was performed in aliquots of these sample preparations.
40
3.6.2 Muscle glycogen (Study I, II, IV)
Levels of muscle glycogen was determined according to the procedure described by
Leighton et al. (181) in 2-3 mg of lyophilized muscle in study I and IV, and in the pellet
and supernatant following muscle homogenization (see below) in study II, due to tissue
limitations. In brief, 150 µl of 1 M KOH was added to each muscle sample and pellet
followed by 15 min digestion at 70 °C, after which the digest was transferred to new
tubes and the pH adjusted to 4.8 with glacial acetic acid. In study II, 20 µl of super-
natant from each sample was also adjusted to the same pH using 10% acetic acid. After
acidification, 0.1 M NaAcetate-buffer with amyloglycosidase was added to all samples
and enzymatic glycogen degradation was carried out during 2 hours at 40 °C, after
which the glucose concentration was measured spectrophotometrically as described by
Bergmeyer (178).
3.6.3 Amino acids (Study II, III, IV)
In study II and III free amino acids from 2-3 mg lyophilized muscle samples were ex-
tracted with ice-cold 5% trichloroacetic acid (40 µL per mg). Following acidification
samples were maintained on ice for 30 min, centrifuged at 10 000 g for 3 min and the
concentrations of free amino acids in the supernatants were measured by HPLC as
above. In study IV, free amino acids were liberated from 5 mg of muscle tissue using
perchloric acid and analyzed as above using LC-MS/MS (180).
3.6.4 Immunoblotting
Here follows a general description of the western blot protocol according to the proce-
dure in all four studies. Freeze dried muscle samples were homogenized in ice-cold
buffer (80-100 µl ∙ mg dry weight-1
) constituting of 2 mM HEPES (pH 7.4), 1 mM
EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM β-glycerophosphate, 1% TritonX-100, 1
mM Na3VO4, 2 mM dithiothreitol, 1% phosphatase inhibitor cocktail and 1 % (v/v) Halt
Protease Inhibitor Cocktail utilizing a BulletBlender with 0.5 mm ZrO beads or a
ground-glass homogenizer (study III). Next, homogenates were rotated for 30 min at
4°C and cleared from myofibrillar and connective tissue debris by centrifugation at 10
000 g for 10 min at 4°C after which the resulting supernatant was collected. The protein
concentration of the supernatants was determined in aliquots diluted 1:10 in purified
water using a colorimetric protein assay. Samples were then diluted in Laemmli sample
buffer and homogenizing buffer to obtain a final protein concentration of 1.0-1.5 µg ∙
µl-1
. All samples were then heated to 95°C for 5 min to denature proteins present in the
supernatant, and then kept at -20°C until further separation on SDS-Page. For protein
separation, 20-30 µg of protein from each sample were loaded on pre-cast gradient gels
(4-20% acrylamide) and electrophoresis performed on ice at 250-300V for 30-40 min.
Next, the gels were equilibrated in transfer buffer (25 mM Tris base, 192 mM glycine,
41
and 10% methanol) for 30 min at 4° C, after which proteins were transferred to polyvi-
nylidine fluoride membranes at a constant current of 300 mA for 3 h at 4º C. Thereafter,
successful transfer and loading was confirmed by staining the membranes for total pro-
tein content. After imaging and destaining, the membranes were blocked in Tris-
buffered saline (TBS; 20 mM Tris base, 137 mM NaCl, pH 7.6) containing 5% non-fat
dry milk for 1 h at room temperature. Thereafter, the membranes were incubated over-
night with commercially available primary antibodies diluted in TBS supplemented
with 0.1% Tween-20 containing 2.5% non-fat dry milk (TBS-TM). Following primary
antibody incubation, membranes were washed with TBS-TM and incubated for 1 h at
room temperature with secondary antibodies conjugated with horseradish peroxidise.
Next, the membranes were washed with TBS-TM and TBS, and finally, the target pro-
teins were visualized by applying chemiluminescent substrate, thus enabling quantifica-
tion. Following image capturing of phosphorylated proteins, membranes were stripped
of the phosphospecific antibodies, after which the membranes were re-probed with
primary antibodies for each respective total protein, as described above. In study I, II
and IV, all phosphoproteins were normalized to their corresponding total protein and
when only total protein was measured, values were normalized against the total protein
stain. In study III, levels of both total and phosphorylated proteins were normalized to
the level of α-tubulin in that sample. To standardize the immunoblotting procedure, all
samples from each subject were loaded on the same gel and all gels were run simultane-
ously, and prior to blocking, membranes were cut and assembled so that membranes for
the entire sample set were exposed to the same blotting conditions.
3.6.5 Immunoprecipitation (Study I, II, IV)
In study II and IV the protein interaction between 4E-BP1 and eIF4E was assessed by
immunoprecipitating (IP) eIF4E and blotting for co-IP 4E-BP1. In study II, tissue sam-
ples were homogenized in ice-cold CHAPS-lysis buffer containing 40 mM Hepes (pH
7.5), 120 mM NaCl, 1mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 0.5 mM
Na3VO4, 10 mM β-glycerophosphate, 1 % (v/v) Halt Protease Inhibitor Cocktail and 0.3
% (w/v) CHAPS detergent. In study IV, muscle tissue was homogenized in
immunoblotting (IB) buffer as described above. All homogenates were then rotated 30
min at 4°C, cleared by centrifugation at 10 000 g for 10 min at 4°C, the resulting super-
natant collected and assayed for protein concentration. In study II, homogenates con-
taining 500 µg of protein was incubated with 5 µg of mouse anti-eIF4E antibody and 15
µl of protein G magnetic beads for 4 hours at 4° C, whereas in study IV, 350 µg of pro-
tein was combined with 4 µg of antibody and magnetic beads overnight. Following
incubation, the beads with the immune-complexes were trapped using a magnetic rack
and washed twice in a high salt variant of the corresponding lysis buffer. The beads
were then suspended in 100 µl 1x Laemmli sample buffer with 100 µM DTT, and
42
heated for 10 min at 70º C and then immunoblotted for eIF4E and 4E-BP1 as described
above.
In study I the interaction of the TSC-complex was assessed by immunoprecipitating
TSC1 from muscle samples that had been homogenized in ice-cold IP lysis buffer con-
taining 50 mM HEPES (pH 7.5), 0.1 mM EGTA, 1 mM EDTA, 1% (vol/vol) Triton X-
100, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 0.27 M sucrose, 0.1%
(vol/vol) β-mercaptoethanol and 1% (vol/vol) Halt Protease Inhibitor Cocktail. Follow-
ing centrifugation and serial IP of S6K1 (described below) and aliquot of the remaining
homogenate containing 175 µg of protein was combined with 1 µg of goat-anti TSC1
antibody and 10 µl of protein G magnetic beads and incubated at 4° C overnight. After
incubation, the magnetic beads were trapped and washed four times in IP lysis buffer,
resuspended in Laemmli sample buffer, boiled for 5 minutes and finally immunoblotted
for TSC1 and TSC2 as described above.
In study I, II and IV immunoprecipitation was performed to analyze the kinase activity
of S6K1 and in study I and IV also that of AMPK. For both proteins different lysis
buffers was used in the three studies, IP lysis buffer in study I, CHAPS lysis buffer in
study II and IB lysis buffer in study IV. All buffers had been controlled for downstream
kinase assay compatibility. For S6K1, homogenates containing 500-750 µg of protein
were incubated with 5-7.2 µg of rabbit anti-S6K1 antibody and 10 µl of protein-A or G
sepharose beads overnight at 4° C. In study I, immunoprecipitation of AMPKα1 and
AMPKα2 was performed following that of S6K1, and in study IV after homogenization
in IB lysis buffer. Aliquots of 225-250 µg protein with 3-4 µg of corresponding anti-
body and 10 µl of protein-G sepharose each, with the addition of 500-800 µl of AMPK
buffer (50 mM Tris·HCl (pH 7.25), 150 mM NaCl, 50 mM NaF, 5 mM
sodiumpyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% (vol/vol) Triton X-
100, and 1% (vol/vol) Halt Protease Inhibitor Cocktail) to adjust for the slightly higher
pH in the IP and IB lysis buffers. After overnight incubation, all bead-immune-
complexes were spun down and washed twice in high salt variants of corresponding
buffer and finally subjected to one wash in kinase specific assay buffer (see below).
3.6.6 Kinase assays (Study I, II, IV)
After the wash in kinase specific assay buffer (S6K1; 50 mM Tris-HCl pH 7.5, 0.03 %
BrijL23, 0.1 % β-mercaptoethanol and AMPK; 50 mM HEPES pH 7.4, 0.03 % BrijL23,
1 mM DTT) the beads from each sample were suspended in 60 µl of this buffer and then
divided into three 20 µl aliquots. Kinase specific substrate was added to two of these
and the third served as a blank without substrate. Kinase assays were initiated by the
addition of 30 µl of a radiolabeled kinase specific reaction mix, run at 30°C on a rotat-
43
ing platform for 60 min in the case of S6K1 and 30 min for AMPK. The assays were
terminated by the addition of 50 µl phosphoric acid (1 % v/v) to each sample. For
S6K1, the final reaction mix (50 µl) consisted of 100 μM ATP, 10 mM MgCl2, 32
γ-ATP
(specific activity: 2.5-3.0 x 106 cpm ∙ nmol
-1), 30 µM S6K1 substrate (KRRRLASLR),
and for AMPK; 200 µM ATP, 200 µM AMP, 5 mM MgCl2, 32-ATP (specific radioac-
tivity ~ 0.4 x 106 cpm ∙ nmol
-1) and 200 µM synthetic substrate (AMARA: AMAR-
RAASAAALARRR). After termination, the reaction mixtures were spotted onto
squares of p81 Whatman filter paper, washed four times in phosphoric acid and once in
acetone, allowed to dry and finally immersed in scintillation fluid. The radioactivity was
counted on a liquid scintillation counter and the average values from the duplicate as-
says with substrate were corrected for background by subtraction of the blank and the
values obtained expressed in pmol · min-1
· mg-1
protein.
3.6.7 mRNA expression (Study I, II, III)
Total RNA was extracted from approximately 3 mg freeze dried muscle by homogeni-
zation in PureZOL RNA isolation reagent in accordance with the manufacturer’s in-
struction using a Polytron (study I) or the BulletBlender and RNAse free 0.5 mm ZrO2
beads (study II, III). The final amount and purity of the RNA was determined by spec-
trophotometry and 1-2 µg of RNA was subjected to reverse transcription to produce 20-
40 µL cDNA, following thermal cycling. The mRNA was quantified using real-time
quantitative polymerase chain reaction (RT-qPCR), using the housekeeping gene
GAPDH as an internal control. To determine a suitable cDNA concentration, annealing
temperature and PCR cycling protocol for each primer the pooled cDNA obtained from
all of the participants in each study was diluted in a five step series and RT-qPCR there-
after performed for each gene. The standard curves for all of the primers exhibited high
efficiency and an R2 value > 0.99. In addition, single melting peaks were observed dur-
ing melt curve analysis, confirming the presence of only a single product. The reliability
of GAPDH mRNA as an internal control was validated by the 2-∆C’t
method, where ∆C’T
= CT time x – CT time 0. All samples from each subject were run in triplicate and analyzed on
a single 96-well plate for direct relative comparisons. The mixtures (25 µl) for RT-
qPCR amplification contained 12.5 µl 2x SYBR Green Supermix, 11.5 µL template
cDNA diluted in RNase-free water and 0.5 µL forward and reverse primers. The reac-
tions were initiated by heating the samples to 95° C for 3 min, followed by thermal
cycling, 40x at 95°C, 58°C and 72°C for 30 seconds each. All of the CT values obtained
ranged between 18 and 29, with the same fixed threshold being employed for all genes
of interest in one study. Relative changes in mRNA expression for all genes of interest
were quantified by the 2-∆Ct
method, where ∆CT = CT Gene of interest – CT GAPDH.
44
3.6.8 Stable isotope enrichment (Study I, II, IV)
In study I, II and IV, muscle free and protein bound enrichment of L-[ring-13
C6]-
phenylalanine was determine to calculate the fractional rate of protein synthesis (FSR)
in mixed muscle. In study IV, the isotope enrichment was also determined in the myofi-
brillar protein pool as well as in plasma protein of the first pre-infusion blood sample.
For that specific purpose, the protein pellet obtained from extraction of the muscle tis-
sue designated for immunoprecipitation and immunoblotting (see above) was washed
once with 500 µl purified H2O, dissolved in 1 ml IB lysis buffer, and centrifuged at
1600 g for 20 min at 4º C. The plasma samples were precipitated by addition of perchlo-
ric acid and pelleted by centrifugation. The resulting supernatants was discarded and the
pellet containing either myofibrillar protein or plasma protein was lyophilized and
stored at -80º C until further analysis. The mixed muscle samples as well as the dried
myofibrillar and plasma proteins was each combined with 100 µl of an internal standard
(L-[ring-13
C9]-phenylalanine, 5 µmol ∙ l-1
) and in study IV 100 µl of a complete U-13
C
and U-13
N labeled amino acid standard solution, after which the samples were pelleted
and extracted twice with 500 µl perchloric acid. To determine intracellular enrichment
of free phenylalanine in the mixed muscle samples the two extracts containing free
phenylalanine were combined and passed through a cation exchange resin column.
Thereafter the eluted amino acids were dervatizied with MTBSTFA (study I, II) or
phenylthiocarbamyl (study IV) and analyzed using GC-MS/MS and LC-MS/MS, re-
spectively. The mixed muscle, myofibrillar and plasma protein pellets were washed
twice with 70 % ethanol; hydrolyzed overnight in 1 ml 6 M HCl at 110° C; dissolved in
500 µl of acetic acid and then passed through a cation exchange column. To determine
enrichment of protein bound phenylalanine, amino acids derived from the purified pellet
were converted to their N-acetyl-n-propyl esters and analyzed by gas chromatography–
combustion–isotope ratio mass spectrometry (GC–C–IRMS). Muscle protein fractional
synthesis rate was calculated using the standard precursor–product method:
FSR = ΔEp phe / (Eic phe × t) × 100
Where ΔEp phe is the difference in protein bound phenylalanine enrichment between two
biopsies or between pre-infusion plasma protein and the first biopsy for calculation of
FSR at rest in study IV, Eic phe is the intracellular average phenylalanine enrichment in
the two biopsies, and t is the time period for tracer incorporation in hours multiplied by
100 to express FSR in percent per hour (% ∙ h-1
).
3.6.9 Statistical analysis
Parametric statistical procedures were employed to calculate the means and standard
errors of the mean (SEM), and unless indicated otherwise, the values presented are
45
means SEM. To compare changes between trials in analyzes with two or more time
points a two-way (trial x time) repeated measures ANOVA was used. In the case of a
comparison between variables with a single point measurement a one-way ANOVA was
employed. Whenever a main or interaction effect was observed in the ANOVA, a Fish-
er’s LSD post-hoc test was performed to determine where the differences resided. For
comparison between two cases with a single variable the Students´ paired t-test was
used. For some positively skewed distributed variables, log-transformation was per-
formed before the formal analyses. Statistical analyses were performed using STATIS-
TICA version 12.0 (StatSoft, Inc., Tulsa, OK, USA). P<0.05 was considered statisti-
cally significant.
Study Type Protocol Biopsies Muscle analysis
I
Acute,
randomized
cross-over
Resistance exercise
(leg) +/- cycling
exercise before
2x Pre, After cycling, 0, 90
and 180 min after exercise
FSR, kinase activity,
protein phosphorylation,
gene expression and
glycogen
II
Acute,
randomized
cross-over
Resistance exercise
(arm) +/- cycling
exercise before
2x Pre, 0, 90 and 180 min
after exercise
FSR, kinase activity,
protein phosphorylation
and interaction, gene
expression, amino acids
and glycogen
III
Acute,
randomized
cross-over
Resistance exercise
(leg)
Pre, 60 and 180 min after
exercise
Protein phosphorylation,
gene expression and amino
acids
IV
Acute,
randomized
cross-over
Resistance exercise
(leg)
Pre, 0, 90 and 180 min
after exercise
FSR, kinase activity,
protein phosphorylation
and interaction, amino
acids and glycogen
Table 2. Summarized overview of the study protocols
46
4 RESULTS
4.1 Study I
In this study, the signaling response, FSR and gene expression was examined during
one trial with cycling intervals followed by resistance exercise (ER) and one trial with
resistance exercise only (R). The subjects performed an equal number of repetitions,
with the same load and matching time under tension during resistance exercise in both
trials. The interval cycling induced a peak plasma lactate level of 8.7 ± 0.8 mmol ∙ l-1
and decreased muscle levels of glycogen by 32%. Directly following resistance exercise
in both trials plasma levels of lactate were at 11.3 ± 0.8 mmol ∙ l-1
, and with a total de-
crease in glycogen of 49% and 16%, in the ER- and R-trial, respectively.
Immediately after cycling in the ER-trial, the phosphorylation of S6K1 at Thr389
was
increased ~4-fold above baseline, with a similar increase also in the R-trial following
the resistance exercise. During recovery the phosphorylation increased gradually with
an average 12-fold increase noted 180 min after the exercise session, with no differ-
ences between trials at any time point. The activity of S6K1 was unaltered immediately
after exercise in both trials, but increased by an average of 55% and 110% after 90 and
180 min of recovery, respectively, with no differences between trials. The phosphoryla-
tion of 4E-BP1 at Thr37/46
was decreased by an average of 58% in the biopsies taken
directly after exercise in both trials and then returned to the levels at rest during recov-
ery. For eEF2, the phosphorylation at Thr56
was increased by ~100% after cycling in the
ER-trial, and following resistance exercise in both trials a similar increase was noted.
However, following 90 min of recovery the phosphorylation was reduced by an average
of 55% in both trials and remained at that level throughout the entire recovery period.
The activity of AMKPα2 increased by ~90% above baseline directly after cycling in
the ER-trial, and the activity was maintained also after completion of the resistance
exercise, but returned to baseline during recovery. In the R-trial, AMKPα2 activity
remained at resting values during the entire trial, which was also the case for AMKPα1
activity during both trials. The phosphorylation of TSC2 at Ser1387
, a downstream target
of AMPK, increased by ~50% after cycling in the ER-trial, with a similar response
noted directly after resistance exercise in both trials. The phosphorylation of raptor at
Ser792
, a target of AMPK, was increased by an average of 30% after resistance exercise
in both trials and remained elevated throughout the entire recovery period.
Mixed muscle protein FSR during the three hours of rest prior to exercise was 0.050 ±
0.008 % ∙ h-1
, in the first trial (see section 5.4). During the three hours of recovery from
exercise the rates were 0.057 ± 0.008 % ∙ h-1
in the R-trial and 0.074 ± 0.017 % ∙ h-1
in
the ER-trial, with no differences between the two.
47
Figure 7. Phosphorylation of S6K1 at Thr389 (A), kinase activity of S6K1 (B), phosphorylation of
4E-BP1at Thr37/46 (C) and eEF2 at Thr56 (D), before and after exercise. Phosphorylation levels
are normalized to the total levels of corresponding protein. All values are presented as means ±
SE for 8 subjects. Representative bands are shown above the appropriate graph. *P < 0.05 vs.
Rest; #P < 0.05 vs. R-trial.
A B
Pre 90 min 180 min
Ph
osp
ho
ryla
tio
n o
f S6
K1
at
T38
9(a
rbit
rary
un
its)
0
2
4
6
8 RER
*
**#
*
*
*
*
Rest/E-Ex R-Ex
p-S6K1
t-S6K1
Pre 90 min 180 min
S6K
1 a
ctiv
ity
(pm
ol∙
min
-1∙
mg-1
)
0.00
0.05
0.15
0.20
0.10
0.25
0.30 RER
Rest/E-Ex R-Ex
*
*
Pre 90 min 180 min
Ph
osp
ho
ryla
tio
n o
f 4
E-B
P1
at
T37
/46
(arb
itra
ry u
nit
s)
0
2
4
6
8 RER
**#
*
*
Rest/E-Ex R-Ex
p-4EBP1
t-4EBP1
Pre 90 min 180 min
Ph
osp
ho
ryla
tio
n o
f eE
F2 a
t T5
6(a
rbit
rary
un
its)
RER
0
2
4
6
8
*
*#
*
* ** *
Rest/E-Ex R-Ex
p-eEF2
t-eEF2
C D
48
Figure 8. Kinase activity of AMPKα2 (A), phosphorylation of TSC2 at Ser1387 (B) and phosphory-
lation of raptor at Ser792 (C). Phosphorylation levels are normalized to the corresponding total
levels of each protein and presented as means ± SE for 8 subjects. Representative bands are
shown above the appropriate graphs. For raptor only, bands were cut and repositioned to fit the
graph. *P < 0.05 vs. Rest; #P < 0.05 vs. R-trial.
Pre 90 min 180 min
AM
PK
α2
act
ivit
y(p
mo
l∙ m
in-1
∙ m
g-1)
0
2
6
8
14
4
10
12
16
18 RER
*#*#
Rest/E-Ex R-Ex Pre 90 min 180 min
Ph
osp
ho
ryla
tio
n o
f TS
C2
at
S13
87
(arb
itra
ry u
nit
s)
0
2
4
6
8 RER
**#
*
Rest/E-Ex R-Ex
p-TSC2
t-TSC2
Pre 90 min 180 min
Ph
osp
ho
ryla
tio
n o
f R
apto
r at
S7
92
(arb
itra
ry u
nit
s)
0
2
4
6
8 RER *
*
Rest/E-Ex R-Ex
p-Raptor
t-Raptor
A B
C
49
Figure 9. mRNA expression of MuRF-1 (A), MAFbx (B) and PGC-1α (C) before and after exer-
cise. The mRNA expression is in relation to GAPDH and analyzed using the 2-ΔCT method. All
values are presented as means ± SE for 8 subjects. *P < 0.05 vs. Rest; #P < 0.05 vs. R-trial.
The mRNA expression of MuRF-1 was unchanged in the R-trial, whereas it increased
by 120% and 60% at 90 and 180 min after resistance exercise, respectively, in the ER-
trial. The mRNA expression of MAFbx was unaltered in both trials following 90 min of
recovery, but reduced by ~50% following 180 min of recovery in the R-trial. Following
90 min of recovery, the mRNA expression of PGC1-α increased by 8-fold only in the
ER-trial. At 180 min of recovery, the expression was still elevated 7-fold and with a 3-
fold increase also noticed in the R-trial.
4.2 Study II
In study II, mTORC1 signaling, FSR and gene expression was assessed in connection
with two session of arm extension resistance exercise (R-Arm), were one was preceded
by high intensity cycling intervals (ER-Arm). Resistance exercise performance, in terms
of load, number of repetitions and time under tension were similar in both trials. Plasma
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Pre 90 min 180 min
R
ERm
RN
A e
xp
ressio
n o
f M
uR
F-1
(arb
itra
ryu
nits)
* #
* #
A
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
*
#
Pre 90 min 180 min
mR
NA
exp
ressio
n o
f M
AF
bx
(arb
itra
ryu
nits)
B R
ER
0.0
1.0
2.0
3.0
4.0
5.0
mR
NA
expre
ssio
n o
f P
GC
-1α
(arb
itra
ryu
nits) * #
* #
C
Pre 90 min 180 min
R
ER
*
50
levels of lactate peaked at 10.4 ± 0.6 mmol ∙ l-1
after cycling in the ER-Arm trial, and
lactate levels were higher than in the R-Arm trial until 90 min following resistance
exercise. The cycling exercise profoundly increased plasma levels of cortisol which
reached a 3.4-fold higher level than in the R-Arm trial during the resistance exercise,
and returned to baseline values after 90 min of recovery. Plasma levels of cortisol were
unaltered in the R-Arm trial.
In both trials, S6K1Thr389
phosphorylation increased largely immediately after exercise,
but this increase was greater in the R-Arm trial, 11-fold versus 5-fold in the ER-Arm
trial. Following 90 and 180 min of recovery, S6K1 phosphorylation were similar in the
two trials, but still 4 and 6-fold higher than at rest, respectively. The effect on Thr389
was well reflected in the activity of S6K1 which, immediately after exercise, increased
by 205% in the R-Arm trial and only by 69% in the ER-Arm trial. S6K1 activity was
similar between trials during the entire recovery, with a ~80% average increase above
rest. The increase in S6K1 phosphorylation was significantly correlated with the in-
crease in kinase activity (R=0.77).
The phosphorylation of 4E-BP1 at Thr37/46
decreased immediately after exercise by
36% in the ER-Arm trial, whereas it was unchanged compared to baseline in the R-Arm
trial and at all other time points in both trials. This effect was mirrored when phos-
phorylation of only the Thr46
residue was measured. The phosphorylation of 4E-BP1
Ser65
increased by 31% immediately after exercise in the R-Arm trial, but decreased by
36% in the ER-Arm trial. During recovery, the degree of Ser65
phosphorylation returned
to baseline levels in the R-Arm trial, but remained decreased by 33% in the ER-Arm
trial at 90 min, while returning to baseline values after 180 min. The protein interaction
between 4E-BP1 and eIF4E increased by 33% directly following exercise in the ER-
Arm trial, but was unaltered in the R-Arm trial.
The phosphorylation status of Akt at Ser473
did not change at any time point in any of
the trials. The phosphorylation of mTOR at Ser2448
increased by an average of ~90%
immediately after exercise in both trials, and remained elevated compared to baseline
the entire recovery period. The phosphorylation of eEF2 at Thr56
responded in a similar
fashion to exercise in both trials, with an average increase of 27% directly after exer-
cise, which was reversed to an average 29% decrease during 90 and 180 min of recov-
ery.
Immediately following exercise, the degree of AMPK at Thr172
phosphorylation in-
creased by 43 and 71% in the R-Arm and ER-trial, respectively, with the level in the
ER-Arm trial being statistically different from the R-Arm trial. In both trials, the levels
were at baseline after 90 and 180 min of recovery. The phosphorylation of p38 at Thr180
/Tyr182
increased in both trials directly after exercise by an average of 92%. The degree
of phosphorylation decreased during recovery and returned to the values at rest follow-
ing 180 min of recovery in both trials.
51
Figure 10. Phosphorylation of S6K1 at Thr389 (A), kinase activity of S6K1 (B), phosphorylation of
4E-BP1 at Thr37/46 (C) and the amount of total 4E-BP1 interacting with eIF4E (D). Representa-
tive bands, both phosphorylated (upper panel, expect for IP) and total protein from one subject is
shown above each graph. Bands have been rearranged to fit the order of trials in the graph.
Values in graphs are presented as means ± SE for 8 subjects. *P < 0.05 vs. Rest, #P < 0.05 vs. R-
Arm trial.
Mixed muscle FSR during the three hours of rest prior to exercise was 0.058 ± 0.006 %
∙ h-1
in the R-Arm trial and 0.044 ± 0.006 % ∙ h-1
in the ER-Arm trial, with a lower rate
of synthesis in six out of eight subjects in the latter. FSR during 180 min of recovery
was 0.076 ± 0.018 and 0.079 ± 0.007 % ∙ h-1
in the R-Arm and ER-Arm trial, respec-
tively, with no differences between the two. FSR calculated over 300 min of exercise
and recovery were 0.072 ± 0.005 % ∙ h-1
and 0.083 ± 0.007 % ∙ h-1
in the R-Arm and
ER-Arm trial, respectively, detected as a main effect of time and with an increase above
rest in the ER-Arm trial
0
100
200
300
400
500
600
700
800
900
1000
Pre Post 90 min 180 min
p-S6K1
t-S6K1
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 min
Phosp
hory
lation o
f S
6K
1 a
t T
389
(arb
itra
ryu
nits)
R-Arm
ER-Arm *
#
A
0.0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
S6
K1
activity (
pm
ol∙m
in-1
∙m
g-1
) *
#
Pre Post 90 min 180 min
R-Arm
ER-Arm
B
0
200
400
600
800
1000
1200
1400
p-4EBP1
t-4EBP1
Phospho
ryla
tion o
f 4
E-B
P1 a
t T
37/4
6
(arb
itra
ryu
nits)
* #
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 min
Pre Post 90 min 180 min
R-Arm
ER-Arm
C
0
20
40
60
80
100
120
140
160
Pre Post
4E
-BP
1:e
IF4
E a
sso
cia
tio
n
(arb
itra
ryunits)* #
R-Arm ER-Arm
Pre Post Pre Post
IP: eIF4E
t-4EBP1
R-Arm
ER-Arm
D
52
Figure 11. Phosphorylation of mTOR at Ser2448 (A), eEF2 at Thr56 (B), AMPK at Thr172 (C) and
p38 MAPK at Thr180/Tyr182 (D). Representative bands, both phosphorylated (upper panel, expect
for IP) and total protein from one subject is shown above each graph. Bands have been rear-
ranged to fit the order of trials in the graph. Values in graphs are presented as means ± SE for 8
subjects. *P < 0.05 vs. Rest, #P < 0.05 vs. R-Arm trial.
The mRNA expression of MuRF-1 was unaltered during recovery in the R-Arm trial,
but increased by 226% and 123% after 90 and 180 min of recovery, respectively, in the
ER-Arm trial. For MAFbx, the mRNA expression was increased by ~30% during re-
covery in both trials. The mRNA expression of PGC-1α increased by 4.2-fold following
90 min of recovery only in the ER-Arm trial. At 180 min of recovery, the expression
was increased 3.5-fold also in the R-Arm trial and with an even greater, 8.7-fold in-
crease, observed in the ER-Arm trial.
0
200
400
600
800
1000
1200
p-mTOR
t-mTOR
Ph
osp
hory
lation
of m
TO
Ra
t S
24
48
(arb
itra
ryunits)
*
R-Arm
ER-Arm
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 min
Pre Post 90 min 180 min
A
0
400
800
1200
1600
2000
p-eEF2
t-eEF2
*
*
Phosp
hory
lation
of eE
F2 a
t T
56
(arb
itra
ryu
nits)
R-Arm
ER-Arm
Pre Post 90 min 180 min
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 minB
0
200
400
600
800
1000
1200
p-AMPK
t-AMPK
Phosp
hory
lation
of A
MP
K a
t T
172
(arb
itra
ryun
its)
* #
*
R-Arm
ER-Arm
Pre Post 90 min 180 min
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 minCp-p38
t-p38
Phosp
hory
lation o
f p38 a
t T
180/Y
182
(arb
itra
ryu
nits)
*R-Arm
ER-Arm
0
200
400
600
800
1000
1200
Pre Post 90 min 180 min
R-Arm ER-Arm
Pre Post 90 min 180 min Pre Post 90 min 180 minD
53
Figure 12. mRNA expression of MuRF-1 (A) and PGC-1α (B) before and after exercise.. The
mRNA expression is in relation to GAPDH and analyzed using the 2-ΔCT method. All values are
presented as means ± SE for 8 subjects. *P < 0.05 vs. Rest; #P < 0.05 vs. R-Arm trial.
Figure 13. Individual values for mixed muscle protein fractional synthetic rate at rest and during
180 min of recovery from exercise in study I (A) and study II (B).For study I the values at rest are
from the first trial. White bullets represent Rest, grey bullets the R-trial and R-Arm trial, black
bullets the ER-trial and ER-Arm trial and black bars the mean values.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Pre 90 min 180 min
mR
NA
expre
ssio
n o
f M
uR
F-1
(arb
itra
ryu
nits)
R-Arm
ER-Arm
P = 0.07
* #
*
A
0.00
0.01
0.02
0.03
0.04
0.05
mR
NA
exp
ressio
n o
f P
GC
-1α
(arb
itra
ryun
its)
**
* #
R-Arm
ER-Arm
Pre 90 min 180 min
B
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
Mix
ed
mu
scle
FS
R (
% ∙ h
-1)
A R
ER
Rest Recovery Rest Recovery
Study I
Rest Recovery Rest Recovery0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
Mix
ed
mu
scle
FS
R (
% ∙ h
-1)
R-Arm
ER-Arm
B Study II
54
4.3 Study III
This study investigated the effects of oral supplementation of EAA with or without
leucine on mTORC1 signaling after resistance exercise. Plasma levels of insulin were at
its highest during exercise, 42 ± 10 mU ∙ l-1
with EAA and 32 ± 6 mU ∙ l-1
with EAA-
Leu, with no differences between trials. After 60 min of recovery levels of insulin were
at baseline in both trials. With EAA, muscle levels of leucine increased by 64% after 60
min of recovery and remained higher than at rest the entire recovery period. In the
EAA-Leu trial, muscle level of leucine decreased by an average of 41% during recov-
ery. Muscle levels of EAA (not including leucine) increased in both trials during recov-
ery but were 37% higher in the EAA-Leu trial at 180 min.
The phosphorylation of Akt at Ser473
did not change at any time point during both
trials. At 60 min after exercise, the degree of phosphorylation of mTOR at Ser2448
in-
creased to a greater extent with EAA, 145%, than with EAA-Leu, 45%. These differ-
ences between trials were not present at 180 min of recovery, but the values still ~70%
higher than at rest with both supplements. The phosphorylation of S6K1 at Thr389
mir-
rored that of mTOR, with a 59-fold increase with EAA and 8-fold with EAA-Leu, fol-
lowing 60 min of recovery. The increase at 180 min of recovery was similar in both
trials, ~30-fold above the level at rest. Phosphorylation of eEF2 at Thr56
was not sensi-
tive to the different supplements but decreased by ~40% during recovery in both trials.
The mRNA expression and protein levels of MuRF-1 and MAFbx did not change in
response to exercise and was not different between trials.
55
Figure 14. Phosphorylation of Akt at Ser473 (A), mTOR at Ser2448 (B), S6K1 at Thr389 (C) and
eEF2 at Thr56 (D) before and after resistance exercise. Representative bands for the phosphory-
lated proteins and for α-tubulin from one subject are shown above each graph. Bands have been
rearranged to fit the order of trials in the graph. The values presented are in arbitrary units
relative to the level of α-tubulin and represent the mean ± SE for 8 subjects. *P < 0.05 vs. Rest, #P < 0.05 vs. EAA-Leu.
4.4 Study IV
In study IV, the effects on mTORC1 signaling and FSR was examined in response to
oral supplementation of Leucine, BCAA, EAA or Placebo, in connection with re-
sistance exercise. The peak insulin concentration was similar in all four trials, reaching
a level of 13 - 16 mU ∙ l-1
at 15-30 min after exercise, compared to ~5 mU ∙ l-1
. The area
under curve for insulin was greater with EAA compared to Leucine and Placebo. Mus-
cle levels of leucine increased to the same extent at all time points in all trials with ami-
no acid supplementation, with a maximal average increase of 48%. With placebo, mus-
cle levels of leucine decreased by ~30% during recovery. Muscle levels of BCAA in-
creased to the same extent in the BCAA and EAA trials, whereas it decreased with
Leucine and Placebo. Following 180 min of recovery, muscle levels of isoleucine and
0
200
400
600
800
1000
Pre 60 min 180 min
EAA-Leu
EAA
p-Akt
α-tubulin
A
Pre 1h Post 3h Post
EAA - Leu EAA
Pre 1h 3h Pre 1h 3h
Phosphory
lation o
f A
kt at
S 4
73
(arb
itra
ry u
nits)
0
200
400
600
800
1000
1200
1400
Pre 60 min 180 minPre 1h Post 3h Post
p-mTOR
α-tubulin
* #
*
EAA - Leu EAA
Pre 1h 3h Pre 1h 3h
EAA-Leu
EAA
Phosphory
lation o
f m
TO
R a
t S
2448
(arb
itra
ry u
nits)
B
0
300
600
900
1200
1500
1800
Pre 60 min 180 min
p-p70
α-tubulin
* #
*
Pre 1h Post 3h Post
EAA - Leu EAA
Pre 1h 3h Pre 1h 3h
EAA-Leu
EAA
Pho
sph
ory
latio
n o
f S
6K
1 a
t T
389
(arb
itra
ry u
nits)
C
0
200
400
600
800
1000
Pre 60 min 180 min
p-eEF2
α-tubulin
EAA - Leu EAA
Pre 1h 3h Pre 1h 3h
**
Pre 1h Post 3h Post
EAA-Leu
EAA
Phosphory
lation o
f eE
F2
at T
56
(arb
itra
ry u
nits)
D
56
valine were reduced by 55% and 21% with Placebo, and 77% and 42% with Leucine,
respectively. Muscle levels of EAA, not including BCAA, increased during recovery in
the EAA-trial, were unchanged with Placebo and decreased in the Leucine- and BCAA-
trials.
Figure 15. Kinase activity of S6K1. The values presented are means ± SE for 8 subjects. *P <
0.05 vs. rest, †P < 0.05 vs. Placebo, ‡P < 0.05 vs. Leucine, +P < 0.05 vs. BCAA.
Phosphorylation of S6K1 at Thr389
increased in a hierarchal manner 90 min after exer-
cise with all three amino acid supplements; Leucine 16-fold, BCAA 22-fold and EAA
36-fold. Following 180 min of recovery, the level of phosphorylation was elevated
above rest to a similar extent with Placebo and Leucine, while the level with BCAA and
EAA were also higher than with Placebo. The activity of S6K1 followed the pattern of
the Thr389
phosphorylation, with a higher degree of activity with each supplement after
90 min of recovery (Placebo 2-fold, Leucine 4-fold, BCAA 6-fold and EAA 9-fold). At
180 min, the activity was 3-fold greater than at rest with Placebo and Leucine, and 6-
fold with BCAA and EAA, with the latter to being statistically different from the former
two trials. The absolute level and the increase in S6K1 phosphorylation was significant-
ly correlated with the absolute level and the increase in kinase activity, R=0.77 and
R=0.70, respectively.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Pre Post 90 min 180 min
S6
K1
activity (
pm
ol·
min
-1·
mg
-1)
*† ‡
**
*
†*
*†‡
*†‡ +
Placebo
Leucine
BCAA
EAA
57
The phosphorylation of 4E-BP1 at Thr37/46
, as well as Thr46
only, decreased by both
~55% immediately after exercise in all four trials. For Thr37/46
there was a slight in-
crease in phosphorylation degree during recovery in all trials. For that of Thr46
only,
phosphorylation increased by 28% with BCAA after 90 min of recovery, and with an
even greater, 52% increase with EAA. At the end of the recovery the phosphorylation of
Thr46
remained increased in the two latter trials, however with no difference between
the two. The phosphorylation of the Ser65
residue of 4E-BP1 responded in a similar
fashion to that of Thr47
, without the reduction directly after exercise. The protein inter-
action between 4E-BP1 and eIF4E increased in all trials immediately after exercise,
whereas it was reduced ~30% with the three amino acid supplements 90 min after exer-
cise. Following 180 min of recovery, the interaction was reduced in relation to baseline
in all four trials, but with a significantly greater reduction noted in the EAA-trial.
The phosphorylation of the Ser473
residue of Akt, was unaltered directly after exer-
cise, but increased in all trials by 31-73% following 90 min of recovery, and returned to
the levels at rest after 180 min. Phosphorylation of eEF2 at Thr56
did not respond to
amino acid supplementation, but increased immediately after exercise by an average of
126%, followed by a ~40% reduction at both 90 and 180 min of recovery.
The rate of protein synthesis during the 180 min of recovery exhibited a vast degree
of variation in all four trials, with values ranging from -0.065 to 0.157 % ∙ h-1
. Accord-
ingly, the effect of supplementation on FSR could not be determined. When utilizing
pre-infusion plasma protein enrichment to calculate FSR at rest during the first trial it
averaged 0.030±0.007 % ∙ h-1
, and in that first trial, FSR increased to 0.059±0.005 % ∙ h-
1 during the recovery, not taking the type of supplementation into account. In all four
trials, the activity of AMPKα2 increased immediately after exercise by an average of
135%, with no impact of the supplements, and returned to the levels at rest following 90
and 180 min of recovery. The increase in AMPKα2 activity correlated well with the
increase in AMPK Thr172
phosphorylation (R=0.79).There was no change in the activity
of AMPKα1 at any time point during the four trials. There was also no change in the
protein levels of MuRF-1 and MAFbx at any time point during the four trials.
58
Figure 16. Phosphorylation of 4E-BP1 at (A) Thr37/46, (B) Thr46, (C) Ser65 and (D) the amount of
total 4E-BP1 interacting with eIF4E. Above each graph representative immunoblots of both
phosphorylated (upper panel, expect for IP) and total protein (lower panel) for one subject are
shown (rearranged to match the order of trials in the graph). The values presented are means ±
SE for 8 subjects. *P < 0.05 vs. rest, †P < 0.05 vs. Placebo, ‡P < 0.05 vs. Leucine, +P < 0.05 vs.
BCAA.
0
100
200
300
400
500
600
700
800
900
1000
Phosp
hory
lation o
f 4E
-BP
1 a
t T
37/4
6 (
AU
)t-4E-BP1
p-4E-BP1
*
*
Pre Post 90 min 180 min
Placebo Leucin BCAA EAA
Pre Post 90 180 Pre Post 90 180 Pre Post 90 180Pre Post 90 180
Placebo
Leucine
BCAA
EAA
A
0
200
400
600
800
1000
1200
t-4E-BP1
p-4E-BP1
Pho
sph
ory
lation
of4
E-B
P1 a
t T
46
(A
U)
*
†*
*†‡ +
*
Pre Post 90 min 180 min
Placebo Leucin BCAA EAA
Pre Post 90 180 Pre Post 90 180 Pre Post 90 180Pre Post 90 180B
Placebo
Leucine
BCAA
EAA
0
200
400
600
800
1000
1200
t-4E-BP1
p-4E-BP1
Phosp
hory
lation o
f4
E-B
P1 a
t S
65 (
AU
)
*†‡ +
*†‡ *
†‡
Pre Post 90 min 180 min
Placebo Leucin BCAA EAA
Pre Post 90 180 Pre Post 90 180 Pre Post 90 180Pre Post 90 180C
Placebo
Leucine
BCAA
EAA
0
20
40
60
80
100
120
140
160
180
Pre Post 90 min 180 min
*
†*
*†‡*
4E
-BP
1 : e
IF4
E p
rote
in in
tera
ctio
n (
AU
)
IP: t-eIF4E
t-4E-BP1
Placebo
Leucine
BCAA
EAA
Placebo Leucin BCAA EAA
Pre Post 90 180 Pre Post 90 180 Pre Post 90 180Pre Post 90 180D
59
5 METHODOLOGICAL CONSIDERATIONS
5.1 Variability of immunoblotting
To examine the variability of the immunoblotting process, 7.5 mg of homogenous mus-
cle sample from one biopsy was divided in three aliquots. These samples were then
homogenized and the supernatants diluted to the same protein concentration in Laemmli
sample buffer. Samples were run in quadruplicate and blotted for one loading control
(α-tubulin), one total protein (eEF2), one phosphorylated protein (4E-BP1) and con-
trolled for total protein stain using MemcodeTM
. The mean value of the quadruplicate
for each of the three homogenates is shown in table 3. The coefficient of variation for
each quadruplicate was calculated as the standard deviation divided by the mean, times
100, which provides an estimate of the variation between the loading of the same sam-
ple.
Table 3. Signal intensity for the three homogenates analyzed for three proteins and the total
protein stain. The values are in arbitrary units expressed as mean of the quadruplicate for each
homogenate. The coefficient of variation of each quadruplicate is in parentheses (CV%).
MemcodeTM
α-Tubulin Total eEF2 Phospho 4E-BP1
Homogenate I 95 (6.8%) 91 (4.4%) 103 (5.5%) 121 (4.9%)
Homogenate II 93 (2.4%) 91 (2.1%) 108 (1.9%) 124 (1.1%)
Homogenate III 96 (2.4%) 85 (4.7%) 93 (9.3%) 126 (4.1%)
To further test the run to run and sample to sample variability, 16 muscle samples from
one subject in study IV was used. These samples were prepared for immunoblotting, as
described previously, and analyzed for levels of phosphorylated Akt. On the second
occasion, new muscle tissue from the same sample was prepared for immunoblotting.
These “new” samples were analyzed together with the “old” samples under new blotting
conditions e.g. new gel, fresh buffers and freshly diluted antibody. The results from the
experiments are depicted in figure 17, where the degree of phosphorylation is normal-
ized to sample number 1, which is a sample taken at rest. This shows a close relation in
the pattern of phosphorylation for the three conditions. For the samples with a high
degree of phosphorylation there was a closer relationship for the different muscle sam-
ples in the same run than for the same sample in two different runs.
60
Figure 17. The variability between two separate runs and two fractions of the same muscle sam-
ple in the immunoblotting analysis of phosphorylated Akt. The degree of phosphorylation in each
sample is normalized to sample no. 1 for each set of samples.
5.2 Muscle sample homogenization
In study III, the muscle samples were homogenized using a ground glass homogenizer.
In all subsequent studies a BulletBlender was utilized, which is a type of mini bead
beater. To control for the compatibility of the BulletBlender a total of 16 samples divid-
ed in two, were homogenized using the glass homogenizer and the BulletBlender, using
a few slightly different protocols. These samples were immunoblotted for phosphory-
lated S6K1, mTOR and eEF2, as well as for α-tubulin. In general, the signal for proteins
in the mTORC1 signaling pathway was slightly higher with the BulletBlender, whereas
the signal for α-tubulin was slightly lower. As the protein concentration in the superna-
tant of the homogenates did not differ between methods the BulletBlender seemed to
yield a slightly higher relative concentration of proteins of interest and a lower relative
concentration of structural proteins. Importantly, there were no differences in relative
increases in phosphorylation for pre to post exercise samples with the different homog-
enization methods. For example, the average increase in S6K1 phosphorylation was 17-
fold with the glass homogenizers and 16-fold with the BulletBlender. Accordingly, the
BulletBlender was determined suitable for homogenization.
Following homogenization all samples were centrifuged for 10 min at 10 000 g to re-
move cellular debris and the supernatant collected for immunoblotting. This procedure
could potentially remove a subset of proteins that are to be analyzed. To address this
matter, pre and post exercise samples were homogenized after which one aliquot was
centrifuged and the other aliquot not. The uncentrifuged samples had a 2 to 2.5-fold
0
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Original
Re-run
Re-run:New muscle
Sample no.
De
gre
eof phosphory
late
d p
rote
in (
arb
itra
ry u
nits)
61
higher protein concentration. The samples were immunoblotted for several proteins in
the mTORC1 signaling pathway as well as for actin and α-tubulin. It could be conclud-
ed that un-centrifuged samples have a profoundly lower relative concentration of the
proteins in the mTORC1 pathway and that the high concentration of structural protein in
those samples weakens the signal of the proteins of interest. For some proteins i.e. 4E-
BP1, a signal could not be detected in the uncentrifuged samples due to their low con-
centration. The procedure of centrifugation is more likely to generate more accurate
sample to sample differences due to lower background to signal ratio. Results from the
comparison are exemplified by levels of phosphorylated mTOR and actin, in figure 18.
Figure 18. Comparison between centrifuged and uncentrifuged muscle homogenates in the levels
of phosphorylated mTOR at Ser2448 and actin.
In study I and II, different buffers were used when homogenizing muscle samples for
immunoblotting and the immunoprecipitations. In study IV, the same homogenization
buffer was used in all assays since this would enable the use one homogenate from a
larger aliquot of muscle and thus streamline the analysis. The use of the standard buffer
for immunoblotting (IB-buffer) purposes was compared to the corresponding homoge-
nization buffer for immunoprecipitation of each protein (IP-buffer). This was performed
with 16 muscle samples for S6K1 kinase activity (4 x Pre, Post, 90 and 180 min), 8 of
those samples for eIF4E protein interaction (2 x Pre, Post, 90 and 180 min) and 2 of
those samples for AMPK activity (Pre and Post). The results of the comparisons are
shown in figure 19. The IB-buffer was compatible for all assays and generated a higher
signal to background ratio for all. This is most likely because lysis with the IB-buffer
generates a relative higher concentration of the proteins of interest a lower relative con-
centration of structural proteins.
0
5
10
15
20
25
Pre A Post A Pre B Post B
Centrifuged
Not centrifuged
Phospho-mTOR Actin
Imm
unoblo
ttin
g s
ignal (a
rbitra
ry u
nits)
Pre A Post A Pre B Post B
62
Figure 19. Comparison of the standard immunoblotting lysis buffer (IB) to the specific lysis
buffer for immunoprecipitation used in studies I and II for (A) S6K1 activity, (B) 4E-BP1:eIF4E
protein interaction and (C) AMPKα2 activity. The 16 samples are collected from one subject in
study IV, where number 1; Rest, 2; Post exercise, 3; 90 min of recovery, 4; 180 min of recovery.
5.3 Myofibrillar protein extraction
In study IV, analysis enrichment of phenylalanine tracer was made in mixed muscle
protein and in a myofibrillar protein pool. For myofibrillar protein extraction the pellets
following centrifugation of the muscle sample homogenates were recovered. The pellet
was washed once in purified water, dissolved in buffer and subjected to centrifugation at
1 600 g for 20 min. The remaining pellet was freeze dried and analyzed for tracer en-
richment using GC-C-IRMS. To confirm that specific protein pools were attained, the
primary supernatant used for immunoblotting and kinase assay were compared to the
myofibrillar pellet with regard to content of myofibrillar, sarcoplasmic and mitochon-
drial proteins. The blots for the different protein are presented in figure 20A. A compar-
ison was also made between a directly dissolved pellet and the final pellet that was
washed and additionally centrifuged. The myofibrillar pellet was confirmed of be highly
enriched with myosin and actin and very low levels of sarcoplasmic GAPDH and mito-
chondrial COX IV.
0.0
0.3
0.5
0.8
1.0
1.3
1.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
S6
K1
acitiv
ity
(pm
ol∙ m
in-1
∙ m
g-1
)
IB-Buffer
IP-Buffer
Sample no.
A
0
0.4
0.8
1.2
1.6
2
1 2 3 4 5 6 7 8
4E
-BP
1:e
IF4E
inte
raction
Sample no.
IB-Buffer
IP-Buffer
B
1 2
Sample no.
0
5
10
15
20
25
30
AM
PK
α2
acitiv
ity
(pm
ol ∙ m
in-1
∙ m
g-1
)
IB-Buffer
IP-Buffer
C
63
Figure 20. (A) Western blot analysis of the myofibrillar protein pellet used for phenylalanine
tracer enrichment (Pellet-washed), the pellet before purification (Pellet-raw) as well as the re-
maining supernatant. GAPDH is a marker for cytosolic proteins and COX IV for mitochondrial
proteins. (B) Correlation between mixed muscle FSR and myofibrillar FSR in study IV, regression
analysis included 8 subjects measured at four occasions.
5.4 Fractional synthetic rate
Fractional synthetic rate is a widely used method to determine the rate of protein syn-
thesis. There are several methodological approaches, but it is considered preferable to
use a multi-labeled tracer (+5-6 atoms), long duration between biopsies (180 min or
more), the intracellular precursor pool and quantification of tracer utilizing GC-C-
IRMS. Despite the fact that these criteria were met in all three studies where FSR was
assessed, the data was highly variable. For example calculated FSR during 180 min of
recovery from exercise varied from 0.025 to 0.165 % ∙ h-1
in study I, from 0.011 to
0.161 % ∙ h-1
in study II and from -0.065 to 0.157 % ∙ h-1
in study IV. The same degree
of variation was also noticed for myofibrillar protein FSR measured in study IV, which
also correlated well with mixed protein FSR.
Moreover, FSR measured at rest was higher during the second trial in study I
(P<0.05). Therefore, FSR at rest from the first trial was chosen to be included in the
result since FSR at rest in the second trial was 68% higher than during the first trial, and
even higher than FSR during recovery. Moreover, this approach is comparable to that of
other studies in which resting FSR was assessed only in the first of two trials (39, 43).
Due to the counterbalanced randomized experimental design, the FSR at rest was thus
obtained equally distributed between the R- and ER-trial. In study II, FSR at rest was
Myosin
Actin
GAPDH
COX IV
A
-0.10
-0.05
0.05
0.10
0.15
0.20
-0.10 -0.05 0.05 0.10 0.15 0.20
Myofibrilla
r pro
tein
FS
R
Mixed muscle protein FSR
BR= 0.74, p<0.001
64
higher in six out of eight subjects in the second trial, but not statistically different from
the first trial. The CV% for repeated measurement of FSR at rest was 71% in study I
(51% not including the extreme outlier) and 26% in study II (figure 21). Here and for
the following calculations CV% was defined as:
Figure 21. Repeated measurement of FSR at rest in study I (A) and II (B). The subjects were
studied under the same conditions between 8 AM and 11 AM, following an overnight fast. In study
I, one subject is excluded from the illustration due to an extremely outlying value during the
second trial (First; 0.049 % ∙ h-1, Second; -0.092 % ∙ h-1)
The variability of the GC-C-IRMS per se with L-[ring-13
C6]-phenylalanine as tracer is
reported to be 3.0% with low enrichments and 2.1% with high enrichments (182), and is
thus not likely to be a major contributor to the variability in the calculated rates of pro-
tein synthesis.
Using 64 muscle samples from study I, the variability of the analysis of tracer en-
richment was assessed. Two extracts (termed A and B) of each sample were prepared
for GC-C-IRMS analysis of tracer enrichment as described previously. The CV% be-
tween the duplicates for tracer to trace ratio (TTR) in muscle protein was 34%, while
that for the free amino acid pool (used as precursor in the FSR calculations) was 15%
and 35% for calculated FSR (Figure 22). Accordingly the variability in incorporation of
the tracer into muscle protein is greater than that of the enrichment of the intracellular
free amino acid pool. Although it cannot be concluded if the variability is mainly sam-
ple or analysis related, the fact that the two extracts came from the same homogenous
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18A
FS
R a
tre
st 2
nd
tria
l (%
∙ h
-1)
FSR at rest 1st trial (% ∙ h-1)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18
FS
R a
tre
st 2
nd
tria
l (%
∙ h
-1)
B
FSR at rest 1st trial (% ∙ h-1)
65
lyophilized muscle preparation, indicates that it is largely analysis related. This reason-
ing is supported by the close correlation between TTR of myofibrillar and mixed protein
that emerged from two fractions of the same muscle sample.
Figure 22. Repeated measurement of intracellular tracer enrichment (A), muscle protein tracer
enrichment (B) and FSR (C) for 64 muscle samples. Intracellular tracer levels were analyzed
using GC-MS/MS and muscle protein tracer levels using GC-C-IRMS. TTR; Tracer to tracee
ratio.
In study IV it was noticed that the variation in TTR in the muscle protein increased
noticeably and progressively with each trial. During the first trial in study IV the in-
creases in enrichment were stable but as the trials progressed, major increases and even
decreases in enrichment, which is physiologically infeasible, started to appear. Although
only two trials were performed a similar pattern was also noticed in study I, and to a
0
0.02
0.04
0.06
0.08
0 0.02 0.04 0.06 0.08
Intr
acellu
lar
enri
ch
mentana
lysis
A (
TT
R)
Intracellular enrichment analysis B (TTR)
A
0
2.5∙10-4
0
2.0∙10-4
1.5∙10-4
1.0∙10-4
0.5∙10-4
0.5∙10-4 1.0∙10-4 1.5∙10-4 2.0∙10-4 2.5∙10-4P
rote
in e
nrichm
entanaly
sis
A (
TT
R)
B
Protein enrichment analysis B (TTR)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
FS
R a
naly
sis
A (
% ∙ h
-1)
FSR analysis B (% ∙ h-1)
C
66
lesser degree in study II (figures 23A-C). It seems that local differences in muscle pro-
tein turnover rates, that could be affected by fiber type as well as exercise, nutrition and
hormones between trial periods, results in major local differences in baseline enrich-
ment for each new trial. One fact that argues for this reasoning is the large individual
variability in the change in TTR during the period between trials. TTR is expected to
increase between trials due to redistribution of tracer from organs with rapid turnover to
the skeletal muscle where the protein turnover is relatively slow. Since this varied be-
tween subjects and also over time, as seen in study IV, it results in major differences in
baseline enrichment for each subsequent trial. The increase in TTR variation over time
is thus likely to be a large contributor to the variation in FSR, especially in study IV and
to some degree in study I (figure 23D).
In all three studies, the intracellular enrichment of the free amino acid pool increased
immediately after each exercise with an average of 50% compared to the enrichment at
rest. During recovery in all studies the intracellular enrichment decreased slightly but
was still higher than the level at rest by the end of recovery. In theory, the higher intra-
cellular enrichment (precursor pool) should result in a relative higher rate of incorpora-
tion and be accounted for in the FSR equation. However, the biopsy collected immedi-
ately after exercise might not be a representable baseline for the recovery measurement
due to the disturbances of steady state during exercise that cannot be accounted for. It
can thus be argued that the baseline for recovery should be collected e.g. 30 min after
exercise as steady state gets re-established. But since the FSR for example varied from
0.013 to 0.166 % ∙ h-1
at rest and from 0.025 to 0.165 % ∙ h-1
during recovery in study I,
argues that this is not a major contributor to the overall variations in FSR.
In summary regarding FSR, variations in the analysis of tracer enrichment in muscle
protein and sample variability, along with the progressive increase in TTR variation
over time, seem to have had the largest influence on the major variations in FSR. In
study II, although variable, the fluctuations in FSR were less than in study I and IV.
This was particularly reflected in the changes in TTR over time (figure 23B) and be-
tween trials in study II. It might be that the smaller triceps brachii muscle which is gen-
erally more homogenous in terms of fiber type than the vastus lateralis muscle and less
involved in daily activities and exercise is more suitable for tracer studies. The conclu-
sion from the present evaluation of the FSR measurements is to perform not more than
two interventions, and if two interventions are conducted, the period in between should
be highly controlled in terms of exercise and nutrition. Moreover, it might be preferable
to use a small and homogenous muscle in terms of fiber type and to postpone the base-
line biopsy for the recovery period 30-60 min after completion of exercise. Regardless,
the sample to sample variability and the error of enrichment measurement is a disturb-
ing factor.
67
Figure 23. Individual muscle phenylalanine tracer to tracee ratios (TTR) for all biopsies in study
I (A), study II (B) and study IV (C). Dots connected with a line represent one individual subject
during each trial. The change in TTR per day of rest in the period between each trial (D). The
Arabic numerals in study IV represent the days between the first – second, second – third and
third – fourth trial.
0.0252
0.0254
0.0256
0.0258
0.0260
0.0262
0 1 2 3 4 5 6 7 8 9 10 11 12
Muscle biopsy no.
Mu
scle
pro
tein
phe
nyla
lan
ine
tra
cer
to t
racee
ratio
1st Trial 2nd Trial
Study IA
0.0252
0.0254
0.0256
0.0258
0.0260
0.0262
0 1 2 3 4 5 6 7 8
Mu
scle
pro
tein
phe
nyla
lan
ine
tra
cer
to t
racee
ratio
Muscle biopsy no.
1st Trial 2nd Trial
Study IIB
0.0252
0.0254
0.0256
0.0258
0.0260
0.0262
0.0264
0.0266
0.0268
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Muscle
pro
tein
phenyla
lanin
e t
racer
to t
racee
ratio
Muscle biopsy no.
1st Trial 2nd Trial 3rd Trial 4th Trial
Study IVC
0
4∙10-6
Study I Study II Study IV-1 Study IV-2 Study IV-3
ΔT
TR
pe
r d
ay
of re
st b
etw
ee
ntr
ials 3∙10-6
2∙10-6
1∙10-6
-1∙10-6
-2∙10-6
D
68
6 DISCUSSION
The skeletal musculature is of great importance for athletes as well as during states of
disease, and its mass is determined by the rate of protein synthesis and the rate of pro-
tein breakdown. Two major factors that are able to affect these two processes are exer-
cise and amino acids. Their effect on protein synthesis is integrated by the mTORC1
signaling pathway, which is the silver thread of this thesis. The effects of concurrent
exercise and the particular role of leucine on this signaling pathway were identified as
uncharted, and are the main theme of this thesis. In addition, given the limited
knowledge about the regulation of protein breakdown, the aim was to expand the under-
standing of how and if different modes of exercise and amino acids affect this process.
6.1 The effects of concurrent exercise on mTORC1 signaling
The AMPK responds to exercise and energy stress and several studies in rodent skeletal
muscle have shown that both pharmacological and exercise induced activation of
AMPK blunts both basal and stimulated mTORC1 signaling and protein synthesis (169-
171, 173), most likely a mechanism to inhibit the energy consuming process of synthe-
sizing new proteins under conditions of local energy stress. This cross-talk between
AMPK and mTORC1 could explain why some training studies show compromised
hypertrophy in response to concurrent exercise compared to resistance exercise only
(158-160). Therefore, study I was undertaken to explore this potential mechanism in
human skeletal muscle. Previous studies on this topic in human muscle were performed
in the fed state and/or did not actually activate AMPK (43, 175, 176). Therefore, study I
involved a high intensity interval cycling protocol to promote a significant AMPK acti-
vation, which was in fact accomplished. An almost 100% increase in AMPKα2 activity
was noted immediately after interval cycling as well as after resistance exercise in the
ER-trial, a robust increase considering the trainings status of the subjects. However, no
increase in AMPKα2 activity was noted after resistance exercise only. This was a bit
surprising as in study IV there was a 2-fold increase in AMPKα2 immediately following
the resistance exercise session. The resistance exercise induced AMPKα2 activity in
study IV could be related to likely lower oxidative capacity of the subjects, the slightly
more intense exercise protocol and the greater decrease in glycogen (25% vs. 15%) in
comparison to study I. In contrast to the robust stimulation of AMPKα2 activity in study
I, there was no change in the activity of the α1 subunit. This is however reasonable as
α2 and not α1 seems to be the isoform that predominantly responds to exercise (152,
183, 184).
Despite a robust activation of AMPK activity in the ER-trial there were no differ-
ences between trials in the resistance exercise induced stimulation of mTORC1 signal-
69
ing. In fact, concomitant with the increase in AMPKα2 activity after interval cycling
there was a 250% increase in S6K1 phosphorylation. Immediately after cycling exercise
in the ER-trial, as well as after resistance exercise in both trials, there was however a
substantial hyperphosphorylation of eEF2 and hypophosphorylation of 4E-BP1, sugges-
tive of a down-regulation of translation. This effect, in very close proximity to the exer-
cise, was also noticed in study I, II and IV, as well as by others (33, 185, 186), and
seems related to the molecular events during exercise and not the recovery. Importantly,
Rose and colleagues (187) reported that the effect on eEF2 and 4E-BP1 during exercise
is not mediated by AMPK, but rather by exercise induced Ca2+
-CaM signaling. This
notion supports the idea that exercise induced AMPK does not inhibit mTORC1 signal-
ing and also comply with the observation of an increased S6K1 phosphorylation directly
after exercise. In conclusion, the data in study I strongly suggested that exercise induced
activation of AMPK does not inhibit resistance exercise stimulation of mTORC1 signal-
ing in human muscle.
The obvious subsequent question is why does our data not cohere with the studies in
rat skeletal muscle? There are three suggested mechanisms by which AMPK inhibits
mTOR kinase activity. First, AMPK has the ability to phosphorylate TSC2 at Ser1387
which promotes the conversion of Rheb ∙ GTP to Rheb ∙ GDP, thus decreasing
mTORC1 activity (102, 107). TSC2Ser1387
phosphorylation was indeed enhanced after
interval cycling but surprisingly also after resistance exercise in both trials. There is no
obvious explanation to the inconsistency between AMPK activity and TSC2Ser1387
phos-
phorylation, but it suggests that other kinases could be responsible for this phosphoryla-
tion. Putting the upstream regulation aside, as TSC2 phosphorylation was only in-
creased in close proximity to exercise it could actually be argued that TSC2 in fact
dampened mTORC1 kinase activity at those time points in both trials, but had no impact
during recovery. A second mechanism by which AMPK is suggested to inhibit
mTORC1 is by direct phosphorylation of Raptor at Ser792
(108). The fact that Raptor
phosphorylation increased slightly after exercise and during recovery to a similar extent
in both trials, could explain the lack of mTORC1 signaling inhibition, especially as the
phosphorylation is shown to be essential for the AMPK inhibition to occur (108).
AMPK has also been suggested to directly mTOR at Thr2446
which leads to inhibition of
its kinase activity (188). Although Thr2446
phosphorylation was not assessed, the study
that characterized the mechanism showed that an increase in Thr2446
cohered with a
decrease in Thr2448
and vice versa. Since mTORSer2448
phosphorylation increased after
cycling exercise in the ER-trial and after resistance exercise in both trials argues that
AMPK did not likely phosphorylate the inhibitory Thr2446
residue of mTOR. Lastly,
some studies suggest that it is AMPKα1 activity that is responsible for the inhibition of
mTORC1 and consequently muscle cell growth (172, 189). While this could explain the
findings in study I, it is questionable since pharmacological activation of AMPK by
70
AICAR, which is shown to inhibit mTORC1 (169-171), stimulates α2 activity. If how-
ever assumed that it is indeed AMPKα1 that inhibits mTORC1, it would have little
relevance to exercised human skeletal muscle since α2 is the isoform shown to be acti-
vated during exercise in humans.
It may be that the mechanistic cell and animal models are not representative of the
exercise-induced signaling response in human skeletal muscle. For example in the stud-
ies by Inoki et al (107) and Gwinn et al (108), showing the involvement of TSC2 and
Raptor in AMPK mediated mTORC1 inhibition, they utilized loss of function ap-
proaches with amino acid mutated variants of TSC2 and Raptor, which is arguably a
very different model compared to exercise in vivo. Such a difference is clearly illustrat-
ed by the study Pruznak et al (171) in which AICAR administration in rats induced a
more than 2-fold increase in Raptor phosphorylation compared to the minor increase
seen in study I. Thus, despite the major increase in AMPKα2 activity seen after the high
intensity cycling intervals, the magnitude of this increase may not have been large
enough to sufficiently increase TSC2 and/or Raptor phosphorylation in order to nega-
tively affect mTORC1 activity. Therefore it is likely that AMPK related mechanisms
have little practical relevance in exercising human skeletal muscle, as they would re-
quire a much more intense exercise than that performed in study I, which given its ex-
haustive and metabolically demanding nature would be very difficult to achieve, espe-
cially on a regular basis for a prolonged period of time.
One limitation with study I is that the interval cycling compromised resistance
exercise performance, so that the load and repetitions performed during the combined
protocol had to be used in the R-trial. Therefore, one objective with study II was to
examine a concurrent exercise model where resistance exercise performance is not
compromised. The separation of the two modes of exercise in different muscle groups is
also more applicable in a practical point of view for athletes. Finally, the experimental
set up enabled the novel studying of the systemic effect of concurrent exercise, which
was of interest since we and others have previously reported alterations in cell signaling
in the resting muscle following unilateral exercise (38, 128, 151).
Intriguingly, when preceded by interval cycling, resistance exercise induced S6K1
activity was attenuated, AMPK phosphorylation potentiated and the eIF4E:4E-BP1
interaction increased in the triceps immediately after the exercise session. This immedi-
ate reduction in mTORC1 signaling and the increase in AMPK phosphorylation were
resolved during recovery, where all markers of mTORC1 signaling as well as protein
synthesis were stimulated to a similar extent in both trials. Although the acute reduced
response of S6K1 and 4E-B1 had no appreciable influence on the overall outcome, the
underlying mechanism is intriguing. The most apparent explanation for the negative
influence on S6K1 and 4E-BP1 is the potentiating of AMPK phosphorylation in the ER-
Arm trial. The factor responsible for the induced AMPK phosphorylation is not obvious
71
but could potentially by endurance exercise induced IL-6 (190). Although the phos-
phorylation of Raptor was not assessed there were no differences between trials in the
phosphorylation of TSC2Ser1387
or mTORSer2448
which, as discussed above, suggests that
AMPK did not inhibit mTORC1. It must also be acknowledged that the degree of
AMPK phosphorylation does not necessarily reflect the degree of activity but limita-
tions in muscle tissue prevented the assessment of AMPK activity. Finally, the differ-
ence in AMPK Thr172
was only 20%, which could be argued is not substantial enough to
induce the observed differences in S6K1 and 4E-BP1. Taken all together it is question-
able that AMPK is responsible for the immediate inhibitory effect on mTORC1 signal-
ing in study II.
Another speculation is that the substantial increase in plasma levels of cortisol in the
ER-Arm trial is responsible for the effect on S6K1 and 4E-BP1, since injection of glu-
cocorticoid is reported to blunt both basal and stimulated mTORC1 signaling in rat
skeletal muscle (191, 192). This effect seems to depend on activation of the glucocorti-
coid receptor (193) and the following transcription of the mTOR inhibitory protein
REDD1 (194). As we found no change in the protein levels of REDD1, and since the
time frame for this mechanistic event to occur does not cohere, it suggests that cortisol
is not responsible for the finding. In summary, no definite conclusion can be drawn
regarding the mechanism responsible for immediate effect on S6K1 and 4E-BP1 in the
ER-Arm trial, but could be due to an uncharted mechanism, maybe involving activation
of specific phosphatases (195).
Based on the data from study I and II it can be concluded that concurrent exercise,
performed in separate or the same muscle group, does not compromise resistance exer-
cise induced mTORC1 signaling during the three hour recovery from exercise. A mus-
cle biopsy collected in a minute or less after cessation of contractions seem to exhibit
signaling events that are more reflective of the situation during exercise than during
recovery, which in the aspect of signaling is vastly different. This is particularly evident
for eEF2 and 4E-BP1 which are hyper- and hypophosphorylated, respectively, immedi-
ately after exercise but then become hypo- and hyperphosphorylated, respectively, dur-
ing recovery. Although the inhibitory effect of eEF2 and 4E-BP1 during exercise is
suggested not to mainly involve AMPK (186, 187), it could be that AMPK plays a role
in retaining signaling through S6K1 and 4E-BP1 during and in the close proximity to
muscle contractions. Since the acute energy stress, if defined as increased muscle
[AMP] and reduced [ATP], can be resolved within 10 min of recovery from intense
exercise (196), together with the finding that resistance exercise increases AMPKα2
activity (33) (Study IV) argues against the notion that an energy stress activation of
AMPK should inhibit resistance exercise induced mTORC1 signaling and protein syn-
thesis during recovery. The data from study I and II follow this reasoning and suggest
72
that it is the resistance exercise stimuli per se that determines the mTORC1 signaling
and protein synthetic response during recovery.
6.2 Concurrent exercise increases the expression of PGC-1α
The PGC-1α is a master regulator of mitochondrial biogenesis and oxidative adapta-
tions, and its crucial role is well established in both overexpression- and knockout mod-
els in rodent muscle (197-199). Moreover, in human muscle, a clear relationship be-
tween exercise induced mRNA levels of PGC-1α, subsequent increases in its protein
levels and amount of mitochondrial proteins (200). In study I, there was a robust in-
crease in PGC-1α expression with concurrent exercise, compared to only a slight induc-
tion with resistance exercise only. There was also a greater expression of PGC-1α in the
triceps with concurrent exercise in study II, which is intriguing considering that endur-
ance exercise was performed with the legs.
It is conceivable that the mechanism behind the induced PGC-1α expression in study
I was mediated by the increased AMPK activity (201). It is also possible that greater
levels of intracellular Ca2+
and subsequent signaling events during concurrent exercise
could be responsible for the induced expression (202, 203), but we were not able to
accurately determine levels of phosphorylated CaMKii, which limits our conclusion.
Finally, there were no differences in phosphorylated levels of p38 MAPK, which also
has been suggested to control the expression of PGC-1α (204). In study II, there was a
slight difference in AMPK phosphorylation between trials immediately after exercise,
but it is difficult to appreciate the significance of that difference with regard to PGC-1α
expression. However, what is interesting is that the effect of PGC-1α is likely to be
systemically mediated, given that resistance exercise performance and levels of glyco-
gen were similar in both trials. Systemic factors such as catecholamines, reactive oxy-
gen species, corticosteroids, IL-6 and lactate have all been shown to induce PGC-1α
expression in muscle cells (190, 205-208), effects that could be both dependent and
independent of AMPK, and they could all be regarded as potential mediators of the
effect. Future studies are however clearly needed to determine the precise mechanism
responsible for the induction by systemic factors. The ability of exercise to promote
oxidative adaptations in another exercised muscle, or potentially non-exercised muscle,
is exciting. This potential as well as the identification of the factor or mechanism re-
sponsible could have clinical implications in states of disease and during recovery from
injuries. It can be speculated that muscle fiber size may be limited to prevent muscle
fiber anoxia (155), an increase in PGC-1α with concurrent exercise could thus be bene-
ficial to accommodate the oxidative demand in a hypertrophying muscle.
73
6.3 The effects of amino acids on mTORC1 signaling
Leucine seems to have unique anabolic properties among the EAA, having been shown
to alone stimulate mTORC1 signaling and protein synthesis in human skeletal muscle
(62). In study III we set out to determine the importance of leucine among the EAA in
their ability to stimulate mTORC1 signaling. In previous studies on this topic when
extra leucine was added to an amino acid mixture (209, 210) that set up could not de-
termine a particular role of leucine. Hence, we utilized a novel experimental design in
which subjects ingested an EAA supplement, with or without the inclusion of leucine, in
combination with resistance exercise. Following 60 min of recovery from exercise there
was a robust stimulation of mTORC1 signaling when leucine was present, but only a
minor stimulation was noticed when leucine had been removed. At the end of the 180
min recovery period the differences between supplements were no longer present and
mTORC1 signaling was stimulated to a similar extent. This was likely an of to the re-
sistance exercise as amino acids have been shown to have a transient (~2h) stimulation
of mTORC1 signaling and protein synthesis, while that of resistance exercise is more
prolonged (20, 41, 211, 212). However, the lack of a placebo trial in the experiment
prevented a comparison between the sole effect of exercise and the remaining EAA.
Therefore, our group subsequently, in the study of Apró et al. (213), added a placebo
situation as well as a leucine only trial to the experimental design. Here it was conclud-
ed that EAA without leucine is no more effective than flavored water in stimulating
mTORC1 signaling after resistance exercise, and while the response to leucine alone
was highly potent, an even greater response was obtained with the mixture of EAA.
To try and elucidate which EAAs that are responsible for the additive effect of
leucine on mTORC1 signaling, study IV was undertaken. In part since we previously
have shown that ingestion of BCAA potently induces mTORC1 signaling (127, 128) it
was hypothesized that valine and isoleucine mediates the additional effect of the EAA.
Interestingly, 90 min after exercise in study IV, the BCAA were indeed more potent
than leucine in stimulating S6K1 activity and 4E-BP1 phosphorylation, but an even
greater response was seen with EAA.
From study III, along with the study of Apró et al. (213), it can be concluded that
leucine is highly potent in stimulating mTORC1 signaling and that the remaining EAA
exert minimal effects alone, but intriguingly exert a synergistic effect when leucine is
present. Although there is no data in human skeletal muscle, the individual effect of
isoleucine and valine on mTORC1 signaling in cell cultures and animal muscle has been
shown to be relatively small (109, 117, 129, 214) or in some cases absent (215-217).
This sparse effect on mTORC1 signaling has also been noticed for the other individual
EAA (109, 214, 215, 218). It is thus conceivable that the additional effect of the remain-
ing EAA is the sum of their small individual effect, which surprisingly is present only
when leucine is included in the supplement.
74
In study IV, BCAA was shown to have a more sustained effect on mTORC1 signal-
ing than leucine, and comparable to the effect of EAA following 180 min of recovery.
This could be due to the fact that leucine supplementation caused a robust decrease in
the muscle levels of valine and isoleucine and to a certain degree that of the sum of
EAA. Maintenance of muscle levels of BCAA, in particular, might thus be an important
factor in preserving signaling through mTORC1. The up-stream mechanisms by which
amino acids mediate their input to mTORC1 is not well understood. Work conducted in
cell lines has however proposed that leucyl-tRNA synthetase (117) and Sestrin 2 (219)
are leucine sensors that mediate mTORC1 activation, while the latter has also been
shown to sense isoleucine and valine which could explain the present findings of the
potentiating effects of the BCAA. However, the mechanism by which leucine and the
other EAA mediate their stimulatory effect in human skeletal muscle remains to be
determined.
6.4 The role of insulin in amino acid induced stimulation of mTORC1
signaling
Essential amino acids, and in particular leucine, stimulate insulin secretion from the
pancreas (220). It is thus possible that differences in mTORC1 signaling between the
amino acid supplements in study III and IV could in part be an indirect effect mediated
by insulin. It is suggested that insulin has a permissive role in amino acid induced
mTORC1 signaling (69, 70), which could be explained mechanistically by the finding
that insulin and amino acids signal in parallel to mTORC1, insulin via the TSC-Rheb
axis (104) and amino acids through the Rag-proteins (113). In the suggested model,
amino acids promote translocation of mTORC1 to Rheb which, when activated by insu-
lin, promote mTORC1 kinase activity (113). The key questions are then, what level of
insulin is required for sufficient Rheb ∙ GTP loading, and does increasing levels of insu-
lin increase mTORC1 signaling under amino acid stimulation? In perfused rat liver and
skeletal muscle of piglets, it has been shown that increasing insulin above fasting levels
has no further impact on mTORC1 signaling under stimulation by prandial levels of
amino acids (111, 221). In relation to human muscle, Glynn and co-workers (74)
showed that increasing plasma insulin from 15 to 50 mU ∙ l-1
by adding carbohydrates to
an EAA supplement had no additional effect on the anabolic response induced by amino
acids following exercise. In contrast, under amino acid infusion at rest, increasing levels
of insulin by infusion from fasting (5 mU) up to varying prandial and really high levels
(30, 72 and 167 mU) potentiates S6K1 phosphorylation (71).
So how does this all relate to the findings in this thesis? On a general note it must be
recognized that the repeated bolus administration of the amino acid supplements em-
ployed in our studies only mildly induces insulin secretion. For example, in two of our
previous studies (127, 128) there were no or only small differences in plasma levels of
75
insulin following BCAA ingestion or placebo, all at a level below 20 mU ∙ l-1
. In study
IV, the peak insulin concentration was 16 mU ∙ l-1
with EAA and 14 mU ∙ l-1
with place-
bo. In study III, the peak insulin concentration was however 40 mU ∙ l-1
with EAA and
30 mU ∙ l-1
EAA-Leu, but the fasting level was almost 3-fold higher than in study IV,
differences that are most likely related to the different analytical procedures. With this
in mind it is not likely that insulin is responsible for the potentiating of mTORC1 sig-
naling attained by leucine inclusion in study III, or by BCAA and EAA in study IV. As
insulin signals to mTORC1 through Akt, and as there were no difference in Akt phos-
phorylation in any trial in the two studies it supports the idea that insulin is not likely
involved in the potentiating of mTORC1 signaling. It must however be acknowledged
that differences in Akt phosphorylation might have been present in closer proximity to
exercise at a time point we did not assess.
It could however be speculated that exercise increases insulin sensitivity and that the
small differences in insulin thus results in a relatively greater signaling response that
contributes to a greater extent. But as this should be mediated by Akt signaling argues
against this idea since Akt signaling did not differ between trials. On another note, re-
sistance exercise is suggested to in similarity with insulin activate Rheb by disabling
TSC2-function (119). It could be argued that resistance exercise would then in contrast
reduce the significance of insulin signaling to Rheb, since Rheb already would be
stimulated by resistance exercise. This could explain the fact that Greenhaff and co-
workers (71) showed an effect of increasing insulin levels on mTORC1 signaling at rest
while Glynn and colleagues (74) showed no such effect after resistance exercise. In
summary, the different stimulation of mTORC1 signaling induced by the amino acid
supplements in studies III and IV are most likely direct effects of the specific amino
acids and not indirectly mediated by insulin.
6.5 Details in exercise and amino acid regulation of mTORC1 signaling
During the course of data collection in this thesis some interesting aspects concerning
specific regulation of components in the mTORC1 signaling pathway became apparent.
One such aspect was the divergent response of S6K1 and 4E-BP1 phosphorylation in
study I, II and IV, which was apparent in the biopsies taken immediately after exercise.
This might seem puzzling as both S6K1 and 4E-BP1 are well characterized down-
stream targets of mTORC1 (86) and respond in a similar manner to insulin (222). How-
ever, the mechanism by which mTORC1 is suggested to meditate the phosphorylation
of these two substrates offer an explanation. Both S6K1 and 4E-BP1 contains TOR
signaling (TOS) motifs (specific amino acid sequence) to which Raptor binds and re-
cruits them for phosphorylation by mTOR (76, 77). The drug rapamycin inhibits S6K1
and 4E-BP1 binding to mTOR and interestingly these two substrates exhibit different
sensitivity to rapamycin, with S6K1 being substantially more sensitive than 4E-BP1
76
(223). It was recently shown that the TOS-motif of 4E-BP1 is more suitable for the
mTOR interaction and exhibits a higher affinity for raptor than S6K1, and is conse-
quently less sensitive to rapamycin to inhibit this interaction (224). This notion would
explain the fact why 4E-BP1 is highly phosphorylated at rest while S6K1 is almost
completely dephosphorylated. Accordingly, only a modest increase in mTOR kinase
activity will have a substantial notable difference in the phosphorylation status of S6K1.
The finding that exercise, in study I, II and IV, only stimulated S6K1 phosphoryla-
tion while resistance exercise and amino acid ingestion, in study IV, also stimulated 4E-
BP1 phosphorylation is likely a result of the different substrate specificity and the great-
er mTORC1 activity with the combined stimuli. The dephosphorylation of 4E-BP1
noted directly after exercise in studies I,II and IV is not likely due to a reduced
mTORC1 signaling, as S6K1 Thr389
increased. It could be speculated that this is an
effect of 4E-BP1 dephosphorylation by specific phosphatases, such as PP2A (195, 225),
and a consequence of competition with S6K1 for binding to Raptor (226), as S6K1 must
have interacted with mTORC1, thus making 4E-BP1 more readily available for phos-
phatases and less susceptible for mTOR.
In study IV, additional interesting aspects concerning the regulation of 4E-BP1 in
human muscle emerged. We found that amino acid ingestion had no impact on phos-
phorylation on the combined Thr37/46
residues, but increased that of Thr46
. The lack of
effect by amino acids ingestion on Thr37/46
has also been reported previously in our
laboratory (213). In addition, resistance exercise did not decrease the phosphorylation of
Ser65
, which however responded in a similar fashion to amino acids as Thr46
. The Thr37
and Thr46
sites are shown to be direct targets of mTOR (86, 87), but while this is not
shown for Ser65
, this site is clearly mTOR-dependent and rapamycin sensitive (85, 88,
89). In relation to 4E-BP1 function, all Thr sites are proposed to be important for resolv-
ing the eIF4E interaction, while Ser65
is dispensable for this function (83, 90, 91). The
data in study IV suggests that Thr37
is readily phosphorylated by mTORC1 in the basal
state and that only Thr46
respond to the increased mTORC1 activity following amino
acid ingestion. Moreover, Thr46
was more reflective of the 4E-BP1:eIF4E interaction
than Ser65
, which is in line with the mechanistic studies and the suggestion that Ser65
may function to prevent eIF4E re-association (92).
Further insight to the regulation of 4E-BP1 was made in study II. Due to limitations
in muscle tissue, 4E-BP1:eIF4E interaction was only assessed before and immediately
after exercise, but in line with study IV, a decrease in Thr46
phosphorylation was mir-
rored by and increased interaction. Surprisingly, Ser65
phosphorylation increased in the
R-Arm trial and decreased in the ER-Arm trial. The increase in Ser65
but not Thr46
is
confounding and implies that other kinases than mTOR are able to phosphorylate this
site. Collectively, our data support that a decrease in 4E-BP1 phosphorylation at Thr37/46
results in an increased 4E-BP1:eIF4E interaction and argues that an increase in Thr46
is
77
required for the interaction to decrease, thus promoting translation initiation. The fact
that exercise only increases S6K1 while exercise combined with amino acid ingestion
also stimulate 4E-BP1 could, in addition to the degree of S6K1 activity, explain the
additive effect on protein synthesis by exercise and amino acids.
The data in this thesis clearly support the previous studies showing that eEF2 re-
sponds to exercise but not to amino acid stimulation (128, 130, 213, 227, 228). This fact
is somewhat confounding as previous work in cells has shown that insulin decreases
eEF2 Thr56
phosphorylation (increases its activity) in a rapamycin-sensitive manner and
that S6K1 inhibits the eEF2 kinase, thus reducing eEF2 Thr56
(100, 229). The increase
in eEF2 phosphorylation (decreases its activity) immediately after exercise is however
most likely an effect of increased Ca2+
-CaM signaling (187, 230) and not an effect of a
reduced mTORC1 signaling, as S6K1 was activated. Moreover, a complete blunting of
Ca2+
- signaling or increased S6K1 activity could thus not likely be responsible for the
eEF2 hypophosphorylation during recovery, especially since S6K1 activity generally
differed between trials but eEF2 phosphorylation did not. The stimulation of eEF2
could instead by an effect of activation of stress signaling pathways, as discussed by
Browne and Proud (231). Taken all together, the data in this thesis clearly suggest that
S6K1 is not in major control the phosphorylation of eEF2 in response to exercise and
amino acid ingestion in human skeletal muscle. Accordingly, the phosphorylation status
of eEF2 is not a suitable marker of mTORC1 signaling under these conditions.
6.6 The effect of exercise and amino acids on markers of protein
breakdown
The majority of proteins in the muscle are broken down in the ubiquitin-proteasome
pathway (134) where the two proteins MAFbx and MuRF-1 are key regulators and the
expression of these two proteins are shown to be readily up-regulated in over 30 models
of atrophy (136). Interestingly, the expression of these two genes is also affected by
exercise (37, 137-140), to which they exhibit a divergent response. The general observa-
tion is that exercise increases the expression of MuRF-1 while that of MAFbx is unal-
tered or decreased. The data obtained in study I and II is in overall agreement with the
previous findings except for the minor increase in MAFbx expression in study II. The
expression of both genes was unaltered in study III, which could be due to the amino
acid supplementation or possibly the relatively light resistance exercise protocol. The
interpretation of the data in study IV is limited by the fact that only total protein and not
mRNA expression of these genes were assessed. On a general note it must be
acknowledge that 180 min might be a too short time period to detect changes in protein
levels using immunoblotting, which would require a change of at least 10-15%.
We and others have previously shown that intake of protein-carbohydrate drink
(232), BCAA (140) or amino acid infusion (233) reduces the mRNA expression of
78
MuRF-1 after exercise. Although we did not include a placebo situation in study III it is
possible that both amino acid supplements prevented an exercise induced increase of
MuRF-1. This reasoning is supported by the fact that we previously found an increase in
MuRF-1 after completion of the same exercise protocol in young males (137). In that
case it would be argued that the effects of amino acids are not mediated by leucine per
se, which is in accordance with the findings of Herningtyas and colleagues (234) in
C2C12 muscle cells. However, we found no effect of amino acids on protein levels of
MuRF-1 in study IV.
In study I and II, the expression of MuRF-1 was readily induced by concurrent exer-
cise but unaltered by resistance exercise only. The latter was unexpected since we have
previously shown an increase after resistance exercise only (137, 140), but could be
explained by the more exercise accustomed subjects in study I and II. The mRNA ex-
pression of MAFbx in these studies was inconsistent, with a minor increase in both
trials in study II, and a decrease with resistance exercise in study I, while being unal-
tered after concurrent exercise. The reason for the inconsistent MAFbx expression is
difficult to appreciate, but the divergency between MuRF-1 and MAFbx could be a
result of their different functions, or different molecular targets to be precise. MAFbx is
proposed to target regulatory proteins such as MyoD (235) and eIF3-f (236) for degra-
dation. Since the eIF3 complex is involved in ribosome assembly and suggested to in-
teract with S6K1 and mTORC1 in the signaling transduction (96), a reduction in
MAFbx would be beneficial for the protein levels of eIF3 and thus in coherence with
the stimulation of anabolic signaling following resistance exercise. MuRF-1is suggested
to target myofibrillar proteins (237, 238) and an increased expression would thus reflect
an increased breakdown of contractile proteins with exercise, which seems reasonable.
It must be acknowledged that there is no data suggesting that the increase of MuRF-1
expression is mechanistically responsible for the acute exercise induced increase in
protein breakdown. This is rather an effect to support and maintain an increased demand
for protein breakdown. This could indicate both a reduced protein balance effect, i.e. “a
negative outcome”, and an increased muscle cell remodeling, i.e. “a positive outcome”,
and the findings that concurrent exercise increases MuRF-1 expression could actually
support both these functional outcomes. There is thus a clear demand for studies exam-
ining the functional role of MAFbx and MuRF-1 in response to exercise, training and
muscle growth in humans.
While it is reasonable that both atrophy and exercise induce the expression of
MuRF-1, and that the divergent expression of MuRF-1 and MAFbx might be due to
their different functions, the molecular mechanisms responsible for these effects are not
well defined and seem rather complex. A number of transcription factors have been
proposed to control the expression of these ubiquitin ligases, but the first to be identified
was the class-O forkhead transcription factors (FOXO), and in particular FOXO1 and
79
FOXO3a (239, 240). Moreover, it has been shown that IGF-1 is able to inhibit the ex-
pression of MuRF-1 and MAFbx, thus preventing atrophy, by Akt or mTORC1 signal-
ing towards the FOXOs (239-241). The involvement of Akt or mTORC1 in the exercise
induction of MuRF-1 is questionable since it would accordingly be due to a reduced Akt
and mTORC1 signaling, which was not the case. This does not however exclude the
involvement of the FOXOs, but it seems appropriate to assume that Akt and mTORC1
is not in major control of MuRF-1 in connection with exercise. However, it has been
shown in C2C12 muscle cells that amino acid induced suppression of MAFbx but not
MuRF-1 expression is sensitive to rapamycin (234), suggesting that mTORC1 can con-
trol the expression of MAFbx, which could explain their divergent response to exercise.
Moreover, pharmacological activation of AMPK in myotubes and in rat skeletal muscle
has been shown to, via FOXOs, induce the expression of both these ubiquitin ligases
(242-244). This mechanism could explain the induced expression of MuRF-1 in the ER-
trial and the unaltered expression in the R-trial in study I, as there were significant dif-
ferences in AMPK activity between trials. Furthermore, the down regulation of MAFbx
in the R-trial and the unaltered expression in the ER-trial could be due to activated
mTORC1 signaling, of which effect was counteracted by AMPK in the latter trial.
In study II, where endurance and resistance exercise were separated in different
muscle groups we expected that the concurrent exercise induced expression of MuRF-1
would not occur. Surprisingly, as described, this was not the case and although there
were minor differences in AMPK phosphorylation these are not likely to explain the
robust differences in MuRF-1 expression. Given that glucocorticoid treatment is a well
described atrophy stimulus shown to induced MuRF-1 and MAFbx expression (245),
we choose to determine plasma levels of cortisol. Notably, the level of cortisol was 3.4-
fold higher during exericse in the ER-Arm trial and was elevated for over two hours,
and is therefore a potential candidate responsible for the induced MuRF-1 expression.
Waddel and colleagues (246) have provided detailed information about the glucocorti-
coid regulation of the ubiquitin ligases, and intriguingly the MuRF-1 promoter, but no
MAFbx, contains a perfect glucocorticoid receptor response element. Although, both
MAFbx and MuRF-1 respond to cortisol, the effect on MuRF-1 can be direct and more
rapid while the effect on MAFbx is indirect and likely requires glucocorticoid induced
expression of FOXOs. This mechanism is in accordance with the findings in study II
and would indicate that MAFbx expression could have been induced at a later, non-
assessed, time point.
In summary, exercise affects the expression of MuRF-1 and MAFbx, generally in a
divergent manner, which might be a result of their different functions. The molecular
mechanism responsible for the divergent response is unknown, but could be due to
different susceptibility to mTORC1 activity or differences in their promoter region.
Concurrent exercise increases MuRF-1 expression which could be a consequence of
80
increased AMPK activity and/or elevated levels of plasma cortisol. The increased
MuRF-1 expression likely reflects and increased demand for protein breakdown or
muscle cell remodeling. While it has been previously shown that amino acids reduced
the expression of these ubiquitin ligases, the data in study III suggest that this effect is
not primarily mediated by leucine.
6.7 Differences between the vastus lateralis and triceps brachii muscles
The mTORC1 signaling response to resistance exercise in the vastus lateralis muscle
has been explored in numerous studies, but that in the triceps brachii had not been de-
scribed prior to study II. Although a direct comparison cannot be made, study I and II
involved subjects with similar training status and involved a resistance exercise protocol
with matching intensities as well as number of repetitions and sets, thus justifying an
appreciation of the potential differences in the two muscle groups. One striking differ-
ence was noted in the temporal response of S6K1 phosphorylation and activity. In the
vastus, S6K1 phosphorylation was only increased to a minor extent immediately after
exercise, but increased gradually with the highest degree of phosphorylation and activity
noted at the end of recovery. While in the triceps, the maximal activation was noted
immediately after exercise and then it remained significantly elevated above rest
throughout the 180 min of recovery. One potential explanation for this could be fiber
type distribution, which for vastus lateralis generally is equal between slow- and fast
twitch fibers and for triceps brachii is dominated by fast twitch fibers (247, 248). It has
been previously shown that resistance exercise induced activation of S6K1 mainly oc-
curs in type II fibers (249), but this does however not explain the large temporal differ-
ences, which thus lack an apparent explanation. Besides the temporal difference in
S6K1 activation there were no other obvious differences between vastus and triceps
with regard to protein signaling, gene expression or protein synthesis.
6.8 The relationship between mTORC1 signaling and protein synthesis
The main objective of this thesis was to examine the molecular mechanisms regulating
muscle protein synthesis in response to different modes of exercise and ingestion of
specific amino acids. Here, signaling offers potential understanding and is of great im-
portance for future research ideas and directions, while protein synthesis is the physio-
logical hard endpoint. Which event that is of most importance is determined by the
researchers’ specific interest, but determination of their relationship is regardless of
major value. However, as discussed previously, methodological issues limit a distinct
overall conclusion of the protein synthetic response, but it is nevertheless of importance
to reflect on the relationship between mTORC1 signaling and protein synthesis.
81
First of all it must be acknowledged that mTORC1 signaling is shown to be required
for resistance exercise and amino acid induced protein synthesis in human skeletal mus-
cle (122, 130). The major question then is if the degree of mTORC1 signaling reflects
the rate of protein synthesis? Most studies examining the acute anabolic response to
exercise and nutrition tend to focus on one outcome, which limits the present under-
standing. However, a number of studies in human muscle have shown a relationship
between the degree of mTORC1 signaling, assessed by S6K1 phosphorylation, and the
rate of protein synthesis (49, 63, 211, 212, 250), while others do not (71). Methodologi-
cal errors, differences in methodological approaches between labs and number of biop-
sies used for signaling assessment are likely confounding factors in the interpretation of
the relationship. It is also of utter importance to recognize that signaling is just a snap-
shot, a static measurement, while FSR is the summarized effect over a period of time, a
dynamic measurement. This notion stresses the importance of multiple muscle biopsies
for signaling assessment when trying to establish a relationship between these two out-
comes. This was apparent in all four studies were differences between trials were pre-
sent only at certain time points. Although we were not able to establish a relationship
between the degree of mTORC1 signaling and FSR in study I and II, the similar signal-
ing response during three hour recovery was reflected by similar rates of protein synthe-
sis between trials in both studies.
Intuitively, the degree of phosphorylation/activity should be of some relevance from
a biological point of view. If the proteins in the signaling pathways were merely on and
off switches the wide dynamic range of S6K1 kinase activity for example seems rather
futile. If we consider that a highly potent stimulus by resistance exercise and amino acid
ingestion induces a ~40-fold increase in S6K1 phosphorylation, a ~10-fold increase in
S6K1 activity and a ~150% increase in protein synthesis (251), it indicates that the
magnitude of change is scaled down for the different outcomes. Ergo, a certain differ-
ence in degree of S6K1 phosphorylation is needed for a detectable change in FSR, and
if this relationship is constant, a ~4-fold increase in phosphorylation would be needed to
acquire a ~15% increase in FSR. This is subject to change depending of which signaling
protein that is under consideration. 4E-BP1 for example has a much lower dynamic
range of phosphorylation from the level at rest, and this notion is important to consider
when reflecting on the significance of certain signaling responses. Furthermore, with
regard to different proteins in the signaling pathway, as discussed previously, a certain
stimulation of mTORC1 activity may only activate S6K1, whereas a higher activity
could be needed to affect 4E-BP1. Since 4E-BP1 affects cap-dependent translation
initiation (capacity) and S6K1 the efficiency of translation initiation, their combined
effect on the rate protein synthesis is difficult to appreciate. This could mean that the
effect of S6K1 on FSR is not linear and could change dependent on the effects of 4E-
82
BP1. Accordingly, a number of factors need to be considered when relating mTORC1
signaling to the rate of protein synthesis.
Finally, only a few human studies have examined the mTORC1 signaling response
for a period longer than 5-6 hours after stimulation, and there are no time course studies
above that point. As the rate of protein synthesis following an exercise bout can be
sustained for more than 24 hours (11) there is an obvious lack of knowledge concerning
the underlying mechanism for the sustained effect. However, it was recently shown in
rodent muscle that mTORC1 signaling following resistance type exercise stimulation
peaked after 3 hours, but was elevated for 18 hours (252). Interestingly, the rate of pro-
tein synthesis was still elevated beyond that point, suggesting that other mechanisms are
responsible for the prolonged response. However, given that mTORC1 signaling can be
elevated for at least 18 hours after exercise, indicates that potential differences between
interventions could be present later than the most commonly assessed 3 hour post inter-
vention period. This view advocates a 24 h time course of the mTORC1 signaling re-
sponse following an intervention to enable a proper determination of its relation to the
rate of protein synthesis.
6.9 Gender differences
To consider differences between genders is an essential aspect in all areas of physiolog-
ical research. To conduct a valid comparison between the sexes in the present studies,
preferably eight or more male and female subjects would have been required to detect
potential gender differences in mTORC1 signaling and protein synthesis. Such large
group of subjects was not within the scope of the present studies, and to avoid potential
confounding factors the studies were performed with females (study III) or males (study
I, II, and IV) only. The inclusion of males in study I, II was due to methodological issue
with triceps muscle biopsies and the intention to have a very similar group of subjects in
both trials for comparisons. In study IV it was due to biopsy sampling aspects together
with the wider range of males in the particular population of interest. Since no firm
conclusion concerning gender differences in the present thesis can be drawn it is im-
portant to reflect on this aspect. Previous studies in the field have mostly concluded that
basal rates of muscle protein synthesis is similar between males and females (253-258),
while one study displayed 15% higher basal rates in women (259). Moreover, the stud-
ies that have evaluated the aspect of gender on the effects of resistance exercise (257),
amino acid provision (256) and the combination (258) on mTORC1 signaling and FSR
have found no differences in the response between males and females. Accordingly, the
results in study III and IV are not likely to differ between males and females. Since we
are unable to decisively determine which systemic factor(s) that influenced the signaling
and mRNA expression response in study II, it is difficult to discuss potential gender
differences. However, it is plausible the endurance exercise induced levels of hormones,
83
cytokines and myokines differ between men and women, which may have had an im-
pact on the outcome. Moreover, with regard to study I, AMPKα2 activity has been
shown to be higher in men than in women following 90 min of cycling exercise (260).
This finding was attributed to the higher rates of fat oxidation in the women, and it is
thus uncertain if differences between genders would have been present after the anaero-
bically challenging interval cycling protocol. Nevertheless, as mTORC1 signaling was
not influenced by AMPK activity, a lower AMPK response in women would only have
weakened the conclusion but not influenced the outcome per se. To conclude, in con-
cern of the previous studies in the field there is no reason to suppose that the results in
the thesis would differ between men and women within the study specific population.
6.10 Representativeness and implications
The study population for each study was quite specific, especially in study I, II and IV
which involved trained males in the age of 20-35, and in study III, recreationally active
women of the same age. It is therefore of interest to discuss whether the results in the
studies are relevant for other populations. The aspect of gender has been addressed in
the foregoing section but another vital aspect is training status. In general untrained
subjects seem to exhibit a more profound acute response to exercise, as exercised in-
duced increases in AMPK activity (261, 262), mTORC1 signaling (263, 264) and pro-
tein synthesis (40, 48) are reduced following a period of training. A more dramatic and
variable signaling response in untrained could then have confused the interpretation of
the response in study I and II. However, the conclusion that concurrent exercise does
not interfere with the anabolic response to resistance exercise would reasonably hold
true in untrained as well. An assumption that is based on the fact that untrained subjects
exhibit a more unspecific response to exercise (16) and that concurrent training increas-
es hypertrophy compared to resistance training only in untrained (166, 167, 265). In
study IV, and to some extent in study III, more untrained subjects would likely have had
a greater response to the resistance exercise, which may resulted in a less distinct differ-
ence between the different supplements, if thought in terms of a reduced signal to back-
ground ratio where exercise sets the level of background.
What are then the implications and significance of this thesis for the scientific com-
munity and the public? Study I is of great value for the understanding of the interplay
between AMPK and mTORC1 in human skeletal muscle, which seem to differ from cell
and animal models. Given that AMPK and mTORC1 are central players in the regula-
tion of muscle metabolism the understanding of their interplay is of high importance. In
a more applied point for training adaptations, study I argues that the different contrac-
tion modes per se are compatible for the anabolic signaling response, and suggest that
other factors like residual fatigue, increased energy requirements and neuronal adapta-
tions are more likely to compromise longterm resistance training adaptations. Study II is
84
likely the one of most significance for the general public as it utilized an exercise model
that can be applied both in athletes and recreationally active people. As mentioned pre-
viously, the understanding of the factor/mechanism responsible for the systemic in-
crease in PGC-1α could have clinical implications in states of disease and during recov-
ery from injuries where oxidative capacity is compromised or the mitochondria are
dysfunctional. The significance of an increased MuRF-1 expression with concurrent
exercise is difficult to evaluate due to the lack of knowledge of its distinct function, but
could be beneficial in the aspect of cell remodeling and maintenance, or detrimental in
the aspect of protein net balance. Study III shows that lack of leucine diminishes amino
acid stimulation of mTORC1 signaling. This is vital from a mechanistic point of view
but of less relevance from a practical point of view since most meals contain ample
amounts of leucine, but could be of consideration for certain vegetarian meals. In study
IV, EAA supplementation was shown to have the greatest overall effect on mTORC1
signaling after resistance exercise. While this also is interesting from a mechanistic
point of view it has some practical relevance. Leucine and BCAA are common sport
nutrition supplements and are also in some use in clinical nutrition. It could however be
argued based on study IV that EAA supplementation is preferable for the anabolic stim-
ulus in the aspect of mTORC1 signaling and presumably for providing ample amount of
substrates for protein synthesis.
6.11 Limitations
All study designs and analysis have limitations, but recognition of these will strengthen
the interpretation of the data obtained as well as improve the quality of future work. One
obvious limitation, that has already been addressed, is the lack of reliable FSR data in
study IV. Although the signaling was the principal interest and the data is solid, espe-
cially in study IV with the S6K1 activity measurement, the conclusion is limited by the
absence of a hard end point measurement. Moreover with regard to study III, here it
cannot be concluded if the similar effects on mTORC1 signaling at 180 min post exer-
cise are due to exercise or amino acids, and why a presumed increase in MuRF-1 ex-
pression was absent. The inclusion of a placebo trial in study III would however have
resolved these matters. A similar point can be made in study I and II, where it cannot be
concluded if the increases in gene expression with concurrent exercise is an effect of the
combined stimulus or merely the endurance exercise. This could have been addressed
by including a cycling exercise trial, but we were nevertheless able to answer our spe-
cific research question with the present study design.
In study I and II, the fasting potentially had an impact on the results which limits the
interpretation from an applied point of view. For example plasma levels of cortisol and
mRNA expression of MuRF-1 may have been exaggerated, and post exercise rates of
protein synthesis reduced, all due to the prolonged fasting. However, nutrient ingestion
85
during these trials would have prevented us to answer the research question of interest.
In study II it would also have been beneficial to assess activation of the triceps brachii
with EMG in relation to the pectorals and the deltoids which were also involved in the
specific exercise. Finally, our signaling data is overall limited by the lack of a muscle
sample taken in relative close proximity (~30 min) to the exercise bouts. A 30 min post
exercise biopsy could have detected a potential increase in Akt phosphorylation in study
III and IV. Furthermore, this would have permitted a closer estimation of when the
differences between trials in S6K1 and 4E-BP1 (study II) as well as in AMPK (study I)
were resolved.
6.12 Future directions
Study I, and study II in particular, has offered the greatest potential for further explora-
tion. The most obvious questions that need to be addressed are; what are the systemic
factors behind the acute effects on S6K1 and 4E-BP1, as well as the induced MuRF-1
and PGC-1α expression? It is also of major interest to characterize the signaling path-
ways that mediate the input. One possible way to address these questions could be to
infuse physiological high doses of various systemic factors during resistance exercise in
humans and examine the subsequent signaling and mRNA expression response. Fur-
thermore, it would be of importance to resolve as to why exercise induced AMPK does
not inhibit mTORC1 in human skeletal muscle, and if other AMPK activators such as
metformin could have a different effect. It would also be of interest to explore how
nutrient/amino acid ingestion might affect the outcome in study I and II. One major
issue that requires further exploration is the mechanism controlling the expression of
MAFbx and MuRF-1 in response to exercise, and importantly what is the physiological
function of their altered expression to this stimuli?
While major work is being performed in cells regarding how different amino acids
are sensed within the cell and how this is mediated to mTORC1 this exploration must be
continued in human muscle. Finally, more methodological data on FSR measurements
are needed. The variability commonly presented in the literature as well as in this thesis
is far too high to likely reflect biological variation of such a highly controlled process as
protein synthesis. The field is also in need of refined methods to determine protein syn-
thesis and in particular protein breakdown.
86
6.13 Conclusions
The major conclusions of this thesis are as follows,
Exercise induced activation of AMPK does not inhibit resistance exercise in-
duced mTORC1 signaling or protein synthesis in the vastus lateralis muscle
during recovery
Interval cycling with the legs performed prior to resistance exercise of the tri-
ceps muscle does not influence mTORC1 signaling and protein synthesis dur-
ing recovery from resistance exercise, but reduces S6K1 activity and increases
4E-BP1:eIF4E interaction in the immediate connection with exercise
Concurrent exercise increase the mRNA expression of MuRF-1 and PGC-1α
compared to resistance exercise only, even when endurance exercise is per-
formed with a different muscle group
Absence of leucine in an EAA supplement substantially reduces the activation
of mTORC1 signaling following resistance exercise
BCAA supplementation following resistance exercise stimulates mTORC1
signalling more potently than ingestion of leucine alone, but not as effectively
as EAA supplementation. However, the effect of EAA seem to be primarily
attributed to the BCAA
87
7 SAMMANFATTNING
Vår skelettmuskulatur har en unik förmåga att förändra sin fenotyp beroende på inver-
kan av externa stimuli så som olika typer av träning och nutrition. Olika träningsformer
ger upphov till olika anpassningar hos muskeln, där konditionsträning ökar antalet mi-
tokondrier och kapillärer som medför en ökad kapacitet oxidera fett i muskeln, vilket
bidrar till en ökad uthållighet. Styrketräning däremot ökar mängden kontraktila protei-
ner och glykolytiska enzymer vilket ger upphov till muskeltillväxt (hypertrofi) och en
ökad anaerob kapacitet. Inuti muskeln pågår det konstant en proteinsyntes samt en ned-
brytning av protein, och för att muskeln ska kunna öka i massa måste syntesen vara
kraftigare än nedbrytningen. Två mycket potenta stimuli för att öka syntesen är styrke-
träning och intag av aminosyror/protein, och deras gemensamma effekt på syntesen
krävs för att den ska bli kraftigare än nedbrytningen vilket därmed kan leda till hyper-
trofi på sikt. På molekylär nivå handlar proteinsyntes om translationsprocessen där
mRNA transkripten från generna översätts till aminosyrasekvenser som bildar proteiner.
Den så kallade mTORC1 signalvägen har övergripande kontroll över denna process och
integrerar samt aktiveras av och olika stimuli så som styrketräning, aminosyror och
insulin.
Aminosyrornas stimulatoriska kapacitet av mTORC1 signalvägen och proteinsynte-
sen är tillskriven gruppen av essentiella aminosyror (EAA), där den grenade aminosyran
leucin har fått specifik uppmärksamhet på grund av sin kapacitet att enskilt kunna sti-
mulera till en ökning av proteinsyntesen. Det är dock inte känt hur betydelsefull leucin
är bland de essentiella aminosyrorna och hur stor roll de övriga aminosyrorna spelar i
aktiveringen av mTORC1 och proteinsyntes. När det gäller styrketräning har en del
studier påvisat att samtida konditionsträning, så kallad kombinationsträning, leder till
hämmad styrkeökning samt muskeltillväxt jämfört med enbart styrketräning, och det
råder en ovisshet om de potentiella bakomliggande mekanismerna. När man konditions-
tränar uppstår en energistress i muskeln vilket leder till aktivering av proteinet AMPK,
som i sin tur medför ett ökat uttryck av genen PGC-1α som övergripande styr tillväxten
av mitokondrier. När AMPK är aktiverat så kommer det enligt teorin hämma mTORC1
signalering och proteinsyntes efter styrkträning, vilket då skulle leda till hämmad mus-
keltillväxt efter en tids kombinationsträning. Denna teori kommer från studier där man
farmakologiskt aktiverat AMPK i celler eller använt sig av elstimuleringsmodeller hos
råttor, men det är okänt om denna potentiella mekanism är relevant i samband med
träning i human muskel.
Då relativt mycket kunskap existerar om hur träning och nutrition påverkar protein-
syntesen och de bakomliggande molekylära mekanismerna är kunskapen mycket be-
gränsad vad gäller detta i relation till proteinnedbrytning. Mest uppmärksamhet har
88
givits den så kallade ubiquitin-proteasome signalvägen där de två proteinerna MuRF-1
och MAFbx har identifierats som kritiska komponenter. Dessa proteiner har visats öka
vid flertalet tillstånd som ger upphov till atrofi och intressant nog även visats påverkas
av träning. Dock är kunskapen om deras reglering och respons på olika typer av träning
och nutrition mycket begränsad i human muskel.
Syftet med denna avhandling är att genom fyra delstudier utöka kunskapen om hur olika
träningsformer samt olika aminosyror påverkar mTORC1 signalvägen, proteinsyntesen
samt markörer för proteinnedbrytning i human skelettmuskulatur. De specifika syftena
med avhandlingen är att:
Undersöka om AMPK aktiverat genom konditionsträning hämmar direkt efter-
följande styrketräningsinducerad mTORC1 signalering och proteinsyntes
Utforska akuta systemiska effekter av konditionsträning på styrketräningsinduce-
rad mTORC1 signalering, proteinsyntes och genuttryck, genom att separera de
olika träningsformerna till ben- och armmuskulatur
Bestämma aminosyran leucins roll bland EAA i förmågan att stimulera
mTORC1 signalering i samband med styrketräning
Undersöka de separata effekterna av leucin, grenade aminosyror (BCAA) och
EAA på mTORC1 signalering och proteinsyntes i samband med styrketräning
Utforska hur markörer för proteinnedbrytning påverkas av olika träningsformer
samt olika aminosyror
I studie I fick vältränade och unga försökspersoner genomföra två försöksdagar innehål-
landes ett styrketräningspass i form av benpress, där ett ut av passen direkt föregicks av
ett högintensivt pass med cykelintervaller. Denna intervallträning medförde som ämnat
en påtaglig aktivering av AMPK, som var förhöjd även vid avslutandet av den efterföl-
jande styrketräningen. Trots denna kraftiga aktivering var det ingen skillnad i styrketrä-
ningsinducerad aktivitet av enzymet S6K1, en väletablerad markör för mTORC1 signa-
lering, mellan de två försöken. Det var därtill ingen skillnad i hastigheten på proteinsyn-
tesen under den tre tim långa återhämtningsperioden från de olika passen. Däremot
medförde kombinationsträningen ett kraftigt ökat genuttryck av MuRF-1 samt även det
av PGC-1α, jämför med enbart styrketräning.
Delstudie II hade i princip samma upplägg som studie I, med den skillnaden att
styrkträningen genomfördes med armmuskulaturen och att biopsierna tog från triceps
brachii muskeln. Cykelintervallerna medförde att aktiveringen av mTORC1 signale-
ringen efter styrkträningen var något hämmad jämfört med enbart styrketräning i de
muskelprover som togs i direkt anslutning till träningen. Dessa skillnader var inte kvar
under återhämtningsperioden, där mTORC1 signalering och hastigheten av proteinsyn-
tesen var lika i båda situationerna. Trots att träningsformerna separerades mellan ben
89
och arm så gav kombinationsträningen även här en kraftig ökning av uttrycket av
MuRF-1, och även till en viss utsträckning det av PGC-1α.
I studie III fick moderat tränande och unga försökspersoner genomföra två pass med
styrketräning i form av benpress under vilka de, i randomiserad ordning, fick inta ett
tillskott av EAA med eller utan aminosyran leucin. Intag av EAA medförde en mycket
kraftig aktivering av mTORC1 signalering efter styrketräningen och denna stimulering
var kraftigt reducerad då leucin inte fanns med i tillskottet. Uttrycket av MuRF-1ökade
inte efter träningen vilket kan tolkas som att träningen inte var tillräckligt potent i detta
avseende eller att uttrycket hämmades av tillförsel av aminosyror, vilket då inte är spe-
cifikt medierat av leucin.
I den avslutande delstudien fick styrketräningsvana och unga försökspersoner
genomföra fyra försök med styrketräning i form av benpress, där de i randomiserad
ordning fick inta ett tillskott med leucin, BCAA, EAA eller placebo. Efter halva åter-
hämtningsperioden ökade aktiviteten av S6K1 i ett hierarkiskt mönster (Placebo < Leu-
cin < BCAA < EAA), men i slutet av återhämtningen var effekten lika mellan grenade
och essentiella aminosyror som fortfarande var mer potenta än leucin och placebo. Ett
liknande mönster uppvisades även för fosforyleringen av 4E-BP1, den andra väletable-
rade nedströms markören för mTORC1. Metodlogiska problem, troligen beroende av
upprepade infusioner av isotopmärkt fenylalanin, förhindrade en utvärdering av effekten
på proteinsyntesen.
För att sammanfatta, högintensiva cykelintervaller utförda direkt innan styrketräning
utav ben- eller armmuskulatur påverkar inte mTORC1 signalering eller proteinsyntes
under den tre timmar långa återhämtningsperioden. Kombinationsträning ökar genut-
trycket av markören för proteinnedbrytning MuRF-1 jämfört med enbart styrketräning,
och detta sker även om träningsformerna separeras mellan arm och ben. Det är dock
oklart vad denna ökning har för praktisk betydelse. Ett liknande fynd gjordes även för
uttrycket av genen för PGC-1α, vilket indikerar en anpassning för ökad oxidativ kapaci-
tet i muskeln. Aminosyran leucin är ytterst viktig bland EAA i deras förmåga att stimu-
lera mTORC1 signalering efter styrketräning. Intag av alla tre BCAA ger en kraftigare
respons på anabol signalering efter styrketräning än intag av enbart leucin, men störst
respons erhålls av de essentiella aminosyrorna, vars effekt är till stor del medierad av
BCAA som en grupp.
90
8 ACKNOWLEDGEMENTS
I would like to express my sincerest gratitude to The Swedish School of Sport and
Health Sciences for enabling my pursuit for a doctoral degree and for providing a stimu-
lating research environment. I would also like to direct a special recognition to the Swe-
dish National Center for Research in Sports for the financial support of the experiments
in this thesis. There are a number of persons that made it possible for this thesis to be
more than just blank pages. I owe my deepest gratitude to:
Madeleine, my wife and love of my life. Thank you for your tremendous support, for
always staying positive and having a smile on your face despite my many early morn-
ings and late evenings and while doing so carrying two beautiful children, you are made
of steel!
Jacqueline, my baby girl and apple of my eye. You make life worth living!
Eva Blomstrand, my supervisor. Thank you for giving me the opportunity to become a
PhD, for providing such freedom and trust as well as showing me what meticulous sci-
entific work is all about.
William Apró, my “scientific brother from another mother”. Although you were not
my supervisor you were the one that thought me the most and I owe most of my lab
skills to you. I am absolutely inspired by your devotion to science and tremendously
grateful for your drive to develop our methodology, it definitely raised the bar. Thank
you for your friendship and bearing with my wailing in times of trouble. I truly hope for
a future collaboration!
HC-Holmberg, my co-supervisor. You are an amazing source of inspiration. Your
drive and “can-do” approach is contagious. Thank you for all your support, for always
being there in times of need and for keeping me in track of the finer things in life.
Björn Ekblom, the Åstrand laboratory’s amazing Prof. Emeritus. I can literally say that
the studies in my thesis would not have happened if it was not for you. I am amazed by
your passion and words cannot describe how grateful I am for you being in the lab with
me at 5 AM time and time again to perform our invasive trials. You are one of a kind!
Marjan Pontén, lab manager. Thank you for taking me under your wings when I first
came to the lab. I am so grateful for your kindness and support throughout my years in
the lab.
Per Frank, past PhD-student and roommate. Thank you for the glory days, you made
sure my time in the lab was a blast. We had the most awesome conference trips that will
be remembered for life. You are a great friend with an awesome sense of humor.
Niklas Psilander, past PhD-student and roommate. Mr “I did not sleep last night”, you
have your heart in the right place and you are a great friend. Thank you for creating a
nice atmosphere in the lab and for inspiring me to aim for long distance running.
91
Daniele Cardinale, PhD student and olive oil enthusiast. I am inspired by your passion
and drive, the sky is not a limit for you. Thank you for a great time in the lab and for the
awesome days in Toronto. I almost feel half Italian since I, like you, can appreciate the
difference between orecchiette, farfalle, bucatini, pappardelle and etc.
Gustav Olsson, “one of those epidemiologists”. I totally admire your positive attitude
and social skills. Thank you for making sure that the lab is more than just a work place
and for all remarkable topics for discussion.
Frida Björkman, PhD student and “kötta i skogen” fanatic. Thank you for bearing
(almost) with our guys in the room, for pushing me while on the cross-trainer and
being so sincere.
Jane Salier-Eriksson, PhD student and dancing passionate. Thank you for bringing an
amazing spirit to the lab and making sure that we Swedes mind our manners.
Filip Larsen and Thomas Schiffer, the labs’ own Chip and Dale. Thank you for letting
me know how much I dislike hardcore heavy metal music! Jokes aside, I truly admire
your knowledge and accomplishments.
Mikael Mattson, lecturer, adventure racer and always jetlagged, Thank you for involv-
ing me in your project before I became a PhD-student and for showing how a modern
scientist should work.
Kent Sahlin, for flawlessly running the lab and for being a remarkable source of
knowledge, Elin Ekblom-Bak and Örjan Ekblom, for both being a source of inspira-
tion and role models of research, Karin Söderlund, for assistance whenever needed,
Malin Åman, Amanda Ek, Alexandra Jernberg and Kate Bolam, for sheering me on
and making the lab a happier place, Eva Andersson, for your remarkable spirit, Mats
Börjesson, for a positive attitude and for showing what hard work is all about, Robert
Boushel, for bringing your expertise and international touch to the lab, Peter Schantz,
for showing what it is to be a real scientist, Mikael Flockhart, for all the good times in
the lab, Camilla Norrbin, for all the help you have provided to me as a confused PhD-
student
Henrik Mascher, for the help and good times in the dawn of my PhD-studies
Gerrit van Hall, for your expertise and help with the tracer work and for your hospitali-
ty in the CMCF. Nina Pluszek and Andreas Bornø, for your kindness and assistance
with the tracer analysis
Lee Hamilton, for your remarkable help with setting up the kinase assays!
Inger Ohlsson and Antonio Villanueva, for running the trials in study III
Hedvig Samuelsson, for all the biopsies you dissected in study IV
92
Johan Hurtigh, for the biopsies you dissected before “realizing that research was not
your thing”
Oscar Olson, for your enormous help with creating the illustration on the front page
Chocolate, coffee, diet coke and protein bars, for making life a little more worth
living, for lifting my spirits in times of need and simply for keeping me awake
The GIH gym, for enabling me to maintain my physique despite many hours of work
and immense amounts of chocolate
My mother Ulla and father Göran, for all your love and support throughout the years
and for always having faith in me. Your pride in my accomplishments, regardless of
what they have been, has always kept me going! My grandmother Agda, for love, sup-
port and being the strongest person I know, I owe my fighting spirit to you. My sisters
Jessica and Helena, for always being there for me. My second family Sven, Elisabet,
Katarina and Isabel, for taking me in with open arms, for showing me support, for
taking me to distant places in the world and always staying positive.
Last but absolutely not least, an enormous thanks to all you brave subjects. I know I
pushed you hard, I know it hurt a lot sometimes, I know you were hungry and exhausted
but I knew you would make it through. You guys were truly amazing and I am honored
to have met you!
93
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