European Journal of Sport Science

ISSN: 1746-1391 (Print) 1536-7290 (Online) Journal homepage: https://www.tandfonline.com/loi/tejs20

It's not just about protein turnover: the role of

ribosomal biogenesis and satellite cells in the
regulation of skeletal muscle hypertrophy

Matthew Stewart Brook, Daniel James Wilkinson, Ken Smith & Philip James
Atherton

To cite this article: Matthew Stewart Brook, Daniel James Wilkinson, Ken Smith & Philip James
Atherton (2019): It's not just about protein turnover: the role of ribosomal biogenesis and satellite
cells in the regulation of skeletal muscle hypertrophy, European Journal of Sport Science, DOI:
10.1080/17461391.2019.1569726

To link to this article: https://doi.org/10.1080/17461391.2019.1569726

Published online: 09 Feb 2019.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tejs20

Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88

 European Journal of Sport Science, 2019
https://doi.org/10.1080/17461391.2019.1569726

REVIEW ARTICLE

It’s not just about protein turnover: the role of ribosomal biogenesis and
satellite cells in the regulation of skeletal muscle hypertrophy

MATTHEW STEWART BROOK1,2, DANIEL JAMES WILKINSON1,2, KEN SMITH1,2, &

PHILIP JAMES ATHERTON1,2
1
MRC-ARUK Centre for Musculoskeletal Ageing Research, University of Nottingham, Derby, UK & 2National Institute for
Health Research (NIHR), Nottingham Biomedical Research Centre (BRC), Clinical, Metabolic and Molecular Physiology,
University of Nottingham, Derby, UK

Abstract
Skeletal muscle has indispensable roles in regulating whole body health (e.g. glycemic control, energy consumption) and, in
being central in locomotion, is crucial in maintaining quality-of-life. Therefore, understanding the regulation of muscle mass
is of significant importance. Resistance exercise training (RET) combined with supportive nutrition is an effective strategy to
achieve muscle hypertrophy by driving chronic elevations in muscle protein synthesis (MPS). The regulation of muscle
protein synthesis is a coordinated process, in requiring ribosomes to translate mRNA and sufficient myonuclei density to
provide the platform for ribosome and mRNA transcription; as such MPS is determined by both translational efficiency
(ribosome activity) and translational capacity (ribosome number). Moreover, as the muscle protein pool expands during
hypertrophy, translation capacity (i.e. ribosomes and myonuclei content) could theoretically become rate-limiting such
that an inability to expand these pools through ribosomal biogenesis and satellite cell (SC) mediated myonuclear addition
could limit growth potential. Simple measures of RNA (ribosome content) and DNA (SC/Myonuclei number)
concentrations reveal that these pools do increase with hypertrophy; yet whether these adaptations are a pre-requisite or a
limiting factor for hypertrophy is unresolved and highly debated. This is primarily due to methodological limitations and
many assumptions being made on static measures or correlative associations. However recent advances within the field
using stable isotope tracers shows promise in resolving these questions in muscle adaptation.

Keywords: Muscle, hypertrophy, ribosomal biogenesis, satellite cell, myonuclei

Highlights
. Resistance exercise training (RET) combined with supportive nutrition is an effective strategy to achieve muscle
hypertrophy by driving chronic elevations in muscle protein synthesis (MPS).
. The regulation of MPS is a complex process, requiring ribosomes to translate mRNA and sufficient myonuclei density to
provide the platform for ribosome and mRNA transcription.
. As the muscle protein pool expands during hypertrophy, translation capacity (i.e. ribosome number and myonuclei
content) could theoretically become rate-limiting such that an inability to expand these pools through ribosomal
biogenesis and satellite cell (SC) mediated myonuclear addition could limit growth potential. Yet, wether these
processes are a pre-requisite or obligatory for muscle hypertrophy is unknown.
. Recent advances using sable isotope tracers show promise in resolving these fundamental questions.

Introduction
Skeletal muscle is essential for movement, enabling
the completion of everyday tasks that underpin inde-
pendence. Furthermore, muscle hypertrophy is desir-
able among athletes and in the general population due
to the rising interest in muscle health and wellbeing.
Skeletal muscle is important in whole body metab-
olism, having significant roles in the storage and
metabolism of proteins, lipids and glucose (Brook
et al., 2016b; Wolfe, 2006). These roles are critical
in maintaining whole-body metabolic and functional
health; this is typified by the notion that progressive

Correspondence: Matthew Stewart Brook, Division of Metabolic and Molecular Physiology, Postgraduate Entry Medical School, Royal
Derby Hospital, Uttoxeter Road, Derby, DE22 3DT, UK. E-mail: mszmsb@nottingham.ac.uk

© 2019 European College of Sport Science

Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88

 2 M. S. Brook et al.
declines in muscle mass in disease and ageing propa-
gate frailty, metabolic comorbidities and mortality
(Brook, Wilkinson, & Atherton, 2017a). As such
there has been considerable research efforts from our
lab (Atherton & Smith, 2012) and others’ (Phillips,
Tipton, Ferrando, & Wolfe, 1999) aimed at unravel-
ling the regulation of muscle mass. However, many
aspects of hypertrophy remain unclear and debated,
with a confusing array of optimal nutritive/exercise
practices available (Aragon & Schoenfeld, 2013;
Hulmi, Lockwood, & Stout, 2010). Understanding
the mechanisms and most effective strategies to
induce skeletal muscle hypertrophy would offer great
resource in combating muscle wasting and add
clarity to optimal exercise practices.
Placing skeletal muscle under regular mechanical
overload known as resistance exercise training
(RET), is a well-known stimulator of muscle hypertro-
phy and its regulation is impacted based upon inten-
sity (Kumar et al., 2009; Mitchell et al., 2012),
volume (Burd et al., 2010; Kumar et al., 2012), fre-
quency (Schoenfeld, Ogborn, & Krieger, 2016) and
contraction mode (i.e. eccentric vs concentric
[Franchi et al., 2014]). Intriguingly, muscle adap-
tation has shown to be most active during the early
phases of a RET, with increases of 5–10% in muscle
mass/size achieved by the first 3–6 weeks, at which
point continued increases are considerably slower
(Brook et al., 2015, 2016c; DeFreitas, Beck, Stock,
Dillon, & Kasishke, 2011). Moreover, hypertrophic
responses are markedly heterogeneous, with some
individuals showing negligible changes in muscle
volume in response to regular progressive RET, and
others may increase up to 10% or more (Phillips
et al., 2013; Stec et al., 2016). With skeletal muscle
being composed of many long, multinucleated, post
mitotic fibres, overall changes in muscle size occur
through an increase in muscle fibre area, rather than
fibre number (hyperplasia). Irrespective of loading
combinations and RET paradigms employed, the fun-
damental mechanism driving this fibre hypertrophy is
an increase in protein content, which principally must
occur by muscle protein synthesis (MPS) exceeding
muscle protein breakdown (MPB).
Protein synthesis essentially relies on the eukary-
otic central dogma, which in its simplest form sees
the flow of genetic information from DNA>RNA>
Protein. However, as a muscle fibre continues to
grow and the protein pool expands, the foundations
that support protein synthesis (i.e. DNA>RNA) will
become diluted and presumably the so called
“capacity” for protein synthesis could become
strained. An increase in cellular RNA is regulated
by ribosomal biogenesis (translational capacity),
whereas an increase in DNA represents satellite cell
(SC) division and myonuclear addition, that in turn
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
provides the platform for ribosome production and
mRNA availability. Yet despite considerable progress
in understanding MPS responses, those of RNA and
DNA have somewhat lagged, especially in humans.
As such, fundamental aspects of muscle physiology
are unresolved, with one of the most rudimentary
questions being can muscle hypertrophy occur in
the absence of ribosomal biogenesis and myonuclear
addition, or are they a pre-requisite for adaptation? In
this review, we will discuss evidence of the roles of
ribosomal biogenesis and SC mediated myonuclear
addition in hypertrophy and highlight new methods
which can further knowledge in this area.
The regulation of skeletal muscle protein
turnover
Protein synthesis is the process by which ribosomes
create polypeptide chains through linking amino
acids together in a specific order according to
mRNA. As such, rates of protein synthesis can be
modulated by the rate of mRNA translation, known
as “translational efficiency”. A primary control
point regulating translational efficiency and therefore
protein synthesis in the majority of eukaryotic cells is
by cap dependent translation. This involves the
assembly of many eukaryotic initiation factors
(eIF’s) to form a preinitiation complex (PIC) that
interacts with the 5′ end of an mRNA to instigate
protein synthesis (for more detail readers are directed
to [Jackson, Hellen, & Pestova, 2010]). However,
with protein synthesis being an energy demanding
processes (e.g. through peptide bonding) it is unsur-
prising that there is myriad of regulating signaling
cascades, many of which culminate on the mamma-
lian target of rapamycin (mTOR), that integrates
signals such as exercise, AA availability and energy
status to coordinate cellular metabolism (Goodman
et al., 2011). Some of the best understood targets of
mTOR are those directly involved in cap-dependent
translation, including P70S6K1, 4E-BP1, and
RPS6 that can enhance translation initiation and effi-
ciency in the absence of ribosomal biogenesis
(Chesley, MacDougall, Tarnopolsky, Atkinson, &
Smith, 1992).
Two of the most well documented stimulators of
MPS are the protein components of a meal (particu-
larly EAA) and RE. In the absence of nutrition, MPB
exceeds MPS, such that AA’s from muscle are
released to support protein synthesis and energy
needs in other organs (Pozefsky, Tancredi, Moxley,
Dupre, & Tobin, 1976). Upon consumption of a
meal, the protein components drive a robust
(>100%) yet transient (60–90 mins) increase in
MPS to replenish muscle protein lost during

nutritional absence (Atherton et al., 2010). Once this
is achieved the muscle becomes “full” and any
additional AA’s are excreted such that excess
protein consumption in healthy individuals would
not result in muscle growth (Witard et al., 2014). A
bout of RE in the absence of AA’s results in a
similar potentiation of MPS (Kumar et al., 2009),
yet due to greater increases in MPB the overall net
effect is negative (Phillips et al., 1999). However,
upon consumption of dietary protein in proximity
to RE, MPS is enhanced further than RE or feeding
alone, achieving overall positive balance and extend-
ing the muscle full point that when repeated over
extended periods results in muscle hypertrophy
(Atherton & Smith, 2012).
Beyond nutritive/mechano-sensitive pathways,
muscle mass and RE induced hypertrophy are also
regulated by hormones. Testosterone (T), growth
hormone (GH) and Insulin-like growth factor 1
(IGF-1), are considered pro-anabolic, however only
T has been definitively demonstrated to clearly
promote MPS and subsequent hypertrophy (Griggs
et al., 1989). With regard to GH and IGF-1 in
humans (in young adult muscle), evidence is
lacking for a direct role for these hormones in
driving MPS, yet GH does increase collagen syn-
thesis supporting a role in extracellular matrix remo-
deling (Doessing et al., 2010). However, that is not to
say GH and IGF-1 do not hold important roles in
skeletal muscle maintenance and hypertrophy, with
in-vivo rodent studies demonstrating hypertrophy
with IGF1 infusion (Adams & McCue, 1998) or
overexpression (Musarò et al., 2001) and atrophy
with IGF-1R knockout (Spangenburg, Le Roith,
Ward, & Bodine, 2008). Whilst the importance of
IGF-1/IGF1-R has been brought into question
during overload induced hypertrophy (Spangenburg
et al., 2008), IGF-1/IGF-1R may hold critical roles
in muscle repair and maturation (Heron-Milhavet,
Mamaeva, Leroith, Lamb, & Fernandez, 2010). In
humans RE temporarily increases systemic hor-
mones, yet associations between elevated individual
hormones and resulting muscle mass have been ques-
tioned (West & Phillips, 2012). However, the entire
endocrine response (i.e. T, GH, IGF1, Insulin, corti-
sol) has been related to muscle hypertrophy
(Mangine et al., 2017) suggesting coordinated
elevations in multiple hormones may have a comp-
lementary effect in stimulating enhance muscle
growth.
Repeated bouts of RE supported by sufficient EAA
consumption that enhance MPS is undoubtedly the
major driver of protein accretion and hypertrophy.
However, as myofibres increase in size and as the
demand for increased protein content continues,
the foundations that permit MPS could be
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
It’s not just about protein turnover 3
constrained. Taking simple proxy measures of ribo-
some number and myonuclei content (i.e. RNA
and DNA concentration) indicate these pools
expand over time in response to RET which must
be due to greater changes in the synthesis of these
pools during this period of expansion. That being
said whether changes in ribosomal and myonuclei
content dictate the extent of muscle hypertrophy
capacity is unclear and has remained a controversial
and highly debated topic.
The role of ribosomal biogenesis in the
regulation of muscle mass and hypertrophy
As ribosomes serve as the protein synthetic machin-
ery of the cell, the rate of protein synthesis is not
only determined by translational efficiency (discussed
above) but also the total number of ribosomes (trans-
lational capacity) (Millward, Garlick, James, Nna-
nyelugo, & Ryatt, 1973). Ribosomes are themselves
composed of RNA (ribosomal RNA (rRNA)), tran-
scribed by RNA polymerase 1 (POL1) as a 47S pre-
rRNA which is then cleaved into 28S, 18S, 5.8S
and subsequently assembled with ribonuclear pro-
teins to from a mature ribosome (Figure 1) (Chaillou,
Kirby, & Mccarthy, 2014). POL1 therefore serves as
a primary control point in ribosomal biogenesis, with
the transcription factors TIF-1A, TIF-1B and UBF
key in forming a PIC with POL1 at the rDNA promo-
ter. The regulation of TIF-1A and UBF transcrip-
tional activity is achieved through multiple signaling
proteins, including ERK, AMPK, mTORc1 and
P70S6K1 (reviewed in Kusnadi et al., 2015) enabling
the control of ribosomal biogenesis to be influenced
by multiple pathways such as hormones, nutrients
and contractile activity. Another key regulator of
ribosomal biogenesis is c-MYC, an oncoprotein
involved in regulating cell growth and virtually all
aspects of ribosome formation. c-MYC directly upre-
gulates many of the proteins involved in rDNA tran-
scriptional control including UBF, TIF-1A, TIF-1B,
Pol1 and many other proteins involved in the for-
mation, processing and export of mature ribosomes.
Further, c-MYC enhances POL1 transcription by
remodeling rDNA chromatin structure and directly
interacting with the SL1 complex, stabilising Pol1
recruitment at the promoter (van Riggelen, Yetil, &
Felsher, 2010). Day-to-day fluctuations in MPS
that maintain muscle mass in healthy individuals are
predominantly achieved by transient increases in
translational efficiency, without changes in RNA
content (Chesley et al., 1992). RNA content and
therefore ribosome number is likely to be adequately
maintained to sustain the protein synthetic needs of
habitual activity and nutritional intake, whilst being
 4 M. S. Brook et al.

Figure 1. Overview in the signaling of muscle translational efficiency and translational capacity. Muscle protein synthesis (MPS) Is deter-
mined by both the rate of mRNA translation by ribosomes (i.e. translational efficiency) and the number of available ribosomes (i.e. transla-
tional capacity). Ribosomal biogenesis is the process of ribosome production, beginning with the transcription of rDNA in the nucleus to form
a 47s pre-RNA, that is subsequently cleaved and assembled into a mature ribosome. During habitual activity and dietary behaviours, the
primary driver of MPS is that of AA, increasing translational efficiency whilst maintaining a sufficient number of ribosomes through the stimu-
lation of ribosomal biogenesis. Resistance exercise (RE), along with sufficient AA intake enhances translational efficiency greater than RE or
AA alone such that positive muscle protein balance is maintained. In addition, RE stimulates ribosomal biogenesis and satellite cell mediated
myonuclear addition increasing translational capacity, that is likely to be key in supporting and or permitting hypertrophic adaptations. SC,
satellite cell. AA, amino acid. ↑ increase. ↔ no change. T, testosterone. G, growth hormone. I, insulin-like growth factor 1.

continually replenished to maintain functional ribo-
somes. In support of this, ribosomal biogenesis path-
ways are entwined with those regulating translational
efficiency (i.e. mTORc1) (Mayer & Grummt, 2006)
(Figure 1) and in response to chronic muscle
loading or nutritional modulation, bio-markers of
increased ribosomal biogenesis are observed (i.e.
Increased c-MYC / 47s pre-RNA) (Stec, Mayhew,
& Bamman, 2015).
With rRNA comprising 85% of the total RNA
pool, increases or decreases in the size of this pool
are indicative proxies of changes in the rates of ribo-
somal biogenesis. Early models of overload induced
hypertrophy in animals demonstrated rapid and sub-
stantial increases in RNA, with attempts to prevent
RNA synthesis in vivo inhibiting muscle growth
(Goldberg & Goodman, 1969). However, the speci-
ficity of these compounds to inhibit RNA synthesis
and not DNA synthesis is a confounding factor.
More recent preclinical models to approach this
subject have utilised synergist ablation (SA) tech-
niques combined with regular sampling to create a
time-course of molecular events. At the onset of
SA, there is an early increase in RNA concentration,
45S-pre-rRNA and a coordinated expression in regu-
lators of POL1 transcription, indicative of ribosomal
biogenesis and increased translational capacity at the
onset of hypertrophy (von Walden, Casagrande,
Ostlund Farrants, & Nader, 2012). Early increases
in RNA content with SA have been demonstrated
previously (Miyazaki, Mccarthy, Fedele, & Esser,
2011) however, whether this precedes or limits

Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88

 It’s not just about protein turnover 5

muscle hypertrophy is unclear as muscle mass
increases similarly at this time. Moreover, early
increases in muscle weight may be influenced by
water (oedema), such that increased ribosome
content may occur before increases in muscle mass
(Marino et al., 2008). Supporting this notion, the
use of electrical stimulation to replicate RE in vivo
showed that 12 h after RE there was a significant
increase pre-rRNA and a trend for greater total
RNA- again supporting early active ribosomal bio-
genesis prior to hypertrophy (West et al., 2016). Fur-
thermore, using EU labelled RNA, Kirby showed
that early increases in ribosomal biogenesis were
due to an increase in translational output per myonu-
clei, not an increase in active myonuclei, suggesting
that myonuclei already possess a capacity to maintain
greater levels of transcription in the absence myonu-
clei addition and meet the transcriptional demands
for hypertrophy (Kirby et al., 2016).
In agreement with the rapid onset of ribosomal bio-
genesis in animal models, contractile stimulation in
humans also causes a rapid expression in markers of
rDNA transcription, including increased expression
of 45S-pre-rRNA 24 h hours after RE (Stec et al.,
2016). Further RNA content increased after two
bouts of RE (Bickel et al., 2005), a response which
is sustained at 3 and 6-weeks of continued RET
(Brook et al., 2015). Some of the strongest evidence
supporting a requirement of ribosome biogenesis in
muscle hypertrophy has been demonstrated using k-
means cluster analysis. After 4-weeks of RET, 42
older adults (60–75y) were categorised into three
responder groups based on the extent of hypertrophy
of type II fibres, showing either no change (-7%), a
modest increase (22%) or extreme increase (83%).
At the onset of training all groups had the same
RNA content, whilst after training there was no
increase in RNA concentration in the “no change
group”, a trend for an increase in the “modest
group” (+9%) and a significant increase in the
“extreme group” (+26) (Stec et al., 2016); a finding
recently reproduced in young individuals when clus-
tered by change in vastus lateralis thickness (Mobley
et al., 2018). Similarly we have shown that age-
related deficits in long term MPS and resulting
hypertrophy were allied with inferior ribosomal bio-
genesis and a lack of c-MYC induction (Brook
et al., 2016a). Together, these studies have demon-
strated that increases in translational capacity are
often present in those with the greatest hypertrophy,
yet this does not necessarily mean it is a prerequisite
or limiting factor (Figure 2). However, taking a large
group (n=66) of mixed gender and age individuals,
only extreme hypertrophic responders increased
total RNA content after the first exercise bout, see-
mingly positioning those that can rapidly expand
ribosomal capacity at a hypertrophic advantage
(Table I). That being said, after 16-weeks, both

Figure 2. Mechanisms of increased translational capacity and fibre CSA. Skeletal muscle hypertrophy occurs through an increase in protein
synthesis, protein content and therefore overall fibre CSA. Presumably this would lead to a dilution in the foundations of protein synthesis (i.e.
translational capacity) in both Ribosomal content (RNA) and myonuclei number (DNA). However, whether fibre hypertrophy can occur
without an increase in translational capacity via ribosomal biogenesis or satellite cell mediated myonuclear addition remains a contentious
issue. Further, as shown above, an expansion in translation capacity can result from varied adaptations. 1 – Lack of fibre hypertrophy due
to an absence of increased in translational capacity, 2 – Fibre hypertrophy in the absence of an increase in translational capacity, 3 – Fibre
hypertrophy driven by an increase in translational capacity from existing myonuclei, 4 – Fibre hypertrophy driven by an increase in transla-
tional capacity from satellite cell mediated myonuclear addition, 5 – Fibre hypertrophy driven by an increase in translational capacity from
both existing new myonuclei. CSA, cross sectional area. SC, satellite cell. ↑ increase. ↔ no change.

Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88

 6 M. S. Brook et al.
Table I. Analysis of human subjects after undergoing 16 weeks of progressive resistance exercise training clustered by vastus lateralis fibre
cross sectional area (Bamman, Petrella, Kim, Mayhew, & Cross, 2007) with their respective increases in muscle satellite cell/myonuclear
(Petrella, Kim, Mayhew, Cross, & Bamman, 2008) and RNA content (Kim et al., 2007). ↑ increase. – no change.
Protein
Δ
Baseline fibre Fibre Baseline Δ Baseline
Cluster area area SC SC
Non – – – –
Mod – ↑ – –
Xtr – ↑↑ ↑ ↑↑
Ref (Bamman et al., 2007)
moderate and non-responders showed equals
increases in RNA content despite diminished hyper-
trophy, highlighting muscle growth is a coordinate
process and cannot rely on increases in translational
capacity alone (Kim, Petrella, Cross, & Bamman,
2007).
Role of satellite cells (SC) in the regulation of
skeletal muscle mass and hypertrophy
Skeletal muscles cells are multinucleated but termin-
ally differentiated cells (Heron & Richmond, 1993).
However, skeletal muscle also possess a residual
pool of resident muscle stem cells – SC (Mauro,
1961). Unlike their sub-sarcolemma Myonuclei
counterparts, SC are located in the extracellular
matrix between the sarcolemma and the basal
lamina and in being a type of stem cell (unlike myo-
nuclei) (Bintliff & Walker, 1960), SC possess
mitotic potential. The physiological role of SC is
thought to be the provision of “new” nuclei to exist-
ing myofibers, supporting transcriptional capacity,
while at the same time ensuring through division, sus-
tainment of an extracellular SC population. Upon
activation, SC exist quiescence and enter the cell
cycle where they can proliferate as myoblasts and
potentially terminally differentiate. A population of
these cells will undergo asymmetric cell division
where by one daughter cell remains quiescent main-
taining SC number and a continued source of myo-
nuclei (Moss & Leblond, 1971; Troy et al., 2012).
The contended role of SC in the regulation of hyper-
trophy was derived from the concept that each nuclei
of a multi-nucleated myofibre appears to synthesise
protein for a close vicinity domain (Figure 2) (Gun-
dersen, Sanes, & Merlie, 1993; Hall & Ralston,
1989) and that this karyoplasmatic ratio is held con-
stant (Allen, Roy, & Edgerton, 1999). Obviously,
this means that as a muscle hypertrophies, the fixed
nuclei content of terminally differentiated muscle
cells essentially becomes “diluted” to a point that a
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
DNA RNA
Δ Myonuclear RNA RNA 16
myonuclei Myonuclei domain 24 h wk.
– – – – ↑
– ↑ ↑ – ↑
– ↑↑ ↑↑ ↑ ↑
(Petrella et al., 2008) (Kim et al., 2007)
new source of nuclei is needed to support the tran-
scriptional requirements of supporting a larger myofi-
ber volume. Therefore, the potential role for SC in
regulating and/or limiting muscle hypertrophy has
many foundations that warrant detailed consideration.
Numerous pre-clinical studies have been carried
out with the intention to resolve the “mechanistic”
role of SC as determinants of muscle hypertrophy,
using genetic, chemical or irradiation techniques
(Rosenblatt, Yong, & Parry, 1994). Non-genetic
based animal studies aimed at delineating the role
of SC in post-natal muscle growth tended to focus
on DNA synthesis inhibition by gamma-irradiation
or by way of chemical DNA synthesis inhibitors.
Such techniques led to early reports that SC were
indispensable for muscle hypertrophy (Rosenblatt
et al., 1994). However, such approaches were
blighted by lack of specificity in targeting SC activity;
instead targeting all cells displaying mitotic activity
(e.g. fibroblasts, endothelial cells). Since skeletal
muscle hypertrophy is the summation of complex
and coordinated tissue remodeling involving multiple
cell types, it could be argued that impaired muscle
hypertrophy in these studies was not a result of ablat-
ing SC activity per se, but rather of suppressing
mitotic activity in multiple cell types involved in
hypertrophy. This thesis is supported by genetic
approaches, creation of mouse strains in which con-
ditional ablation of SC (i.e. PAX7 expressing cells)
could achieve SC depletion (-90%) in adult mice.
To determine the role of SC in hypertrophy, SC
depleted animals and wild-type counterparts were
exposed to SA. Intriguingly, it was shown that
>90% depletion of SC did not limit muscle hypertro-
phy despite an absence of myonuclei accretion
(McCarthy et al., 2011). Instead, there was an expan-
sion of myonuclear domains (lack of BrdU (DNA)
positive myonuclei) in SC-depleted muscle, a
finding arguing against there being a critical upper
limit for myonuclear domain. In a second study,
using the same model, it was reported that SC-
depletion also did not impact on skeletal muscle re-
 It’s not just about protein turnover 7

growth following hind limb suspension-induced force behind the lack of increase in myofibre
atrophy (Jackson et al., 2012). While this also nuclear number such that one’s ability to sustain
points to a non-obligatory requirement for SC in positive increases in net protein balance rather than
muscle growth, this was in direct contradiction to to stimulate SC activity, is a rate limiting physiologi-
the early Rosenblatt studies and, moreover, recent cal step for human skeletal muscle hypertrophy.
inducible PAX7 depletion studies contesting a non-
obligatory role of SC e.g. due to methodological con-
cerns (mis-quantification of myofibre hypertrophy Perspectives on the roles of ribosomal
and inclusion of regenerating fibres in the data ana- biogenesis and SC in muscle hypertrophy
lyses [Egner, Bruusgaard, & Gundersen, 2016]). As
Over two decades on from initial studies, this leaves
such, there remains uncertainty around the role of
one to ponder, how might these results be reconciled?
SC in muscle hypertrophy, despite significant
Firstly, it should be pointed out that the SA model is a
advances in animal models. However, PAX7
blunt tool of accelerated surgically-induced muscle
depletion studies have demonstrated SC to be indis-
growth in rodents, which cannot accurately represent
pensable in muscle regeneration (Lepper, Partridge,
RE-induced hypertrophy for which there exist no
& Fan, 2011), with SC also showing key roles in reg-
robust pre-clinical models. As such, whether or not
ulating the muscle fibre micro-environment during
ribosomal biogenesis and myonuclear addition are
hypertrophy and ageing (Fry et al., 2014, 2015),
“required” or “obligatory” in experimental models
highlighting the important roles SC play in processes
of rapid muscle hypertrophy, is not really as critical
other than hypertrophy.
an issue as whether they are involved in human phys-
Consistent with the notion of constant myonuclear
iological muscle hypertrophy induced by resistance
domains and a role for SC in muscle hypertrophy,
exercise. At present only tentative conclusions can
recruitment of new nuclei from SC fusing with the
thus be made: (i) increased ribosomal content and
pre-existing muscle fibre syncytia has been noted as
myonuclear incorporation into muscle may be associ-
a feature of muscle hypertrophy in humans (Allen
ated with human muscle hypertrophy, (ii) increases in
et al., 1999). Moreover, altered regulation in numer-
muscle CSA area, ribosome content and myonuclear
ous myogenic regulatory factors (MRF’s), as proxies
number may be subject to inter-individual variation
for SC activation, proliferation and differentiation
in the contribution of MPS and SC in humans, and
have been shown to be up-regulated in the hours fol-
(iii) while ribosomal biogenesis and SC incorporation
lowing a bout of RE (McKay et al., 2008), with
are a feature of hypertrophy, according to genetic
expansion of the SC pool and Myonuclei addition
models, SC activity may or may not be dispensable
accompanying muscle fibre hypertrophy in the early
for muscle hypertrophy, whilst equivalent studies
stages of RET (Snijders et al., 2016). However, an
investigating ribosomal biogenesis are yet to be per-
important and contentious question remains to be
formed. Collectively, these data suggest caution in
fully resolved: during “normal” skeletal muscle
interpreting data derived from studies on this topic
hypertrophy- is myonuclear addition a physiological
and highlight that the involvement of ribosomal bio-
pre-requisite for muscle hypertrophy or adaptation?
genesis and SC incorporation, particularly in
While there are few studies in humans to address
human physiological hypertrophy, remains somewhat
this, Petrella et al. (2008) applied a cluster analysis
of an open question; one that requires new technol-
to examine relationships between inter-individual
ogies for use in vivo, in humans.
variance in hypertrophic responsiveness to resistance
exercise and that of SC activity. In this study, it was
demonstrated that so-called “high-responders” for
Advances in isotopic techniques to quantify
RET-induced hypertrophy displayed an increased
nucleic acid polymerisation: the future?
SC number at baseline, and also increased myonuclei
number in response to resistance exercise (vs. less Despite assumptions and limitations relating to so-
responsive individuals) (Table I). Since they called “obligatory requirements” for ribosomal bio-
observed that high-responders expanded their myo- genesis and myonuclear addition in muscle hypertro-
nuclear domains (i.e. cytoplasm-to-nucleus-ratio), phy, their physiological roles remain a highly
the authors suggested this was the driving force contentious issue, primarily due to methodological
behind demand for myonuclear addition from SC and experimental limitations. Our current under-
sources to support myofibre hypertrophy in success- standing relies heavily on inference from static
ful growth-adaptors. Which factors communicate measures such as histological staining and correlative
this need for increased “transcriptional capacity” is changes of cell size with myonuclei number or DNA/
unknown. Nonetheless, it could also be that a poor RNA content (Kadi et al., 2004; Petrella et al., 2006).
“intrinsic” capacity for hypertrophy, is the driving Moreover, the use of unrepresentative animal models

Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88

 8 M. S. Brook et al.
of hypertrophy (i.e. SA) alongside irradiated or
genetic models of SC ablation to determine contri-
bution of SCs to hypertrophy has further clouded
our understanding (McCarthy et al., 2011; Rosen-
blatt et al., 1994). Therefore, there is a need to
develop novel dynamic measures of ribosomal bio-
genesis and myonuclei addition in vivo.
Historical measures of DNA and RNA synthesis
used radiolabeled pyrimidine analogues such as 3H-
thymidine or BrDU (Waldman et al., 1991).
However due to their highly toxic nature and the
ability to alter cell proliferation in vivo, there use
has been limited (Asher et al., 1995). Stable isotope
tracers offer an attractive alternative, being safe for
human use and (in most cases) have the same chemi-
cal behaviour as their naturally abundant cousin with
their use in in vivo metabolism is far reaching (Wilk-
inson et al., 2017). There are many sites for potential
stable isotope incorporation during nucleotide syn-
thesis, however ∼80–90% of deoxyribose arises
from extracellular glucose providing a route for con-
tinuous reliable uptake of an isotopically enriched
compound (Neese et al., 2002). For instance by
using labelled glucose the turnover of a range of
blood cells has been made (Macallan et al., 2009);
however due to the need to maintain a constant inges-
tion or intravenous infusion of stable isotopically
labeled glucose, these techniques are only useful for
fast turning over cell populations i.e. 24–48 h (Macal-
lan et al., 2009). However, for cells with much slower
proliferation rates, such as that of the SC of post-
mitotic skeletal muscle cells, an alternative technique
is required, and recent novel developments in the
application of the “universal” stable isotope tracer
deuterium oxide (D2O) has led to great step
forward in this field (Brook et al., 2017b).
D2O has some unique properties that make it
ideally suited for the measurement of skeletal
muscle nucleic acid pools. Administered orally,
either as a single bolus or continuously (daily or
weekly), D2O rapidly equilibrates with body water
creating a homogenous, slowly turning over, precur-
sor pool available for use by multiple substrates. The
deuterium from the body water can then be incorpor-
ated onto different substrates at stable C–H positions
through biological reductions during de novo syn-
thesis, and the metabolic flux of these substrate
pools can be calculated from the measurement of
the amount of the label that is incorporated (Heller-
stein, 2004). Furthermore, the homogenous distri-
bution among the body water pool provides an
easily accessible and measurable surrogate precursor
pool (Dufner & Previs, 2003), which due to the rela-
tively slow turnover of the body water pool, allows for
the measurement of metabolic turnover over periods
of hours–days–weeks–months (Brook et al., 2015;
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
Wilkinson et al., 2014). These unique properties of
D2O are ideal therefore for application to studies of
human skeletal muscle where relatively limited cellu-
lar proliferation occurs.
Work in animals in vivo has already highlighted the
utility of these D2O methods for understanding the
metabolic control of skeletal muscle, with DNA and
RNA synthesis shown to be active in mature skeletal
muscle of rats (Brook et al., 2017c; Drake et al.,
2015). Building on this, using D2O Robinson and
colleagues have been able to detect rates of DNA
turnover in adult human skeletal muscle, again
showing slow yet detectable rates of DNA turnover
of ∼0.03%/d, and a suggestion for increased rates
with exercise (Robinson, Turner, Hellerstein, Hamil-
ton, & Miller, 2011). However, few studies have
investigated human muscle beyond this, in part due
to lack of appropriate validation and technical devel-
opment of these D2O techniques. However, we have
made recent advances within the field to rectify this.
Developing and validating highly sensitive GC-MS/
MS techniques we were able to detect very low
levels of deuterium incorporation into newly syn-
thesised ribonucleotides, enabling us to make the
first measures of RNA synthesis in humans in
muscle. Further we showed that dynamic increases
in RNA synthesis (i.e. ribosomal biogenesis) were
active at the onset of RE, presumably playing an inte-
gral role in the prevailing hypertrophy (Brook et al.,
2016a, 2017c). Further, rates of ribosomal biogenesis
were strongly associated with rates of MPS, highlight-
ing that changes in translational efficiency and trans-
lational capacity are a coordinated process during
hypertrophy (Brook et al., 2017c). The enhanced
sensitivity of these techniques are not limited to just
RNA synthesis, if new DNA has been synthesised
then deuterium will be incorporated and isolation of
SC/myonuclear fractions will have the potential to
show without doubt the occurrence of cellular pro-
liferation and therefore myonuclear addition. These
newly developed techniques are widely applicable
and if combined with appropriately designed studies
have the potential to definitively determine the
likely physiological role of ribosomal biogenesis and
myonuclear addition in human muscle growth.
Conclusion
Understanding how ribosomal biogenesis and SC
mediated myonuclear addition influence MPS and
ultimately muscle hypertrophy would make signifi-
cant advances in the field of muscle physiology.
However, despite many years of research and techno-
logical advances, many of these questions remain
unresolved. The use of overload induced hypertrophy

in animal models has provided valuable information
in clearly demonstrated that an expansion of transla-
tional capacity and an increase in myonuclei are
aspects of, and correlated with muscle growth
(Nakada, Ogasawara, Kawada, Maekawa, & Ishii,
2016). However further progress to determine if
these adaptations are a pre-requisite or dictate hyper-
trophy are lacking and often rely on a time course of
associated measures. The development of genetic
animal models to explore SC have and will continue
to show great promise, yet currently contrasting
results are obtained (Egner, Bruusgaard, & Gunder-
sen, 2017), whilst equivalent models to investigate
ribosomal adaptations are lacking. Still, a confound-
ing problem with SA is the lack of physiological rel-
evance to RE induced hypertrophy. In human
studies, conclusions are often based on associations
between cellular changes and the extent of hypertro-
phy. The rapid technical advances in recent years
with regards to D2O stable isotope tracer methods
based on the work our lab and others (Brook et al.,
2017c; Mathis et al., 2017; Neese et al., 2002),
finally provides researchers with the tools necessary
to hone in on the dynamics of RNA and DNA turn-
over in relation to maintenance and growth of human
muscle mass and in ageing and catabolic diseases.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors would like to acknowledge the Dunhill Medical Trust
(R364/1112), The Physiological Society, a Medical Research
Council Confidence in Concept award and the MRC-Arthritis
Research UK Centre for Musculoskeletal Ageing Research (MR/
R502364/1 and MR/P021220/1) for supporting our work in this
area. All authors contributed equally to this manuscript.
References
Adams, G. R., & McCue, S. A. (1998). Localized infusion of IGF-I
results in skeletal muscle hypertrophy in rats. Journal of Applied
Physiology, 84, 1716–1722.
Allen, D. L., Roy, R. R., & Edgerton, V. R. (1999). Myonuclear
domains in muscle adaptation and disease. Muscle & Nerve,
22, 1350–1360.
Aragon, A. A., & Schoenfeld, B. J. (2013). Nutrient timing
revisited: Is there a post-exercise anabolic window? Journal of
the International Society of Sports Nutrition. doi:10.1186/1550-
2783-10-5
Asher, E., Payne, C. M., & Bernstein, C. (1995). Evaluation of cell
death in EBV-transformed lymphocytes using agarose gel elec-
trophoresis, light microscopy and electron microscopy. II.
Induction of non-classic apoptosis (“para-apoptosis”) by tri-
tiated thymidine. Leukemia & Lymphoma, 19, 107–119.
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
It’s not just about protein turnover 9
Atherton, P. J., Etheridge, T., Watt, P. W., Wilkinson, D., Selby,
A., Rankin, D., … Rennie, M. J. (2010). Muscle full effect
after oral protein: Time-dependent concordance and discor-
dance between human muscle protein synthesis and
mTORC1 signaling. The American Journal of Clinical Nutrition,
92, 1080–1088.
Atherton, P. J., & Smith, K. (2012). Muscle protein synthesis in
response to nutrition and exercise. The Journal of Physiology,
590, 1049–1057.
Bamman, M. M., Petrella, J. K., Kim, J., Mayhew, D. L., & Cross,
J. M. (2007). Cluster analysis tests the importance of myogenic
gene expression during myofiber hypertrophy in humans.
Journal of Applied Physiology, 102, 2232–2239.
Bickel, C. S., Slade, J., Mahoney, E., Haddad, F., Dudley, G. A., &
Adams, G. R. (2005). Time course of molecular responses of
human skeletal muscle to acute bouts of resistance exercise.
Journal of Applied Physiology, 98, 482–488.
Bintliff, S., & Walker, B. E. (1960). Radioautographic study of
skeletal muscle regeneration. American Journal of Anatomy,
106, 233–245.
Brook, M. S., Wilkinson, D. J., & Atherton, P. J. (2017a). Nutrient
modulation in the management of disease-induced muscle
wasting: Evidence from human studies. Current Opinion in
Clinical Nutrition and Metabolic Care, 20, 433–439.
Brook, M. S., Wilkinson, D. J., Atherton, P. J., & Smith, K.
(2017b). Recent developments in deuterium oxide tracer
approaches to measure rates of substrate turnover:
Implications for protein, lipid, and nucleic acid research.
Current Opinion in Clinical Nutrition and Metabolic Care.
doi:10.1097/MCO.0000000000000392
Brook, M. S., Wilkinson, D. J., Mitchell, W. K., Lund, J. L.,
Phillips, B. E., Szewczyk, N. J., … Atherton, P. J. (2017c).
A novel D2O tracer method to quantify RNA turnover as a
biomarker of de novo ribosomal biogenesis, in vitro, in
animal models, and in human skeletal muscle. American
Journal of Physiology-Endocrinology and Metabolism, 313,
E681–E689.
Brook, M. S., Wilkinson, D. J., Mitchell, W. K., Lund, J. N.,
Phillips, B. E., Szewczyk, N. J., … Atherton, P. J. (2016a).
Synchronous deficits in cumulative muscle protein synthesis
and ribosomal biogenesis underlie age-related anabolic resist-
ance to exercise in humans. The Journal of Physiology, 594,
7399–7417.
Brook, M. S., Wilkinson, D. J., Mitchell, W. K., Lund, J. N.,
Szewczyk, N. J., Greenhaff, P. L., … Atherton, P. J. (2015).
Skeletal muscle hypertrophy adaptations predominate in the
early stages of resistance exercise training, matching deuterium
oxide-derived measures of muscle protein synthesis and
mechanistic target of rapamycin complex 1 signaling. The
FASEB Journal, 29, 4485–4496.
Brook, M. S., Wilkinson, D. J., Phillips, B. E., Perez-Schindler, J.,
Philp, A., Smith, K., & Atherton, P. J. (2016b). Skeletal muscle
homeostasis and plasticity in youth and ageing: Impact of nutri-
tion and exercise. Acta Physiologica, 216, 15–41.
Brook, M. S., Wilkinson, D. J., Smith, K., & Atherton, P. J.
(2016c). The metabolic and temporal basis of muscle hypertro-
phy in response to resistance exercise. European Journal of Sport
Science, 16, 633–644.
Burd, N. A., West, D. W. D., Staples, A. W., Atherton, P. J.,
Baker, J. M., Moore, D. R., … Phillips, S. M. (2010). Low-
load high volume resistance exercise stimulates muscle protein
synthesis more than high-load low volume resistance exercise
in young men. PLoS ONE, 5, e12033.
Chaillou, T., Kirby, T. J., & Mccarthy, J. J. (2014). Ribosome bio-
genesis: Emerging evidence for a central role in the regulation of
skeletal muscle mass. Journal of Cellular Physiology, 229, 1584–
1594.
 10 M. S. Brook et al.
Chesley, A., MacDougall, J. D., Tarnopolsky, M. A., Atkinson, S.
A., & Smith, K. (1992). Changes in human muscle protein syn-
thesis after resistance exercise. Journal of Applied Physiology, 73,
1383–1388.
DeFreitas, J. M., Beck, T. W., Stock, M. S., Dillon, M. A., &
Kasishke, P. R. (2011). An examination of the time course of
training-induced skeletal muscle hypertrophy. European
Journal of Applied Physiology, 111, 2785–2790.
Doessing, S., Heinemeier, K. M., Holm, L., Mackey, A. L.,
Schjerling, P., Rennie, M., … Kjaer, M. (2010). Growth
hormone stimulates the collagen synthesis in human tendon
and skeletal muscle without affecting myofibrillar protein syn-
thesis. The Journal of Physiology, 588, 341–351.
Drake, J. C., Bruns, D. R., Peelor, F. F., Biela, L. M., Miller, R.
A., Miller, B. F., & Hamilton, K. L. (2015). Long-lived Snell
dwarf mice display increased proteostatic mechanisms that are
not dependent on decreased mTORC1 activity. Aging Cell, 14,
474–482.
Dufner, D., & Previs, S. F. (2003). Measuring in vivo metabolism
using heavy water. Current Opinion in Clinical Nutrition and
Metabolic Care, 6, 511–517.
Egner, I. M., Bruusgaard, J. C., & Gundersen, K. (2016). Satellite
cell depletion prevents fiber hypertrophy in skeletal muscle.
Development, 143, 2898–2906.
Egner, I. M., Bruusgaard, J. C., & Gundersen, K. (2017). An
apparent lack of effect of satellite cell depletion on hypertrophy
could be due to methodological limitations. Response to
‘Methodological issues limit interpretation of negative effects
of satellite cell depletion on adult muscle hypertrophy’.
Development, 144, 1365–1367.
Franchi, M. V., Atherton, P. J., Reeves, N. D., Flück, M.,
Williams, J., Mitchell, W. K., … Narici, M. V. (2014).
Architectural, functional and molecular responses to concentric
and eccentric loading in human skeletal muscle. Acta
Physiologica, 210, 642–654.
Fry, C. S., Lee, J. D., Jackson, J. R., Kirby, T. J., Stasko, S. A., Liu,
H., … Peterson, C. A. (2014). Regulation of the muscle fiber
microenvironment by activated satellite cells during hypertro-
phy. The FASEB Journal, 28, 1654–1665.
Fry, C. S., Lee, J. D., Mula, J., Kirby, T. J., Jackson, J. R., Liu, F.,
… Peterson, C. A. (2015). Inducible depletion of satellite cells
in adult, sedentary mice impairs muscle regenerative capacity
without affecting sarcopenia. Nature Medicine, 21, 76–80.
Goldberg, A. L., & Goodman, H. M. (1969). Relationship between
cortisone and muscle work in determining muscle size. The
Journal of Physiology, 200, 667–675.
Goodman, C. A., Frey, J. W., Mabrey, D. M., Jacobs, B. L.,
Lincoln, H. C., You, J. S., & Hornberger, T. A. (2011). The
role of skeletal muscle mTOR in the regulation of mechanical
load-induced growth. The Journal of Physiology, 589, 5485–
5501.
Griggs, C., Herr, E., Robert, C., Kingston, W., Jo-, R. F., Herr, B.
E., & Forbes, G. (1989). Effect of testosterone on muscle mass
and muscle protein synthesis. Journal of Applied Physiology, 66
(1), 498–503.
Gundersen, K., Sanes, J. R., & Merlie, J. P. (1993). Neural regu-
lation of muscle acetylcholine receptor epsilon- and alpha-
subunit gene promoters in transgenic mice. The Journal of Cell
Biology, 123, 1535–1544.
Hall, Z. W., & Ralston, E. (1989). Nuclear domains in muscle
cells. Cell, 59, 771–772.
Hellerstein, M. K. (2004). New stable isotope–mass spectrometric
techniques for measuring fluxes through intact metabolic path-
ways in mammalian systems: Introduction of moving pictures
into functional genomics and biochemical phenotyping.
Metabolic Engineering, 6, 85–100.
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
Heron, M. I., & Richmond, F. J. (1993). In-series fiber architec-
ture in long human muscles. Journal of Morphology, 216, 35–45.
Heron-Milhavet, L., Mamaeva, D., Leroith, D., Lamb, N. J., &
Fernandez, A. (2010). Impaired muscle regeneration and myo-
blast differentiation in mice with a muscle-specific KO of IGF-
IR. Journal of Cellular Physiology, 225, 1–6.
Hulmi, J. J., Lockwood, C. M., & Stout, J. R. (2010). Effect of
protein/essential amino acids and resistance training on skeletal
muscle hypertrophy: A case for whey protein. Nutrition &
Metabolism, 7, 51–31.
Jackson, R. J., Hellen, C. U. T., & Pestova, T. V. (2010). The
mechanism of eukaryotic translation initiation and principles
of its regulation. Nature Reviews Molecular Cell Biology, 11,
113–127.
Jackson, J. R., Mula, J., Kirby, T. J., Fry, C. S., Lee, J. D., Ubele,
M. F., … Dupont-Versteegden, E. E. (2012). Satellite cell
depletion does not inhibit adult skeletal muscle regrowth follow-
ing unloading-induced atrophy. American Journal of Physiology-
Cell Physiology, 303, C854–C861.
Kadi, F., Schjerling, P., Andersen, L. L., Charifi, N., Madsen, J.
L., Christensen, L. R., & Andersen, J. L. (2004). The effects
of heavy resistance training and detraining on satellite cells in
human skeletal muscles. The Journal of Physiology, 558, 1005–
1012.
Kim, J.-S., Petrella, J. K., Cross, J. M., & Bamman, M. M. (2007).
Load-mediated downregulation of myostatin mRNA is not suf-
ficient to promote myofiber hypertrophy in humans: A cluster
analysis. Journal of Applied Physiology, 103, 1488–1495.
Kirby, T. J., Patel, R. M., McClintock, T. S., Dupont-
Versteegden, E. E., Peterson, C. A., & McCarthy, J. J.
(2016). Myonuclear transcription is responsive to mechanical
load and DNA content but uncoupled from cell size during
hypertrophy. Molecular Biology of the Cell, 27, 788–798.
Kumar, V., Atherton, P. J., Selby, A., Rankin, D., Williams, J.,
Smith, K., … Rennie, M. J. (2012). Muscle protein synthetic
responses to exercise: Effects of age, volume, and intensity.
The Journals of Gerontology Series A: Biological Sciences and
Medical Sciences, 67, 1170–1177.
Kumar, V., Selby, A., Rankin, D., Patel, R., Atherton, P.,
Hildebrandt, W., … Rennie, M. J. (2009). Age-related differ-
ences in the dose-response relationship of muscle protein syn-
thesis to resistance exercise in young and old men. The
Journal of Physiology, 587, 211–217.
Kusnadi, E. P., Hannan, K. M., Hicks, R. J., Hannan, R. D.,
Pearson, R. B., & Kang, J. (2015). Regulation of rDNA tran-
scription in response to growth factors, nutrients and energy.
Gene, 556, 27–34.
Lepper, C., Partridge, T. A., & Fan, C.-M. (2011). An absolute
requirement for Pax7-positive satellite cells in acute injury-
induced skeletal muscle regeneration. Development, 138,
3639–3646.
Macallan, D. C., Asquith, B., Zhang, Y., de Lara, C., Ghattas, H.,
Defoiche, J., & Beverley, P. C. L. (2009). Measurement of pro-
liferation and disappearance of rapid turnover cell populations
in human studies using deuterium-labeled glucose. Nature
Protocols, 4, 1313–1327.
Mangine, G. T., Hoffman, J. R., Gonzalez, A. M., Townsend, J.
R., Wells, A. J., Jajtner, A. R., … Stout, J. R. (2017).
Exercise-induced hormone elevations are related to muscle
growth. Journal of Strength and Conditioning Research, 31(1),
45–53.
Marino, J. S., Tausch, B. J., Dearth, C. L., Manacci, M. V.,
McLoughlin, T. J., Rakyta, S. J., … Pizza, F. X. (2008). Β 2
-Integrins contribute to skeletal muscle hypertrophy in mice.
American Journal of Physiology-Cell Physiology, 295, C1026–
C1036.

Mathis, A. D., Naylor, B. C., Carson, R. H., Evans, E., Harwell, J.,
Knecht, J., … Price, J. C. (2017). Mechanisms of in vivo ribo-
some maintenance change in response to nutrient signals.
Molecular & Cellular Proteomics, 16, 243–254.
Mauro, A. (1961). Satellite cell of skeletal muscle fibers. The
Journal of Biophysical and Biochemical Cytology, 9, 493–495.
Mayer, C., & Grummt, I. (2006). Ribosome biogenesis and cell
growth: mTOR coordinates transcription by all three classes
of nuclear RNA polymerases. Oncogene, 25, 6384–6391.
McCarthy, J. J., Mula, J., Miyazaki, M., Erfani, R., Garrison, K.,
Farooqui, A. B., … Peterson, C. A. (2011). Effective fiber
hypertrophy in satellite cell-depleted skeletal muscle.
Development, 138, 3657–3666.
McKay, B. R., O’Reilly, C. E., Phillips, S. M., Tarnopolsky, M. A.,
& Parise, G. (2008). Co-expression of IGF-1 family members
with myogenic regulatory factors following acute damaging
muscle-lengthening contractions in humans. The Journal of
Physiology, 586, 5549–5560.
Millward, D. J., Garlick, P. J., James, W. P. T., Nnanyelugo, D. O.,
& Ryatt, J. S. (1973). Relationship between protein synthesis
and RNA content in skeletal muscle. Nature, 241, 204–205.
Mitchell, C. J., Churchward-Venne, T. A., West, D. W. D., Burd,
N. A., Breen, L., Baker, S. K., & Phillips, S. M. (2012).
Resistance exercise load does not determine training-mediated
hypertrophic gains in young men. Journal of Applied
Physiology, 113, 71–77.
Miyazaki, M., Mccarthy, J. J., Fedele, M. J., & Esser, K. A. (2011).
Early activation of mTORC1 signalling in response to mechan-
ical overload is independent of phosphoinositide 3-kinase/Akt
signalling. The Journal of Physiology, 589, 1831–1846.
Mobley, C. B., Haun, C. T., Roberson, P. A., Mumford, P. W.,
Kephart, W. C., Romero, M. A., … Roberts, M. D. (2018).
Biomarkers associated with low, moderate, and high vastus
lateralis muscle hypertrophy following 12 weeks of resistance
training. PLoS ONE, 13, 1–20.
Moss, F. P., & Leblond, C. P. (1971). Satellite cells as the source of
nuclei in muscles of growing rats. The Anatomical Record, 170,
421–435.
Musarò, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolny,
G., Molinaro, M., … Rosenthal, N. (2001). Localized Igf-1
transgene expression sustains hypertrophy and regeneration in
senescent skeletal muscle. Nature Genetics, 27, 195–200.
Nakada, S., Ogasawara, R., Kawada, S., Maekawa, T., & Ishii, N.
(2016). Correlation between ribosome biogenesis and the mag-
nitude of hypertrophy in overloaded skeletal muscle. PLoS
ONE, 11, e0147284.
Neese, R. A., Misell, L. M., Turner, S., Chu, A., Kim, J., Cesar,
D., … Hellerstein, M. K. (2002). Nonlinear partial differential
equations and applications: Measurement in vivo of prolifer-
ation rates of slow turnover cells by 2H2O labeling of the deox-
yribose moiety of DNA. Proceedings of the National Academy of
Sciences, 99, 15345–15350.
Petrella, J. K., Kim, J., Cross, J. M., Kosek, D. J., &
Bamman, M. M. (2006). Efficacy of myonuclear addition
may explain differential myofiber growth among resist-
ance-trained young and older men and women. American
Journal of Physiology-Endocrinology and Metabolism, 291,
E937–E946.
Petrella, J. K., Kim, J.-S., Mayhew, D. L., Cross, J. M., &
Bamman, M. M. (2008). Potent myofiber hypertrophy during
resistance training in humans is associated with satellite cell-
mediated myonuclear addition: A cluster analysis. Journal of
Applied Physiology, 104, 1736–1742.
Phillips, S. M., Tipton, K. D., Ferrando, A. A., & Wolfe, R. R.
(1999). Resistance training reduces the acute exercise-induced
increase in muscle protein turnover. American Journal of
Physiology, 276, E118–E124.
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
It’s not just about protein turnover 11
Phillips, B. E., Williams, J. P., Gustafsson, T., Bouchard, C.,
Rankinen, T., Knudsen, S., … Atherton, P. J. (2013).
Molecular networks of human muscle adaptation to exercise
and age. PLoS Genetics, 9, e1003389.
Pozefsky, T., Tancredi, R. G., Moxley, R. T., Dupre, J., & Tobin,
J. D. (1976). Effects of brief starvation on muscle amino acid
metabolism in nonobese man. Journal of Clinical Investigation,
57, 444–449.
Robinson, M. M., Turner, S. M., Hellerstein, M. K., Hamilton, K.
L., & Miller, B. F. (2011). Long-term synthesis rates of skeletal
muscle DNA and protein are higher during aerobic training in
older humans than in sedentary young subjects but are not
altered by protein supplementation. The FASEB Journal, 25,
3240–3249.
Rosenblatt, J. D., Yong, D., & Parry, D. J. (1994). Satellite cell
activity is required for hypertrophy of overloaded adult rat
muscle. Muscle & Nerve, 17, 608–613.
Schoenfeld, B. J., Ogborn, D., & Krieger, J. W. (2016). Effects of
resistance training frequency on measures of muscle hypertro-
phy: A systematic review and meta-analysis. Sports Medicine,
46, 1689–1697.
Snijders, T., Smeets, J. S. J., van Kranenburg, J., Kies, A. K., van
Loon, L. J. C., & Verdijk, L. B. (2016). Changes in myonuclear
domain size do not precede muscle hypertrophy during pro-
longed resistance-type exercise training. Acta Physiologica, 216,
231–239.
Spangenburg, E. E., Le Roith, D., Ward, C. W., & Bodine, S. C.
(2008). A functional insulin-like growth factor receptor is not
necessary for load-induced skeletal muscle hypertrophy. The
Journal of Physiology, 586, 283–291.
Stec, M. J., Kelly, N. A., Many, G. M., Windham, S. T., Tuggle,
S. C., & Bamman, M. M. (2016). Ribosome biogenesis may
augment resistance training-induced myofiber hypertrophy
and is required for myotube growth in vitro. American Journal
of Physiology-Endocrinology and Metabolism. doi:10.1152/
ajpendo.00486.2015
Stec, M. J., Mayhew, D. L., & Bamman, M. M. (2015). The effects
of age and resistance loading on skeletal muscle ribosome bio-
genesis. Journal of Applied Physiology, 119, 851–857.
Troy, A., Cadwallader, A., Fedorov, Y., Tyner, K., Tanaka, K. K.,
& Olwin, B. B. (2012). Coordination of satellite cell activation
and self-renewal by par-complex-dependent asymmetric acti-
vation of p38α/β MAPK. Cell Stem Cell, 11, 541–553.
van Riggelen, J., Yetil, A., & Felsher, D. W. (2010). MYC as a reg-
ulator of ribosome biogenesis and protein synthesis. Nature
Reviews Cancer, 10, 301–309.
von Walden, F., Casagrande, V., Ostlund Farrants, A.-K., &
Nader, G. A. (2012). Mechanical loading induces the
expression of a Pol I regulon at the onset of skeletal muscle
hypertrophy. American Journal of Physiology-Cell Physiology,
302, C1523–C1530.
Waldman, F. M., Chew, K., Ljung, B. M., Goodson, W., Hom, J.,
Duarte, L. A., … Mayall, B. (1991). A comparison between
bromodeoxyuridine and 3H thymidine labeling in human
breast tumors. Modern Pathology, 4, 718–722.
West, D. W. D., Baehr, L. M., Marcotte, G. R., Chason, C.
M., Tolento, L., Gomes, A. V., … Baar, K. (2016). Acute
resistance exercise activates rapamycin-sensitive and insensi-
tive mechanisms that control translational activity and
capacity in skeletal muscle. The Journal of Physiology, 594,
453–468.
West, D. W. D., & Phillips, S. M. (2012). Associations of exercise-
induced hormone profiles and gains in strength and hypertro-
phy in a large cohort after weight training. European Journal of
Applied Physiology, 112, 2693–2702.
Wilkinson, D. J., Brook, M. S., Smith, K., & Atherton, P. J.
(2017). Stable isotope tracers and exercise physiology: Past,
 12 M. S. Brook et al.
present and future. The Journal of Physiology. doi:10.1113/
JP272277
Wilkinson, D. J., Franchi, M. V., Brook, M. S., Narici, M. V.,
Williams, J. P., Mitchell, W. K., … Smith, K. (2014). A
validation of the application of D2O stable isotope tracer tech-
niques for monitoring day-to-day changes in muscle protein
subfraction synthesis in humans. American Journal of
Physiology-Endocrinology and Metabolism, 306, E571–E579.
Tiago França de Farias - tiagoddroid@gmail.com - CPF: 082.020.113-88
Witard, O. C., Jackman, S. R., Breen, L., Smith, K., Selby, A., &
Tipton, K. D. (2014). Myofibrillar muscle protein synthesis
rates subsequent to a meal in response to increasing doses of
whey protein at rest and after resistance exercise. The
American Journal of Clinical Nutrition, 99, 86–95.
Wolfe, R. R. (2006). The underappreciated role of muscle in health
and disease. The American Journal of Clinical Nutrition, 84, 475–
482.