on Muscle Protein Synthesis (MPS)

Attiyyah Omerjee P11279592 BSc Hons Biomedical Science

BIOM3006 – Dissertation Supervisors: Dr V.H. Pavlidis, Dr M. Morris, Dr M. C. Limb

Combating Sarcopenia: The Effects of BCAA (Branched Chain Amino Acids) on Muscle Protein Synthesis (MPS) in Elderly Women, in Comparison to Whey Protein.

Attiyyah Omerjee P11279592 BSc Hons Biomedical Science ‐ 2014 – 2015 BIOM3006 – Dissertation Supervisors:  Dr V.H. Pavlidis  Dr M. Morris  Dr M. C. Limb

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Acknowledgements . . . My sincerest thanks go to the Division of Metabolic Physiology Research Group at the University of Nottingham (Derby) for granting me the opportunity to work with some wonderful people, and to Dr Kenneth Smith, Debbie Rankin and Dr Anna Selby for training me with so much patience. Thank you also to Amanda Gates and Margaret Baker for diligently teaching me how to survive studies, and for the afternoon Tea Party of legend. And of course to the PhD bunch for the laughs and sage advice.

So much gratitude goes to my parents and family – for not going as nutty as I did, and providing me with a warm home to retreat to for shelter each day. To (Dr) Colleen Deane – for helping clear up the mysteries To Sabba Nasar – my laboratory partner‐in‐crime. We propped each other up! To Dr Vasilios H. Pavlidis – Sir you are a font of wisdom and kind guidance. Your words still raise my spirits and keep me believing To Dr Mhairi Morris – for seeing the value of human creativity To Dr Marie C. Limb – for the endless pestering and patient explanations, as well as the repeated proofreading and painstaking corrections. Thank you for being awesome

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Abstract . . . Introduction – Sarcopenia is a global phenomenon that dates back to Ancient Greece. Muscle is a dynamic tissue capable of adapting to stresses and modifying its abilities. Nutrition driven hypertrophy is of great interest, specifically with the introduction of EAA leucine as a dose‐dependent anabolic stimulator. Measurement of MPS can be achieved through the use of stable isotope tracers, and provide can provide an in vivo representation of muscle metabolism when measured using spectrometric techniques. Aim – Little data has been gathered around muscle mass‐regulation in elderly women. The aim is to investigate the dosage effects of 1.5g and 3g Leu EN supplement to determine their effects at upregulating MPS in elderly women. Methods and Materials – (n=18) Elderly females (n=8/group, 66±3yr, BMI 29±1) were recruited to be studied at fasted state prior to and upon ingestion of Leu EN supplements, at rested state. Results – Signficant increases in MPS were observed with the 3g Leu EN supplement during EF, but no overall significance observed with the administration of 1.5g Leu EN supplement. Neither produced any noticeable effects during LF. Comparison of 3g data to 20g Whey Protein from literature (under identical conditions) showed no overall significant difference between feeds during EF. Conclusion – 3g Leu EN supplementation could be designated the minimal stimulatory dose, with further investigations needed to deduce the effects of higher doses, and the coupling of supplementation to exercise.

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Combating Sarcopenia: The Effects of BCAA (Branched Chain Amino Acids) on Muscle Protein Synthesis (MPS) in Elderly Women, in Comparison to Whey Protein. 1.0 Introduction ................................................................................................................... 6 1.1 The Timelessness of Ageing _____________________________________________________ 6 1.2 Structure and Function of Human Skeletal Muscle____________________________ 7 1.3 Skeletal Muscle Architecture __________________________________________________ 10 1.4 Muscle Metabolism ____________________________________________________________ 12 1.5 Nutrition‐driven Hypertrophy ________________________________________________ 13 1.6 The Balance of Synthesis and Breakdown in the Context of Ageing _______ 13 1.7 The ‘Muscle Full’ Phenomenon of MPS _______________________________________ 14 1.8 Realising Leucine as a Potent Activator of MPS _____________________________ 14 1.9 Intracellular Signalling Effects of Leucine ___________________________________ 14

2.0 The in‐vivo measurement of Muscle Metabolism ........................................ 16 2.2 Tracking Leucine’s Stimulation of MPS ______________________________________ 17 2.3 Tracing Synthesis and Breakdown ___________________________________________ 18 2.4 Fractional Synthetic Rate (FSR) ______________________________________________ 19 2.5 Aim ______________________________________________________________________________ 20

3.0 Methods and Materials ............................................................................................ 20 3.1 Ethical Statements _____________________________________________________________ 20 3.2 Subject Recruitment ___________________________________________________________ 21 3.3 Subject Demographics _________________________________________________________ 21 3.4 Studying Participants __________________________________________________________ 21 3.5 Obtaining Blood and Muscle Samples ________________________________________ 24 3.6 pAA Preparation and Initial Numerical Analysis ____________________________ 24 3.7 Muscle Protein Extraction _____________________________________________________ 25 3.8 Intracellular Phenylalanine Enrichment (IC Phe EN) Determination _____ 25 3.9 Myofibrillar Sample Preparation and Analysis ______________________________ 25 Page 4 of 36


3.10


Data and Statistical Analysis ________________________________________________ 26

4.0 Results and Discussion ............................................................................................ 26 4.1 The Changes in Leucine Concentration (µM) Over Time ___________________ 26 4.2 Statistical Analysis of FSR Results ____________________________________________ 27 4.3 Comparison to Whey Supplement (20g) Taken from Literature __________ 29

5.0 Critical Analysis of the work conducted .......................................................... 30 5.1 Comparisons Made To Men ___________________________________________________ 30 5.2 Exercise as a Potent Stimulator of MPS ______________________________________ 30 5.3 Modelling Women’s Data Collection on Clinical Procedures and Data Collection Developed from Male Studies ___________________________________________ 30

6.0 Suggestions for the improvement and pursuit of further work ............ 31 6.1 Long Term Analysis ____________________________________________________________ 31 6.2 Metabolites of Leucine ________________________________________________________ 32 6.3 Understand Leucine further __________________________________________________ 32 6.4 Genomic Analysis ______________________________________________________________ 32 6.5 Projected Future Developments ______________________________________________ 33

7.0 Conclusion .................................................................................................................... 33 7.1 Overview _______________________________________________________________________ 33 7.2 Further Work __________________________________________________________________ 33

8.0 References .................................................................................................................... 35 Key abbreviations used throughout MPS

Muscle Protein Synthesis

FSR

Fractional Synthetic Rate (% h‐1)

EAA

Essential Amino Acids

AA

Amino Acid

EF

Early feed – this is the immediate 2hr period postprandial

LF

Late feed – this extends 2‐4hr postprandial.

EN

Enriched or Enrichment, depending on context

BCAA

Branched Chain Amino Acid

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Combating Sarcopenia: The Effects of BCAA (Branched Chain Amino Acids) on Muscle Protein Synthesis in Older Women, in comparison to Whey. 1.0

Introduction

1.1

The Timelessness of Ageing

1.1.1

In the days of Ancient Greece, ageing was regarded with a mixture of hatred and fear strong enough for it to be described as a progressive, chronic disease by Aristotle in 384 BC (Narici and Maffuli, 2010). Sarcopenia is derived from the Greek words ‘sarcos’, meaning ‘flesh’ and ‘penia’, meaning ‘lack of’, and was first used by Rosenberg in 1989 (Mitchell et al., 2012; Hairi et al.,2012; Narici and Muffalli, 2010). He used it to describe the loss of lean muscle mass with age, but there is still no definite global consensus on what exactly constitutes sarcopenia (Mitchell et al., 2012). The Oxford Dictionary (2015) definition is “the loss of skeletal muscle mass and strength as a result of ageing”.

1.1.2

The last two decades have seen births increase over deaths, and an upward trend also in life-expectancy. The number of UK residents aged 90 years and older has tripled since the early 1980s. Overall, the UK population is increasing and ageing, with a projected population of 73.3 million people by 2037 (ONS, 2015). In extreme cases, old age can mean a quarter of the elderly over 90 years require long-term residential, nursing or hospital care (Office of Fair Trading, 2005; Bajekal et al., 2006 as taken from Mitchell et al., 2012). With increased life expectancy, an increased quality of life is essential to enjoying one’s later years and reducing the cost of healthcare burdens on the NHS and the economy as a whole.

1.1.3

Cicero (44 BC) himself said in his treatise ‘Cato maiore de senectute’ that “it is our duty… to resist old age, to compensate for its defects, to fight against it as we would fight against a disease; to adopt a regimen of health; to practice modern exercise; and to take just enough food and drink to restore our health” (Narici and Muffalli, 2010). Retention of muscle mass has profound benefits for the elderly aged sixty and above, such as increased mobility, a lower risk of falling/injury, greater independence and a better sense of wellbeing and happiness. Page 6 of 36

Attiyyah Omerjee P11279592 BSc Hons Biomedical Science 1.1.4


Research into developing clinical interventions is therefore essential to improving quality of life for the elderly. One avenue to pursue is the formulation of nutritional supplements that can enhance skeletal muscle protein synthesis (MPS), based on the exploitation of biochemical understandings of physiological muscle regulation.

1.2

Structure and Function of Human Skeletal Muscle

1.2.1

Movement is one of the characteristic functions of living organisms (Tortora and Grabowski 2003). At its most basic; our forms are held by our skeletons, and movement is produced when muscles pull on these bone frames. Almost all bodily muscle is derived from the primary germ cell layer known as the mesoderm. Multinucleated muscle fibres are the result of fusion of many progenitor cells, illustrated in Figure 1. The immediate, surrounding regions regulated by myonuclei (the nuclei of myofibres) are referred to as nuclear domains. They are not constant in size and shape, but allow for coordinated activity across adjacent segments, for example in myosin production. This is not always the case though, and non-uniformity in contractile abilities has been reported (Frontera and Ochala, 2014). Figure 1 – Multinucleated myofibre stained with fluorescent DNA‐binding dye, visualised with confocal microscopy. Taken from Allen et al. (1995), as used by Frontera and Ochala (2014).

1.2.2

Comprising approximately 40% of total body weight, skeletal muscle is one of three muscle types, and the body’s primary motile facilitator. It exists to convert chemical energy to mechanical energy in order to achieve movement through limb interaction, generate force and power and maintain posture (Frontera and Ochala, 2014). Additionally, it functions as a storage site for myoglobin and other substances, as well as facilitating transport of lymphatic fluid, aiding vascular circulation and generating heat to maintain core body temperature (Tortora and Derrickson, 2011). Skeletal muscle is readily distinguished by distinct markings, in comparison to Page 7 of 36



cardiac and smooth muscle. Striations characterise its cells, demarcating the contractile units (sarcomeres) within each fibre. Muscle is surrounded and permeated by three protective layers of connective tissue that hold its shape and attach it to bone. They extend beyond the muscle tissue itself, merging to form tendons. See Figure 2

Figure 2 – Components of skeletal muscle Taken from Tortora and Derrickson (2011)

1.2.3

75% of skeletal muscle is composed of water, with the remaining 25% composed of proteins (20%), and other minerals, inorganic salts, lipids and carbohydrates making Page 8 of 36



up the remaining (5%) (Frontera and Ochala, 2014). The key structural component of muscle is undoubtedly protein, of which there are a few significant types: 

Actin (thin filament) constitutes 20% of total myofibre protein (Goodsell 2001), appearing as two-stranded, helical chains of globular monomers, with a twist every 37nm (Alberts et al. 2004).



Nebulin is entwined around actin filaments (Frontera and Ochala, 2014), and is thought to help stabilise development (Tortora and Derrickson, 2011).



Myosin (thick filament) is found in skeletal muscle; composed of “two ATPase heads, and a rodlike tail” (Alberts et al. 2004). It appears as a bipolar filament formed of identical myosin dimers joined at their tails. Several types of myosin can be found in a single muscle cell, owing to uncoordinated myonuclear domain synthesis. These hybrid fibres are found to increase with exercise, ageing and certain diseases (Frontera and Ochala, 2014). Their effects on force-generation and contractile ability, amongst other phenomena, have yet to be elucidated.



Dystrophin links actin to integral proteins of the sarcolemma, which is then attached to the connective tissue around muscle. Dystrophin is responsible for force transmission to tendons (Tortora and Derrickson, 2011).



Titin – An elastic protein that holds myosin to the Z line, stabilising the thick filament, and has been implicated in force generation (Frontera and Ochala, 2014). It is so named, after the word ‘Titan’, to reflect its size (Tortora and Derrickson, 2011). Appearing as a 4.2MDa protein, it is also known as ‘Connectin’ and runs continuously through each myofibril. It is the third most abundant protein in muscle, after actin and myosin (Nishikawa et al., 2012).



Desmin and α-Actinin are other proteins involved in sarcolemma and extracellular matrix association and Z disc composition (Frontera and Ochala, 2014).

Combined, titin and nebulin are thought to have effects on myofibril assembly, as well as later effects on overall sarcomeric stiffness and integrity (Frontera and Ochala, 2014). 1.2.4

Sarcomeres are the contractile units of muscle. Visualised with electron microscopy, they extend between two adjacent Z-discs (Tortora and Derrickson 2011). Mammalian sarcomeres are (~2.5m) long, and contract simultaneously throughout Page 9 of 36



each muscle fibre (Alberts et al 2004). Not all the molecules needed for regular functioning of muscle are found in the myofibres. A closely associated plasma membrane layer sheathing each myofibre is known as the ‘sarcoplasmic reticulum’. It contains, amongst other things, Calcium, that is released through electrical stimulation and Myoglobin - a red oxygen-binding pigment that provides a store of oxygen for aerobic respiration (Tortora and Derrickson, 2o11). A direct correlation exists between the degree of development of the sarcoplasmic reticulum and the contractile ability of a muscle (Frontera and Ochala, 2014). Deep invaginations of the sarcoplasmic reticulum, called Transverse (T) tubules are responsible for ubiquitous nervous transmission through muscle. Together with nearby terminal cisternae, the T tubules form a ‘triad’ structure that optimises neurone-mediated electrical stimulation of muscle. 1.2.5

Constituting 20% of skeletal muscle mass (Eisner 2013), mitochondria are the respiratory powerhouses of muscle where ATP is oxidatively synthesised. Mitochondria can be positioned beneath the sarcolemma to reduce distances for oxygen-diffusion. However, in light of new structural data, they have also been found to exist in the inter-myofibrillar space, forming 3D networks between each other. Mitochondrial populations correlate directly to the muscle’s ability to resist fatigue and oxidatively respire (Frontera and Ochala, 2014), a predictable phenomenon because of their known role in generating ATP for muscle function. It must be said, not all energy is produced in the mitochondria. The intensity and duration of activity required of muscle influences the energy-obtainment pathways taken by muscle. Other avenues followed by muscle to generate energy substrates depend on a wide range of environmental factors.

1.3

Skeletal Muscle Architecture

1.3.1

Skeletal Muscle architecture can be defined as “the arrangement of muscle fibers within a muscle relative to the axis of force generation” (Lieber, Fridén 2001), where the force generating axis (FGA) refers to the aponeurosis/tendon that fibres attach to and exert force on when contracting. See Figure 2 for illustration of muscle attachment to bone.

1.3.2

Muscle length (Lm) is defined as “the distance from the origin of the most proximal muscle fibers to the insertion of the most distal fibers”. Fibre length is variable, averaging out in all muscles at ~60% maximum, and terminating end-to-end. (Lieber and Fridén 2001). Page 10 of 36



There are three categories of fibre orientation: 

Parallel muscle – These muscles possess fibres that extend longitudinally from the FGA, (Lieber and Fridén 2001) in parallel, as the name implies.



Unipennate – Fibres here attach at a single, constant angle to the FGA known as the ‘angle of pennation’, varying between (0-30°) (Lieber and Fridén 2001).



Multipennate - The most common structure found in the body; fibres extend at various angles relative to the FGA (Lieber and Fridén 2001).

Figure 3 – Illustration of skeletal muscle architecture, in the context of human physiology. A – Parallel muscle as seen in the biceps brachii B – Unipennate muscle running at a constant angle to the FGA, as seen in vastus lateralis muscle C – Multipennate muscle extending at various angles to the FGA, here illustrated in the gluteus medius muscle. Lm – Muscle length

1.3.4

Lf – Muscle fibres

Muscle fibres vary not just in orientation, but in terms of their metabolic and contractile abilities too, with certain types of fibres capable of producing either faster, slower, shorter or prolonged contractions. Page 11 of 36



Muscle is a complex tissue, essential to life and the maintenance of health through movement, chemical storage and heat generation that contribute to homeostatic mechanisms. The structure of muscle influences its functional ability, and heterogeneity exists at many structural levels between different types of muscle, depending on their composition and use.

1.4

Muscle Metabolism

1.4.1

Muscle undergoes a phenomenal amount of wear and tear on a constant basis. Skeletal muscle is perhaps the worst affected, being the most abundant type of muscle found in the body. As a result, muscle is forced to adapt to the levels of motional/locomotory and metabolic duress imposed upon it. Adaptation occurs at a molecular level, characterised by alterations in the sarcoplasmic, myofibrillar and mitochondrial subfractions of skeletal muscle. It is directed by the type of contractions performed, their intensity and duration, and the genetic makeup of the individual that defines their “responder status” (Timmons, 2011 taken from Atherton and Smith (2) , 2012).

1.4.2

Skeletal muscle adapts to stresses by increasing in size through hypertrophy: muscle cells grow larger, rather than increasing in number. This can occur either by upregulating RNA production and protein synthesis from existing myonuclei, or through the acceptance of donated nuclei from satellite cells. Satellite cells are myogenic precursor cells located in the basal lamina, outside the sarcolemma of muscle (Blaauw and Reggiani, 2014). By donating nuclei to myofibres, the amount of myonuclei available for synthesis of new proteins to induce hypertrophy increases too. The overall role of satellite cells in the induction of hypertrophy is complex, and has yet to be elucidated. In a review by Blaauw and Reggiani (2014), the compilation of data from recent investigations led to some conflicting conclusions. Broadly speaking, in light of the evidence covered, the authors summarised with: satellite cells are not required per se for the stimulation of muscle hypertrophy, but definitely play an important role because satellite cell activation has been consistently observed alongside all models of hypertrophy.

1.4.3

Two major external factors govern the regulation of skeletal muscle hypertrophy in the body (Atherton and Smith(2) , 2012; Bukhari et al., 2015). They are:

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Attiyyah Omerjee P11279592 BSc Hons Biomedical Science 


Nutrient availability, specifically that of o

Essential amino acids (EAAs) that cannot be synthesised de novo by the body. Other non-essential amino acids can be synthesised in vivo when required.

o 

Insulin

Physical Exercise

1.5

Nutrition‐driven Hypertrophy

1.5.1

Nutrition-driven hypertrophy occurs as a result of amino acid (AA) incorporation into new muscle proteins, without the involvement of satellite cells. A popular supplement used as a concentrated source of amino acids is whey protein. It is a byproduct extracted from the production of dairy foods, composed of β-lactoglobulins and α-lactalbumin and soluble in water. Whey is lactose-free and available in powdered form (Davisco Foods, 2015). Recent work repeatedly compares whey to EAAs at differing doses as an assessor of efficacy and to contextualise expected outcomes (Churchward-Venne et al., 2012; Atherton (1) et al., 2012).

1.6

The Balance of Synthesis and Breakdown in the Context of Ageing

1.6.1

Muscle proteostasis exists in a diurnal balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Whether increased rates of MPB with static MPS, or constant rates of MPB and a decreased rate of MPS are responsible, sarcopenia must ultimately favour overall muscle loss according to Atherton et al. (2012(2)). They have also shown that overall, with age; muscles have a blunted ability to respond to EAA/whey protein availability and exercise. The term ‘anabolic resistance’ was coined to describe this tendency to mount a dampened response as we age, and they concluded that it is not increased MPB that is responsible for sarcopenia, but decreased MPS (Atherton (2) et al., 2012).

1.6.2

The fasted state, also known as basal or post-absorptive, precedes feeding. It can be divided into two distinct phases, as defined by Bukhari et al. (2015). The early feed phase lasts approximately 2hr postprandial (0-2hr), and the late feed begins after it, lasting 4hr postprandial (2-4hr).

1.6.3

During the fasted state, MPS occurs at postabsorptive levels that diminish the longer this period stretches. MPB begins to exceed MPS, leading to an overall breakdown muscle state. Postabsorptive (basal) rates of MPS are thought to exist for approximately 67% of the day (Bukhari et al., 2015). Upon consumption of EAAPage 13 of 36



containing food, MPS rises to make up for the muscle protein lost during fasted periods so that muscle mass is maintained. Physical exercise too, as mentioned earlier, is vital to the maintenance of muscle mass (Bukhari et al., 2015).

1.7

The ‘Muscle Full’ Phenomenon of MPS

1.7.1

The ‘Muscle Full’ phenomenon observed with nutrition-driven increases in MPS is characterised by muscle’s inability to be further stimulated once a set point in nutrition-availability has been reached. It was observed that 2h after stimulation of MPS with 10g EAAs (and the whey equivalent ~20g of protein), MPS returned to baseline rates, despite the continued availability of nutrition. One theory for this full set-point was proposed by Joe Millward; wherein further synthesis of more muscle is physically hampered by the inelastic collagen and connective tissue of the endomysium that sheaths each fibre. The term ‘Muscle Full’ is used to describe this expression of the law of limiting returns in nutrient-driven skeletal MPS (Atherton (1) et al., 2012).

1.8

Realising Leucine as a Potent Activator of MPS

1.8.1

Amino acids were defined as the sole contributors toward nutrient-induced muscle anabolism. They were then discovered to have dose-dependent effects on MPS (Churchward-Venne et al., 2012; Atherton (2) et al., 2012). In recent years, this understanding has been refined to exclude the other branched-chain essential amino acids Valine and Phenylalanine as the causative agents, with Leucine now regarded as the primary source of this response (Atherton (1) et al., 2012; Bukhari et al., 2015). Indeed, Leucine is used by many as a paradigm for the stimulation of MPS in skeletal muscle metabolic research (Wilkinson et al., 2013).

1.8.2

Leucine in particular has been found to exert a more than passive bystander effect on muscle: acting not only as a substrate for MPS, but as a potent chemical activator (Wilkinson et al., 2013). Leucine is capable of stimulating acute up-regulation of MPS, as stated by Layman et al. (2006), of studies performed by Biolo (1997) and Volpi (2003) because: The innate muscle metabolism equilibrium was shown to swing in net favour of MPB after intensive bouts of exercise or overnight fasts. The equilibrium only shifted back in favour of MPS after adequate protein, or “specifically (when) leucine is consumed to increase plasma… leucine concentrations”.

1.9

Intracellular Signalling Effects of Leucine

1.9.1

Leucine is thought to play a very active role in regulating MPS by actually stimulating intracellular signalling pathways that are involved in the initiation and up-regulation Page 14 of 36



of MPS. The mTORc1-p70S6K1 (mammalian target of rapamycin and phosphorylated ribosomal kinase at Serine6 residue respectively) signalling pathway is thought to be affected in particular (Katsanos et al., 2006; Atherton et al., 2010b taken from Wilkinson et al., 2013). 1.9.2

Leucine is a branched-chain amino acid (BCAA) that is considered to induce phosphorylation (activation) of AKT, P70S6K1 and 4EBP1. In particular, Leucine is known to be a key player in the activation of the mTORc1 signalling receptor (Atherton et al., 2012 (1); Layman et al., 2006). It stimulates mTOR kinases of the Insulin Signalling Cascade to phosphorylate 4EBP1; an inhibitor of eukaryotic Initiation Factors (eIF). 4EBP1, through phosphorylation, subsequently dissociates from the eIF (eurkaryotic Initiation Factor – a transcription factor). The eIFs, in particular eIF4E, are then free to initiate translation of proteins related to increasing muscle mass (Layman et al., 2006). Leucine also causes p70S6 kinases to phosphorylate ribosomal proteins, thereby further aiding translation initiation. Figure 4 illustrates some of the pathways followed, with reference to the mTORc1 pathway of muscle hypertrophy.

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Figure 4 ‐ An illustration of the anabolic pathways that induce hypertrophy. mTOR (mammalian target of Rapamycin) can be seen immediately downstream of the AKT pathway, which ultimately begins at the Insulin receptor. Phosphorylation of 4EBP‐1 leads to phosphorylation and subsequent liberation of eIF4E, a known initiator of protein synthesis. Additional activation of mTOR causes a cascade via the P70s6K pathway. Taken from Zudin et al. (2014).

2.0 2.1.1

The in‐vivo measurement of Muscle Metabolism

Very few studies to date have been done in older women to investigate the subtleties of muscle metabolism when EAAs (specifically Leucine)/whey protein supplements are fed at submaximal doses, with the aim being to increase MPS. Women show markedly more varied tendencies in hormonal regulation and sex chromosomal differences in comparison to men, with monthly cycles rotating their bodies through a vast range of signalling molecules that will certainly vary according to gravidity. Biological age in women tends to be of far greater significance when determining bodily development than in men. Page 16 of 36



There is indeed controversy around the inclusion of women in clinical trials due to theoretical concerns about age and gender differences in treatment effects. A legitimate fear also exists about the potential harm to foetuses with the use of investigational drugs. Broadly speaking, the main reason for the variation in biological aspects between men and women likely stems from the differences in gene possession and expression that may in/directly modify the effects of certain treatments on certain individuals.

2.1.3

In order to somehow capture this proteostatic dynamic, an involved metabolic marker had to be used to track changes. The use of stable isotope tracers for the measurement of muscle metabolism dates back to the methods first proven by Rennie and Wolfe in 1982 (Atherton et al., 2012 (2)). Stable isotopes are safe, nonradioactive, naturally occurring labelled variations of existing molecules. The difference lies in the number of neutrons found in their constituent atoms. These ‘heavier’ molecules act almost exactly like their regular counterparts, but can be distinguished by their mass difference using mass spectrophotometric techniques. Good tracers need to be safe for use in humans and mimic the actions of their natural counterparts. Uptake and incorporation of these isotopes can be measured in bodily tissues/humours, such as blood (plasma) and muscle, whereupon they can be used to gauge the size of free AA pools, and the rate of breakdown and synthesis occurring in a particular body of muscle.

2.2

Tracking Leucine’s Stimulation of MPS

2.2.1

A [1, 2 13C2] Leucine isotope can replace natural Leucine to track its metabolic sojourn, and discern its role even as an involved metabolic stimulator. Carbon13 is a non-radioactive isotope of Carbon12. In a protein supplement being developed to enhance MPS for example, labelling the Leucine in this supplement would allow Leucine’s dosage-related regulation of

Figure 5a – The chemical structure of the [1, 2 13C2] Leucine isotope.

muscle mass to be followed with clarity. Figure 5a illustrates the structure of [1, 2 13C2] Leucine.

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[13C6] Phenylalanine is an isotope of the BCAA Phenylalanine. It differs in the possession of six atoms of C13 present in the benzene ring. Figure 5b illustrates.

Figure 5b – The chemical structure of 13C6 Phenylalanine. It is used as a tracer to map muscle protein synthesis (MPS). Taken from Sigma Aldrich (2015).

2.2.3

Phenylalanine is a better tracer to make use of than Leucine for example. The reason lies behind the metabolic simplicity of its role as a precursor for protein synthesis. When Phenylalanine enters the body, it is simply uptaken and converted to phenyl tRNA as a precursor to be used for protein translation, via ribosomal assembly. [13C6] Phenylalanine to be used in the synthesis of new muscle proteins is represented by its plasma amino acid concentration [pAA] measured in arterial samples – this is the amino acid concentration of Phenylalanine available in blood, and is subsequently diluted by its entry into different biological pools e.g. bone, liver etc. Furthermore, the amount of Intracellular [13C6] Phenylalanine enrichment (IC Phe EN) found in the sarcoplasmic fraction of muscle biopsies indicates the amount actually available to the myofibrillar fraction of muscle to be utilised in protein synthesis. The change in FSR can be plotted alongside concentrations of (labelled) Leucine in supplements to deduce its dose-dependent effects in elderly women.

2.3

Tracing Synthesis and Breakdown

2.3.1

Tracers are introduced into subjects as primed infusions – a large bolus of tracer is fed into the hand antecubital fossa vein, and left to equilibrate while the individual reaches steady state. They must remain relatively inactive at this stage, until required, because exercise is also known to be a stimulator of MPS, and would Page 18 of 36



therefore interfere with measurements of the response to Leucine activity. Steady state is where the processes that dilute/excrete the tracer are equal to the processes introducing it. Tracer is diluted by entry into free AA pools, but once at steady state, the only thing then capable of inducing a change is an increase in MPB (muscle protein breakdown). The movement of molecules into and out of different pools causes an overall change to the net balance of that molecule in a specific location. In the case of muscle breakdown, AAs leave the myofibrillar, sarcoplasmic, collagenic and mitochondrial etc. pools, causing an increase to the net balance of AAs in the immediate vicinity of that muscle. Figure 6 illustrates the differences in time taken to reach steady state when simple and primed infusions are used. Primed infusions take far less time, and are therefore desirable.

Figure 6 – Chart to show the differences in time taken for the subject to reach steady state, comparing simple and primed infusions.

2.4

Fractional Synthetic Rate (FSR)

2.4.1

The Fractional Synthetic Rate (FSR%h-1) is a measurement of synthesis in a particular fraction. It is commonly used to calculate the percentage rate of skeletal muscle myofibrillar synthesis. FSR is calculated using the classic precursor-product method, where enrichment levels are used to measure the incorporation of tracer into the myofibrillar fraction of muscle biopsy samples. The equation for FSR can be found below, as taken from Wilkinson et al. (2013) and Burd et al. (2011):

Page 19 of 36



The change over the total, calculated as a percentage over time. Where:  δEm – The change in [13C6] Phenylalanine enrichment (E) in atoms per excess (IC Phe EN APE) between the (m – myofibrillar) fraction of the previous and current biopsy sample. This represents the actual amount of phenylalanine that has been incorporated into the muscle as new protein.  t – The time period over which the biopsy samples in question were taken. (This is used to correct for and standardise the FSR value to % per hour).  Ep – The mean enrichment (E) of the total precursor over the aforementioned time period. This refers to the amount of Phenyl tRNA that is present in the sarcoplasmic fraction for incorporation. [pAA] is used as a proxy for the precursor available in the sarcoplasm, as IC Phe.

2.5

Aim

2.5.1

The proposed study aims to investigate the changes in metabolism in elderly women, when given Leucine-enriched EAA supplementation (Ajicomix – Ajinomoto Inc.) at doses of (1.5g) and (3g), under rested conditions. To date, little is known about the effects of leucine at stimulating MPS in elderly women, with the majority of current understanding founded in data gathered from studies conducted in men. Both genders age, and a holistic understanding of how best to prolong anabolism is required if we are to develop interventions to reduce the severity of sarcopenia, and improve the quality of life for the elderly in our ageing populations across the world.

3.0

Methods and Materials

3.1

Ethical Statements

3.1.1

Ethical Approval was obtained from the University of Nottingham’s Medical School Research Ethics Committee and the study complied with the Helsinki Declaration, along with preregistering at clinical trials (reg no. NCT02053441). All subjects were invited with postal letters (mailshot), and all procedures were explained before obtaining written consent. Participants had the option to abort the study at any stage, and contact details for relevant staff were provided for pre- and post-study communication to voice concerns/queries etc.

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3.2

Subject Recruitment

3.2.1

Female participants (n=18) were selected between the ages of 60 - 69yrs, and screened for diabetes, renal disease, cardiovascular/respiratory disease, malignancy and those taking medications known to affect muscle metabolism (e.g. NSAIDs, statins etc.). Questionnaires were used to investigate medical history, and body composition was measured using DEXA (Dual-energy X-Ray Absorptiometry) and had blood tests to exclude those with anaemia/haematological problems and with a BMI >30.

3.2.2

A power calculation stated a minimum of 8 participants would be required to detect differences between groups of 25% in biological variables (α=0.05, β=0.95) (Atherton (1)

et al., 2012).

3.3

Subject Demographics

3.3.1

Elderly females (n=8/group, 66±3yr, BMI 29±1). When comparing dose responses between two groups, it is essential that the participants studied are homogenous when assessed according to essential parameters, such as age, BMI and weight etc. that define their sarcopenic status.

Group 0 Leucine (1.5g)

Group 2 Leucine (3g)

Mean Age (years)

66 ± 2

64 ± 4

Height (m)

1.63 ± 0.84

1.62 ± 0.064

Lean Body Mass (kg)

77.3 ± 14.6

65.3 ± 9.8

BMI (kg/m2)

28.9 ± 3.92

25.1 ± 3.81

Mean Sarcopenic Index

6.43 ± 0.81

6.32 ± 0.32

Table 1 – Subject demographics showing equivalence for all parameters. This is essential for any comparisons to be made between the two groups.

3.3.2

Sarcopenic Indices were calculated for each participant: ALM / Height2

Where:

ALM – Lean mass of arms and legs (kg) – this is measure by DEXA. Height is measured in metres. Page 21 of 36



3.4

Studying Participants

3.4.1

Subjects were studied in the overnight fasted state over a 3hr period, having been asked to refrain from heavy exercise 48hr prior to attending. Subjects had 18-g cannulas inserted into the antecubital vein of the right arm for infusion of tracer. A hotbox was used to create an arterio-venous shunt - actually cannulating an artery would be extremely uncomfortable for participants. Using a hotbox on one arm ‘arterialises’ the blood by increasing venous dilatation, opening the arterio-venous shunts in the hand and raising oxygen saturation levels. ‘Arterialised’ venous blood obtained from the forearm has been asserted as near-indistinguishable from actual arterial blood (Winter et al. 2007; Nauck et al. 1992).

3.4.2

Temporal muscle biopsies were conducted using the conchotome technique by a certified clinician. The branched-chain EAA supplement (Ajicomix – Ajinomoto Inc.) for both groups 0 (3g) and 2 (1.5g) was composed of 40% Leucine, Valine and Isoleucine, dissolved in 200mL of water. See Figure 7 for the study protocol followed.

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Figure 7 – Study protocol used for Group 0 and 2 participants. Obtained with permission, courtesy of the University of Nottingham, MRC‐ARUK Metabolic Physiology Group

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3.5

Obtaining Blood and Muscle Samples

3.5.1

Blood samples were taken for both arterialised measurements of pAA. Blood was taken into Lithium/Heparin and EDTA Vacutainer tubes and stored on ice before being centrifuged at 32,000 rpm for 20 minutes. Plasma was aliquotted into tubes and frozen at -80°C until needed.

3.5.2

Muscle biopsies were taken using the conchotome biopsy technique (5mL, 1% lignocaine), washed in ice-cold phosphate-buffered saline, rapidly picked free of fat and connective tissue by a registered laboratory technician and flash-frozen in N2(l). Biopsy samples were stored at -80°C for later analysis.

3.6

pAA Preparation and Initial Numerical Analysis

3.6.1

Plasma amino acid concentrations were determined according to their derivatisation as amino esters, using MTBSTFA. NorLeucine was used as an Internal Standard for the measurement of BCAAs [1, 2 13C2] Leucine.

3.6.2

D2 Phenylalanine (deuterated Phenylalanine – 2H2) was used as the internal standard for the analysis of L-[ring-13C6] Phenylalanine for measurement in the plasma samples.

3.6.3

Standard curves were generated using pAA sequential dilutions (pAA curve generation and leucine interpretation), and using increasing enrichments 0%-10% D5 Phenylalanine (Phe enrichment curve generation).

3.6.4

(200L) of arterial and venous plasma had (10L) of urease (Sigma Aldrich Corporation) added to destroy any urea that would have caused interference. Samples were deproteinised in ice-cold Ethanol, incubated at 5°C for (20 minutes) and centrifuged to remove precipitated plasma proteins. Remaining supernatant was decanted and dried down under N2(g) at 90°C. Removal of lipids from the sample was performed using a Hydrochloric acid (500L) - Ethyl acetate (2mL) extraction, whereupon the sample was evaporated to complete dryness again to remove residual moisture. ACN (acetonitrile) (100L) solvent was used to suspend the amino acids. To this was added the derivatisation reagent MTBSTFA (N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide) (Sigma), which is used as commonly used for silylation of primary and secondary amines. Samples were derivatised at 90°C for (60 minutes), before being decanted into autosampler vials (Agilent Technologies©) and analysed using using a GC-MS (Gas Page 24 of 36



Chromatography machine coupled to a Mass Spectrophotometer; Thermo Scientific), by SIM (selected ion monitoring) at m/z 302 for leucine, isoleucine, valine and norleucine. Phenylalanine was measured at 234 (simple phenylalanine), 236 (L[ring-13C6] Phenylalanine) and 240 (2H2 phenylalanine). Corrections were made for overlapping spectra. Concentrations of Phenylalanine were derived from 2H2 (D2) phenylalanine, and BCAAs derived from measurement of norleucine, with reference to a standard curve of sequential volumes within each batch (generated with pAA and D5 Phenylalanine).

3.7

Muscle Protein Extraction

3.7.1

The myofibrillar and sarcoplasmic fractions of biopsies were separated: muscle was minced finely with sharp scissors in ice-cold extraction buffer to liberate the soluble sarcoplasmic components. Centrifugation of the homogenate produced the myofibrillar pellet, with the sarcoplasm suspended in the supernatant. The supernatant was decanted off to be stored separately to be used for the IC Phe EN calculations.

3.8

Intracellular Phenylalanine Enrichment (IC Phe EN) Determination

3.8.1

(200µL) of sarcoplasm was filtered of its AAs by adding 0.2M PCA (Perchloric acid) and 2µL of D2 Phenylalanine (Internal Standard). This was chilled and neutralised, with urea abolished upon addition of urease. AAs were eluted using Dowex ionexchange resin. AAs were derivatised as discussed below. These samples are required for measurement of IC Phe EN.

3.9

Myofibrillar Sample Preparation and Analysis

3.9.1

Myofibrillar concentrations of [13C6] Phenylalanine were determined by N-acetyl-Npropyl ester (NAP) derivatisation. The soluble myofibrillar and insoluble collagen pellet was rinsed in homogenisation buffer twice to remove any remaining sarcoplasm, and rinsed with (500L) 0.3M NaOH (30mins at 37˚C) twice. The myofibrillar pellet was precipitated with 1mL (1M Perchloric Acid – PCA) before being washed twice with 70% Ethanol.

3.9.2

Subsequent hydrolysation of the myofibrillar fraction was achieved by incubating in 0.05M HCl and Dowex ion-exchange resin (1mL) at 110°C overnight. Constituent amino acids released by the overnight hydrolysis were eluted in 2M NH4OH(l), before being evaporated and resuspended in Acetonitrile. Derivatisation to esters with MTBSTFA (N-Methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide was performed to allow GC-C-IRMS analysis. Page 25 of 36



3.10 Data and Statistical Analysis 3.10.1 Data was analysed using Xcalibur software (Thermo Scientific) and Microsoft Excel 2013 (Microsoft Corporation) to generate graphical representations of mean [pAA] and [IC Phe] and mean FSR changes across both groups of participants. Additional statistical testing was conducted on FSR data to determine significance (p