Quake3World

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Chapter 1
Introduction
1.1 Populations that would benefit from this research
Athletic performance and vanity aside, there are many important reasons for wanting to
know more about how to build muscle; not the least being the underestimated role of muscle mass
in healthy ageing. Sarcopenia is the unexplainable, age-related loss of muscle mass which has a
negative impact on strength, power, functional ability and daily living (Evans, 1997). This
phenomenon is wide spread among apparently healthy older adults; recent estimates indicate that
approximately 45% of people 65 years or older in the United States exhibit sarcopenia (Janssen et
al., 2004) and 20% are functionally disabled (Manton, 2001). An accumulating amount of evidence
suggests that sarcopenia underlies many other undesirable conditions that are associated with
ageing, such as osteoporosis, diabetes, unwanted weight gain, an increased susceptibility to illness,
falls and related injuries (Scheibel, 1985; Dutta, 1997; Evans, 1997; Doherty, 2003;). The direct
healthcare costs attributable to sarcopenia each year in the United States could be as high as $18.5
billion (Janssen et al., 2004).
There is little doubt that sarcopenia is a multifaceted phenomenon and its mechanisms are
yet to be clearly identified (Marcel, 2003; Roubenoff, 2003). However, the reduction in
functionality can be attributed to reduced motor unit activation (Jakobi & Rice 2002), muscle mass
(Frontera et al., 1991; Lynch et al., 1999) and quality (Frontera et al., 2000a; Klein et al., 2001).
The quality of skeletal muscle (from here on known as muscle), can be defined as the efficiency to
perform its various functions per unit of mass (Balagopal et al., 1997a). While there appears to be a
relationship between a quantitative loss of muscle and diminished functional capacity (Frontera et
al., 1988; 1990; 2000b), cross-sectional data on older adults also suggests that the rate of decline in
muscle strength accelerates with age (Hughes et al., 2001). Of even greater concern is the
longitudinal data (spanning 10-12 years) that shows the decline in leg muscle strength can be 60%
greater than estimates from a cross-sectional analysis in the same population (Hughes et al., 2001).
Although functional capacity depends on both the quality and quantity of muscle mass, a major
problem is that the minimal amount required to maintain health and independent living with
advancing age is unknown. However, the rate of muscle mass decline with age is thought to be
fairly consistent; approximately 1–2% per year past the age of 50 years (Sehl et al., 2001), but a
reduction of 30% or more is thought to limit normal function (Bortz, 2002). Some researchers
suggest that sarcopenia may be reversible (at least to a certain extent) (Roubenoff, 2003). Others
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believe that tomorrows older adults should be concerned with building a greater “starting reserve
capacity” of lean body mass (LBM)a today to ensure they avoid the unknown threshold that
precedes physical frailty and compromised health (Marcel, 2003). In the year 2000, there was an
estimated 600 million people on the planet aged 60 and over. This figure will rise to 1.2 billion by
2025, and 2 billion by 2050 (W.H.O. 2003). Therefore, the prevention, early detection and
treatment of sarcopenia is needed to alleviate the load of a rapidly ageing population.
Ageing is also associated with changes in body composition that may carry severe
metabolic consequences. While muscle mass declines dramatically between the age of 50 and 75
years by approximately 25%, this is accompanied by a substantial increase in body fat. The results
of cross-sectional research (Baumgartner et al., 1995; Van Loan, 1996; Gallagher et al., 1997;
Forbes, 1999) suggest that the average adult can expect to gain approximately 1 pound (0.45kg) of
fat every year between ages 30 to 60, and lose about a half pound of muscle over that same time
span; a change that is equivalent to a 15 pound (6.8kg) loss of muscle and a 30 pound (13.6kg) gain
in fat (Forbes, 1999). These age-related changes in body composition have metabolic
repercussions. Muscle tissue has a large working range of ATP turnover rates and tremendous
potential to consume energy. Due to its mass, muscle is a highly important thermogenic tissue and
a prime determinant of basal metabolic rate (BMR) (which for most of us is the largest single
contributor to daily energy expenditure) (Elia et al., 2003). Therefore, muscle tissue is not only
important for maintenance of a healthy weight, by virtue of its mass and mitochondrial content it is
also the largest site of lipid oxidation (Perez-Martin et al., 2001; Heilbronn et al., 2004). This
means that muscle not only plays an integral role in fat metabolism but also the maintenance of
lipoprotein and triglyceride homeostasis (Thyfault et al., 2004). Muscle is also the primary site of
glucose disposal in the post-prandial state (Perez-Martin et al., 2001). Exercised muscle promotes
healthy glucose metabolism (Henriksen, 2002). Therefore, maintenance of this metabolically active
tissue (with elevated mitochondrial potential) would also reduce the risk for the development of
type-II diabetes (Perez-Martin et al., 2001).
To confirm these assumptions, cross-sectional data shows that older men and women
generally have a decreased ability to mobilize and oxidize fat, as well as possess a slower BMR in
comparison to their younger counterparts (Calles-Escandon et al., 1995; Nargy et al., 1996;
Levadoux et al., 2001). Additionally, this age-related decline in BMR and fat metabolism is
suggested to be related more to a reduction in LBM than ageing per se (Calles-Escandon et al.,
1995; Nargy et al., 1996; Levadoux et al., 2001). In fact, the preservation of muscle mass
a As muscle mass constitutes approximately 60% of all lean body mass, these terms are often used
interchangeably, particularly with regard to alterations in body composition.
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throughout ageing may reduce the decline in BMR and possibly reduce the degree of body fat
accumulation that is characteristically observed in older adults (Evans 1997; Marcel 2003). Unlike
aerobic fitness capacity (Broeder et al., 1992), LBM is an important determinant of BMR (Calles-
Escandon et al., 1995; Nargy et al., 1996; Levadoux et al., 2001). For this reason, strategies that
preserve LBM are thought to be the cornerstone of any successful attempt at weight loss (Broeder
et al., 1992; Poehlman et al., 1998). The prevalence of overweight adults in the United States
increased from 47% in 1976-1980 to 65% in 1999-2002 (DHHS, 2004). (These percentages are
thought to be similar in Australia although no accurate data has been obtained in recent years).
Therefore, it is clear that lifestyle strategies that focus on maintaining muscle mass will enhance
health and may prevent or reduce the severity of many ageing-related illnesses, as well as reduce a
significant economic burden on the health care system (Janssen et al., 2004).
Besides its locomotive and metabolic implications, muscle tissue is the body’s largest
reservoir of bound and unbound proteins (amino acids) and constitutes 50-75% of all proteins in
the human body (Waterlow, 1995). While quantitative estimates suggest that about 1-2% of muscle
is synthesized and broken down on a daily basis, the mass of this tissue means that it accounts for
up to 50% of whole body protein turnover (Rennie & Tipton 2000). This impact (on whole body
protein turnover) is a clear reflection of the essential role that muscle tissue plays in the regulation
of whole body amino acid metabolism. Muscle is the main reservoir and synthesis site of mino
acids (AA) that are constantly exported to meet an array of physiological demands. One example is
glutamine, the essential fuel that powers many aspects of immune function and cell turnover (Curi
et al., 2005; Rutten et al., 2005). Therefore, the preservation of muscle mass is critically important
to populations living with conditions such as HIV, various forms of cancer and intestinal conditions
such as Crohn's disease and Colitis. Although clinically unrelated, all of these conditions promote
cachexia; muscle wasting that manifests from a systemic inflammatory response (Hack et al., 1997;
Kotler, 2000). This chronic immune response causes a dramatic increase in whole body protein
turnover that leads to excessive breakdown of muscle tissue in an attempt to provide the chemical
energy that is required. A decline in muscle mass signals a progression in the illness but also
underlines mortality (Hack et al., 1997; Kotler, 2000). Despite significant advances in the
medication strategies used to combat these illnesses, cachexia is still a major problem that has not
been resolved (Hack et al., 1997; Kotler, 2000).
Whether it’s to out perform the competition or maximize personal potential, athletes
(recreational and professional) are competitive by nature. This drive to succeed and a growing
awareness that nutritional choices can influence exercise performance has fuelled an explosion in
the interest of nutritional ergogenic aids; dietary compounds that may enhance muscle strength,
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size and athletic performance (Bradley et al., 2001). Recent decades have also seen a growing
awareness of exercise as part of a healthy lifestyle (DHHS, 2004; CSEPHC, 1998). The sports
nutrition industry has expanded beyond its non-commercial roots into a $2 billion a year growth
market. With revenue sales of dietary supplements projected to reach in excess of $4.5 billion in
the United States alone by 2007 (Tallon, 2003), this defines an enormous expectation for potential
benefit. However, very few dietary supplements that are marketed as ergogenic aids have sciencebased
evidence of their effectiveness. Despite the public’s interest and the previously discussed
clinical implications, there is a paucity of research-based information on cost-effective, nutritional
strategies that may help build and maintain muscle mass.
1.2 Factors that regulate the size of human skeletal muscle mass
The mechanisms that regulate the maintenance of human muscle mass are complex and
influenced by numerous factors such as physical activity, nutrition, hormones, disease and age
(Rennie et al., 2004). Once fully differentiated, the number of muscle cells stays constant (within a
few percent) so that, with a few exceptions, a gain or loss of muscle tissue can only occur via an
increase (hypertrophy) or a decrease (atrophy) in size of the existing muscle cells (Hargreaves &
Cameron-Smith 2002). The quality and quantity of muscle protein is essentially maintained
through a constant remodelling process (protein turnover) that involves continuous synthesis and
breakdown (Rasmussen & Phillips 2003). Protein turnover is a complex, regulated process but is
essentially controlled by the initiation of protein synthesis and proteolytic pathways. There are a
number of regulators that affect protein turnover either by stimulation or inhibition of protein
synthesis and protein degradation (Liu et al., 2006) (figure 1.1). For example, insulin and IGF-1
prevent contractile protein degradation (IGF-1 inhibits degradation via the ubiquitin pathway).
Cortisol and myostatin are known inhibitors of protein synthesis; cortisol, cytokines and ubiquitin
proteins activate protein degradation. Conversely, insulin, amino acids, mechanical loading and the
anabolic hormones are considered stimulators of protein synthesis. The focus of this chapter is to
discuss protein turnover and its regulators in relation to the factors that may increase the size of
human muscle mass. Specifically, those factors are resistance exercise (RE) and macronutrient
intake. However, when considering all aspects that may influence the size of human muscle mass,
one must keep in mind that the regulatory network that controls this process may not reside
exclusively within muscle.
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contractile
proteins
cortisol
insulin
amino acids
mechanical
loading
testosterone
GH, IGF-1/MGF
myostatin
cortisol
cytokines
ubiquitin
insulin IGF-1
extracelluar intracellular
inhibitor
activator
sa tellite
ce ll
amino
acids
synthesis
degradation
Figure 1.1 Regulators that affect muscle protein turnover.
A number of regulators affect protein turnover either by stimulation or inhibition of protein
synthesis and protein degradation. For example, (shown in red) insulin and IGF-1 prevent
contractile protein degradation whereas cortisol and myostatin inhibit protein synthesis. Cortisol,
cytokines and ubiquitin proteins (shown in green) activate protein degradation. Conversely, insulin,
amino acids, mechanical loading and the anabolic hormones (in green) activate protein synthesis.
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Some rather convincing evidence suggests that the regulation of whole body protein
metabolism (and the size of muscle mass) involves a regulatory circuit between muscle, blood
(plasma) and liver AA metabolism (Hack et al., 1997). As mentioned previously, cachectic
conditions fail to conserve muscle proteins and convert abnormally large amounts of AA into
glucose, and release large amounts of nitrogen as urea. The conversion of AA into glucose is
linked to the rate at which ammonium ions in the liver are converted into either urea or glutamine
(Gln). In contrast to Gln biosynthesis, urea production requires hydrogen carbonate anions (HCO3
-)
and thereby generates protons (figure 1.2). Carbamoylphosphate is the rate-limiting step for urea
biosynthesis, and is limited by the availability of HCO3
- anions, which are scavenged by protons. A
series of studies involving humans living with and without cachectic conditions strongly suggest
that the hepatic catabolism of cyst(e)ine (both Cys and its disulphide twin, cystine) is a key
regulator of whole body protein metabolism (Kinscherf et al., 1996; Hack et al., 1996; 1997; 1998;
Holm et al., 1997). The hepatic catabolism of cyst(e)ine into sulphate (SO4
2-) and protons (H+)
down-regulates urea production in favour of Gln biosynthesis, thereby retaining nitrogen in the
muscle AA reservoir (figure 1.3). The necessary hepatic catabolism of cyst(e)ine is an essential
proton-generating process that inhibits carbamoylphosphate synthesis and thus, shifts whole body
protein metabolism toward the preservation of the muscle AA pool and maintains the size of
muscle mass (Hack et al., 1997). This pathway is essentially textbook biochemistry. Nevertheless,
a substantial amount of data suggests that the availability of cyst(e)ine in plasma determines the
threshold at which AA are converted into other forms of chemical energy that ultimately influences
body composition (Kinscherf et al., 1996; Hack et al., 1996; 1997; 1998; Holm et al., 1997).
The term “controlled catabolism” is used to explain this regulatory circuit (figure 1.3)
(Hack et al., 1997). The controlled release of AA by muscle in the post-absorptive state is
compensated by AA uptake and protein synthesis in the post-prandial state (Holm et al., 1997). The
most important function of this regulatory circuit is to ensure that any time urea production is too
high and plasma AA are accordingly too low, controlled muscle catabolism is triggered. This leads
to export of cyst(e)ine, an increase in plasma cyst(e)ine and a down regulation of the hepatic urea
production. However, in cachectic conditions this process is thought to be disturbed. The
etiologically unrelated illnesses mentioned previously are characterized by high glycolytic activity
(increased lactic acid production) in muscle, increased hepatic urea production and abnormally
high post-absorptive venous plasma glutamate (Glu) levels combined with a low plasma Gln/Cys
ratio. Often, these characteristics are observed before wasting becomes evident (Droge et al., 1996;
Lundsgaard et al., 1996; Dworzak et al., 1998; Hack et al., 1997; 1998). However, it is also
interesting that similar biochemical symptoms (and subsequent alterations in body composition)
have been observed in healthy adults.
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Cyst(e)ine
glutamate
glutamine
arginine
ornithine
UREA
HCO3
-
CO2
muscle
amino acids
a-ketogluterate
NH4
+
carbamoylphosphate
H2O
SO4
2- H+
citrulline
Figure 1.2 Urea biosynthesis and the influence of the amino acid cysteine.
Urea production requires hydrogen carbonate anions (HCO3
-) and thereby generates protons.
Carbamoylphosphate is the rate-limiting step for urea biosynthesis, and is limited by the
availability of HCO3
- anions, which are scavenged by protons. The hepatic catabolism of cyst(e)ine
is an essential proton-generating process that inhibits carbamoylphosphate synthesis and downregulates
urea production (Hack et al., 1997).
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Figure 1.3 Regulatory circuit of whole body protein metabolism (featuring cysteine).
The most important function of this regulatory circuit is to ensure that any time urea production is
too high and plasma AA are accordingly too low, controlled muscle catabolism is triggered. This
leads to the export of cyst(e)ine that is catabolized within the liver into sulphate and protons; a
process that promotes glutathione (GSH) synthesis, down regulation of urea production and shifts
whole body nitrogen economy towards the preservation of the muscle AA pool. Adapted from
research by Hack et al. (1997).
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In several trials that collectively involved 75 moderately well-trained men (between 20-60
years of age), correlations were detected between changes in body composition (both LBM and fat
mass) and post-absorptive plasma AA Gln, Glu and Cys (Kinscherf et al., 1996). In these trials, the
participants undertook 4-8 weeks of high-intensity (anaerobic-based) exercise training while
plasma AA were monitored. The participants with a high plasma Gln/Cys ratio maintained or
increased LBM and decreased fat mass during the program. Conversely, a low Gln/Cys ratio,
accompanied by a significant increase in Glu (25 to about 40 mM), was indicative of a reduction in
LBM and changes in T lymphocyte numbers (Kinscherf et al., 1996). Additionally, the participants
with the lowest Gln/Cys ratio at the start of the exercise programs lost the most LBM and the
reductions in LBM was accompanied by body fat accumulation. In one of these trials, a group
supplemented with a cyst(e)ine precursor, N-acetyl-cysteine (NAC) (400mg/day) during the
exercise program increased plasma cysteine levels and demonstrated a beneficial change in body
composition (i.e., an increase in LBM and or a decrease in fat mass) (Kinscherf et al., 1996).
Aside from these longitudinal investigations, cross-sectional data on AA exchange rates in healthy
adults aged from 28 to 70 years also reveal a number of conspicuous relationships between postabsorptive
plasma Gln, Cys and body composition.
Firstly, this cross-sectional data demonstrates that plasma Cys levels exhibit by far the
strongest age-dependant change of any AA (Kinscherf et al., 1996; Holm et al., 1997; Hack et al.,
1997; 1998). Secondly, older adults (60 years and over) demonstrate lower Gln exchange rates and
Gln/Cys ratios than their younger counterparts (Hack et al., 1997; 1998). Thirdly, a highly
significant (P < 0.001) correlation between a low Gln/Cys ratio and increased body fat was
observed across all age groups (Hack et al., 1997). A highly significant correlation between
decreased LBM and plasma Cys/thiol ratio was also observed in older adults (Hack et al., 1998).
Collectively, these results underline the importance of Cys in the regulation of LBM (Hack et al.,
1996). However, this data also points toward an age-related decrease in the efficiency of cyst(e)ine
catabolism to regulate hepatic urea production (Hack et al., 1997). That is, the liver of an older
person with a given plasma Cys level appears to convert less AA to Gln than a young, healthy
individual. Therefore, muscle stores would be relied upon increasingly with advancing age to meet
the metabolic demands for Gln. Some have speculated that the end result of this malfunction is a
steady but aggressive catabolism of muscle tissue throughout the lifespan (Hack et al., 1997; 1998).
Based on the findings of these investigations, a regulatory relationship between muscle, plasma,
liver cyst(e)ine concentrations and whole body protein metabolism is evident. In particular, the
concentration of cyst(e)ine appears to determine the threshold at which other AA are converted into
other forms of chemical energy that ultimately influences body composition.
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One mechanism that may disturb this regulatory circuit and induce excessive muscle loss is
the high level of circulating cytokines such as interleukin-6 (IL-6) and tumour necrosis factor-alpha
(TNF- ) which are a feature of cachectic conditions such as cancer and HIV (Strassmann et al.,
1992; Kotler, 2000) but are also characteristic of older adults (Visser et al., 2002; Krabbe et al.,
2004; Toth et al., 2005). These cytokines increase the requirements for L-gamma-glutamyl-Lcysteinylglycine
(glutathione); the centrepiece of all cellular antioxidant defences. Glutathione
(GSH) regulates re-dox status, cytokine production and many aspects of metabolism such as DNA
and protein synthesis, cell proliferation and apoptosis (Townsend et al., 2003; Wu 2004). Chronic
inflammation increases -glutamyl-cysteine synthetase activity in the liver. As Cys is the rate
limiting AA in GSH synthesis (Wu 2004), conditions that promote a chronic inflammatory
response are thought to create an unfavourable competition for a limited hepatic cyst(e)ine
reservoir — an underlying mechanism of oxidative stress and a cachectic environment that
promotes muscle wasting (Hack et al., 1997; Bounous & Molson 2003; Townsend et al., 2003).
However, no matter how tempting it maybe to speculate, the probability is that no single
mechanism may be solely responsible for changing the size of human muscle mass. Therefore,
clinical interventions that focus on increasing or maintaining muscle mass may need to be
multifaceted. The application of stable isotope tracer technology, advances in
immunohistochemical techniques, more accurate body composition assessment and powerful
microarray methods are all contributing to a better understanding of the aspects that ultimately
control the size of human muscle mass. Advancements in these techniques have also provided
greater insights into the physiological responses from two major factors that serve to positively
affect protein turnover and increase the size of human muscle mass; RE and macronutrient intake.
1.3 Resistance exercise:
Adaptations and influences that may affect muscle hypertrophy
RE is regarded as fundamental to the development and maintenance of muscle mass in
adults (Feigenbaum & Pollock 1999). Conventional RE typically involves the controlled movement
of weighted devices such as barbells, dumbbells and machines (with fulcrums and loaded weight
stacks) where muscles undergo concentric (shortening), isometric (static) and eccentric
(lengthening) actions against a constant external load — the magnitude of which is limited by the
individual's concentric strength. Conventional RE training typically involves the use of heavy loads
lifted in sets of 1 to 12 maximum-effort repetitions (Feigenbaum & Pollock 1999; Kraemer et al.,
2002). This form of exercise has been studied quite intensively over the last 25 years as an
intervention to offset age-related changes in body composition, strength and functional ability
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(Barry & Carson 2004; Hunter et al., 2004) as it is a most potent activator of the cellular and
molecular mechanisms that promote muscle anabolism (Rasmussen & Phillips 2003). The
functional adaptations that occur with conventional RE (from here on referred to as simply, RE) are
typically interpreted within the context of two components; a central component that accounts for
training-induced changes in motor unit recruitment (Enoka, 1998), and a peripheral component that
describes changes occurring at the level of the muscle tissue (Jones et al., 1989). Both aspects will
be discussed in the following section.
The value of RE training for increasing functional strength and muscle mass became a
topic of increasing interest within the scientific community once DeLorme and Watkins (1948)
demonstrated the importance of progressive loading for the rehabilitation of injured World War II
military personnel. A major reason for this interest is the remarkable plasticity of muscle tissue
(Booth & Baldwin 1996). Muscle responds specifically to current functional demands; fibres can
undergo extensive remodelling within their contractile apparatus to meet new functional
requirements (Booth et al., 1998). Improvements in strength and/or muscle mass are the result of
this remodelling process which involves transformations at the cellular and molecular levels
(Staron et al., 1990; Booth & Thomason 1991). For example, it has been proposed that fibre type
classifications, based on myosin ATPase (mATPase) histochemistry, represent a continuum that
span the functional demands of the muscular system. During contractile activity muscle fibres are
recruited from type-I (slow twitch)  Ic  IIc  IIac  IIa  IIab  IIb (fast twitch) (Staron &
Johnson 1993). RE typically involves large contractile efforts but slow shortening velocity (Staron
et al., 1984). Therefore, initial adaptations to heavier contractile loads occur via the expression of
slower isoforms of contractile proteins (Williams & Neufer 1996; Dunn & Michel 1997). Of the
contractile proteins within muscle, the myosin heavy chains (MHCs) are the largest subunits. The
MHCs make up the majority of the myosin filament that forms the cross bridge according to the
sliding filament theory and is the site of mATPase activity. The MHC isoform expressed within a
muscle fibre reflect the contractile properties of the histochemically-delineated fibre type (Staron &
Johnson 1993). Nine different MHC isoforms have been identified in mammalian muscle; four of
which are expressed in adult rodent muscle (Booth and Baldwin 1996) (MHC type-I, IIa, IIx, and
IIb). Three, (type-I, IIa, and IIx) are expressed in most human adult musclesb (Adams et al., 1993).
The differing isoforms with their unique mATPase activity reflect the fibre type and correlate with
speed of contraction; MHC I being the slowest and MHC IIx the fastest and most powerful
(Bottinelli et al., 1994; Fry et al., 1994; Williamson et al., 2001).
b A gene for a type-IIb MHC has not been identified in humans. Fibres that have been classified as
type-IIb express the type-IIx isoform rather than type-IIb. Therefore, human muscle fibres are often
classified as type-I, IIa and IIx (Smerdu et al., 1994; Ennion et al., 1995).
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The unique adaptability of muscle tissue appears to reside in the ability of the fibres to
transcribe different isoforms of MHC protein (Staron & Johnson 1993; Wright et al., 1997). It is
these alterations in the phenotypic expression of the MHCs that provide a mechanism of adaptation
to stresses placed upon the muscle, such as increased and decreased usage. The polymorphism of
the MHCs play a major role in the adaptability and contractile efforts necessary for various types of
exercise (Wright et al., 1997). Furthermore, MHC isoform expression does appear to change along
with the fibre type transitions that are a consequence of chronic RE (Fry et al., 1994; Staron, et al.,
1994; Williamson et al., 2001). Fibres containing predominantly MHC IIx are not well suited for
consistent activity; the available literature suggests that these fibres readily convert to type-IIa
during heavy contractile loading (Fry et al., 1994; Staron et al., 1994; Williamson et al., 2001). As
a result, muscle fibre recruitment profiles related to training intensity (i.e., the load used) and speed
of contraction, play an important role in dictating down-regulation in the expression of the type-IIx
gene (Willoughby & Nelson 2002). While MHC isoform (and subsequent fibre type) transitions
tend to go in the direction from type-IIx to type-IIa, there is little or no change to type-I regardless
of the modality of exercise (Staron et al., 1984; Staron & Johnson 1993). Changes in the relative
abundance or rate of translation of the mRNAs encoding the different MHC isoforms must precede
major changes in the protein isoform composition by several weeks. This is due to the slow
turnover of MHC in human muscle (Balagopal et al., 1997). This delay between exercise-induced
changes in MHC mRNA isoform expression and alterations in protein isoform distribution is the
basis for mismatches between mRNA and protein expression in individual fibres during RE
training (Andersen et al., 1997). Thus, MHC protein isoform concentrations may not be
representative of gene expression early in a training program (Staron & Johnson 1993; Fry et al.,
1994; Willoughby & Nelson 2002). It is clear that functional changes occur within muscle in
response to RE training and these peripheral adaptations involve extensive remodelling of
qualitative or intrinsic contractile properties such as MHC isoform composition (Duchateau et al.,
1984; Jones & Rutherford 1987; Alway et al. 1989) and alterations in fibre type distribution
(Staron et al., 1984). However, due to its apparent locomotive and metabolic benefits, muscle
hypertrophy is the peripheral adaptation that has received an increasing amount of attention in
recent years (Rennie et al., 2004; Volek, 2004; Phillips et al., 2005).
Hypertrophy can be described as an increase in the cross-sectional area (CSA) of the
muscle itself (determined by magnetic resonance imaging) or the individual fibres (Staron et al.,
2000). Muscle fibre hypertrophy is commonly determined by needle biopsy and ATPase staining
(Hather et al., 1991; Green et al., 1999; Ahtiainen et al., 2003). RE-induced muscle hypertrophy is
essentially the result of a net increase in muscle proteins (Phillips, 2000). It is presumed that
contractile (myofibrillar) protein volume increases in direct proportion with exercise-induced fibre
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hypertrophy (MacDougall et al., 1977; Always et al., 1989; Shoepe et al., 2003). It is also
presumed that sarcoplasmic or non-contractile proteins (such as alpha-actinin, myomesins, desmin,
dystrophin, nebulin, titin, and vinculin) increase in proportion with the increased synthesis of
myofibrillar protein (Alway et al., 1989; Phillips, 2000). However, one recent study has shown that
RE training “refines” the acute stimulus so that it is preferentially directed towards contractile
protein synthesis more so than non-contractile proteins (Kim et al., 2005). The increase in
myofibrillar protein that is associated with hypertrophy includes an increase in the number of
myosin and actin filaments inside each sarcomere (Booth & Thomason 1991) as well as the
addition of new sarcomeres in a parallel force-producing arrangement (Roman et al., 1993; Staron
et al., 1994; McCall et al., 1996). As the contractile proteins make up at least 80% of the fibre
space (Phillips, 2000), minimal increases in contractile proteins may contribute significantly to
muscle fibre hypertrophy (Wilborn & Willoughby 2004). Aside from athletic populations,
significant muscle hypertrophy during RE training has been documented in male and female adults
of all age groups (Frontera et al., 1988; Staron et al., 1991; Donnelly, 1993; Häkkinen et al., 2000;
Bamman et al., 2003), including the frail elderly (90+ years old) (Fiatarone et al., 1990; 1994).
However, the exact mechanical prerequisites that are responsible for this adaptation remain elusive.
Under maximal activation, each type of muscle contraction (concentric, isometric and
eccentric) is capable of stimulating hypertrophy (Adams et al., 2004). However, conventional RE
involves voluntary contraction of muscles as they undergo concentric, isometric and eccentric
actions against a constant external load, the magnitude of which is limited by the individual's
concentric strength. Eccentric strength is generally 20-50% greater than concentric strength
(Bamman et al., 1997). This means that eccentric loading is always submaximal during
conventional RE. Nonetheless, high-overload RE is shown to cause significant myofibrillar
disruption (including Z-band disruption) (Paul et al., 1989), even in individuals that lift weights
regularly (Staron et al., 1992). Furthermore, submaximal eccentric loading (approximately 80% of
maximum concentric strength) is shown to provide more severe, prolonged myofibrillar disruption,
soreness, and force deficit than concentric-only lifting with the same load (Gibala et al., 1995), a
finding that has also been confirmed in RE-trained individuals (Gibala et al., 2000). From the
research available, eccentric loading appears to be essential to obtaining an optimal hypertrophy
response from RE (Hortobagyi et al., 1990; Hather et al., 1991; Higbie et al., 1996; Hortobagyi et
al., 1996). Also, the mechanical damage that results from RE seems to be an important stimulus for
remodelling and hypertrophy (MacDougall 1986; Gibala et al., 2000). However, whether the
damage obtained from RE is causally related to the growth response has not been clarified;
nowhere in the literature has it been shown that muscle damage is an essential component of
hypertrophy (Kraemer et al., 2002).
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Although hypertrophy has been documented in a variety of populations that have
undertaken RE training, the magnitude of this response may vary considerably between individuals
in both men and women (Hubal et al., 2005). One study that utilized a large cohort (over 500
participants) report that some participants demonstrated increases in muscle size over 10 cm2
combined with 100% improvements in strength while others undertaking the exact same program
showed little or no change in strength or hypertrophy (Hubal et al., 2005). The hypertrophy
response can extend to each of the major fibre types (Green et al., 1999). However, the magnitude
of the response is typically much greater in the type-IIa and IIx subgroups (Staron et al., 1990;
Hather et al., 1991; Kraemer et al., 1995; McCall et al., 1996). Therefore, inherent fibre type
proportions can influence an individual’s potential for hypertrophy. However, hypertrophy is a
multifaceted phenomenon that appears to be influenced by a multitude of factors such as age;
gender; endocrine profiles; nutrition (that particularly affects anabolic processes such as energy
production and protein turnover); strength development; training status; and possible genetic
differences as well as the type of RE program performed (Kraemer et al., 1999; Roth et al., 2001;
Newton et al., 2002; Beunen & Thomis 2004). Unfortunately, very few studies have directly
assessed the impact that these variables may have on the hypertrophy response to RE training.
Some of these aspects are discussed briefly in following paragraphs of this section, others such as
protein turnover (1.4), the molecular events (1.5), nutrition (1.6 & 1.7) and RE programming
(1.10) are the focus of separate sections within this chapter.
Ageing
It is clear that ageing changes muscle physiology. Advancing age appears to reduce
maximum voluntary muscle strength (and power), protein and enzyme synthesis rates,
sarcoplasmic reticulum (SR) Ca2+ uptake and the expression of myo-specific genes (Yarasheski
2003; Nair 2005). However, what is truly fascinating is that RE training can reverse or at least
improve each of these aspects (Yarasheski et al., 1993; Hasten et al., 2000; Jubrias et al., 2001;
Newton et al., 2002; Hunter et al., 2004). In line with these findings, other studies have shown that
ageing per se does not diminish the capacity for muscle fibre hypertrophy (Frontera et al., 1988;
Hikida et al., 2000; Hagerman et al., 2000) or the ability to gain muscle mass (Frontera et al 1988;
Hunter et al 2004; Binder et al., 2005). A major contributor to the age-related decline in muscle
mass is thought to be the loss of alpha-motor neuron input that occurs with ageing (Brown, 1972).
Yet the research available on this topic suggests that older adults respond with strength and power
improvements in a similar manner to young adults (Häkkinen et al 2001; Newton et al., 2002).
Also, ageing does not appear to change absolute and relative BMR alterations in response to RE
(Lemmer et al., 2001). As discussed in the previous section, an age-related decline in muscle mass
15
is associated with chronic, low-grade inflammation that is characterized by increased levels of
TNF- and other pro-inflammatory cytokines (such as IL-6), and markers of inflammation (such as
C-reactive protein) (Visser et al., 2002; Krabbe et al., 2004). However, RE training is shown to
reduce muscle TNF- protein levels in older adults (Greiwe et al., 2001a). This training-induced
reduction in muscle TNF- protein was associated with an increase in muscle protein synthesis.
Additionally, Bautmans et al. (2005) report that 6 weeks of RE training resulted in favourable
changes in heat-shock proteins (such Hsp70) in monocytes and lymphocytes in older adults, and
these changes were associated with strength gains and modulation of circulating cytokines. The
heat-shock proteins protect cellular integrity during stressful situations such as oxidative stress and
infection. The Hsp70 in particular is thought to play a role in RE-induced muscle hypertrophy
(Kilgore et al., 1998). However, its production is negatively correlated to an age-related increase in
circulating IL-6 and TNF- (Njemini et al., 2002). Therefore, RE training appears to be an
effective stimuli to preserve muscle mass in older adults (Hunter et al., 2004), and this may partly
be due to the ability of RE to modulate the production of cytokines and heat-shock proteins
(Greiwe et al., 2001a; Bautmans et al., 2005). However, when hypertrophy-related responses in
younger and older adults (60 years and over) have been compared directly, some clear differences
have been observed.
Some (Balagopal et al 1997; Toth et al., 2005), but not all (Volpi et al., 2001), studies
report that older adults possess basal muscle protein synthesis (MPS) rates that are 19-40% lower
than younger adults. Also, older adults appear to possess a diminished capacity to synthesize new
muscle in response to anabolic stimuli when compared directly to their younger counterparts. For
example, in response to a single bout of RE (of the same relative intensity) the stimulation of MPS
is shorter-lived in older adults (Sheffield-Moore et al., 2005). This response is also accompanied by
a greater release of muscle AA and a more vigorous acute-phase response of plasma proteins
(particularly albumin), suggesting a differential hepatic and muscle response to RE between young
and older adults (Sheffield-Moore et al., 2005). When compared directly to younger adults, older
adults also demonstrate a diminished anabolic sensitivity to the consumption of protein via
decreased intramuscular expression, and activation (phosphorylation), of the signalling proteins
that initiate muscle protein synthesis (Cuthbertson et al., 2005). Additionally, these anabolic
deficits were associated with marked increases in NFκB, an inflammation-associated transcription
factor (once again providing a link between ageing-related inflammatory responses and a decline in
muscle mass). These acute-response investigations are supported by the few longitudinal RE
training studies that have directly compared chronic adaptations between young and older adults.
For instance, hypertrophy, strength and LBM gains in older adults are smaller in magnitude when
compared directly to younger adults (Häkkinen et al 1998b; Lemmer et al., 2001; Dionne et al.,
16
2004). The most recent example is that by Dionne et al. (2004) who assessed the impact of a 6-
month RE program on LBM, resting energy expenditure (REE) and insulin sensitivity (glucose
disposal) in 19 younger (18-35yrs) and 12 older (55-70yrs) non-obese caucasian women. In the
younger women, the RE program resulted in a gain in body mass (due to an increase in LBM),
increased REE and glucose disposal. Conversely, the older women showed a reduction in fat mass
and a lesser capacity to gain LBM with no improvement in insulin sensitivity or REE. Thus,
younger women appear to possess a greater capacity for metabolic changes in response to RE
compared to the older women (Dionne et al., 2004). Aside from a diminished response to anabolic
stimuli, other age-related aspects that may influence the hypertrophy response include; a higher
concentration of circulating cytokines that retard protein synthesis rates (Visser et al., 2002; Toth et
al., 2005), lower concentrations of anabolic hormones in circulation (Kraemer & Ratamess, 2005;
Toth et al., 2005), and differences in muscle gene expression (Welle et al., 2003). Although an agerelated
diminished capacity for hypertrophy is evident, no studies have attempted to assess at what
stage in life these chemical and physical alterations take place. Also, it is not known what the main
instigators are. For example, could it be hormonal, physical activity or quality of nutrition? Do
these factors have a combined effect on gene regulation? If so, what are the genes that are
modulated? Recent work has confirmed that a lifelong exercise program offers real protection
against the increasing levels of oxidative stress that damage cellular structures and cause the ageing
of tissue (Rosa et al., 2005). However, it is not known whether a life-long commitment to RE
training may provide a preventative effect against an age-related decline in muscle mass. These are
important questions that need to be answered before wide scale prescriptions can be made to an
ageing population.
Gender
With regard to sex-specific differences, earlier studies suggested little difference between
men and women in their capacity to build muscle mass (Cureton et al., 1988; Staron et al., 1994;
O’Hagen et al., 1995). However, a recent study (Hubel et al., 2005) that utilized a large cohort (342
women, 243 men) reported some clear gender-specific differences in strength and hypertrophy
development. This longitudinal training study assessed the hypertrophic response to 12 weeks of
RE in the upper arm muscles of untrained men and women. Results revealed a 2% difference
between men (20%) and women (18%) in hypertrophy that was considered highly significant (P <
0.001). Previous studies that examined this topic showed sex-specific differences of 6-7% but due
to the smaller (n), these differences were deemed not statistically significant (Cureton et al., 1988;
O’Hagen et al., 1995). Therefore, sex-specific differences in hypertrophy may have gone
undetected in previous studies due to a lack of statistical power. Hubal et al. (2005) also reported
17
that the women in their study outpaced the men considerably in relative gains in strength.
Therefore, although males probably experience greater absolute increases in hypertrophy, strength
and muscle mass (Lemmer et al., 2001), women may experience equivalent or greater changes in
strength (Hubel et al., 2005). However, this may also be due to a female’s lower initial starting
strength level (Hubel et al., 2005). From this study it’s also clear that the large variations in
hypertrophy that are evident among individuals do not appear to be sex-specific, at least not in
healthy young adults (Hubel et al., 2005). No long-term studies (greater than 12 weeks) have
examined sex-specific hypertrophy responses to RE. However, cross-sectional data on male and
female bodybuilders (Alway et al., 1992) combined with the results of longer term (6 month)
training studies on females suggest that the hypertrophy response in females, in the long term, is
generally lesser in magnitude than what has been characteristically observed in males (Kraemer et
al., 2000; Nindl et al., 2001b). This difference in capacity for hypertrophy could be due to apparent
differences in anabolic hormonal concentrations (Kraemer et al 1991; Häkkinen et al., 2000b). For
example, circulating testosterone concentrations are generally 10 times higher in males than in
females (Wright, 1980).
Endocrine responses
Anabolic hormonal responses are integral in the regulation of tissue growth and energy
substrate metabolism and therefore, are thought to play an important role in the hypertrophy
response to RE (Kraemer & Ratamess 2005). Plasma concentrations of circulating anabolic
hormones such as growth hormone (GH), testosterone, and IGF-1 diminish with age and this has
been associated with the age-related decline in muscle mass (Kraemer et al., 1999). However, the
contribution of these age-associated hormonal alterations to the size of muscle mass is unclear
(Shroeder et al., 2005). While GH is shown to stimulate protein synthesis in humans (Fryburg et
al., 1992), cross-sectional (Gallagher et al., 1997; Melton et al., 2000) and longitudinal studies
(Morley et al., 1997; Harman et al., 2001) have shown that (serum total and free) testosterone
concentrations in particular are, important regulators of net myofibrillar protein balance. Muscle
fibre hypertrophy is also shown to be proportional to increases in circulating testosterone
concentrations, even in the absence of RE (Sinha-Hikim et al., 2002). The action of growth factors
(such as IGF-1) are thought to be secondary to androgen activity (Bamman et al., 2001). Therefore,
the acute elevations in circulating testosterone (bound and unbound) that are consistently observed
after RE (Kraemer et al., 1990; 1995; 1999; Staron et al., 1994; Häkkinen et al., 1995; McCall et
al., 1999; Raastad et al., 2000; Ahtiainen et al., 2005a) appear to be an important component in the
development of muscle hypertrophy. However, the role these acute perturbations play in the
remodelling/adaptation process is yet to be fully elucidated (Kraemer & Ratamess 2005). What is
18
clear is that the acute increase in circulating anabolic hormones such as GH, testosterone and IGF-1
that are observed in response to RE are not gender specific — although responses are typically
higher in men than women (Kraemer et al., 1991; Häkkinen & Pakarinen 1995; Linnamo et al.,
2005). It is also interesting to note that the magnitude of these acute increases are independent of
the individual’s absolute level of strength (Kraemer et al., 1998a). Some training variables such as,
the amount of load used, volume (Kraemer et al., 1990; 1991; Gotshalk et al., 1997; Similos et al.,
2003), and exercise selection (Kraemer et al., 1990; Volek et al., 1997a; Hansen et al., 2001), have
also been shown to influence the magnitude of the acute hormonal response to RE. What these
differences may contribute to chronic adaptations is uncertain as physiological outcomes were not
documented in these studies.
The data on RE’s ability to influence hormonal responses over the long term, and how this
may contribute to changes in muscle mass, is equivocal. For example, while RE training is thought
not to alter resting GH or testosterone concentrations (Häkkinen et al., 1987; 2000b; Hickson et al.,
1994; Ahtiainen et al., 2003; Kraemer & Ratamess 2005), some studies report an increase followed
by a reduction in circulating testosterone in response to different training phases (Raastard et al.,
2000; Ahtiainen et al., 2003). Other research suggests that training may improve the acute anabolic
hormonal response to an RE workout. That is, higher levels of GH (Rubin et al., 2005) and
testosterone and/or lower cortisol concentrations (Kraemer et al., 1995; 1999b; Ahtiainen et al.,
2005b) have been observed in trained as opposed to untrained individuals after a workout.
Conversely, others report no differences in acute training responses between trained and untrained
participants (Ahtiainen et al., 2003). However, it is clear that the hypertrophic response is specific
to the loaded muscle(s) (Kraemer & Ratamess 2005). Therefore, activation by a systemic hormone
would require load-mediated modulation of the hormone's efficacy in the exercised muscle. The
load-mediated modulation of receptor expression and/or binding affinity in muscle has been
demonstrated in rodents (Deschenes et al., 1994) and humans during RE training (Bamman et al.,
2001; Willoughby & Taylor 2004). This may explain localization of the growth response with
elevated blood anabolic hormones. In healthy humans, at least one study has shown that sequential
bouts of heavy RE increase blood testosterone concentrations and muscle androgen receptor
expression that correspond to subsequent increases in myofibrillar protein (Willoughby & Taylor
2004).
Energy production
Aside from a localized modulation by anabolic hormones, RE is shown to promote other
peripheral adaptations that may contribute to, or be a product of, muscle hypertrophy. An earlier
examination (McDougall et al., 1977) of biochemical changes within muscle revealed that 5
19
months of RE increased resting concentrations of ATP (18%) in previously sedentary individuals.
An increase in resting ATP from exercise training is thought to be a function of increased
mitochondrial number and size. However, some (Hather et al., 1991; Wang et al., 1993), but not all
studies (Alway et al., 1988; Chilibeck et al., 1999) have shown that RE-induced hypertrophy is
accompanied by a proportional increase in mitochondrial proteins. Unlike endurance training, RE
training does not appear to improve the capillary-to-fibre ratio, with similar capillary densities in
bodybuilders and sedentary subjects documented (Tesch 1984). Conversely, an increase in fibre
CSA in response to an RE training program does not appear to compromise fibre capillarization or
oxidative potential in healthy adults (Tesch et al., 1990; Green et al., 1999). The study by
MacDougall et al. (1977) also demonstrated that RE may promote an increase in resting
concentrations of creatine (Cr) (39%), phosphocreatine (PCr) (22%) and muscle glycogen levels
(66%) in previously untrained participants. However, more recent work suggests that chronic RE
training probably does not induce further increases in the phosphagen (ATP-ADP and PCr-Cr)
(Rawson & Volek 2003), or muscle glycogen (Haff, 2003) reservoirs. Nevertheless, muscle
contraction and athletic performance during maximal effort, short term activity (such as RE) is
dependant on the maximum rate of ATP regeneration via the phosphagen and
glycolysis/glycogenolysis systems.
In this regard, the ATP-ADP system is considered a “co-factor” (albeit an essential one)
necessary for biological function within key compartments of the cell that require energy.
However, it is now generally accepted that the availability of PCr is most critical to the
continuation of muscle force production and performance during high intensity exercise (Balsom et
al., 1994; Greenhaff 1997). The PCr-Cr system (encompassing its site-specific CK isoenzymes)
plays a pivotal, multifaceted role in muscle energy metabolism. The PCr-Cr system integrates all
the local pools (or compartments) of adenine nucleotides; the transfer of energy from mitochondrial
compartments to that in myofibrils and cellular membranes as well as the feed back signal
transmission from sites of energy utilization to sites of energy production. The main roles of the
PCr-Cr system are illustrated in figure 1.4. The first main function of the PCr-Cr system is that of a
temporal energy buffer for ATP regeneration achieved via anaerobic degradation of PCr to Cr and
rephosphorylation of ADP. This energy buffering function is most prominent in the fasttwitch/
glycolytic fibres; these fibres contain the largest pool size of PCr (Tesch et al., 1989). The
energy (ATP) required for high intensity exercise is met by the simultaneous breakdown of PCr
and anaerobic glycolysis of which the PCr-Cr system provides up to one-third of the total energy
required (Greenhaff et al., 1994). The second major function of the PCr-Cr system is that of a
spatial energy buffer (or transport system) and involves aerobic metabolism. In this capacity, the
20
PCr-Cr system serves as an intracellular energy carrier connecting sites of energy production
(mitochondria) with sites of energy utilization (Na+/K+ pump, myofibrils and the SR) (figure 1.4).
Figure 1.4 The main functions of the Cr-PCr system in a muscle fibre.
The first is that of a temporal energy buffer for the regeneration of ATP via anaerobic degradation
of PCr to Cr and rephosphorylation of ADP. The second major function of this system is that of a
spatial energy buffer or transport system that serves as an intracellular energy carrier connecting
sites of energy production (mitochondrion) with sites of energy utilization, such as Na+/K+ pump,
myofibrils and the SR.
21
This process has been coined the Cr-Pi shuttle (Bessman & Geiger 1981) to describe the
specificity of this system. Meaning, Cr literally shuttles energy from the mitochondrion to highly
specific sites and then returns to regenerate energy exactly the equivalent to its consumption at
those sites (Bessman & Geiger 1981). These reactions can only occur via compartment-specific CK
isoenzyme located at each of the energy producing or utilizing sites that transduce the PCr to ATP
(Wallimann et al., 1992). The mechanism of action of all the CK isoenzymes involves functional
coupling with adenine nucleotide translocase (ANT) in the mitochondria and with ATPases in
myofibrils and cellular membranes based on the metabolic channeling of adenine nucleotides
(Vendelin et al., 2004). This mitochondrial-based energy transport system may be more prominent
in the slow-twitch fibres as these fibres characteristically contain a smaller PCr pool and a
relatively higher mitochondrial CK isoenzyme compared to the fast-twitch fibres. Conversely, the
percentage of cytosolic CK is smaller in the slow-twitch fibres compared to the more glycolytic
fibres (Wallimann et al., 1992). Another function of the PCr-Cr system is the prevention of a rise in
ADP that would have an inhibitory effect on a variety of ATP-dependant processes, such as crossbridge
cycling. A rise in ADP production would also activate the kinase reactions that ultimately
result in the destruction muscle adenine nucleotides (Greenhaff, 1997). Therefore, the removal of
ADP via the CK reaction-induced rephosphorylation serves to reduce the loss of adenine
nucleotides while maintaining a high intracellular ATP/ADP ratio at the sites of high energy
requirements (Hultman & Greenhaff 1991).
The CK reaction during the resynthesis of ATP takes up protons (Wallimann et al., 1992)
and therefore, another function of this PCr-Cr system is the maintenance of pH in exercising
muscle. In a reversible reaction (catalysed by the site specific CK), Cr and ATP form PCr and ADP
(figure 1.5). The formation of the polar PCr "locks" Cr within the muscle and maintains the
retention of Cr because the chargeprevents partitioning through biological membranes (Greenhaff,
1997). When pH declines (i.e., during exercise when lactic acid accumulates), the reaction will
favour the generation of ATP. Conversely, during recovery periods (i.e., periods of rest between
exercise sets), where ATP is being generated aerobically, the reaction will proceed toward the right
and increase PCr levels. The notion that maintenance of PCr availability is crucial to continued
force production and performance during high intensity exercise is further supported by research
that demonstrates the rate of PCr utilization is extremely high during the first seconds of intense
contraction— high anaerobic ATP regeneration rates result in a 60-80% fall in PCr (Bogdanis et
al., 1995). Not only is the depletion of muscle PCr associated with fatigue (Hultman & Greenhaff
1991), the resynthesis of PCr and the restoration of peak performance are shown to proceed in
direct proportion to one another, despite low muscle pH during recovery (Bogdanis et al., 1995).
22
Figure 1.5 The reversible phosphorylation of Cr by ATP to form PCr and ADP
23
In fact, PCr availability correlates with performance during short term power production
(Bogdanis et al., 1995). Therefore, energy supply appears to be more critical for generating power
than the direct effect of protons on contractile mechanisms. While the Cr-Pi shuttle clearly plays an
integral role in the efficient delivery of energy for muscle contraction, this system may also serve
another important anabolic function and that is the supply of energy for the synthesis of muscle
proteins during hypertrophy (Bessman & Savabi 1988).
Once Ingwall et al. (1974; 1976) demonstrated that increasing Cr availability selectively
stimulated the rate of synthesis of contractile proteins (actin and myosin) in culture, Bessman et al.
(1980) proposed that the Cr-Pi shuttle may play a role in contractile-specific protein synthesis via
an increase in PCr "traffic" within the intervening space between the mitochondrion and the
contractile apparatus during muscle contraction. These researchers postulated that a protein
synthesizing microsome lay adjacent to, or may even be a part of creatine-phosphokinase (CPK)
site at the myofibril where it would receive some of the ATP liberated when this CK isoenzyme
transfers ATP to the cross-bridge binding site (Bessman et al., 1980). Therefore, this protein
synthesizing complex would benefit from an increase in Cr-Pi shuttle activity that would occur
during contractile activity. The researchers demonstrated evidence of this protein synthesizing
component via two experiments. Firstly, protein synthesis was measured in muscle and in
hepatocytes (in vitro) as affected by fluorodinitrobenzene (FDNB). Increasing concentrations of
FDNB inhibited muscle protein synthesis in parallel with the inhibition of CPK activity but had no
effect on protein synthesis in hepatocytes (which have little CPK activity) (Carpenter et al., 1983).
A second set of experiments by this group were even more specific; they demonstrated that PCr
was a better energy donor for protein synthesis by microsomes than the ATP-ADP system (Savabi
et al., 1988). The general concept put forward by Bessman’s group is that net protein synthesis is
affected only by the increase of energy supplied to the contractile apparatus (Bessman & Savabi
1988). This is supported by studies that suggest increasing Cr availability increases myosin
synthesis and/or differentiation of myogenic satellite cells (Ingwall et al. 1974; 1976; Young &
Denome 1984; Vierck et al., 2003). Therefore, while PCr availability appears to be all-important to
maintenance of muscle force production and performance during high intensity exercise, it may
also play a role in the synthesis of muscle protein, and therefore, hypertrophy during RE. A
revelation in exercise physiology in recent decades has been the confirmation that resting
concentrations of PCr and Cr can be increased via dietary supplementation (Balsom et al., 1994).
This strategy is shown consistently to provide an ergogenic benefit, particularly during repeated
bouts of high intensity muscle contraction (Terjung et al., 2000). Supplementation to specifically
increase PCr availability and augment muscle hypertrophy during RE is an important aspect of this
dissertation and is covered in greater detail later in this chapter (section 1.9).
24
Along with the phosphagen system, glycolysis and glycogenolysis are considered to be
important energy contributors during RE (Lambert & Flynn 2002). In fact, during one set of 12
maximum-effort repetitions in the arm curl exercise, just over 82% of ATP demands were
estimated to be met by glycogenolysis (MacDougall et al., 1999). A single bout of high-intensity
RE characteristically results in a significant reduction in muscle glycogen of 30-40% (Robergs et
al., 1991; Tesch et al., 1998; MacDougall et al., 1999; Haff et al., 2000). As little as 3 (maximumeffort)
sets of 12 repetitions in the barbell curl exercise can reduce glycogen stores in the biceps by
25% (MacDougall et al., 1999). This RE-induced reduction in muscle glycogen is particularly
evident in type-II muscle fibres (Tesch et al., 1998). While these fibres contain a higher
concentration of glycogen than the type-I fibres (approx. 26%), the type-II fibres also exhibit a
much higher rate of glycogenolysis (64%) during intense activity (Greenhaff et al., 1991). A
preferential depletion of glycogen in type-II fibres has also been demonstrated after other forms of
high-intensity, short term exercise (Green, 1978; Vollestad et al., 1992). The type-II fibres are
responsible for maximum force production, and low glycogen levels in these fibres have been
associated with compromised performance during RE (Lambert et al., 1991; Haff et al., 1999;
Leveritt & Abernethy 1999). Muscle damage is reported to increase the requirement for
carbohydrate (CHO) for optimal glycogen synthesis (Costill et al., 1990), and the muscle damage
that occurs from RE may reduce the capacity to store glycogen (O’Reily et al., 1987).
Alternatively, high muscle glycogen stores appear to offer an ergogenic benefit during high
intensity exercise (Balsom et al., 1999). Increasing CHO ability before (Lambert et al., 1991),
during (Haff et al., 2001), and after RE (Haff et al., 1999) results in improved work capacity.
Therefore, the implementation of a CHO supplementation regime during RE training may prevent
decreases in performance and stimulate an increase in muscle glycogen resynthesis as well as
improve the potential for greater physiological adaptations. However, dietary strategies that
promote the maintenance or increase of muscle glycogen during RE is a topic that has received
little investigation (Haff, 2003). Clearly, muscle glycogen is an important fuel source during RE,
the amount of glycogen stored within muscle has the potential to influence force production, work
capacity and therefore chronic adaptations (i.e. hypertrophy and strength). For these reasons,
strategies that promote the maintenance or increase in muscle glycogen during RE training warrant
investigation.
The relationship between strength and hypertrophy
Maximal voluntary strength is typically measured by repetition maximum (RM) or
isometric/dynamic torque (Kraemer et al., 1995; Aagaard et al., 2002). Muscle hypertrophy and
strength development are closely related. For example, force production is usually proportional to
25
muscle fibre CSA (Kraemer, 2000). An increase in muscle fibre CSA is thought to underline most
of the improvements in force production and strength that are achieved during RE training (Shope
et al., 2003). However, hypertrophy also contributes to improved force production by altering
muscle architecture such as, an increase in the pennation angle and fascicle length (increase in the
number of sarcomeres in series) of pennate muscles (Kawakami et al., 1993; 1995; Kearns et al.,
2000; Kumagai et al., 2000; Aagaard et al., 2001). These alterations in muscle architecture appear
to play an important role in expression of functional strength (Brechue & Abe 2002). Hypertrophy
increases the pennation angle of pennate muscles that results in an increase in force production
(Kearns et al., 1998). Although it is the changing pennation angle that allows for greater sarcomere
packing per CSA, the benefit of greater fascicle length in these muscles is the maintenance of an
increase in force per CSA; greater fascicle length limits the change in pennation angle associated
with muscle hypertrophy to improve force production per CSA (Kearns et al., 1998, 2000;
Kumagai et al., 2000). To underline the influence of fascicle length in functional strength, a study
of experienced weight-lifters demonstrated that greater fascicle lengths in the triceps and vastus
lateralis was closely related to greater 1RM strength in the barbell squat, deadlift and bench press (r
values ranged from 0.63 to 0.54; P < 0.01) (Brechue & Abe 2002). While an increase in fascicle
length is an alteration in muscle architecture that is considered an important contributor to
improved force production and functional strength (Abe et al., 1999; Kearns et al., 2000; Kumagai
et al., 2000), the magnitude of this adaptation is positively associated with increases in LBM
(Brechue & Abe 2002). Therefore, it is clear that muscle morphology provides important
contributions to the development of strength during RE training. However, neural adaptations are
also thought to play a prominent role in strength devel

development, both in the initial stages of a training
program (Morianti & DeVries 1979; Carson & Reik 2001; Carroll et al., 2002) and in the longer
term (Sale, 1992; Häkkinen et al., 1988; 1998b).
The capacity to generate force is essentially dependent on motor unit activation (Sale,
1992). The motor unit serves as the functional unit of the neuromuscular system. In most
mammalian skeletal muscles, motor units are comprised of a single motor neuron and the multiple
muscle fibres that it innervates (McComas, 1996). Motor unit populations differ between muscles;
in general, small muscles such as the external rectus of the eye, lumbricals, and interossei muscles
have few muscle fibres per motor unit (100 or less), whereas larger muscles such as the medial
gastrocnemius can have up to nearly 2,000 muscle fibres per motor unit (Lee et al., 1975). To
initiate movement, motor units are recruited according to their size; from small to large (Henneman
1965; Sale, 1992). Maximal force production requires the recruitment of all motor units. This
includes the high threshold motor units that must be recruited at a high-enough firing rate to
produce maximal force (Sale, 1992). Untrained individuals appear not to be able to voluntarily
26
recruit the highest-threshold motor units (Moranti, 1992). Training often results in greater force
production, in the presence or absence of hypertrophy (Morianti & DeVries 1979; Abernethy et al.,
1994; Phillips, 2000). Therefore, a large part of the strength improvements observed in response to
RE is thought to be a result of an improved ability to recruit all motor units that initiate and control
movement (Rutherford & Jones 1986; Akima et al., 1999; Leong et al., 1999). RE training is shown
to enhance neural drive (i.e., recruitment and rate of firing) (Aagaard et al., 2002), improve
recruitment order efficiency (Sale, 1992) and increase the synchronization of the motor units
(Milner-Brown et al., 1973; Felici et al., 2001). This aspect is an important component of force
development as greater synchronization ensures that a greater number of motor units are firing at
any one time. Strength improvements from RE training can also be linked to an improvement in
the deactivation of antagonist muscles along with the improved activation of agonist muscles
(Häkkinen et al., 1998a) as well as decreased Golgi tendon organ inhibition (Sale, 1992). Training
can also alter the manner in which muscles are recruited by the central nervous system (Carroll et
al., 2001). This is associated with a change in the input–output properties of the corticospinal
pathway, such that a greater degree of muscle activation is generated by the same amount of
cortical input (Carroll et al., 2002). A reduction in the cortical input is necessary to elicit a given
level of force that may serve to benefit the production of coordinated movements by reducing the
level of central drive and thus minimizing the potential for functional interference within the motor
cortex (Carson et al., 1996; 1998). Therefore, it is apparent that both hypertrophy and neural factors
influence the expression of strength. However, the magnitude to which each of these aspects
contributes to strength development during a training program is not clear.
Training status
Untrained individuals or “novices” (those with no RE training experience or who have not
trained for several years) generally respond readily with significant improvements in strength and
hypertrophy to a wide variety of RE protocols (Gettman et al., 1978; Morganti et al., 1995; Campos
et al., 2002; Goto et al., 2005). Strength gains develop rapidly in untrained participants; they are
clearly evident within the first 4-8 weeks of training (Hickson et al., 1994; Staron et al., 1994;
O'Bryant et al., 1998) and neural adaptations are thought to play a prominent role in this early
stage. It is presumed that muscle hypertrophy follows improvements in strength as hypertrophy
becomes evident 6 to 8 weeks into an RE program (Phillips, 2000). However, the contribution of
neural and morphological adaptations to strength development can be influenced by the complexity
of the exercise (Chilibeck et al, 1998). For example, one study has shown that over a 20 week
training period, hypertrophy of the upper extremity muscles occurred during the first 10 weeks
whereas the lower extremity and trunk muscles did not hypertrophy until the last 10 weeks
27
(Chilibeck et al, 1998). The authors of this research believed that for simple motor tasks such as the
biceps curl exercise, early gains in strength may occur concurrently with muscle hypertrophy. On
the other hand, more complex movements associated with the trunk (e.g., bench press) and legs
(e.g., leg press) require a longer period for neural adaptation, and thus delay muscle hypertrophy.
However, the reasons for this are unclear.
“Trained” or “experienced” individuals have been defined as those with approximately 6
months of consistent RE training (Kraemer et al., 2002). The term “advanced” or “highly trained”
refers to those individuals with years of consistent training experience who have also attained
significant strength and/or muscle hypertrophy (Häkkinen et al., 1988). Trained individuals are
generally considered to have much slower rates of improvement than untrained individuals (Giorgi
et al., 1998; Häkkinen et al., 1998b; Schiotz et al., 1998), but very little is known about
neurological and hypertrophy contributions in the development of strength in these participants.
Several time courses in the literature propose that generally, neural adaptations and changes in
protein quality (i.e., myosin isoforms) provide most, if not all the early strength improvements
observed in the initial stages of a training program (Moritani, 1992; Sale, 1992; Wathen et al.,
2000). The contribution of neural factors is thought to diminish as training continues beyond 6 to 8
weeks (Sale, 1992; Moritani 1992; Wathen et al., 2000). Histochemically determined fibre CSA
studies show that hypertrophy becomes evident after 6 weeks of training but this response is also
suggested to plateau after 12 to 16 weeks of training. Therefore, according to the most of the
timelines proposed in the literature (Sale, 1992; Wathen et al., 2000; Fleck & Kraemer 1997), little
or no significant improvements in strength or hypertrophy occur after 16 weeks of training! In fact,
the use of anabolic drugs has been proposed as the only way a trained individual may experience
significant hypertrophy (Sale, 1992). Nevertheless, further (significant) hypertrophy has been
reported with continued training (after the initial 6 weeks) in both men and women (Frontera et al.,
1990; Staron 1990; Kraemer et al., 1995; Green et al., 1999). In a year-long RE training program,
older men and women were shown to increase muscle strength within the first 8 weeks but muscle
fibre hypertrophy did not occur until after 30 weeks of training; well after most of the strength
gains had occurred (Pyka et al., 1994). There is also evidence that suggests the hypertrophic
response can continue with chronic training. For instance, subjects who have participated in
resistance training programs spanning several years (Alway et al., 1988; Klitgaard et al., 1990;
Kadi et al., 1999) typically have larger muscle fibres than previously sedentary individuals who
have trained for only 3–6 months (MacDougall et al., 1980; Kraemer et al 1985; Alway et al.,
1989; McCall et al., 1996). Additionally, significant hypertrophy and strength improvements have
been documented in longitudinal studies involving trained (Häkkinen et al., 1985; Staron et al.,
1991; McCall et al., 1996; Volek et al., 1999; Cribb et al., 2006) and even highly-trained
28
individuals (Häkkinen et al., 1988). However, it also appears as though these athletes are also able
to experience significant neurological adaptations that result in strength improvements over the
longer term (one to two years) (Häkkinen et al., 1988). Therefore, although the adaptations maybe
less in magnitude, clearly RE-trained individuals still possess the capacity for neurological and
morphological adaptations that result in significant improvements in strength and hypertrophy.
While training is presumed to diminish the capacity for significant hypertrophy (Sale,
1992; Kraemer et al., 2002), an alternate consideration is that the methodologies used to estimate
hypertrophy have not been sensitive enough to detect small but significant increases in protein
accretion. For example, more recent AA (isotope) kinetic studies clearly show that a net gain in
muscle protein is possible after a single workout (if AA are supplemented at this time) (Tipton et
al., 1999; 2001; 2003). By definition, this is hypertrophy.
The idea that the hypertrophy response to RE training may be improved under certain
conditions is a consideration that has only received attention by the scientific community in recent
years (Volek, 2004). An aspect that warrants consideration when discussing chronic adaptations to
prolonged RE training is that many earlier longitudinal studies did not control some important
variables that are now known to influence strength and hypertrophy development. For instance, it is
well-established that an improvement in strength will increase the potential for hypertrophy;
strength gains usually lead to increases in muscle CSA (Atha, 1981; Saltin, 1983; Tesch, 1992).
However, the level of supervision of participants during RE training studies has been identified as a
limiting factor in strength development (Mazzetti et al., 2000). A personal training approach to RE
supervision (i.e., one-to-one or one-to-two instruction during each workout) is shown to ensure
better control of workout intensity and greater strength improvements during training (Mazzetti et
al., 2000). Yet, very few training studies document this level of supervision in their research
protocols. It is also clear that the type, timing and quantity of macronutrient intake can potentially
influence strength and hypertrophy development (as discussed in section 1.5 of this chapter).
However, many of the longitudinal RE training studies that form our current body of knowledge
and shape our current perceptions do not mention any attempt to control or monitor dietary intake
(Häkkinen et al., 1985; 1987; 1991; 1995; 1996; 1998; 2001; Staron et al., 1990; 1991; 1994;
Hather et al., 1991; Adams et al., 1993; Ploutz et al., 1994; Hortobagyi et al., 1996; Ostrowski et
al., 1997; Chestnut & Docherty 1999; Takashi et al., 2000; Abe et al., 2000; Campos et al., 2002).
Therefore, it is quite possible that much of the research that has assessed strength and hypertrophy
may have overlooked some important variables that can influence the chronic adaptations to RE
training. To support this view, recent studies that have examined the effects of strategies designed
to optimize the hypertrophy response have revealed a much greater capacity for improvements than
29
previously assumed. For example, although chronic training is presumed to diminish the
hypertrophic response, training studies that have utilized a personal-training approach to
supervision of trained participants and dietary intervention have reported average increases in
muscle fibre CSA of 35% (for all fibre types) (Volek et al., 1999), and gains in LBM of up to 6%
(Cribb et al., 2006). These responses were previously thought only to be possible via the use of
anabolic steroids (Sale, 1992). Obviously, there are limitations to the extent of any physiological
adaptation that can be achieved from physical training. However, it is equally apparent that we
know very little about the aspects that may improve the hypertrophy response from RE. Therefore,
the typical expectations (and limitations) that are currently associated with muscle hypertrophy
may be some what premature.
1.4 Resistance exercise, protein turnover and muscle hypertrophy
Any adaptive change in muscle mass such as hypertrophy must involve alterations in
protein turnover. That is, the difference between rates of MPS and muscle protein breakdown
(MPB) determines net protein balance (NPB) (Rassmussen & Phillips 2003). A single bout of RE
results in the acute stimulation of MPS (50-100% above basal values), that peaks within 3-24 hours
and remains elevated at a diminishing rate for up to 48 hours post-exercise (Chesley et al., 1992;
Biolo et al., 1995; Phillips et al., 1997; Yarasheski et al., 2001). However, studies that have
assessed the rate of MPB and MPS simultaneously after a single bout of RE in both the fed
(Phillips et al., 2002), and fasted state (Biolo et al., 1995; Phillips et al., 1997), demonstrate that
both processes are stimulated equally so that NPB remains negative. A positive NPB is not
obtained until exogenous AA are provided (Biolo et al., 1997). The muscle degradation that occurs
in response to RE is thought to be an important component of the remodelling response (Laurent &
Millward 1980). However, it is the stimulation of MPS that appears to be the facilitating process
that underlines net protein accretion and hypertrophy (Rennie et al., 2004; Cuthbertson et al.,
2005). Early research established that in response to loading, an increase in muscle mass correlated
with the magnitude of stimulation of MPS (Goldberg, 1968). Since then, other studies have
confirmed that the rate of MPS is the critical regulatory event that leads to load-induced
hypertrophy (Wong & Booth 1990; Phillips et al., 1997; Baar & Esser 1999). While these studies
examined the impact of RE on mixed MPS, more recent work has confirmed that strenuous
exercise can stimulate an increase in myofibrillar, sarcoplasmic and connective tissue protein
synthesis rates (Louis et al., 2003; Miller et al., 2005; Mittendorfer et al., 2005). Chronic training
also appears to influence this response. For example, untrained participants demonstrate large
increases in both contractile and non-contractile MPS (Louis et al., 2003; Mittendorfer et al., 2005).
However, after an 8 week training program, this acute response is modulated so that the stimuli
30
becomes preferential toward an increase in contractile synthesis more so than non-contractile
proteins (Kim et al., 2005). This recent finding confirms previous studies that reported the
stimulation of mixed MPS in response to RE was most likely to be an increase in MHC protein
synthesis (Hasten et al., 2000; Balagopal et al., 2001). However, while these studies provide
important insights into the acute physiological responses to RE, very little work has examined the
chronic effects (longer than 8 weeks) of training on muscle protein turnover.
One cross-sectional study has demonstrated a comprehensible training effect on protein
turnover (Phillips et al., 1999). That is, in response to a single workout, RE-trained participants
demonstrated smaller increases in MPS rates and little or no change in rates of MPB, unlike
untrained participants who showed large increases in both rates of MPS and MPB (while working
at the same relative exercise intensity) (Phillips et al., 1999). Data obtained from a subsequent
longitudinal study by the same researchers confirmed that the magnitude of stimulation of mixed
MPS in response to the same absolute load is diminished after 8 weeks of training (Phillips et al.,
2002). However, the training program still provided significant increases in protein turnover rates
(both MPS and MPB) at rest and in the hours after RE (Phillips et al., 2002). These increases were
also noted in the previously mentioned cross-sectional study but did not reach statistical
significance (Phillips et al., 1999). Therefore, a RE training program is capable of evoking a
substantial increase in resting and post-exercise protein turnover. However, recent work by Kim et
al. (2005), suggests that this increase may be modulated by training. Kim et al. (2005), employed a
single leg RE training protocol for 8 weeks (the collateral leg served as the control) and results
showed that the training program attenuated the acute large increase in mixed MPS. However, the
stimulatory response in contractile protein synthesis rates was of similar magnitude in both the
trained and untrained leg muscles after the program (Kim et al., 2005). Another interesting finding
was that training resulted in an increase in resting mixed MPS rates, which is indicative of an
extensive remodelling response (Rassmussen & Phillips 2003) but resting myofibrillar protein
synthesis rates remained unchanged. While these results may appear to be contradictory, it may be
that training attenuates the large acute response in mixed MPS observed in untrained participants
(Phillips et al., 1997; 1999; 2002) but not the stimulatory effect on myofibrillar protein synthesis
rates (Kim et al., 2005). Once again it is important to remember that this 8 week study utilized
(previously) untrained participants. There is no longitudinal data on the effects of RE on muscle
protein turnover in RE-trained individuals. Therefore, we do not know if these responses may
change with prolonged training or if different RE programs are capable of altering these responses
and adaptations.
31
The potent stimulatory effect of RE on protein turnover is indicative of an extensive
muscle protein remodelling process. An important part of this remodelling process is not only the
repair of extensive ultrastructural damage but also degradation and removal of damaged proteins
before new proteins can be incorporated into the contractile machinery. This extensive degradation
process that must occur requires activation of proteolytic pathways (DeMartino & Ordway 1998;
Ordway et al., 2000). Moreover, as damage appears to be reduced after subsequent bouts of muscle
contraction (Ebbling & Clarkson 1989; Clarkson et al., 1992; Willoughby et al., 2003), this
suggests an adaptive response within the actual pathways of protein degradation. Intriguingly, this
“protective response” has been documented after just one subsequent bout of RE performed almost
6 weeks after a previous bout (Stupka et al., 2001). The biochemical characteristics of this
adaptation include an attenuated release of CK, changes in the inflammatory response and
ubiquitin-conjugated protein content (Stupka et al., 2001) as well as reduced expression of
ubiquitin, E2 protein, and 20S proteasome mRNAs (Willoughby et al., 2003). While extracellular
(inflammatory activation such as lysosomes) and intracellular (ubiquitin) proteolytic pathways
exhibit adaptive responses, the mechanism(s) of proteolysis in response to chronic loading activity
such as RE, are not well understood (Reid, 2005).
For instance, the signals generated during muscle contraction that modulate these pathways
(and pathway-related gene transcription) are not clear. In particular, it is uncertain what the
molecular modifications that stimulate ubiquitin conjugation to muscle proteins are, or the ratelimiting
step(s) in targeting and degradation of substrate proteins (Reid, 2005). In comparison, the
mechanisms that underline MPS (the transcription and translation of mRNA into protein) have
been elucidated to a much higher degree than proteolytic responses. This is mainly because the
protein-synthetic machinery forms a cohesive metabolic unit with the ribosome and the
endoplasmic reticulum. For instance, regulatory changes in the machinery of protein synthesis can
be linked relatively clearly with an increase in the synthesis of protein or phosphorylation of a
signalling complex, whereas in the case of proteolytic pathways, a change in the activity of key
components of the system may occur with no, or apparently opposite, changes in the extent of net
protein balance (Attaix et al., 2001). However, it is clear that the autophagic-lysosomal pathway is
responsible for the bulk of proteolysis (Kadowaki & Kanazawa 2003), and the ubiquitinproteasome
pathway plays a significant role in the fine control of the degradation of specific
proteins (Lecker et al., 1999). The ubiquitin-proteasome pathway probably also has a prominent
role in the contraction-induced remodelling of muscle (Ordway et al., 2000). Additionally, the
autophagy system appears to be physiologically controlled by plasma AA. Recently, the amount of
leucine within muscle, but also other AA such as glutamine, tyrosine, phenylalanine, proline,
methionine, and histidine in the liver, has been identified in the regulation of this process
32
(Kadowaki & Kanazawa 2003). However, despite a concerted effort over the last 30 years to
examine this key aspect of muscle protein metabolism (Waterlow 1995; Carson et al., 1997; Baar et
al., 1999; Kadowaki & Kanazawa 2003; Rennie et al., 2004), in general, this appears to be all that
is known about the proteolytic mechanisms that underline the adapatational responses to RE.
1. 5 The molecular events associated with hypertrophy
Significant advances in our understanding of the processes responsible for muscle
hypertrophy are mainly due to a greater understanding of the mechanisms that underline MPS.
Once MPS was recognized as a critical regulatory event for load-induced changes in muscle mass,
this response became a major focus that has lead to an increased knowledge base in the logistics
that control the size of human muscle mass. Therefore, this section will focus exclusively on the
molecular processes and pathways that regulate MPS in response to RE and nutrient consumption.
The paragraph below provides a very simplistic overview of the molecular processes involved and
this is followed by a brief discussion of the main events.
RE induces mechanical stress (tensile, compressive and shear) and transient changes in
sarcoplasmic calcium concentrations, energy substrate levels, the re-dox state as well as increases
the availability of hormones and cytokines (No.1 figure 1.6). Sufficient changes to any (or all) of
these variables are thought to activate a network of signal transduction pathways that transfer the
mechanical stimuli into specific chemical signalling within the cell. Once activated, these pathways
activate transcription factors that alter the expression of muscle genes within the nucleus (No.2
figure 1.6). Active, nuclear transcription together with receptor binding of muscle growth-factors,
androgens and glucocorticoids change the expression of the major muscle growth regulators such
as IGF-1/MGF, myostatin and other muscle genes (No.3 figure 1.6). Whereas IGF, insulin and RE
all appear to (partially) recruit the same pathway which activates protein synthesis via increased
translational initiation (No.4 figure 1.6), AA intersect and stimulate MPS via mTOR; a central
protein in the signal transduction pathway of MPS (No.5 figure 1.6). While AA activate mTOR, an
increased energy demand sensed by AMPK inhibits the phosphorylation of mTOR and the
stimulation of MPS (No.6 figure 1.6). Growth factors (IGF/MGF) and myostatin appear to play a
key regulatory role in the proliferation and fusion of satellite cells to the muscle fibre (No.8 figure
1.6); a process that is considered integral to muscle hypertrophy.
33
extracelluar intracellular
Nutrient Timing
PROTEIN
SYNTHESIS
eIF-2B
4E-BP1
PKB/Akt
ERK1/2
P13K
nucleus
IGF-1/MGF
Myostatin &
other mRNAs
Raptor
mTOR
Insulin / IGF-1
Androgens
satellite
cell
p70S6K
integrins
Cytokines
transcription factors
fiber type adaptations
AMPK
Ca2+
calcineurin
Mechanical
stress
RE
?
Glucocorticoids
Growth factors
amino acids
RE
1
2
3
4
1
5
6
8
7
NFAT pathway
RohA-FAK-SRF
c-jun
Ras/MAPK
pathway
muscle fiber
GSK3
Figure 1.6 Signalling pathways that lead to muscle protein synthesis
33
34
The stimulation of protein synthesis within muscle might be mediated by pre-translational
(alteration in the abundance of mRNA), translational (alteration in protein synthesis per unit of
mRNA) or post-translational (transformation of the protein such as phosphorylation) events (Booth
et al.,1998). However, the rate limiting step of MPS appears to be translation initiation (Mathews et
al., 1996; Sonenberg 1996); a complex, multiple step process in which the mature mRNA transcript
exits the nucleus and is coupled to the ribosomal machinery (No.7 figure 1.6). Translation initiation
is thought to be regulated by three key proteins that are in turn controlled by post-translational
modification (Merrick, 1992). These three proteins are, eukaryotic initiation factor 2 (eIF-2B)
eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and 70-kDa S6 protein kinase (p70-
S6k) (Mathews et al., 1996). While eIF-2B is thought to regulate general protein synthesis, 4E-BP1
and p70-S6k control tissue growth-related protein synthesis (Mendez et al., 1997). In particular,
p70-S6k has been confirmed as a major regulatory kinase in the activation of MPS in response to
RE (Karlsson et al., 2004).
In research that has now proved to be seminal, Baar and Esser (1999) established that
phosphorylation of p70-S6k was not only increased following mechanical loading (designed to
mimic high intensity RE), the increase in p70-S6k activity correlated with increases in muscle
mass. Next it was shown that translation (the actual process of protein synthesis using mRNA as
the template and the signal transduction pathways that regulate it), is selectively activated by
contractions designed to mimic RE (Nader & Esser 2001). Upstream activators of p70-S6k, such as
protein kinase B (PKB) and mammalian target of rapamycin (mTOR) were then identified as
crucial for muscle hypertrophy (Bodine et al., 2001; Rommel et al., 2001). For example, specific
inhibition of mTOR with rapamycin results in a 95% blockade of hypertrophy (Bodine et al.,
2001). Signalling to PKB (also known as Akt) via PI3 kinase is probably involved in this pathway
(Rommel et al., 2001). Evidence obtained from in vivo and in vitro work implicate the
phosphorylation of mTOR with the subsequent activation of not only p70-S6k but also 4E-BP1
which allows the association of the ribosomal scaffolding proteins eIF4E and eIF4G. In rodent
models, this signalling cascade (i.e., PI3/PKB-mTOR-p70-S6k/4E-BP1) has been linked strongly
to the stimulation of MPS in response to mechanical loading designed to mimic RE (Bodine et al.,
2001; Pallafacchina et al., 2002; Bolster et al., 2003; Atherton, 2005). In humans, phosphorylation
of 4E-BP1 and p70-S6k are shown to be stimulated in parallel with both myofibrillar and
sarcoplasmic MPS rates after intense isometric exercise (Rennie et al., 2001). A persistent, longlasting
rise in p70-S6k phosphorylation, with smaller transient rises in PKB/Akt phosphorylation
have been observed in humans after more conventional high-overload RE (Cuthbertson et al., 2002;
personal communication with MJ Rennie). Therefore, the PI3/PKB-mTOR-p70-S6k/4E-BP1
cascade is a likely pathway that activates MPS in humans in response to RE (No.4 figure 1.6).
35
Whereas the PI3/PKB-mTOR-p70-S6k/4E-BP1 pathway is associated with muscle growth,
the calcineurin/NFAT pathway and to a lesser extent the Ras/MAPK pathway, is thought to control
muscle fibre type distribution (No.2 figure 4) (Serrano et al., 2001). Calcineurin is a cytoplasmic
calcium-regulated phosphatase that is thought to be activated in overloaded muscles via the chronic
increases in intracellular calcium that occur under these conditions (Panchenko et al., 1974).
Overload-induced fibre hypertrophy and fibre type transformations are shown to be prevented in
vivo by administration of calcineurin-specific inhibitors (Dunn et al., 1999). Therefore, calcineurin
appears to be crucial in signalling the adaptive responses to RE (Dunn et al., 2000). Once
activated, calcineurin is thought to signal downstream genes involved in regulating muscle fibre
size via dephosphorylation of its substrate transcription factors such as NFAT (nuclear factor of
activated T cells) (Dunn et al., 2000). Additionally, various NFAT isoforms can activate genes
which have been implicated in fibre transitions and hypertrophy (Olson & Williams, 2000).
Another likely candidate that appears to link mechano-chemical transduction to gene expression
and, ultimately, muscle growth is the integrin-meditated RhoA-FAK-SRF pathway (Carson & Wei
2000) (No 2. figure 1.6). The intergrins are membrane-associated proteins that act as primary
sensors for relaying an array of physical or mechanical signals from the surrounding environment
into the interior of the cell. Serum response factor (SRF) is a transcription factor substrate of FAK
that binds to the -actin gene via the serum response element (SRE1) (Lee et al., 1992). SRE1 is a
hypertrophy regulatory element that is thought to activate specific contractile protein genes to
produce more mRNA in response to overload conditions (Carson et al., 1995). This pathway
provides a transcriptional link between membrane, the genome and subsequent expression of
muscle protein (Carson & Wei 2000). Muscle hypertrophy may also require activation of the
mitogen-activated protein kinase (MAPK)-signalling cascade. The MAPK pathway (also
associated with the extracellular signal-regulated kinase-ERK1/2) (No.2 figure 1.6) is thought to be
important to exercise-induced muscle morphology as it activates several myogenic transcription
factors (Widegren et al., 1998). Interestingly, a single bout of RE has recently been shown to
activate some of the MAPK associated signalling proteins (such as p38) in young but not older
adults (Williamson et al., 2003; Karlsson et al., 2004; Creer et al., 2005).
The stimulation of protein synthesis within skeletal muscle by the consumption of a mixed
macronutrient meal is due primarily to the essential amino acids (EAA) (Rennie et al., 2006). Of
the EAA, the branch chain amino acids (BCAA) are the most potent at stimulating protein
synthesis via upregulation of the initiation of mRNA translation (Kimball & Jefferson 2006). The
consumption of EAA-containing meals results in the phosphorylation of p70-S6k and the
eukaryotic initiation factor proteins eIF4E and eIF4G (Cuthbertson et al., 2005; Rennie et al.,
2006); a mechanism that not only promotes global translation of mRNA but also contributes to
36
processes that mediate discrimination in the selection of mRNA for translation (Kimball &
Jefferson 2006) (No.5 figure 1.6). Insulin stimulates glucose uptake, glycolysis, and glycogen
synthesis within muscle via the activation of PI3-PKB(Akt)-GSK-3 signalling pathway (Kimball et
al., 2002). While RE also seems to (partially) recruit the same signalling pathway as insulin (No 4.
figure 1.6), AA can stimulate MPS without a large amount of insulin (Cuthbertson et al., 2005). In
fact, the amount of insulin required for full activation of MPS appears to be quite low (Bennett et
al., 1989; Bohe et al., 2003). Unlike RE or insulin, the presence of AA do not appear to stimulate
MPS via activation of PI3 and PKB(Akt) (Greiwe et al., 2001b; Liu et al., 2002). It is also clear
that the activation of MPS by AA does not involve gene expression (at least in the short term)
(Svanberg et al., 2000). However, recent studies in humans (Cuthbertson et al., 2005) have
confirmed in vitro (Christie et al., 2002) and in vivo (Hara et al., 1998; Anthony et al., 2000) work
that demonstrates AA stimulate MPS directly via activation of the Raptor-mTOR complex (and its
regulatory proteins S6K1 and 4E-BP1) (No.5 figure 1.6).
The large (289 kDa) raptor-mTOR complex is expressed more in muscle than other tissues
(Kim et al., 2002). It is nutrient sensitive and contains multiple binding sites (Proud, 2002). The
major role of mTOR is to integrate various signals of the energetic status within the cell with
environmental stimuli to control cell growth (Deldicque et al., 2005a). The mTOR complex
appears to be a focal intersection point in the MPS signalling cascade, that is, both RE and AA
activate mTOR (Deldicque et al., 2005a). In rodent muscle, phosphorylation of mTOR is shown to
be preferentially localized in the type-IIa (tibialis anterior) fibres (Parkington et al., 2003). This
may be one explanation for the greater hypertrophy that is consistently observed in these fibres in
response to RE training (Kraemer et al., 1995; McCall et al., 1996; Volek et al., 1999). Whereas
AA and RE can activate mTOR, increased energy demand sensed by AMPK inhibits mTOR
(Bolster et al., 2002). This may partly explain the high stimulation of MPS that occurs after and not
during exercise (Bylund-Fellenius et al., 1984). However, MPS (and hypertrophy) can also be
stimulated independently of mTOR (Bodine et al., 2001).
Enhanced activity of eIF-2B is shown to occur after RE (Farrell et al., 1999) or
administration of AA (Kimball et al., 1998). The eIF-2B complex is the only one of the three
regulators of translation initiation (the rate limiting step of MPS) that is not under direct control of
mTOR (Deldicque et al., 2005a). In this pathway (No.4 figure 1.6), PKB phosphorylates and in
this instance, inactivates the protein kinase GSK-3 which in turn phosphorylates eIF-2B.
Phosphorylation of eIF-2B results in the inhibition of its guanine nucleotide exchange activity,
which stimulates global protein synthesis (Bolster et al., 2002). Thus, activation of PKB indirectly
results in enhanced eIF-2B activity through inhibition of GSK-3. Some AA such as leucine can
37
H2O
H2O
catabolic signal
anabolic signal
muscle cells
Cell swelling increases
Protein synthesis
Glycogen synthesis
Lactate uptake
Pentose phosphate shunt
Amino acid uptake
Ketoisocaproate oxidation
Acetyl-CoA carboxylase
MAP kinase activity
Glutathione (GSH) efflux
Actin polymerization
Microtubule stability
mRNA levels of c-jun and inhibits
protein degradation
Cell swelling decreases
Glycogenolysis
Glucose-6-phosphatase activity
Carnitine palmitoylt’ase I activity
Urea synthesis from NH4+
Viral replication
Modulators of cell volume
Insulin/glucogon/IGF-1
Amino acids, particularly Gln
Hydroperoxides (oxidative stress)
Na+/H+ and Cl-/HCO3
- exchange
K+ channel blockers
Conjugated bile acids
Energy substrates such as creatine
cAMP
Adapted from research by Lang et al.,
1998.
also activate MPS independently of mTOR (and phosphorylation of 4E-BP1 and p70-S6k)
(Anthony et al., 2000; 2002). Therefore, it appears that both RE and AA can stimulate MPS via
both mTOR-dependant and independent pathways. Intriguingly, the various AA may differ in how
they activate MPS (Meijer & Dubbelhuis 2004). For example, the BCAA are thought to be
important signalling molecules in translation initiation (Kimball & Jefferson 2006) where as other
AA such as Gln may stimulate MPS via an indirect mechanism such as an increase in cell volume
(figure 1.7).
Figure 1.7 Regulatory effects of cell volume
Very small alterations in cell volume (hydration) act as separate and potent regulators of
cellular function (Haussinger et al., 1993; 1995). For example, an increase in muscle cell volume
acts as an independent, anabolic signal that stimulates a number of anabolic processes such as an
increase in AA transport and protein synthesis (Stoll et al., 1992), inhibition of protein degradation
(Haussinger et al., 1990; Vom Dahl et al., 1996), and stimulation of glycogen synthesis (Peak et al.,
38
1992) (figure 1.7). Conversely, a reduction in cell volume evokes catabolic and anti-proliferative
responses (Lang et al., 1998). Cell volume is dynamic and changes within minutes under the
influence of certain substrates, hormones, AA, glucose and oxidative stress (Lang et al., 1998). In
humans, a close relationship between the cellular hydration state in muscle and nitrogen balance
has been shown in a variety of cachectic conditions irrespective of the underlying disease
(Haussinger et al., 1993). Therefore, cellular hydration’s impact on protein turnover may provide a
better understanding of the catabolic environment that leads to the excessive muscle degradation
that is apparent in various disease states. While the signalling mechanisms that link changes in cell
volume to anabolic and catabolic events is unclear, the pathway is thought to resemble that
activated by growth factors. That is, phosphorylation of the MAP kinases and c-jun (Agius et al.,
1994). The activation of this signalling cascade may also (partly) explain the influence of cell
hydration on gene expression (Lang et al., 1998). However, there also appears to be a relationship
between proteolytic activity and cellular hydration, regardless of whether the latter is modified by
hormones, AA, bile acids, K+ channel blockers (such as Ba2+) or anisotonic exposure (Lang et al.,
1998). Therefore, hydration changes may be the common mechanism that underlines proteolysis
control by these heterogeneous effectors. Gln is considered a most potent activator of increasing
cell volume (Haussinger et al., 1993). Gln triggers an increase in cell volume via an increase in
intra-cellular osmolarity following Na+ dependant transport across the cell membrane (Low et al.,
1996). In vitro and in vivo studies have demonstrated that the concentration of Gln within various
cells (including muscle) regulate cell volume, protein synthesis rates (Haussinger et al., 1995; Low
et al., 1996) and nitrogen balance (Haussinger et al., 1993). Recent in vitro work has demonstrated
the importance of Gln-induced cell swelling in the phosphorylation of mTOR and S6 kinase
(Fumarola et al., 2005). In this study, decreasing cell volume (via depravation of Gln) prevented
the activation of mTOR and S6 kinase, independent of energy status. Conversely, both complexes
were activated by increased AA availability (this was particularly prominent with leucine). The
most important finding from this work was that the activation of mTOR and S6 kinase required the
presence of Gln and the accompanying increase/maintenance of cell volume that Gln provides
(Fumarola et al., 2005).
So far the discussion within this section has identified the signal-transduction pathways
that lead to the activation of MPS via RE, insulin or the presence of AA. However, the combination
of RE and AA is shown to stimulate MPS beyond the rate that can be achieved by either one alone.
The magnitude of this effect appears to be synergistic rather than simply additive (Biolo et al.,
1997; Tipton et al., 1999). Recent work suggests this may be due to enhanced phosphorylation of at
least one of the key signalling proteins that controls translation initiation (Karlson et al., 2004).
When an oral dose of BCAA (total of 100mg/kg) was provided to healthy participants before,
39
during, and after RE, a greater site-specific phosphorylation of the p70-S6k protein in muscle was
observed (Karlson et al., 2004). The bout of exercise alone led to a marked increase in ERK1/2
(p38) MAPK phosphorylation, but this was completely suppressed upon recovery and unaltered by
the BCAA. Furthermore, phosphorylation of the ribosomal protein S6 was increased in the
recovery period only during the BCAA administration. This study provides further evidence that
AA and RE-induced signalling of MPS may converge at one point; the mTOR complex (No 4 and
5. figure 1.6). However, a recent study has shown that muscle glycogen availability can also
influence the activation of MPS during RE (Creer et al., 2005). In this study, two groups of athletes
(cyclists) performed a single bout of RE. One group performed RE with (diet-induced) low
glycogen stores and failed to activate PKB/Akt unlike the group that performed RE with high
muscle glycogen levels. However, in this study, the phosphorylation of ERK1/2 (and a ribosomal
S6 kinase) was unaffected by glycogen availability (Creer et al., 2005). Therefore, the presence of
AA and CHO during RE may provide the best chance of maximal activation of the signalling
pathways that stimulate MPS.
Muscle is a tissue in which gene expression is regulated to a large extent by the loadsensitive
signalling complexes and pathways discussed in the previous paragraphs of this section.
Recovery and adaptation to exercise in general, is associated with transcriptional activation of
specific genes involved with muscle growth, vascularization and metabolism (Hargraves &
Cameron-Smith 2002). In healthy humans, significant elevations in various immune, metabolic and
myo-specific mRNA have been identified in response to a single bout of RE (Chen et al., 2003;
Zambon et al., 2003). While this response could occur within minutes of muscle activation
(Puntschart et al., 1998), gene expression is predominantly thought to take place in the recovery
period; anywhere from 3-12 hours post exercise (Pilegaard et al., 2000) but can be detected up to
48 hours after RE (Hespel et al., 2001). The research to date indicates that large scale changes in
transcription regulation play a key role in the metabolic shifts and muscle remodelling responses to
exercise training (Pilegaard et al., 2000; Richardson et al., 2000; Chen et al., 2002). While recent
work has shown that prior training history can modify the acute gene responses in skeletal muscle
to subsequent exercise (Coffey et al., 2005), it is not understood how such transcriptional changes
are regulated during training. A large proportion of the changes in protein turnover that are
observed in response to RE are probably posttranscriptional, due mostly to increased translational
efficiency (Welle et al., 1999). However, using gene chip microarrays, Chen et al. (2002) revealed
that RE may lead to selective changes in gene expression through both transcriptional and
translational mechanisms.
40
The work by Chen et al. (2002) showed that 65% of the mRNAs whose abundance was
altered by RE, were detected in both the total and polysomal analyses; thus indicating
transcriptional mechanisms. However, another 25 mRNAs whose expression in the total mRNA
analysis was not altered, but their abundance in the polysomal (i.e., active) fraction changed with
exercise; indicating regulation through alterations in translation. Using a small group of young,
healthy males, the same research group (Chen et al., 2003) then revealed a high similarity (~50%)
in the expression responses observed between human undergoing eccentric contractions and the
data obtained from the previous analyses on rats (Chen et al., 2002). Some changes that were
specific to humans included greater inflammatory responses and vascular remodelling as well as
the confirmation of c-fos as an important transcription factor in the response to exercise-induced
damage. However, it is worth noting that the RE protocol used in this research (sitting and rising
from a chair 300 times) was more like a test of muscular endurance rather than a bout of high
overload RE. Therefore, while this data is novel, the exercise protocol used may limit its
application in deciphering the overall molecular program of hypertrophy in response to
conventional RE training.
Adult skeletal muscle fibres are unique in that they are multinucleated cells. Each
myonucleus controls the production of mRNA and protein synthesis over a finite volume of
cytoplasm, a concept known as the DNA unit or myonuclear domain (Cheek, 1985; Hall & Ralston
1989). Essentially, a skeletal muscle fibre consists of a mosaic of overlapping nuclear domains
(Hall & Ralston 1989). Satellite cells are undifferentiated myogenic cells that lie under the basal
lamina of the skeletal muscle fibre. Activation of these satellite cells results in their incorporation
into the muscle fibres as new myonuclei (Moss, 1971). If satellite cell proliferation is inhibited,
muscle fibre growth or recovery from atrophy is inhibited (Rosenblatt et al., 1994). The exerciseinduced
muscle damage-recovery process seems to have a similar cellular mechanism to postnatal
growth in that satellite cells become activated and fuse with the damaged muscle fibres (Goldring
et al., 2002). The process of myoblast fusion is thought to be integral to hypertrophy as it maintains
myonuclear domain size and thus the capacity for muscle protein synthesis. That is, hypertrophy of
a muscle fibre is thought to be dependant upon the insertion of new nuclei to maintain a constant
ratio of nuclei to cytoplasmic volume (McCall et al., 1998). However, few studies have
investigated the possible modulation of satellite cells and myonuclear number to accommodate REinduced
hypertrophy in normal human muscles. The results of a recent study (Kadi et al., 2004)
involving healthy adults confirmed previous work (Kadi & Thornell 2000; Roth et al., 2001) that
RE training does enhance the size of the satellite cell pool. However, the hypertrophy of individual
muscle fibres observed by Kadi et al. (2004) was not accompanied by an enhancement of the
myonuclear number. In this study, 30 days of training was sufficient to induce an increase in the
41
satellite cell pool and an additional 60 days of training further enhanced satellite cell frequency
(Kadi et al., 2004). Interestingly, the increase in satellite cell number following the RE program
was maintained for a long time after the cessation of training (up to 90 days) (Kadi et al., 2004).
These results support the hypothesis that in normal skeletal muscles, the transcriptional activity of
individual myonuclei may not be maximal (Newlands et al., 1998). That is, the transcriptional
capacity of existing myonuclei could sustain increased protein synthesis following RE training, the
size of each myonuclear domain simply increases and controls a greater amount of cytoplasm, but
once the transcriptional activity of existing myonuclei reaches a certain level, additional myonuclei
provided by satellite cell proliferation and fusion to the fibre become required to support a greater
cytoplasmic volume (Allen et al., 1995; Hikida et al., 1998; Kadi & Thornell 2000). In general, it
appears that an increase in myonuclear number only occurs when mammalian (including human)
muscle fibres hypertrophy by more than 26% (Cabric & James, 1983; Allen et al., 1995; Hikida et
al., 1998; Roy et al., 1999; Kadi & Thornell 2000). The existing myonuclei are able to increase
their rate of protein synthesis and support a moderate expansion of the cytoplasmic area in smaller
hypertrophy responses (Giddings & Gonyea 1992; Kadi et al., 2004).
From the extensive literature on myogenic differentiation, it is clear that growth factors
play crucial roles in the formation of muscle (Florini et al., 1991). One growth factor in particular,
insulin-like growth factor-1 (IGF-1) appears to be involved with the expression of a complete
spectrum of muscle-specific proteins (Florini et al., 1991; 1992). IGF-1 stimulates muscle protein
synthesis (Jurasinsk et al., 1995), and myoblast proliferation and differentiation in vitro (Florini
1996). IGF-1 is also capable of promoting muscle growth by activating the regulators of
translational initiation via the PI3K-PKB-mTOR pathway (Song et al., 2005). On the basis of their
mRNA transcripts, three isoforms of IGF-1 have been identified in human muscle, they are; IGFIEa,
IGF-IEb, IGF-IEc (Yang & Goldspink 2002; Hameed et al., 2003a). One of these, IGF-IEa is
expressed both in working and nonworking muscle (McKoy et al., 1999), and is very similar to the
isoform produced by the liver and probably has a similar function (Hameed et al., 2003a). The
isoform IGF-IEc in humans has been dubbed the mechano-growth factor (MGF); not to be
confused with IGF-IEb in rats that has also been called MGF (Hameed et al., 2003b). MGF is the
mechano-sensitive splice variant of IGF-1 that is expressed specifically and rapidly in response to
tissue damage (Haddad & Adams 2002; Hill & Goldspink 2003). MGF is thought to play an
important role in muscle hypertrophy firstly by direct stimulation of myofibrillar protein synthesis
but also activation of satellite cell proliferation and differentiation, followed by fusion of
differentiated myoblasts to hypertrophying fibres (Adams, 1998).
42
One of the special functions of the MGF isoform (and its unique carboxyl peptide
sequence) is to activate the muscle satellite (stem) cells for division (Yang & Goldspink 2002; Hill
& Goldspink 2003). Whereas the IGF-IEa peptide is localized throughout the cytoplasm, the MGF
peptide is localized in the nucleus and is expressed only in response to tissue damage (as it is
thought to prevent apoptosis). MGF has a short half life and does not appear to survive in the
extracellular compartment for any appreciable length of time (Yang & Goldspink 2002; Hill &
Goldspink 2003). For these reasons, MGF is thought to have a unique role in replenishing the stem
cell pool for repair and growth throughout life (Goldspink, 2005). The positioning of the satellite
cells (under the basal lamina) for proximal activation by MGF also adds to the weight of evidence
that suggests a key role of this IGF-splice variant in the muscle regenerative/hypertrophy response
to RE. However, only two studies have examined the expression of the IGF-1 splice variants in
human muscle in response to loading. The data from these studies suggest that in response to RE,
MGF is increased for a short time, soon after exercise, and this response was detected in young
adults, but not older adults (Hameed et al., 2003a). In general, the expression of all IGF-I variants
(a,b and c) appears to be down-regulated during the initial stages of recovery from RE (Psilander et
al., 2003). Unfortunately, a more detailed time course of IGF-I mRNA expression after RE is not
known. However, the small amount of research that has been completed on this topic has shown
that IGF-1 mRNA content measured during recovery from exercise is highly variable among
subjects; some exhibit a marked increase while other show no change (Hameed et al., 2003;
Psilander et al., 2003a). These large variations in IGF-1 expression might help to explain the wide
variation in hypertrophy responses that have been observed in a large group of people undertaking
the exact same RE program (Hubel et al., 2005).
The efficacy of muscle IGF-I is dependent not only on its expression but also on its
availability, which is regulated by a family of six IGF binding proteins (Kraemer & Ratamess
2005). For example, in muscle, IGFBP-4 has a high affinity for IGF-I and thus inhibits its
myogenic effects, whereas IGFBP-5 may facilitate (Florini et al., 1996) or inhibit (James et al.,
1996) IGF-I-stimulated differentiation under certain conditions. Additionally, IGFBP-1 has been
shown to inhibit IGF-I-stimulated protein synthesis (Frost & Lang 1999). In humans, RE is shown
to produce alterations in these binding proteins such as a decreased expression of IGFBP-4
(Bamman et al., 2001), that may alter the availability of active IGF-1 to tissues. These alterations
caused by RE have been detected in circulation over a 13 hour period (Nindl et al., 2001).
Therefore, the impact that RE has on the IGF-I system may not be completely related to the total
amount of IGF-I that is in circulation but rather the manner in which IGF-I is partitioned among its
family of binding proteins. However, another possible mechanism for increasing muscle IGF-I
availability is via androgen activation (Bamman et al., 2001). The results of one study suggest that
43
IGF-I action in muscle is secondary to androgen activity (Bamman et al., 2001). Rodent and human
studies confirm that RE increases androgen receptor expression and number along with IGF-1
mRNA (Deschenes et al., 1994; Bamman et al., 2001). Therefore, although the mechanism(s) are
not clear, muscle androgen and IGF-I activities appear to be related. To summarize the role of IGF-
1 (and its related splice variants) in the hypertrophy process; it is most likely to be multifaceted
involving localized activation via the short-lived MGF and the longer-lasting (~12 hours)
alterations in circulating IGF-1 binding proteins, as well as alterations in androgen receptor
expression. However, the integration of each of these aspects within the overall picture of muscle
accretion during RE training has not been elucidated. In particular, the residual effect that RE
appears to have on serum IGF-1 binding proteins (that appears to last up to 12 hours) may provide
a beneficial “carryover” effect when consecutive days of RE are performed. This may be one
advantage to training a different muscle group each day; a strategy that is characteristic of
bodybuilders.
Whereas growth factors are essential to activating satellite cell proliferation, the
progression of satellite cells into myoblasts requires the regulation of muscle specific proteins
belonging to the basic-helix-loop-helix family of transcription factors collectively called myogenic
regulatory factors (MRFs) (Murre et al., 1989). Members include MyoD, myogenin, myf-5, MRF-4
and MEF-2 which collectively function as dominant activators of muscle differentiation (Perry &
Rudnick 2000). Animal studies show overloaded muscle undergoing hypertrophy contains
increased mRNA levels of the MRFs (Carson & Booth 1998; Lowe et al., 1998; Mozdziak et al.,
1998). More recently, myogenin, MyoD, and MRF4 mRNA levels were shown to be transiently
elevated (from 100-400%) in human muscle in response to RE for up to 24 hours (Psilander et al.,
2003). After dimerization with a ubiquitous E protein, the MRFs bind to an E-box domain and
activate downstream muscle genes, such as MLC, troponin I, and desmin (Lin et al., 1991;
Wentworth et al., 1991; Li & Capetanaki 1993) which are building blocks of larger protein
complexes that synthesize functional and contractile elements. Therefore, the MRFs may contribute
to the hypertrophy response by regulating the transcription of more complex structures. In line with
such a possibility, a correlation between changes in muscle mass and MRF expression have been
observed in three studies (Hespel et al., 2001; Alway et al., 2002a; 2002b). One example is the
study by Hespel et al. (2001) that showed the MRF4 protein content in human muscle correlated (r
= 0.73; P < 0.05) with the concomitant change in muscle fibre CSA after 10 weeks of RE. In
addition, several studies have shown that muscle injury induces satellite cell activation along with
MRF expression (Rantanen et al., 1995; Marsh et al., 1997; Launay et al., 2001). The MRFs appear
to play a role in myoblast proliferation and differentiation (Murre et al., 1989). Myogenin has been
suggested to regulate the expression of a protein that is required for satellite cell fusion (Lowe &
44
Alway 1999). As satellite cell proliferation, differentiation, and fusion with muscle fibres are
necessary for continued hypertrophy, the MRFs might be involved in regulating muscle growth
over the long term. However, the MRFs are also shown to be up-regulated during atrophy (Voytik
et al., 1993; Alway et al., 2001). One explanation for the observed up-regulation of the MRFs
during both hypertrophy and atrophy could be that these myo-specific transcription factors regulate
other processes that coincide with changes in muscle mass such as the phenotypic expression of the
MHCs and fibre type. MyoD and myogenin have been implicated in regulating muscle fibre type
(Hughes et al., 1993; Voytik et al., 1993). In rodent muscle, myogenin has been found in slowtwitch
fibres and MyoD in fast-twitch fibres (Hughes et al., 1993; Voytik et al., 1993). In humans,
one study has shown that type-IIx MHC expression is associated with MyoD mRNA, whereas
type-I and IIa MHC mRNA (the slower twitch isoforms) were associated with myogenin mRNA
expression, 6 hours after a single bout of high overload RE (Willoughby & Nelson 2002). This
research confirmed previous work by Mozdziak et al. (1998) that MyoD and myogenin play a role
in MHC isoform gene expression in response to overload.
When attempting to describe gene transcription regulation in the complex picture of
muscle hypertrophy, it is also important to consider that the process may depend on the suppression
of some genes and increased transcription activity of others. A predominant action of a RE training
program in the overall hypertrophic response may be the selective inhibition of transcription or
degradation of specific mRNA prior to selective expression of other genes (Cameron-Smith, 2002).
One example of this selective inhibition may be the cytokine myostatin, also known as growth
differentiating factor-8 (GDF-8). A member of the transforming growth factor-β (TGF-β)
superfamily, myostatin appears to negatively regulate muscle growth (McPherron et al., 1997). In
adult muscle, myostatin expression is thought to decrease muscle size probably by way of a
glucocorticoid receptor meditated mechanism (Ma et al., 2001) to inhibit satellite cell activation
and induce proteolysis (Ma et al., 2003). However, myostatin may also inhibit myoblast
differentiation by down-regulating MRF expression (Langley et al., 2002). Myostatin mRNA
expression is increased in immobilized (inactive) muscle (Carlson et al., 1999), whereas loading is
shown to reduce the expression of this gene (Wehling et al., 2000). In humans, one study reported
that 9 weeks of concentric-only RE resulted in a decrease in myostatin mRNA (Walker et al.,
2004). Based on this data, one would assume that RE would be an effective activity that downregulates
myostatin expression to promote hypertrophy. However, the role of RE in nullifying
myostatin’s negative effects on muscle hypertrophy may not be so straight forward. Another study
that involved healthy males undertaking 12 weeks of high overload RE training, reported an
increase in glucocorticoid receptor and myostatin mRNA expression despite significant
hypertrophy (Willoughby, 2004). In this study, serum follistatin-like related gene (FLRG) was
45
shown to increase (127%) with a concomitant down-regulation of the activin IIb receptor. These
findings suggest that RE training may increase myostatin mRNA expression (and serum myostatin
levels) in the presence of hypertrophy, and the increase in myostatin expression is nullified by a
concomitant increase in its inhibitory-binding protein and decrease in its cell receptor (Willoughby,
2004). Therefore, while some studies suggest that for hypertrophy to occur, myostatin expression
must be down-regulated, other work suggests that hypertrophy occurs in the presence of an
increase in myostatin via a training-induced mechanism that inhibits this cytokine’s negative effect
on muscle growth.
Clearly, many questions remain unanswered with regard to the molecular events that
ultimately result in muscle hypertrophy. This discussion is concluded by highlighting some of the
controversial or unexplained phenomenon to which clarification would be particularly valuable for
improving the efficacy of RE prescription. Firstly, the initial events within muscle contraction that
activate PI3K/PKB and therefore MPS, are unknown. The IGF-1 response would be too slow and
the influence of insulin is thought to be minimal (Kimball et al., 2002). While calcium release (Ji et
al., 2002) and/or integrin signalling (MacKenna et al., 1998) are the likely candidates, so far there
is no evidence that links either of these to the PKB/m-TOR signalling cascade. Secondly, if one
keeps in mind that chronic RE is supposed to diminish the acute MPS response, then the impact of
training on the signalling pathway(s) that lead to MPS need to investigated. If the molecular
signalling responses that lead to MPS are down-regulated by training, then attempts to identify
possible sites of amplification/up-regulation within these pathways should be considered. Finally,
exactly how the EAA trigger the phosphorylation of mTOR and the MPS cascade remains unclear.
This is particularly important to clarify as recent work by Cuthbertson et al. (2005) has revealed a
diminished anabolic response to protein consumption in older humans via reduced activation of the
initiating protein complexes (such as mTOR). In light of this information, it now appears crucial to
find non-pharmaceutical ways to amplify the molecular signalling process that activate MPS. The
data produced by Karlsson et al. (2004) with regard to the synergistic effect of BCAA and RE on
MPS, via the greater phosphorylation of at least one of the initiatory proteins, underscores the
important role that strategic nutritional intervention may play in enhancing the hypertrophy
response to RE.
46
1.6 Acute responses to nutrient intake & resistance exercise
associated with hypertrophy
Early studies that utilized isotope labelled AA and tracer limb exchange methods
established that the large increase in whole-body synthesis observed after the consumption of a
meal is due mainly to the changes in protein synthesis rates in muscle. In fact, in response to
feeding, muscle mass contributes more than half of the total increase in whole body protein
synthesis (Rennie et al., 1982). Other studies that have used similar methods (Svanberg et al., 1996;
1997; 1999; 2000; Volpi et al., 1999; 2003; Rasmussen et al., 2002; Rennie et al., 2002) have
established the following; 1) the consumption of mixed macronutrient meals alter rates of MPS and
promotes a positive NPB; 2) MPS increases approximately 30-100% in response to a meal and the
major contributing factor to this response is AA; 3) the effect of insulin on MPS is dependant on
the availability of AA; 4) the net accretion of muscle protein that is observed after a meal is due
mostly to the large increase in MPS (inhibition of MPB contributes but to a lesser extent) and 5)
the anabolic response to feeding is rapid but transient. That is, during the post-prandial phase (1-4
hours after the meal) MPS is elevated while MPB is reduced. MPB increases in the post-absorptive
state (approx 5 hours after the meal) and MPS rates decline. Therefore, accretion of protein only
occurs in the fed state. More recently, it has also been confirmed that meal consumption stimulates
both myofibrillar and sarcoplasmic protein synthesis (3-fold compared to basal values)
(Mittendorfer et al., 2005). While there may be some small differences between muscle groups
(such as triceps versus soleus and vastus lateralis), in general, the stimulation of MPS by AA in
humans appears not to be influenced by anatomical location or fibre-type (Mittendorfer et al.,
2005).
The development of muscle hypertrophy would involve repeated disruption/damage to
certain muscle fibres that later must undergo a recovery phase such as a coordinated qualitative and
quantitative change in muscle proteins. The physiological processes responsible for these events
would be influenced by the availability of hormones, cytokines and growth factors which are in
turn, influenced by the presence (or absence) of the macronutrients. For example, it is clear that the
intake of CHO and protein (PRO) at a time close to RE (i.e., the hours just before and/or after a
workout) alters the acute hormonal and protein turnover response patterns to create an environment
that probably helps to optimize conditions for recovery (Kraemer et al., 1998b; Volek, 2004). The
consumption of proteins (or EAA) and RE have a synergistic effect on MPS that results in a
positive net balance (Biolo et al., 1997; Tipton et al., 1999). The strategic intake of CHO (glucose)
before or after RE does not appear to alter the MPS response (Roy et al., 1997; Borshiem et al.,
47
2004) but may reduce myofibrillar breakdown CHO (Roy et al., 1997). The combination of CHO
(35g) with EAA (6g) after RE is shown to provide a synergistic effect on MPS and NPB that is
much greater than either macronutrient alone (Miller et al., 2003). In fact, when this combination is
consumed 1 or 3 hours after a single bout of RE, MPS rates were shown to increase up to 400%
above pre-exercise values; the highest value ever recorded (Rasmussen et al., 2000). The same
small dose of EAA and CHO is also shown to promote a similar anabolic effect within muscle
when administered just before RE (Tipton et al., 2001). While these investigations utilized AA
solutions, other studies have confirmed that whole proteins, such as whey and casein, evoke an
anabolic response that is similar in magnitude to free form AA (Levenhagen et al., 2001; Tipton et
al., 2004; Wilborn et al., 2005). Therefore, it is clear that nutrient-timing (i.e., the consumption of
PRO and CHO before and/or after RE) not only augments the anabolic response, most importantly,
it shifts net protein balance to a positive state (albeit for a tra

transient time). This result can be (at
least partly) attributed to changes in the acute hormonal response pattern.
Few studies have examined the effects of macronutrient intake on RE-induced hormonal
response patterns (Chandler et al., 1994; Roy et al., 1997; Kraemer et al., 1998b; Tarpenning et al.,
2001). This is some what bemusing and disappointing given the large number of studies that have
assessed endocrine responses to RE, and the important role that hormones play in the regulation of
gene expression and protein metabolism. The following paragraphs of this section provide a brief
discussion on the acute endocrine (and associated immune) responses to RE and pre-post
macronutrient intake in relation to muscle hypertrophy.
Compared to a low-caloric (non-insulin stimulating) placebo, a high calorie PRO-CHO
supplement (total; 7.9 kcal/kg, 1.3g CHO/kg, and 0.7g protein/kg per day) consumed 2 hours
before and just after RE is shown to provide higher blood insulin levels in the hour after exercise
(Kraemer et al., 1998b). A macronutrient-induced stimulation of insulin is expected to improve the
anabolic response by increasing the uptake of nutrients (AA and glucose) while decreasing MPB
(Wolfe & Volpi 2001). Thus, the stage is set for the initial recovery phase. However, while insulin
is clearly associated with improving the anabolic response to RE, the impact of nutrient-timing on
other hormones such as GH, testosterone, cortisol and growth factors such as IGF-1 is less clear.
An increase in protein synthesis, lipolysis, and glucose conservation are all hallmark
responses to increased availability of GH (Thissen et al., 1994; Rennie 2003). The nutrient-timing
study by Kraemer et al. (1998b) demonstrated that PRO-CHO supplementation enhanced acute
serum GH responses for 30 minutes after exercise compared to the non-caloric placebo. This
response was observed on the first day but not the following two days of training (Kraemer et al.,
48
1998b). The reason for this is not clear. Chandler et al. (1994) also reported an increase in serum
GH in response to a similar dose of PRO-CHO immediately and 120 minutes after RE. In contrast,
Williams et al. (2002) reported no significant effect of PRO-CHO on the GH response to RE. The
regulation of hepatic IGF-1 is characteristic of GH (Thissen et al., 1994). Kraemer et al. (1998b)
also reported that nutrient-timing with the PRO-CHO supplement elevated serum IGF-1 levels for
30 minutes after RE in 2 out of 3 training days. After a 6 month training program, an increase in
(resting) plasma IGF-1 has been observed in response to the daily consumption of a PRO-CHO
supplement (42g PRO, 24g CHO) as opposed to CHO (70g) alone (Ballard et al., 2005).
Furthermore, Willoughby et al. (2005) recently reported that 10 weeks of heavy RE combined with
protein supplementation before and after each workout was effective at increasing serum IGF-1 and
muscle MGF mRNA expression. Although nutrient-timing during RE may increase serum IGF-1
concentrations, the anabolic action of this growth factor on tissue is thought to reside in alterations
within its binding proteins (Friedlander et al., 2001). Separately, meal consumption (Lee et al.,
1997) and RE (Nindl et al., 2001) appear to influence the regulation of the IGF-1 binding proteins.
However, no studies have examined the impact of nutrient intake and RE on the IGF-1 binding
proteins and muscle anabolism.
Testosterone is known to promote anabolism by augmenting the synthesis of muscle
protein, and the consumption of PRO-CHO before and after RE appears to have an effect on
circulating levels of this hormone. The nutrient-timing study by Kraemer et al. (1998b) also
assessed acute testosterone responses. The researchers reported an acute increase in circulating
testosterone followed by a sharp decrease (to levels that were significantly lower than baseline)
when the PRO-CHO supplement was administered before and after RE. This response was
consistently observed on each of the three training days assessed. Studies by Chandler et al. (1994)
and Bloomer et al. (2000) have also shown a similar response. Collectively, these studies indicate
that although RE evokes an increase in circulating testosterone, nutrient intake close to RE results
in a decrease in circulating testosterone concentrations in the hours post-exercise; a finding that is
directly opposite to RE studies that did not involve nutrient intake. This rapid decrease in blood
testosterone levels in response to PRO-CHO consumption close to RE may be due to increased
metabolic clearance of this hormone such as increased uptake by muscle. At least one study
supports this assumption. Chandler et al. (1994) showed that a decline in circulating testosterone in
response to nutrient-timing after RE was not linked to a decrease in lutenizing hormone production.
Nutrient-timing provides a dramatic increase in MPS in the hours after a workout (Tipton et al.,
1999; 2003). Therefore, the drop in circulating testosterone could be due to increased uptake by
muscle to facilitate this process. To further support this contention, Volek (2004) reported that a
49
meal-induced decrease in post-workout circulating testosterone corresponded with an increase in
muscle androgen receptor content.
The adreno-cortical steroids such as cortisol (or glucocorticoids) serve to induce catabolic
processes within muscle that are thought to be essential to the remodelling response (Nieman et al.,
2004). However, the data on the response pattern of this hormone to RE and macronutrient
consumption is conflicting. Cortisol is regulated by pituitary adrenocorticotropin (ACTH), which in
turn, is under the influence of hypothalamic corticotrophin-releasing hormone (Smith, 2000). An
important action of the glucocorticoids is local and systemic modulation of inflammatory
mechanisms related to cytokine-meditated cortisol secretion that occurs via the hypothalamicpituitary-
adrenal (HPA) axis (Braun & von Duvillard 2004). The HPA axis is sensitive to a variety
of different stressors including exercise and feeding. Exercise causes numerous changes in
immunity that reflects physiological stress and immunosuppression, whereas macronutrient
consumption has the potential to alter this response (Nieman et al., 2004). For example, CHO
supplementation before, during and/or after exercise is proposed to sustain glucose availability and
reduce substrate stress which results in the attenuated stimulation of the HPA axis and lower
cortisol production (Braun & von Duvillard 2004). In turn, this is thought to reduce perturbations in
immune status that lead to lower cytokine production (Febbraio et al., 2003) which attenuates the
inflammatory response (Braun & von Duvillard 2004). However, very few studies have assessed
cytokine responses to RE. One recent study by Neiman et al. (2004) reported that the ingestion of a
6% CHO solution did not alter modest increases in plasma IL-6, IL-10, IL-1ra, and IL-8 (and
muscle gene expression of these cytokines) in a group of strength-trained participants that lifted
weights for 2 hours. Many aspects of protein turnover are controlled by the classic steroid-hormone
binding mechanism. Like testosterone, cortisol also binds to a cytoplasmic receptor and activates a
receptor complex so that it can enter the nucleus, bind specific response elements on DNA and act
directly at the genetic level. In doing so, cortisol (and other hormones) alter the transcription and
subsequent translation of specific proteins (Carson, 1997). These processes are thought to be
integral to tissue remodelling and adaptation to exercise. This may be one reason why the majority
of studies that have assessed cortisol responses to RE show that macronutrient consumption does
not alter the response pattern of this hormone (Kraemer et al., 1998b; Bloomer et al., 2000; Koch et
al., 2001; Williams et al., 2002). However, one study has shown that supplementation with CHO
blunted the cortisol response to RE and this had a positive effect on muscle hypertrophy
(Tarpenning et al., 2001).
In the study by Tarpening et al. (2001), CHO supplementation (6% solution) resulted in
significantly greater gains in both type-I (19.1%) and type-II (22.5%) muscle fibre CSA than a
50
placebo after 12 weeks of RE. The difference in the cortisol response accounted for 74% of the
variance (r = 0.8579, P = 0.006) of change in type-I muscle fibre area, and 52.3% of the variance (r
= 0.7231, P = 0.04) of change in type-II muscle fibre area. This study is unique because it is one of
few that have linked an acute change in the pathway of adaptation (i.e., decrease in post-exercise
cortisol) to a chronic adaptation (i.e., increased muscle size). RE training is shown to reduce the
magnitude of the cortisol response to this exercise (Staron et al., 1994; Kraemer et al., 1999) and
this change appears to be mediated by a reduction in ACTH responses to the exercise stress
(Kraemer et al., 1999). Based on these findings, a reduction in cortisol responses over the longer
term have been proposed as one possible mechanism of protein accretion during RE training
(Kraemer et al., 1999). Therefore, the effects of CHO supplementation reported by Tarpenning et
al., (2001) warrant further investigation.
To summarize the literature regarding the acute responses to macronutrient intake and RE;
firstly, it is clear that the strategic consumption of PRO and CHO close to RE (nutrient-timing)
augments the acute anabolic response but more importantly, shifts protein balance to a positive
state in the hours following exercise. Secondly, nutrient-timing appears to achieve this effect by
providing an abundance of material during high-intensity muscle contraction that alters the acute
hormonal and protein turnover response patterns. However, the chronic affects that may arise from
these acute metabolic perturbations remains largely, unknown. Very little work has linked dietinduced
alterations in protein turnover to chronic adaptations such as strength or hypertrophy
development. The immune and endocrine systems are likely to be critical components in the
pathways that intergrate diet, RE and hypertrophy. Therefore, a greater focus on this topic is
warranted if we are to ascertain a better understanding of how to optimize the hypertrophy response
to nutrient intake and exercise.
1.7 Chronic responses to nutrient intake & resistance exercise
associated with hypertrophy
RE is fundamentally anabolic but is only able to shift NPB to a positive value in the
presence of protein-containing meals. RE and meal consumption interact synergistically to provide
a NPB that is greater than what can be obtained from feeding alone. The chronic response of this
interaction between RE and meal consumption on NPB is thought to be muscle hypertrophy
(Phillips et al., 2005). However, to date, no studies have linked the acute metabolic perturbations
(described previously), to chronic adaptations such as increases in muscle size and strength.
Nonetheless, there is an increasing amount of evidence that supports the theory that nutrient-timing
results in greater muscle hypertrophy. For instance, nutrient-timing with protein (15g of EAA
51
before and after RE) is shown to result in higher net protein accretion over a 24 hour period (Tipton
et al., 2003). In terms of longitudinal investigations, a number of RE training studies report
significantly greater muscle fibre hypertrophy (Esmarck et al., 2001; Andersen et al., 2005) or
statistical trends for greater LBM accretion from nutrient-timing. In a group of older (untrained)
adults (74 ± 2 yrs) undertaking RE for 12 weeks, immediate post-workout provision of a mixedmacronutrient
supplement resulted in significant muscle fibre hypertrophy (22% increase in CSA
of the type-IIa fibres) and a 7% increase in total thigh muscle CSA. In contrast, a matched group
that was fed 2 hours after exercise did not demonstrate hypertrophy (Esmarck et al., 2001). In a
group of physically active (but not RE-trained) young males Chromiak et al. (2004) examined the
effects of post-workout PRO-CHO supplementation on LBM gains during 10 weeks of RE
training. The group given a PRO-CHO supplement (25g PRO, 76g CHO) within an hour of
finishing each RE session (4 times/week for 10 weeks) demonstrated a 3.4kg increase in LBM
compared to a 1.5kg gain seen in a control group given a (92g) dose of CHO (Chromiak et al.,
2004). In another study, Rankin et al. (2004) utilized untrained males and a similar supplement
protocol to Chromiak et al. (2004), and obtained the same result. Only one study has examined the
effects of pre- and post- nutrient consumption on chronic adaptations to RE training (Andersen et
al., 2005). In this study, after 14 weeks, the group of young men provided with a protein
supplement before and after each workout (4x/week, 25g), demonstrated hypertrophy of the type-I
and type-II muscle fibres, whereas no change above baseline was observed in a matched group
given an equivalent dose of CHO.
When interpreting the results of these nutrient-timing studies it’s important to remember
that the investigations restricted the intake of certain macronutrients (namely protein) in the hours
surrounding the workout. That is, the participants were not permitted to consume any other
nutrients other than the designated supplement up to 3 hours before and after each workout
(Esmarck et al., 2001; Chromiak et al., 2004; Rankin et al., 2004; Andersen et al., 2005).
Therefore, the results obtained may have less relevance in a real-world setting. Meaning, strength
athletes and others that desire muscle mass gains would not usually go without consuming a
protein-rich meal for 3 hours after exercise. Nonetheless, based on these findings, it has been
suggested that the strategic use of a PRO-CHO supplement just before and after each workout may
provide the ideal anabolic situation for muscle hypertrophy (Volek, 2004). However, no research
has examined the effects of supplementation with PRO-CHO before and after RE in the presence of
a normal eating pattern. Additionally, no studies have examined whether this supplement-timing
strategy provides greater benefits in terms of muscle hypertrophy or strength development
compared to supplementation at other times during the day.
52
Although nutrient consumption close to RE promotes muscle anabolism in the short term, a
single bout of RE affects (muscle) protein turnover for at least 24 hours, even in trained individuals
(Chesley et al., 1992; MacDougall et al., 1995). Therefore, the type, timing and quantity of the
energy-yielding macronutrients (CHO, PRO and fat) consumed throughout each day also has the
potential to influence net protein accretion and chronic adaptations. For example, meal type,
quantity and frequency is shown to influence the potentially substantial gains and losses of body
proteins that occur during the diurnal cycle of feeding and fasting (Millward et al., 1995; 1996).
Total energy intake also appears to influence the hormonal response patterns associated with
hypertrophy (Forbes et al., 1989; Sallinen et al., 2004). For example, a short term increase in daily
energy intake above maintenance requirements not only increases whole body protein synthesis, it
also elevates resting concentrations of GH and testosterone along with an increase in LBM (Forbes
et al., 1989; Fern et al., 1991; Jebb et al., 1996). Serum IGF-1 concentrations have the potential to
influence chronic adaptations to RE as they are shown to correlate with the synthesis rates of MHC
in both young and older adults (Balagopal et al., 1997b). The restriction of dietary energy and/or
protein is shown to have a negative effect on serum IGF-I concentrations whereas an intake above
maintenance requirements is shown to increase the circulating concentrations of this growth factor
(Thissen 1994). Overall energy needs during any form of exercise training are principally
determined by the individual’s training load (i.e., the intensity x frequency x duration of daily
workouts), body mass and exercise goals (Hawley et al., 1995). As protein synthesis is an ATPdependant
process, it is generally accepted that a slight increase in daily energy (kilojoule) intake
above that needed for weight maintenance would augment muscle mass gains during RE training
(Lambert et al., 2004). While overall energy intake would appear to be an important aspect with
regard to dietary influences on the hypertrophy response, no research has determined what amount
is required to enhance muscle mass gains during RE training (Lambert et al., 2004).
Aside from overall energy intake, it is clear that the type and quantity of macronutrients
consumed each day has the potential to influence adaptations during RE training. For example, the
quantity and composition of dietary fat has been shown to impact resting (Volek et al., 1997a) and
exercise-induced circulating testosterone (free and bound) concentrations (Raben et al., 1992;
Sallinen et al., 2004). Additionally, certain dietary fats such as conjugated linoleic acid (CLA) and
eicosapentaenoic acid (EPA) have shown potential to improve body composition (Gaullier et al.,
2004; Bhattacharya et al., 2005) or promote LBM directly (Barber 2001; Whitehouse et al., 2001).
Therefore, the effects of quantity and composition of dietary fat intake on muscle and strength
development during RE is a topic that deserves further attention. While dietary fat makes an
important contribution to overall energy intake, to date, no direct evidence suggests increasing
dietary fat intake per se, would improve adaptations to RE training (Lambert et al., 2004).
53
Particularly, not at the expense of the PRO and CHO; the macronutrients that predominantly affect
muscle (and whole body) anabolism and catabolism.
CHO alters muscle (and whole body) anabolic and catabolic responses to RE. As these
processes are stimulated on a frequent basis during a RE training program, dietary CHO intake has
the potential to influence chronic adaptations. For example, muscle glycogen stores affect work
capacity during intense exercise (Balsom et al., 1999). The daily maintenance of glycogen stores
appears to be directly related to the CHO in the diet. In particular, the amount of muscle glycogen
synthesis in the 24-hour period post-exercise is directly correlated (r = 0.84) to the amount of CHO
ingested and the timing of that ingestion (Costill, 1991). More specifically related to RE,
inadequate CHO intake may compromise adaptations by impairing glycogen resynthesis and
performance in subsequent bouts (Haff et al., 1999). Also, the extent of exercise-induced protein
degradation is shown to be inversely related to CHO availability (Lemon & Mullin 1980). Aside
from the affect of CHO on protein catabolism, the results of one recent study suggests that
performing RE with low muscle glycogen stores may impair the activation of MPS (via regulation
of the Akt pathway) (Creer et al., 2005). However, despite the obvious importance of an adequate
CHO intake, no research has attempted to define the amount that may improve muscle strength and
hypertrophy development during RE training (Lambert et al., 2004).
Whereas dietary fat and CHO requirements during RE training have received little
attention by the scientific community, protein requirements have been a topic of great controversy
for many years and a clear consensus has still not been reached (Lemon, 2001). In particular, it is
unclear whether or not a high protein intake above the recommended dietary allowance (RDA)
may enhance muscle and strength development (Lemon, 2001). Investigations involving both
strength and endurance athletes indicate that exercise does increase the need for AA/protein;
approximately 1.5 to 2 times the RDA does appear to be advantageous for muscle and strength
development during RE (Lemon, 1992; Tarnopolski et al., 1992). However, at least one
longitudinal study (involving untrained, older adults) demonstrated that a protein intake of twice
the RDA did not enhance LBM accretion during 12 weeks of RE (Campbell et al., 1995).
Conversely, previous work has shown that the quantity of protein in the diet does influence the
potentially substantial gains and losses of body proteins that occur during the diurnal cycle of
feeding and fasting (Pacy et al., 1994; Price et al., 1994; Millward et al., 1995; 1996). Studies that
have examined the impact of protein quantity (from 0.4- 2.5g protein/day) on leucine kinetics (as
an index of whole body protein turnover) have found a greater stimulation of protein synthesis
from high vs. low protein-containing meals (Gibson et al., 1996; Forslund et al., 1998).
Additionally, some (Fern et al., 1991; Forslund et al., 1998), but not all (Tarnopolsky et al., 1992)
54
short-term studies that have investigated protein intake during exercise suggest that up to 3 or 4
times the RDA will enhance whole body protein synthesis and/or protein accretion in young,
healthy adults. These discrepancies in the literature may be due to the methodologies used in each
study. For example, the results obtained from nitrogen balance studies suggest that exercise may
enhance nitrogen efficiency and therefore, possibly decrease protein requirements (Butterfield &
Galloway 1984). Conversely, studies that have utilized the metabolic tracer technique suggest that
dietary protein recommendations (based on the nitrogen status technique) may underestimate
resting needs by 40-90% (Young et al., 1994; 1999). Regardless of the discrepancies in the
literature, reports show that bodybuilders and other strength athletes habitually supplement their
diet with extra protein to achieve intakes that are sometimes up to 4 times the RDA (Kleiner et al.,
1994; Marquart et al., 1998).
The idea that a high protein intake is essential to gaining muscle mass during RE appears
to be imbedded into the culture of strength athletes (Di Paquale 2000; Lemon 2001) and this is
probably due to a number of reasons. Firstly, a variety of factors may influence protein
requirements such as, total energy intake (Gibson, 1996; Young & Borgonha 1999), CHO
availability (Lemon & Mullin 1980), exercise intensity, type and duration (Lemon et al., 1992;
Tarnopolsky et al., 1988), training history (Lemon et al., 1992; Tarnopolsky et al., 1992), age
(Campbell et al., 2001) and timing of macronutrient intake (Roy et al., 1997; Tipton et al., 2003).
Secondly, a high protein intake (up to 3 times the RDA), is reported to be a safe strategy in healthy
humans (Poortmans & Dellalieux 2000) that has favourable effects on body composition (i.e.,
preservation of LBM and greater reduction of body fat) (Fansworth et al., 2003; Layman, 2004).
Additionally, when a higher proportion of overall energy intake is protein (21% or 2.5g/kg/day) a
positive (significant) whole-body protein balance has been reported along with a negative fat
balance (Forslund et al., 1999). Finally, strength athletes may consume a high protein diet simply
because they know that the RDA does not take into account what may be necessary to maximize
athletic performance (Lemon, 2001). Some renowned scientists in this area recommend that high
protein intakes are not necessary for muscle hypertrophy during RE training (Rennie et al., 2004).
Others caution that the underlying biology of maintenance AA needs may be much more
complicated than simply the support of protein metabolism itself; there are so many gaps in our
knowledge of this subject that until the various functions of AA are better understood at both the
mechanistic and quantitative level, current dietary recommendations for both healthy and sick
humans should remain at an intellectually unsatisfactory empirical level (Reeds & Bolio 2002).
An individual’s habitual protein intake may prove to be one of the more important
variables that influence the size of human muscle mass since recent work has confirmed that the
55
concentration of EAA in the blood (plasma) regulates protein synthesis rates within muscle (Bohe
et al., 2003). In particular, it is the extracellular, as opposed to the intracellular, concentration of
EAA that controls the rate of protein synthesis within muscle (Bohe et al., 2003). That is, when
plasma EAA levels are low, MPS rates decline (Kobayashi et al., 2003). Conversely, MPS rates
increase in a linear fashion with increased EAA availability (Bohe et al., 2001). This response
occurs up to a point where very high plasma concentrations (>2.5-fold normal) saturate this
response (Rennie et al., 2002). Once a saturation point is achieved, MPS falls back to basal levels
despite continued AA administration (Bohe et al., 2001). This relationship between plasma EAA
and MPS has been established via infusion studies in the non-exercised state. However, acute
response studies involving oral doses of EAA (Tipton et al., 1999; 2001; 2003), or whole proteins
(Tipton et al., 2004; Paddon-Jones et al., 2005a), show a similar (transient) effect. Aside from
modulating MPS rates, the presence of AA also inhibits MPB, although not as powerfully as they
promote protein synthesis (Rennie et al., 2002). AA inhibit protein breakdown in other tissues such
as the liver, perhaps more powerfully than they do in muscle (Mortimer et al., 1991). A 40g oral
dose of AA appears to temporarily increase the size of the free AA pool (Tipton et al., 2003) so that
while a saturation point may be reached with regard to the stimulatory effect on MPS, an inhibitory
effect on MPB may still be obtained (Rennie et al., 2002). Any inhibitory effect of exogenous AA
on protein breakdown would (theoretically) decrease the size of the free pool available for protein
synthesis, providing a link between the two arms of the processes of protein turnover via the
extracellular pool; this would effectively integrate the action on a whole body basis. The bottom
line is that the stimulation of MPS is the facilitating mechanism of hypertrophy and the
concentration of EAA in the blood is a known regulator of protein synthesis rates within muscle.
Therefore, the type, frequency and quantity of protein consumed during the hours of each day
would influence muscle protein accretion and the magnitude of hypertrophy obtained from a RE
training program. Manipulation of the diet to create and maintain a high concentration of EAA in
the blood stream during RE training that would influence muscle protein accretion and hypertrophy
represents a rather exciting area of research that is yet to receive systematic investigation.
The results of several recent investigations support the theory that dietary manipulation of
EAA availability may influence chronic changes in muscle mass. For instance, there is data that
shows a correlation between acute stimulation of MPS (via protein consumption) and chronic
changes in muscle mass (Paddon Jones et al., 2004). In this study, subjects were given a EAA
supplement three times a day for 28 days during bed rest. Results indicated that acute stimulation
of MPS provided by the supplement on day 1 resulted in a net gain of ~7.5g of muscle over a 24
hour period (Paddon-Jones et al., 2004). When extrapolated over the entire 28 day study, the
predicted change in muscle mass corresponded to the actual change in muscle mass (~210g)
56
measured by DEXA (Paddon-Jones et al., 2004). Additionally, the role of protein supplementation
in promoting muscle tissue accretion has recently been highlighted (Paddon Jones et al., 2005a). In
this study, supplementation (15g of EAA and 30g of CHO) was shown to produce a greater
anabolic effect (increase in net phenylalanine balance) than ingestion of a mixed-macronutrient
meal, despite the fact that both interventions contained a similar dose of EAA. Furthermore, the
consumption of the supplement did not interfere with the normal anabolic response to the meal
consumed 3 hours later. This finding is particularly important; the refractory period when restimulation
of MPS may occur with repeat meal consumption was previously unknown (Rennie et
al., 2004). The results of the investigations by Paddon-Jones et al. (2004; 2005a) suggest that
supplementation between regular meals may provide an additive effect on net protein accretion due
to a more frequent stimulation of MPS. In combination with RE, repeated stimulation of MPS via
the frequent consumption of protein (supplements and meals) would most likely influence the size
of muscle mass, but this has not been investigated directly. When discussing the possible chronic
effects of repeated meal consumption on muscle protein metabolism, the role of insulin must also
be considered. Insulin inhibits muscle proteolysis (Fryburg et al., 1995; Bolio et al., 1999),
probably via the ATP-ubiquitin-dependent proteolytic system that is responsible for myofibrillar
protein breakdown (Reid, 2005). The consumption of a CHO-PRO-containing meal triggers an
insulin response; the extent of which is dependant on the glycemic index but also the glycemic load
of the meal (Alfenas & Mattes 2005). Therefore, the consumption of frequent CHO-PRO meals
throughout a 24 hour period would promote anabolism not only via an increased stimulation of
protein synthesis but also a reduction in protein breakdown (Rennie 2005). While it is logical to
speculate that these responses may improve the rate of whole body protein accretion during RE, the
effects of repeated meal consumption on muscle or whole body protein turnover during RE training
has not been investigated.
Strength and hypertrophy training typically involves multiple workouts throughout the
week that utilize different muscle groups each training session (Kraemer et al., 2002). However,
the impact of daily workouts on net protein balance and accretion within muscle(s) is a topic that
has not been considered. For example, the studies that have assessed the metabolic impact of RE
and feeding have only examined the acute response to one or two small meals consumed close to a
once-off bout of RE within a particular muscle group (Rasmussen et al., 2000; Tipton et al., 1999;
2001; 2004). The measurements obtained may reflect changes in the muscle assessed but they do
not represent changes within other muscle groups that would influence protein metabolism at the
whole body level. For instance, it is clear that a single bout of RE induces an increased
intramuscular "recycling" of AA from protein breakdown and increased inward transport from the
AA pool; muscle protein balance does not become positive until exogenous AA are provided
57
(Biolo et al., 1997). It is also clear that until exogenous AA are provided, the most readily available
source of AA for utilization is from an increase in the rate of muscle protein breakdown (Biolo et
al., 1995, Phillips et al., 1997). Therefore, regular workouts (performed throughout the week)
would provide a systematic stimulation of this process throughout all muscles within the body.
Unless the provision of exogenous AA are provided close to RE on every occasion, consecutive
workouts (involving different muscle groups) could result in a “robbing Peter to pay Paul” scenario
with regard to AA flux and distribution between muscle groups—a response that may reduce or
even eliminate a positive balance obtained from a previous days training in another muscle group.
It is apparent that a single bout of RE can stimulate protein turnover for at least 24 hours
(MacDougall et al., 1995) and a RE program can affect resting protein turnover rates (Kim et al.,
2005). Therefore, the “residual” impact of consecutive days of RE on muscle (and whole body)
protein turnover clearly has the potential to affect protein accretion and the hypertrophy response to
training. The interaction of repeated meal consumption during consecutive days of RE on muscle
protein metabolism (and net accrual) is an important area of research that must receive systematic
investigation if we are to ascertain a better understanding of the processes that may improve muscle
hypertrophy during RE training.
Due to the lack of data that may link acute metabolic perturbations to chronic adaptations
from training and dietary intervention, any prudent discussion of hypertrophy should include the
characteristics of the one population group that represents a living “physiological model” of this
prized adaptation. That is, bodybuilders; men and women that have spent a large portion of their
lives in the pursuit of few other athletic/recreational endeavours except the development of muscle
mass. Although an extreme model (and one that is not without apparent flaws), the dietary and
training characteristics of this group of athletes may provide a better understanding of what is
required to systematically increase muscle mass. To achieve muscle hypertrophy and strength
improvements, bodybuilders characteristically perform daily RE workouts with different muscle
groups and consume high-protein/energy intakes (Lambert et al., 2004). Daily energy intake is
typically divided into a number of small, mixed-macronutrient meals that are consumed frequently
(every 2-3 hours) over the course of their day (24 hour period) (Marquart et al., 1998; Leutholtz &
Kreider 2001). As mentioned previously, this type of eating pattern would provide some important
physiological advantages for promoting hypertrophy. For example, protein synthesis in muscle is a
continuous activity that requires a balanced supply of twenty different AA (Rennie et al., 2004).
The consumption of frequent, protein-rich meals throughout the day may create and maintain a
high level of EAA in the bloodstream that would promote a higher rate of MPS and/or a reduction
in MPB, reduce the potentially substantial losses of body protein that occur during the diurnal cycle
of normal feeding and fasting, and ensure a higher net gain in muscle protein in response to
58
training. This eating pattern would also promote steady-state blood glucose and insulin levels more
so than the traditional three-meals-a-day that most adults follow (Ryan, 2000); thus, minimizing
protein breakdown while promoting the inward transport of nutrients to tissues. Therefore, the
dietary strategies that bodybuilders follow would appear to provide a favourable bio-environment
that is conducive to muscle protein accretion.
1.8 The potential of whey protein to enhance muscle hypertrophy
Although the high protein intakes that many bodybuilders consume can be met easily by
simply increasing energy intake, many strength athletes use protein supplements to achieve high
protein intakes (Brill & Keane 1994; Marquart et al., 1998; Leutholtz & Kreider 2001). Aside from
quantity, one reason for this may be that different types of protein are known to affect whole body
protein anabolism and accretion (Biorie et al., 1997; Bos et al., 2003; Dangin et al., 2003) and
therefore, have the potential to affect muscle and strength development during RE training (Lemon
et al., 2002). The type of protein consumed may influence protein accretion during RE training due
to variable speeds of absorption (Dangin et al., 2001; 2003) differences in AA profiles (Bos et al.,
2003; Phillips, 2005), unique hormonal responses (Bratusch-Marrain & Waldäusl 1979; Carli et al.,
1992) or positive effects on antioxidant defence (Lands et al., 1999). Whey protein (WP) is the
collective term for the soluble protein fractions extracted from dairy milk. WP supplements (80%+
protein concentrates and isolates) generally contain a higher concentration of EAA (45-55g/100g of
protein) than other protein sources and therefore score highly on most evaluations of protein
quality (Bucci & Unlu 2000; Di Pasquale 2000). In particular, they are the richest known source of
BCAA such as leucine (up to 14g/100g protein) (Bucci & Unlu 2000). The BCAA have been
shown to alter concentrations of circulating GH (Bratusch-Marrain & Waldäusl 1979), insulin
(Ferando et al., 1995) and testosterone (Carli et al., 1992) as well as attenuate protein degradation
(Blomstrand et al., 1992; MacLean et al., 1994; Coombes et al., 1995). Leucine in particular, is an
established modulator of muscle protein metabolism and has been identified as a key regulator in
the translation initiation pathway of muscle protein synthesis (Anthony et al., 2001). Oral BCAA
supplementation is shown to augment the phosphorylation of p70-S6k; a major regulatory kinase in
the activation of MPS in response to RE (Karlsson et al., 2004).
The acute response to a single dose of WP is a higher (but transient) blood AA peak and
stimulation of (whole body) protein synthesis when compared to other high quality protein sources
such as casein (the other major bovine milk protein) (Boirie et al., 1997; Dangin et al., 2001; 2003).
A most relevant finding is that the consumption of WP in mixed-macronutrient meals provides a
higher stimulation of MPS and higher net gain in whole body protein in both young and older
59
adults in comparison to isonitrogenous casein meals (Dangin et al., 2003). Aside from its high
concentration of EAA, WP is a rich rare source of Cyst(e)ine residues. WP supplements generally
contain a 3- to 4-fold higher concentration of Cys compared to other protein sources (Bucci & Unlu
2000). As discussed earlier in this chapter (refer to figures 1.2 & 1.3), Cys is thought to play a key
role in the regulation of whole body protein metabolism and LBM. An abundant supply of Cys in
the blood is necessary for hepatic catabolism of this AA into sulphate and protons; a process that
down-regulates urea production, promotes GSH synthesis and shifts whole body nitrogen disposal
in favour of preserving the muscle AA pool. In humans, WP supplementation (up to 1g/kg/day) is
the only protein source shown to augment this pathway of protein metabolism (Lands et al 1999;
Middleton et al., 2004), possibly in a dose-dependant manner (Marriotti et al., 2004). Therefore,
due to its high concentration of EAA, Cys and ability to promote higher net protein accretion, the
incorporation of WP into the diet may enhance muscle strength and hypertrophy development
during RE.
Some studies demonstrate that supplementation with WP does enhance some of the
adaptations desired from RE. WP is shown in several human (Lands et al., 1999; Burke et al.,
2001; Cribb et al., 2006) and rodent (Bouthegourd et al., 2002; Belobrajdic et al., 2004) trials to
improve body composition (i.e., an increase in LBM and/or a decrease in fat mass). During 6
weeks of RE training, WP supplementation (1.2g/kg/day) in RE-trained individuals resulted in an
almost 2-fold higher gain (2.1 vs. 1.2kg) in LBM and a better gain in bench press strength
compared to a CHO control group (Burke et al., 2001). In a double-blinded study that used two
groups of matched, RE-trained young men, our laboratory has previously demonstrated a
significantly greater gain in LBM and strength in a group provided with a hydrolysed WP isolate
(1.5g/kg/day) compared to a matched group given an equivalent dose of casein (Cribb et al., 2006).
Other studies have also reported body composition improvements from WP supplementation. In
direct comparison to other quality proteins such as casein, WP is shown to maintain LBM and
reduce fat mass via enhanced antioxidant (GSH) status (Lands et al., 1999), more efficient fat
oxidation in the hours after exercise (Bouthegourd et al., 2002), improved muscle insulin
sensitivity (Belobrajdic et al., 2004) or suppression of hepatic fatty acid synthesis along with
increased fat utilization by muscle (Morifuji et al., 2005). Recent years have seen an increase in the
number of trials that have involved RE training and dairy protein supplementation. However, while
a number of these studies report changes in strength and body composition (Demling & DeSanti
2000; Antonio et al., 2001; Burke et al., 2001; Chromiak et al., 2004; Rankin et al., 2004; Cribb et
al., 2006) none have compared these changes alongside hypertrophy responses at the cellular level,
such as fibre-specific (i.e., type-I, IIa, IIx) CSA as well as the sub-cellular level, such as contractile
60
protein content. Additionally, no studies have examined the influence of supplementation with WP
exclusively on muscle fibre morphology during RE training.
1.9 The potential of creatine monohydrate to enhance muscle hypertrophy
The rationale for the use of nutritional supplements to enhance exercise capacity is based
on the assumption that they will confer an ergogenic effect above and beyond that afforded by
regular food alone. The proposed ergogenic effect of many nutritional supplements is based on a
presumptive augmentation of a metabolic pathway. However, under the rigor of scientific control,
ingestion of the nutrient often fails to translate into a quantifiable change in exercise performance.
Intriguingly, this does not appear to be the case for creatine monohydrate (CrM)
(n[aminoiminomethyl]-N-methylglycine). Probably due to its vital role in the energy (ATP)
production pathway, investigations involving Cr can be found as early as 1914 (Folin & Denis
1914). However, studies completed in the early 1990’s demonstrated that a CrM “loading phase” (4
x 5g/day for 5 days) consistently elevated muscle Cr concentrations by approximately 25mmol/kg
dry mass (Harris et al., 1992), or 15–40% (Balsom et al., 1994). This sparked a series of
investigations that attempted to link CrM supplementation with improved exercise performance
(Greenhaff et al., 1993; 1994; Birch et al., 1994; Earnest et al., 1995; Casey et al., 1996; Febbraio
et al., 1996; Kreider et al., 1996; 1998; McKenna et al., 1999). Since that time, well over 200
studies have examined the effects of CrM supplementation on exercise performance. A majority of
these studies have reported an ergogenic effect, particularly during exercise involving repeated,
short bouts of extremely powerful activity (Birch et al., 1994; Greenhaff et al., 1993; 1994; Balsom
et al., 1995; Casey et al., 1996; Vandenberghe et al., 1996; Rawson & Volek 2003). However, the
precise physiological mechanisms linking increased muscle Cr content to improved functional
capacity still remain elusive.
Oral supplementation with CrM enters the circulation where active uptake by tissues such
as muscle is facilitated by a Na+ dependent transporter against a concentration gradient (Guimbal &
Kilimann 1993). Supplementation is shown to increase not only muscle Cr and PCr concentrations
but also other tissues with low baseline Cr content such as the brain, liver and kidney (Dechent et
al., 1999; Leuzzi et al., 2000; Ipsiroglu et al., 2001). As discussed earlier in this chapter (in section
1.3 Energy production,) the maintenance of PCr availability within the muscle cell is considered
essential to continued force production and performance during high intensity exercise. Muscular
fatigue during high intensity exercise has been associated with the inability of this tissue to
maintain a high rate of anaerobic ATP production from PCr hydrolysis (Katz et al., 1986;
Hitchcock 1989; Bogdanis et al., 1995). Conversely, improvements in muscular performance
61
during high intensity contractions have been associated with greater ATP resynthesis as a direct
consequence of increased PCr availability via CrM supplementation (Harris et al., 1992; Greenhaff
et al., 1994; Casey et al 1996; Kurosawa et al., 2003). However, CrM loading has also been shown
to delay the onset of neuromuscular fatigue (Stout et al., 2000). This has been demonstrated during
an incremental cycle ergometer test, "the physical working capacity at the fatigue threshold"
(PWCFT); an assessment which utilizes electromyographic (EMG) fatigue curves to identify the
power output that corresponds to the onset of the neuromuscular fatigue (Stout et al., 2000). This
benefit from supplementation was attributed to an elevated muscle PCr concentration on the
transition fromaerobic to anaerobic metabolism during exercise (Stout et al., 2000). However, CrM
loading is also shown to shorten muscle relaxation time (defined as the time for muscle torque to
decrease from 75 to 25% of maximum) during intermittent maximal isometric contraction (Van
Leemputte et al., 1999; Hespel et al., 2002). SR Ca2+ reuptake by virtue of Ca2+/ATPase pump
activity is the rate-limiting step in relaxation of mammalian muscle cells (Dux, 1983). Thus the
effect of CrM on relaxation rate suggests that SR Ca2+ reuptake is facilitated in Cr-loaded muscle
(Van Leemputte et al., 1999; Hespel et al., 2002). This effect may promote an ergogenic action as
the relaxation process accounts for an important fraction of total energy consumption during
repeated contractions (Bergstrom et al., 1988).
As discussed previously (refer to figure 1.5), the formation of the polar PCr (from Cr and
ATP), secures this high-energy phosphate within the muscle and maintains the retention of Cr
because the charge prevents partitioning through biological membranes (Greenhaff, 1997).
Therefore, supplementation with CrM is thought to enhance the cellular bioenergetics of the
phosphagen system by increasing resting PCr concentration within muscle (Hultman et al., 1996;
Greenhaff, 1997). However, according to Bessman & Savabi (1988) supplementation would boost
the efficiency of the Cr-Pi shuttle and the transfer of high-energy phosphates between the
mitochondria and the sites of major energy utilization (via functional coupling with ANT), such as
the myofibrils. Thereby enhancing the availability of energy not only for muscle contraction but
also the synthesis of contractile proteins (figure 1.8). Bessman et al. (1980) proposed that a protein
synthesizing microsome lay adjacent to, or may even be a part of the CK complex at the site of the
myofibril where it would receive some of the ATP liberated when this CK isoenzyme transfers
ATP to the cross-bridge binding site (figure 1.8). This protein synthesizing complex would benefit
from an increase in Cr-Pi shuttle activity that would occur from CrM supplementation and possibly,
enhance the synthesis of contractile proteins during hypertrophy. A number of studies that
examined the ergogenic effects of CrM supplementation also report a significant increase in body
mass and/or LBM (Harris et al., 1992; Balsom et al., 1993; 1995; Earnest et al., 1995). For these
62
reasons, it was suspected that CrM may improve the development of strength and LBM accretion
during RE training (Vandenberghe et al., 1997).
Figure 1.8 The Cr-Pi shuttle and its potential role in contractile-specific protein synthesis.
Adapted from work by Saks et al. (1998); Bessman et al., (1980).
63
Consequently, several studies have confirmed that supplementation with CrM during RE
training does improve gains in LBM in both men and women (Earnest et al., 1995; Vandenberghe
et al., 1997; Bermon et al., 1998; Kreider et al., 1998; Volek et al., 1999; Becque 2000; Chrusch et
al., 2001). Additional evidence suggests that the increase in lean mass and total body water from
CrM during RE training is mainly intracellular (an increase in cell volume) suggesting that body
cell drymass has increased (Poortmans & Francaux 1999; Bemben et al., 2001). That is, CrM may
promote an increase in cell volume and the subsequent activation of the anabolic mechanisms this
phenomenon provides (refer to figure 1.7), resulting in greater accretion of LBM. Surprisingly,
only two studies have quantified the extent of specific muscle fibre hypertrophy in response to RE
training and CrM supplementation (Volek et al., 1999; Tarnopolsky et al., 2001). Only one
managed to demonstrate that supplementation (loading phase followed by 5g/day for 12 weeks)
resulted in significantly greater muscle fibre hypertrophy (all fibre types assessed) compared to a
matched placebo-treated group.
Aside from a beneficial impact on LBM, a review of 22 studies involving CrM and RE
concluded that supplementation does enhance strength and weightlifting performance (Rawson &
Volek 2003). That is, when analysed collectively, CrM supplementation during training provides
an average 8% better gain in muscle strength (as assessed by 1, 3, or 10RM) than placebo treatment
(20 vs 12%)(Rawson & Volek 2003). The average increase in weightlifting performance (maximal
repetitions at a given percent of maximal strength) following CrM supplementation is 14% greater
than the average increase in weightlifting performance after placebo ingestion (26 vs 12%). The
increase in bench press 1RM was as high as 45%, and an improvement in weightlifting
performance in this exercise up to 43% (Rawson & Volek 2003). However, despite a substantial
amount of data that suggests CrM can improve weightlifting performance, the development of
muscle mass and strength, a clear mechanistic explanation for these benefits has remained elusive.
If CrM does enhance skeletal muscle morphology during RE, then one logical explanation
would be that supplementation modulates some aspect of protein turnover; either by stimulating
MPS or by decreasing MPB (section 1.4). Studies that have directly assessed the effect of CrM on
protein turnover have shown that a typical loading phase does not enhance myofibrillar protein
synthesis, leg net amino acid balance or decrease leg muscle protein breakdown either at rest (in
both the fasted and fed state) (Louis et al., 2003a), or after RE (Louis et al., 2003b). Similarly,
Parise et al. (2001) demonstrated that supplementation (loading phase followed by 5g/day for 4
days) did not enhance MPS in men or women but did decrease whole body protein breakdown by
approximately 7.5%. Although this data demonstrates a lack of an acute anabolic effect of CrM on
muscle protein turnover, it is important to remember that this is not necessarily strong evidence for
64
a lack of effect from supplementation on the mechanisms of muscle hypertrophy. For example,
there is evidence that suggests the magnitude of increase in protein synthesis after exercise may be
dependent on the extent of the previous reduction in energy status (a fall in the ATP/ADP ratio)
during muscle contraction (Bylund-Fellenius et al., 1984). Other work has shown that CrM
supplementation can attenuate the exercise-induced fall in the PCr/Cr ratio and maintain the
ATP/ADP ratio during exercise (Kurosawa et al., 2003). Therefore, the CrM loading procedure
performed by the participants in the study by Louis et al., (2003) may have blunted the magnitude
of acute stimulation MPS normally observed after RE. Additionally, assessments in this study only
lasted 4 hours post-exercise. Increasing the availability of PCr (via CrM supplementation) may
enhance the efficiency of the Cr-Pi shuttle to deliver energy required for recovery and the synthesis
of muscle proteins in the days after RE. However, the relationship between energy status and
protein turnover in human muscle is one that has received very little exploration. Further research
to resolve these speculations is warranted.
The effect of CrM on muscle morphology during RE may reside in the stimulation of
transcriptional changes in muscle gene expression that might occur as a result of increased
availability of PCr (and associated ATP/ADP concentration or Ca2+ concentration changes
during/or after contractile activity). The results of which, in terms of protein accretion, would not
be seen for days or weeks after the initial stimuli. To support this notion, Willoughby & Rosene
(2001; 2003) demonstrated that supplementation with CrM (6g/day for 12 weeks of RE) resulted in
greater (relative) strength, LBM and thigh volume. These benefits were observed alongside
increases in the mRNA (type-I, IIa, and IIx), and protein (type-I, IIa and IIx) expression of the
MHC isoforms as well as muscle CK mRNA expression, myogenin and MRF-4 mRNA (and
protein) expression. It is apparent that Cr-loaded muscle is able to perform at a higher capacity
during RE (Rawson & Volek 2003). These transcriptional changes in muscle gene expression that
result in greater strength and hypertrophy from CrM during RE may contribute to, or be a product
of, CrM’s ability to enable muscle to work at a higher capacity. One study by Arciero et al. (2001)
provides a classic example of this senario.
Arciero et al. (2001) compared 1RM strength gains in healthy young males after 28 days of
CrM supplementation (loading phase followed by 10g/day) with or without RE training. Bench
press and leg press 1RM increased 8 and 16% respectively with CrM alone. Compared to the
respective 1RM strength gains demonstrated the by group that took CrM during RE training (18%
for bench press and 42% for leg press), these results suggest that approximately 40% of the
increase in strength over the 4-week period was simply due to the acute effects of CrM on muscle
function (Arciero et al., 2001). CrM’s ability to enable muscle to work at a higher capacity could
65
be due to an increased PCr availability for more efficient ATP resynthesis and/or Ca2+ handling in
muscle, which would allow an athlete to perform more repetitions each set and/or provide more
rapid recovery between sets. Clearly, this would enhance the potential for greater muscle strength
and hypertrophy during a training program. To provide further evidence of this, Syrotuik et al.
(2000) reported that when training volume was kept equal, participants ingesting CrM or placebo
experienced similar increases in muscle strength and weightlifting performance following an 8-
week resistance training program.
The improvements in strength, LBM and muscle hypertrophy that have been reported from
CrM supplementation during RE could be a result of several mechanisms that have been discussed.
These include, improved work capacity via a more efficient supply of ATP; an increased
expression of the musclegenes and regulatory factors associated with hypertrophy; an increase in
satellite cell mitotic activity; or initiation of other anabolic processes which may be secondary to
increased cell volume. The evidence available suggests that CrM is a safe supplement to consume
during exercise training (Poortmans & Francaux 1999; Groeneveld et al., 2005; Poortmans et al.,
2005). This is important as CrM is a widely used supplement among populations that perform RE
training (LaBotz & Smith 1999; Jacobson et al., 2001; McGuine, 2002; Sundgot-Borgen et al.,
2003; Froiland et al., 2004; Morrison et al., 2004; Kristiansen et al., 2005). However, CrM may
also confer a therapeutic benefit (i.e., increased LBM and/or muscle strength) to a wider section of
the population such as older adults (Tarnopolsky, 2000), patients with neuromuscular disorders
(Tarnopolsky et al., 1999; 2004), McArdle’s disease (Vorgerd et al., 2000) and mitochondrial
cytopathies (Tarnopolsky et al., 1997). Since 1993, well over 200 studies have examined CrM’s
ergogenic/therapeutic potential (Rawson & Volek 2003). In comparison, few studies have
examined the effects of CrM supplementation on muscle morphology during RE training (Volek et
al., 1999; Tarnopolsky et al., 2001; Willoughby & Rosene 2001; Fry et al., 2003). In particular,
although a number of studies have assessed changes in strength and body composition in response
to supplementation and training (Earnest et al., 1995; Vandenberghe et al., 1997; Bermon et al.,
1998; Kreider et al., 1998; Volek et al., 1999; Becque 2000; Chrusch et al., 2001), few have
assessed these changes alongside fibre-specific hypertrophy responses (Volek et al., 1999;
Tarnopolsky et al., 2001; Fry et al., 2003). Additionally, no studies involving CrM supplementation
during RE training have reported changes in strength and body composition alongside adaptations
at the cellular level (i.e., fibre-specific hypertrophy) and the sub-cellular level (i.e., contractile
protein content). Finally, no studies have examined the effects of combining CrM with WP on
muscle fibre characteristics during RE training.
66
1.10 Resistance exercise program design for muscle hypertrophy
RE is considered a key component in any health program by organizations such as the
American College of Sports Medicine (ACSM) (Feigenbaum & Pollock 1999) and the American
Heart Association (AHA) (Fletcher et al., 1995; Pollock et al., 2000). RE is also a cost-effective
activity that can be easily implemented into the lifestyles of most adults (Roubenoff, 2003).
However, despite the well documented benefits, research on program design to optimize
hypertrophy is a topic that has received scant attention from the scientific community. RE program
prescription for muscle hypertrophy would involve the correct choice of variables, such as
frequency, intensity, volume and exercise selection to provide the optimal cellular and molecular
responses that promote the desired result. Unfortunately, this information is yet to be clearly
identified. Studies that have compared the effects of manipulating program variables have mainly
been concerned with strength rather than hypertrophy outcomes (Berger 1962; 1965; Anderson &
Kearney 1982; Willoughby, 1993; Stone & Coulter 1994; Tan, 1999). While the development of
strength is an important aspect, this chapter has demonstrated that hypertrophy is a multifaceted
phenomenon that can be influenced by a myriad of factors. Until we have a better understanding of
the various factors that influence hypertrophy (such as age, gender, endocrine profiles, previous
training status, nutrition and genetics), program design will remain at best, an educated guess based
on protocols and principles that would most likely be effective for the population group concerned.
This situation will have to change in the near future. The emerging importance of muscle
preservation for healthy ageing and the spiralling healthcare costs related to sarcopenia in a rapidly
ageing population will eventually force governments to focus greater attention on supporting
research that will determine the most effective exercise programs for hypertrophy. Nevertheless,
this section will attempt to provide the reader with a science-based approach to RE program design
for optimizing muscle hypertrophy.
A science-based approach to RE prescription can be traced to the post World War II era,
when army physician’s DeLorme and Watkins designed progressive RE programs for rehabilitation
of orthopaedically disabled veterans (DeLome, 1945; DeLome & Watkins 1948). Their studies
demonstrated that the use of heavy resistance and a low number of repetitions developed muscular
strength, whereas the use of lighter resistance and a higher number of repetitions developed
muscular endurance. DeLome and Watkin’s research also underscored progression as a
fundamental principle in effective RE program design. Progression is defined as the act of moving
forward or advancing toward a specific goal. Progression in RE entails structuring the training
program for continued improvement in a desired variable over time or until the target goal has been
67
achieved (Kraemer et al., 2002). Although untrained individuals respond favourably to a wide
variety of protocols (Campos et al., 2002; Harris et al., 2004), progression is a fundamental aspect
of program design as physiological adaptations take place in a relatively short period of time
(Staron et al., 1994). For example, it is clear that training reduces the stimuli of MPS for a given
load (Phillips et al., 2002), and less muscle mass is recruited to lift a given load once adaptation has
occurred (Ploutz et al., 1994). Therefore, systematically increasing the demands placed upon the
muscle(s) is necessary for further improvement (Ploutz et al., 1994). Progressive RE program
design involves the proper manipulation of variables to systematically increase the training stress.
This can be achieved by increasing the amount of resistance (overload) used, manipulating the
number of sets and repetitions completed and altering exercise selection (i.e., progressing to more
complex movements). Periodization in RE training refers to planned changes in the training
program in a direct attempt to bring about a peak training response (Fleck & Kraemer 1997). In this
respect, periodization is closely related to progression. For example, the classic linear
strength/power periodization approach follows a general trend of decreasing volume while
increasing intensity during a training program so that strength/power is optimized at the end of the
program (Fleck, 1999). However, periodization can also involve an undulated manipulation of
program variables (Willoughby, 1993). Both approaches are thought to be effective strategies that
help to limit natural training plateaus (that point in time where no further improvements takes
place) and enable higher levels of adaptation to be achieved (Fleck, 1999).
As identified previously in this chapter, the stimulation of MPS is the facilitating
mechanism that underlines hypertrophy (Rennie et al., 2004; Cuthbertson et al., 2005). However, it
is also apparent that the degree of overload placed on muscle determines the magnitude of
stimulation of MPS (Goldberg, 1968; Baar & Esser 1999; Phillips et al., 2002). Additionally, a
close relationship exists between strength and muscle size (Häkkinen et al., 1991; Tesch 1992).
Therefore, the classical prescription for muscle hypertrophy would appear to still hold true. That is,
size and strength gains are directly proportional to the magnitude of overload placed on muscle
(Atha, 1981; Saltin, 1983; Tesch, 1992). An improvement in strength would enable greater
overload to be placed on the muscle(s) and therefore, provide further potential for hypertrophy
(Tesch, 1992; Kraemer, 1996; 1997). This appears to be the basic recommendation from the
leading organizations that prescribe RE, such as the ACSM and the National Strength and
Conditioning Association (NSCA). However, despite a very limited amount of data to draw from,
both organizations also provide rather specific training recommendations for hypertrophy that are
quite distinct from training for strength development. The reason for this is perplexing as a sciencebased
distinction between training for strength and training for hypertrophy is very difficult—the
principles of the former are intertwined within the latter. As a result, the following paragraphs will
68
reveal that the hypertrophy-specific training recommendations made by the leading organizations
are based primarily on anecdotal reports and/or highly speculative observations. And, most of these
recommendations actually contradict the foundation principles required to induce muscle
hypertrophy.
Training load is an important variable as it typically defines the intensity of the exercise
(Kraemer et al., 1996). That is, intensity is typically described as the amount of weight used when
performing an exercise (Kraemer et al., 1990; Häkkinen et al., 1993; Ahtiainen et al., 2003). This is
often defined by the maximum amount of repetitions that can be completed with a given weight
(Tan, 1999). For example, the amount of weight that can be lifted once successfully, with correct
technique is the participant’s 1RM (Stone et al., 2000). Progression in RE is easily implemented by
manipulating the training load. This can be achieved by increasing the load on the basis of a loadrepetition
continuum such as using a percentage of the 1RM (i.e., a higher percentage indicates
fewer repetitions can be performed as the weight is heavier, as opposed to using a lower percentage
with a lighter load where more repetitions can be completed) (Fleck & Kraemer 1997).
Alternatively, progression can be applied by increasing the training load within a prescribed zone
(e.g., 8-12 RM) (Tan 1999). With this method, when a specific RM zone is exceeded, a 2-10%
increase in load is applied so that the individual continues working within the designated RM zone
(Kraemer et al., 2002). Longitudinal studies show that a range of maximal loads (from 1 to 12RM
or 100-60% of 1RM) can provide improvements in strength and hypertrophy in the short term (12-
16 weeks) (Cureton et al., 1988; Staron et al., 1991; 1994; Kraemer, 1996; Goto et al., 2005).
Particularly, untrained individuals respond to a wide variety of loading protocols, even low loads
(15-50% of 1RM) (Gettman et al., 1978; Sale, et al., 1990; Moss et al., 1997; Bemben et al., 2000).
While recent work has confirmed an “RM continuum” for muscle strength and endurance
development (Anderson & Kearney 1982; Stone & Coulter 1994; Tan, 1999), the information on
loading protocols to optimize hype

Stay Tuned.

I didn't read that.

i like kinases.

Scourge wrote:I didn't read that.

Yes you did.

and they lived happily ever after

that actually is a true story

my nick is in that story.

what a nerd

Chapter 1
Introduction
Can you do a preface, a resume or something?
No one is going to go trough all your chapters.