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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