Research and Perspectives in Endocrine Interactions

Bruce Spiegelman Editor

Hormones,
Metabolism
and the
Benefits of
Exercise
Research and Perspectives in Endocrine
Interactions
More information about this series at http://www.springer.com/series/5241
Bruce Spiegelman
Editor

Hormones, Metabolism and

the Benefits of Exercise
Editor
Bruce Spiegelman Fondation IPSEN
Dana-Farber Cancer Institute Boulogne-Billancourt
Harvard Medical School France
Boston, MA
USA

Acknowledgement: The editors wish to express their gratitude to Mrs. Mary Lynn Gage for
her editorial assistance.

ISSN 1861-2253     ISSN 1863-0685 (electronic)

Research and Perspectives in Endocrine Interactions
ISBN 978-3-319-72789-9    ISBN 978-3-319-72790-5 (eBook)
https://doi.org/10.1007/978-3-319-72790-5

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Preface

We gathered in Paris to present new work on exercise and the molecular understand-
ing of its actions and benefits. Exercise has been understood for centuries to provide
benefits to the human body. Diet and exercise have long been prescribed as the first
lines of therapy for metabolic diseases such as obesity and type 2 diabetes. However,
it is perhaps not fully appreciated that exercise is also effective at improving the
function of the liver, muscles themselves, and (importantly) the brain. Endurance
exercise is one of the only ways that adult humans can stimulate de novo neurogen-
esis. If we can understand the precise ways that these benefits occur, we can hope to
both optimize exercise protocols and perhaps find molecular entities that can bring
benefits beyond what can be done by exercise alone.
It is critical to note that the goal of most basic scientists in this area is not to
replace exercise with a pill. Indeed, this seems a very naïve and unlikely goal.
However, it is important to recognize that many people cannot exercise effectively
because of age or infirmities such as cancer, neuromuscular diseases, neurodegen-
eration, and postsurgical trauma. Furthermore, there is a limit to how much exercise
even fit people can do, as exercise takes time and can itself be associated with joint
or muscle inflammation. Perhaps specific molecules regulated in exercise can be
dosed well beyond what is naturally produced during exercise. While this idea is
still at the hypothetical stage, the NIH has responded to these challenges and oppor-
tunities by launching the new program “Molecular Transducers of Physical Activity.”
Exercise is not one thing. It can differ in duration, intensity, and type of activity.
Running and weight lifting are very different things, and they are likely to involve
different signaling systems and bring different benefits. The response to exercise
also has a time element: short-term studies might show a degree of tissue damage,
whereas longer term studies are more likely to show benefits.
Our own work in this area was stimulated by the observations made by us and
others that the transcriptional coactivator PGC1α, which we discovered in brown
fat, is induced in muscle with endurance training; it then stimulates mitochondrial
biogenesis and many of the pathways seen in endurance-trained muscle. Included
here was an observed resistance to muscular dystrophy and muscle atrophy, at least
in mice. Our interest in how PGC1α expression in muscle leads to benefits in distant

v
vi Preface

tissues led to the discovery of irisin, a small secreted protein derived from muscle
(a “myokine”) that is controlled by exercise and PGC1α in mice and humans.
With this as background, this volume contains new work presented at this confer-
ence that attempts to expand the dialogue between scientists focused on human
exercise studies and those who want to understand these processes at the molecular
level. Surely, our ability to develop new therapies in this area will require close col-
laborations between academics and eventually with the biotech and pharmaceutical
worlds.

Dana-Farber Cancer Institute Bruce Spiegelman

Harvard Medical School
Boston, MA, USA
Contents

 uman Brown Adipose Tissue Plasticity: Hormonal and Environmental

H
Manipulation������������������������������������������������������������������������������������������������������   1
Francesco S. Celi
 he Energy Sensor AMPK: Adaptations to Exercise, Nutritional
T
and Hormonal Signals ��������������������������������������������������������������������������������������  13
Benoit Viollet
 lasma Steroids and Cardiorespiratory Fitness Response to Regular
P
Exercise ������������������������������������������������������������������������������������������������������������  25
Zihong He, Tuomo Rankinen, Arthur S. Leon, James S. Skinner,
André Tchernof, and Claude Bouchard
 ending the Signal: Muscle Glycogen Availability as a Regulator
S
of Training Adaptation��������������������������������������������������������������������������������������  43
John A. Hawley
 ptimized Engagement of Macrophages and Satellite Cells
O
in the Repair and Regeneration of Exercised Muscle������������������������������������  57
Regula Furrer and Christoph Handschin
 keletal Muscle microRNAs: Roles in Differentiation, Disease
S
and Exercise����������������������������������������������������������������������������������������������� 67
Rasmus J. O. Sjögren, Magnus H. L. Lindgren Niss, and Anna Krook
 ryptophan-Kynurenine Metabolites in Exercise and Mental Health����������  83
T
Paula Valente-Silva and Jorge Lira Ruas
 he Role of FNDC5/Irisin in the Nervous System and as a Mediator
T
for Beneficial Effects of Exercise on the Brain������������������������������������������������  93
Mohammad Rashedul Islam, Michael F. Young, and Christiane D. Wrann

vii
List of Contributors

Claude  Bouchard  Human Genomics Laboratory, Pennington Biomedical

Research Center, Baton Rouge, LA, USA
School of Nutrition, Laval University, Quebec City, QC, Canada
Francesco  S.  Celi  Division of Endocrinology Diabetes and Metabolism,
Department of Internal Medicine, Virginia Commonwealth University, Richmond,
VA, USA
Regula Furrer  Biozentrum, University of Basel, Basel, Switzerland
Christoph Handschin  Biozentrum, University of Basel, Basel, Switzerland
John  A.  Hawley  Mary MacKillop Institute for Health Research, Australian
Catholic University, Melbourne, VIC, Australia
Research Institute for Sport and Exercise Sciences, Liverpool John Moores
University, Liverpool, UK
Zihong  He  Human Genomics Laboratory, Pennington Biomedical Research
Center, Baton Rouge, LA, USA
Department of Biology, China Institute of Sport Science, Beijing, China
Mohammad  Rashedul  Islam  Massachussetts General Hospital and Harvard
Medical School, Charlestown, MA, USA
Anna Krook  Department of Physiology and Pharmacology, Integrative Physiology,
Karolinska Institutet, Stockholm, Sweden
Arthur S. Leon  Department of Biology, China Institute of Sport Science, Beijing,
China
Magnus Lindgren Niss  Department of Physiology and Pharmacology, Integrative
Physiology, Karolinska Institutet, Stockholm, Sweden
Tuomo Rankinen  Human Genomics Laboratory, Pennington Biomedical Research
Center, Baton Rouge, LA, USA

ix
x List of Contributors

Jorge  Lira  Ruas  Department of Physiology and Pharmacology, Molecular and

Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden
Rasmus J. O. Sjögren  Department of Molecular Medicine and Surgery, Karolinska
Institutet, Stockholm, Sweden
James S. Skinner  School of Kinesiology, University of Minnesota, Minneapolis,
MN, USA
Bruce Spiegelman  Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA, USA
André  Tchernof  Department of Kinesiology, Indiana University, Bloomington,
IN, USA
Paula Valente-Silva  Department of Physiology and Pharmacology, Molecular and
Cellular Exercise Physiology, Karolinska Institutet, Stockholm, Sweden
Benoit Viollet  Institut Cochin, INSERM, U1016, Paris, France
CNRS, UMR8104, Paris, France
Université Paris Descartes, Sorbonne Paris cité, Paris, France
Christiane  D.  Wrann  Massachussetts General Hospital and Harvard Medical
School, Charlestown, MA, USA
Michael F. Young  Massachussetts General Hospital and Harvard Medical School,
Charlestown, MA, USA
Human Brown Adipose Tissue Plasticity:
Hormonal and Environmental
Manipulation

Francesco S. Celi

Abstract  Brown adipose tissue (BAT), brown-in-white (“brite”) and “beige” adi-
pocytes share the unique ability of converting chemical energy into heat and play a
critical role in the adaptive thermogenesis response promoting nonshivering ther-
mogenesis. Uncoupling Protein-1 (UCP1), which allows the uncoupling of sub-
strate oxidation from phosphorylation of ADP, represents the molecular signature of
BAT and beige adipocytes. Until recently, the physiologic role of BAT and beige
adipocytes depots was thought to be limited to small mammalians and newborns.
The discovery of BAT in adult humans and the demonstration of the presence
of inducible BAT activity in white adipose tissue by beige adipocytes have gener-
ated enthusiasm as potential targets for treatment of obesity and other disorders
due to sustained positive energy balance. These findings are particularly important
since in vitro studies have demonstrated that preadipocytes can be directed toward
a common brown phenotype by multiple pathways that, in turn, may be exploited
for therapeutic interventions. In adult humans, BAT activity is more evident in
deep neck fat depots and, to a lesser degree, in subcutaneous adipose tissue, with
a transcriptome signature resembling the rodent beige fat. This observation sup-
ports the hypothesis that human BAT activity and capacity can be modulated. To
this end, we have directed our translational research program toward the charac-
terization of beige fat in humans and on the effects of hormonal and environmen-
tal drivers in the adaptive thermogenesis response. Mild cold exposure, within the
temperature range commonly employed in climate-controlled buildings, is suffi-
cient to generate a significant increase in non-shivering thermogenesis driven by
BAT and beige adipocyte activation. In turn, adaptive thermogenesis generates a
specific hormonal signature and promotes glucose disposal. Chronic exposure to
mild cold induces expansion of BAT mass and activity, whereas exposure to warm
climate abrogates them. Additionally, the metabolic effects of BAT mass expan-
sion are evident only upon stimulation of BAT activity, indicating that both

F. S. Celi (*)
Division of Endocrinology Diabetes and Metabolism, Department of Internal Medicine,
Virginia Commonwealth University, Richmond, VA, USA
e-mail: Francesco.celi@vchuhealth.org

© The Author(s) 2017 1

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_1
2 F. S. Celi

e­ xpansion and activation of BAT are necessary and complementary strategies to

pursue. From an experimental standpoint, human preadipocytes represent a viable
experimental platform to m ­ echanistically interrogate different pathways that are
able to expand and activate browning. Our laboratory has focused on studying
FGF-21, and FNDC5/irisin in their capacity to promote the browning process.
Compared to a white differentiation medium, the addition of either FGF-21 or
FNDC5 results in a reprogramming toward a brown phenotype, as indicated by the
display of brown transcriptome signature and, functionally, by the increase in
oxygen consumption following catecholamine treatment, indicating an increase in
substrate utilization. Collectively, the integration of a detailed assessment of
human physiology with mechanistic observations in cell culture systems can pro-
vide a unique opportunity to translate observations from experimental models to
actionable therapeutic targets.

Introduction

The unique ability of mammalians (and to some degree avians; Vianna et al. 2001)
to maintain their core temperature independently of the environmental temperature
has allowed their evolutionary success and, compared to poikilothermic species, to
the wide distribution of species across climates. This response is defined as “adap-
tive thermogenesis,” which describes the concerted physiologic responses aimed at
defending the core temperature from low environmental temperatures, preserving
the individual’s ideal core temperature for biologic functions (Stocks et al. 2004).
The main components of this response are thermal insulation, non-shivering ther-
mogenesis, and shivering thermogenesis (Celi et al. 2015). From an evolutionary
perspective, the excess of energy expenditure aimed at maintaining the core tem-
perature represents a tradeoff between survival and maintenance of energy stores,
since the availability of energy usually represents the limiting factor to growth and
reproduction for organisms. To this end, moving from an insulative response, which
is energy neutral, to a non-shivering and eventually shivering thermogenesis will
require a progressively greater dissipation of energy stores (Fig. 1). Additionally,
the relative size of the organism and the presence of fur dictate the relative impor-
tance of the insulative versus thermogenic response, with smaller organisms being
biased toward the latter because of their high surface area-to-volume ratio, which,
despite the presence of optimal insulation, will still promote thermal dispersion
(Phillips and Heath 1995). Hence, modulation of energy expenditure plays a critical
role in the maintenance of core temperature in small mammalians (Ravussin and
Galgani 2011). Compared to humans, the capacity of adaptive thermogenesis in
small rodents is significantly greater, resulting in an at least twofold increase in total
energy expenditure, thus enabling survival in a cold environment, albeit at a price of
a compensatory increase in food intake (Ravussin et  al. 2014). Over the recent
Human Brown Adipose Tissue Plasticity: Hormonal and Environmental Manipulation 3

Fig. 1  Model of adaptive thermogenesis. As the environmental temperature decreases from ther-
moneutrality, the adaptive thermogenesis response moves from insulative to non-shivering and
eventually shivering thermogenesis. This progression is mirrored by an increase in the energy
expenditure required to maintain the core temperature. Green, energy expenditure due to basal
metabolic rate; red, expenditure due to adaptive thermogenesis

evolutionary development of the species, humans have gained the ability to modu-
late their environment using garments and climate-controlled buildings. As a result,
exposure to cold is a relatively unusual condition for them and, unless acclimated,
individuals tend to respond by shivering thermogenesis, whereby heat is a side
product of uncontrolled muscle fasciculation. Of interest, upon prolonged exposure
to cold, individuals become resilient and do not display shivering thermogenesis
(Davis 1961), indicating that other mechanisms are recruited.

BAT and the Adaptive Thermogenesis Response

BAT has the unique capability of converting energy stores into heat by virtue of the
Uncoupling Protein 1 (UCP1), which promotes a proton leak in the inner membrane
of the mitochondria, dissociating the oxidative phosphorylation of substrate from
the generation of ATP. This process in essence shunts the chemical energy into heat,
which is in turn dissipated in the circulation (Nedergaard et al. 2001). The rapid
activation and inactivation of the UCP1-driven energy dissipation thus represents a
valuable mechanism that is able to increase efficiently the metabolic rate on demand
by non-shivering thermogenesis, ultimately promoting the survival of the organism
against unfavorable environmental conditions with the least possible consumption
of energy stores (Celi 2009).
4 F. S. Celi

Aside from the anatomically defined intrascapular BAT, in mice non-shivering

thermogenesis is generated in other adipose tissue depots, in particular inguinal, by
“beige” or “brown-in-white (brite)” adipocytes (Ishibashi and Seale 2010; Petrovic
et al. 2010), whose mass and activity can be modulated by various signal hormones
and cytokines, showing a remarkable plasticity (Diaz et al. 2014).

Implications of the Rediscovery of BAT in Adult Humans

Several reports recognized the presence of brown adipocytes in adult humans, either
as incidental findings (Huttunen et al. 1981) or as a nuisance in the interpretation of
18
F fluorodeoxyglucose Positron Emission Tomography (18F FDG-PET) scans
(Hany et al. 2002; Agrawal et al. 2009), but the potential clinical relevance of these
findings was missed until the “rediscovery” of BAT. Three manuscripts published in
the same issue of the New England Journal of Medicine, from the USA (Cypess
et  al. 2009), Finland (Virtanen et  al. 2009), and the Netherlands (van Marken
Lichtenbelt et al. 2009), and followed shortly by a manuscript from Japan (Saito
et al. 2009), clearly indicated that BAT was present in a significant number of adults
and that its presence and activity (measured by proxy as 18F FDG uptake by PET
scanning) correlated with indices of healthy metabolism. Conversely, obesity, dia-
betes and age inversely correlated with BAT activity (Pfannenberg et  al. 2010).
Although the initial observations were purely correlative, the potential of a novel
therapeutic target for the treatment of the metabolic consequences of obesity led to
intense research to expand the capacity and activity of human BAT. Importantly, the
location of adult BAT is different from the intrascapular depots observed in new-
borns (Drubach et al. 2011), and the transcriptome signature resembles more closely
the one observed in rodent inguinal fat (Shinoda et al. 2015). This observation is
relevant from the therapeutic perspective because of the exquisite plasticity of
inguinal adipocytes, providing the rationale to exploit pathways to increase non-­
shivering thermogenesis as a means of promoting energy disposal with consequent
amelioration of obesity and its metabolic consequences.

I ntegrative Physiology Studies of Human Adaptive

Thermogenesis

To better characterize the relevance of the adaptive thermogenesis response, several

integrative physiology studies were carried out in humans, mostly by exposing vol-
unteers to cold. While a short but intense cold exposure (such as immersion of a
limb in ice-cold water) is able to generate maximal 18F FDG-PET uptake and a
significant increase in energy expenditure (Yoneshiro et al. 2011), this method does
not correspond to day-to-day experience and cannot be sustained over time.
Human Brown Adipose Tissue Plasticity: Hormonal and Environmental Manipulation 5

Table 1  Hormonal axes and Hormonal axes and organ systems Response to cold
organ-system response to
Sympathetic nervous system ↑↑
mild cold exposure
Adrenals (medulla, epinephrine) ↑
Adrenal (cortical, cortisol) ↑
Thyroid axis ↑↔
Fasting glucose ↔
Postprandial glucose ↓
Subcutaneous adipose tissue glucose ↔↓
Fasting insulin ↑
Postprandial insulin ↔
Free fatty acids ↑↑
Subcutaneous adipose tissue lipolysis ↔↑
FGF-21 ↑↑

Additionally, intense cold exposure results in muscle fasciculation, with consequen-

tial recruitment of shivering thermogenesis. Conversely, mild cold exposure within
the thermal envelope of climate-controlled buildings can stimulate a sustained phys-
iologic adaptive thermogenesis response. In a crossover study, the adaptive thermo-
genesis response to minimal changes in environmental temperature was assessed in
healthy volunteers who underwent two 12-h energy expenditure (EE) recordings in
a whole room calorimeter (Metabolic Chamber) at 19 and 24 °C (Celi et al. 2010).
The system employed in these studies recorded real-time measurements of EE and
substrate utilization (respiratory quotient) while sealed ports allowed blood sam-
pling during the study. Moreover, study volunteers were fitted with linear acceler-
ometers and subcutaneous abdominal tissue extracellular fluid microdialysis
sampling, and received a standard meal after 6 h of recording, enabling a compre-
hensive assessment of the physiologic response to short-term mild cold exposure.
This complex experimental design allowed to obtain a comprehensive assessment
of the physiology of adaptive thermogenesis.
The data obtained from these studies indicated that a minimal modulation of the
environmental temperature was sufficient to increase EE by approximately 6%,
which corresponds, if projected over 24 h, to 100 kcal in an individual of 70–80 kg.
The magnitude of this change may appear insignificant—only one fifth of the nega-
tive energy balance recommended to achieve sustained weight loss—but it is impor-
tant to note that, over a 1-year period (all things being equal), these differences
would be equivalent to a 20-day fast and to a daily 30-min walk at a moderate pace.
Importantly, these observations also demonstrated that the adaptive thermogenesis
response during mild cold exposure resulted in a significant and potentially relevant
metabolic effect. A summary of the findings is reported in Table 1 (Celi et al. 2010).
Exposure to mild cold is sufficient to drive an adrenergic response, which promotes
lipolysis and increased postprandial glucose disposal. The significant lipolysis
observed during non-shivering thermogenesis is in keeping with a functional imag-
ing study that demonstrated that fatty acids represent the preferred substrate in BAT
6 F. S. Celi

depots. On the other hand, the intervention was also sufficient to generate an
increase in cortisol and a state of relative insulin resistance during fasting, which
indicate an activation of the stress response.
A subsequent study correlated the individual’s BAT activity, as measured by 18F
FDG uptake in the thoracic region, where BAT is commonly observed with the
adaptive thermogenesis response, to mild cold exposure (Chen et al. 2013). In this
case, scans were acquired after a 12-h metabolic chamber recording either at 19 or
24 °C and the EE recordings were performed overnight to allow 18F FDG-PET the
following morning. By comparing the tracer uptake of the two images, the overall
uptake in the region of interest could be calculated and used as a linear variable to
be included in multivariate statistical analyses. This strategy provided the opportu-
nity to explore non-shivering thermogenesis as a diffuse function rather than local-
ized in nests of brown adipocytes, overcoming the signal-to-noise ratio limitation of
functional imaging. In turn, this variable was used to correlate BAT activity with
metabolic and anthropometric parameters. This analysis demonstrated that BAT
activity strongly correlated with the increase in EE during mild cold exposure.
Remarkably, the association was present in individuals both with and without visi-
ble BAT depots on conventional 18F FDG PET imaging, indicating that diffuse (i.e.,
beige adipocytes-driven) uncoupling activity is an important mediator of non-­
shivering thermogenesis in adult humans (Chen et al. 2013).

I ntervention Studies Aimed to Modulate the Human Adaptive

Thermogenesis Response

These correlative studies did not address the question whether human BAT (and
beige adipocytes) had the plasticity displayed by murine inguinal fat-depots, or if
the capacity of BAT was sufficient to modulate a clinically significant endpoint. A
short-term, moderate (10 days acclimatization with exposure at 17 °C daily for 2 h)
cold exposure was sufficient to increase BAT as measured by 18F FDG-PET (van der
Lans et al. 2013), whereas a similar protocol carried out for a longer period of time
(6 weeks acclimatization with exposure at 17 °C daily for 2 h) resulted in significant
fat mass reduction (Yoneshiro et al. 2013). In a longer study, the plasticity of the
adaptive thermogenesis response was assessed by modulating the environmental
temperature overnight (Lee et  al. 2014b). Study volunteers were exposed over a
period of four consecutive months to 24 °C (run-in period), 19 °C (cold acclimatiza-
tion), 24 °C (wash-out period), and 30 °C (heat acclimatization). At the end of each
month the adaptive thermogenesis response was studied using two consecutive met-
abolic chamber recordings at 19 and 24  °C; 18F FDG-PET for visualization and
measurement of BAT activity was performed after the completion of the 19  °C
metabolic chamber stay. After a month-long exposure to mild cold, BAT volume
and activity nearly doubled when compared to the end of the run-in period.
Human Brown Adipose Tissue Plasticity: Hormonal and Environmental Manipulation 7

Conversely, BAT activity was negligible following a 1-month exposure to warm

temperature (30 °C). Remarkably, the increase in BAT activity following the cold
acclimatization was accompanied by a significant increase in postprandial glucose
disposal, but only during mild cold exposure (Lee et al. 2014b). Thus, the data indi-
cate that human BAT is exquisitely plastic but also that activation (i.e., cold expo-
sure or other pharmacologic means) is necessary to generate a significant metabolic
signature.
Collectively, these integrative physiology observations indicate that human BAT
and beige depots represent an ideal pharmacologic target for the treatment of the
consequences of a sustained positive energy balance because of their plasticity and
ability to modulate carbohydrate metabolism.

Manipulation of the Human Adaptive Thermogenesis

Response by Hormones and Myokines

Similarly to white adipocytes, BAT and beige adipocytes are not only targets of
hormones and cytokines but also have an endocrine function, and their secretome
can act in a paracrine and endocrine fashion (Ni et al. 2015). Primary cultures of
preadipocytes derived from the stromal vascular fraction of adipose tissues have
been proven to be a robust experimental model to assess the effects of hormones and
cytokines on their differentiation and function (Diaz et al. 2014). Preadipocytes can
be directed toward a white or beige phenotype by hormonal manipulation of the
culture media, indirectly demonstrating the plasticity of adipose tissue depots.
Moreover, individual hormones, cytokines or drugs can be tested for their ability to
promote “beige-ing” in white-differentiating preadipocytes. Our laboratory has
worked toward defining the role of two regulators of beige adipocytes, FGF-21 and
FNDC5/irisin. FGF-21 is a pleiotropic hormone that is mainly secreted by the liver
and promotes insulin sensitization and browning of adipose tissue depots.
Interestingly, FGF-21 is also secreted by beige adipocytes, creating an autocrine/
paracrine loop (Ni et al. 2015). In humans, FGF-21 follows a circadian rhythm that
is disrupted by cold exposure (Lee et al. 2013). Compared to exposure to 24 °C,
exposure to mild cold resulted in an increase in the FGF-21. Importantly, the
increase in FGF-21 (compared to thermoneutrality) correlated with the non-­
shivering thermogenesis response; this finding was also confirmed by measuring the
changes in FGF-21 in relation to the lipolysis measured in the subcutaneous adipose
tissue mocrodialysate (Lee et al. 2013). Collectively these observations support the
role of FGF-21 in promoting and sustaining the adipocyte role in the non-shivering
thermogenesis response. When FGF-21 was added to the white differentiating cul-
ture medium, human preadipocytes were directed toward a brown phenotype and
were able to increase their oxygen consumption and heat production in response to
norepinephrine treatment (Lee et al. 2013). These findings recapitulate the clinical
8 F. S. Celi

observations that FGF-21 plays a pivotal role in beige adipocyte differentiation and
function. Unfortunately, therapeutic use of FGF-21 has been limited because of
concerns for off-target effects (Kharitonenkov and Adams 2014) possibly due to the
systemic administration of the hormone in pharmacologic doses, unlike the local,
paracrine action within the adipose tissue depots.
The discovery that BAT derives from a myf-5-positive precursor common to
myocytes (Seale et  al. 2008) has brought to the forefront the cross-talk between
skeletal muscle and brown-beige adipocytes. Similar to adipose tissue depots, skel-
etal muscle has the ability to secrete myokines with hormonal activity on distant
tissues. In particular, Irisin, a fibronectin-like peptide released by skeletal muscle
upon contraction, has the ability to promote beige-ing in mouse inguinal adipose
tissue depots and to positively affect energy and carbohydrate metabolism (Bostrom
et al. 2012). The action of Irisin might appear counterintuitive since it is released by
an energy-dissipating organ (skeletal muscle during contraction) and its action pro-
motes the expansion of another energy-dissipating organ tissue (beige adipocytes).
On the other hand, this apparent paradox can be reconciled once it is observed from
the evolutionary perspective of energy conservation: severe cold exposure, which is
able to generate shivering (highly energy inefficient) thermogenesis, promotes the
expansion of the capacity of the more energy-efficient BAT and beige adipocyte-­
driven non-shivering thermogenesis. An integrated physiology experiment was thus
designed to evaluate this hypothesis by characterizing the entire spectrum of the
adaptive thermogenesis response in healthy volunteers exposed to progressively
lower temperatures delivered by cooling blankets (Lee et al. 2014c). This strategy
allowed to capture in the same individual the vasoconstrictive response as well non-
shivering and shivering thermogenesis, while the EE was annotated by indirect
calorimetry (ventilated hood). Study volunteers also underwent basal and post-­
exercise blood sampling following a maximal exercise tolerance test (VO2 Max)
lasting approximately 15 min and a 60-min resistance exercise at 40% VO2 Max.
The findings of this study demonstrated a large inter-individual variability in both
onset of shivering and Irisin secretion either following exercise or during controlled
cooling. Furthermore, during cold exposure the release of Irisin was proportional to
the shivering intensity (measured by surface electromyography). Interestingly,
short-term shivering was sufficient to stimulate the release of a similar amount of
Irisin when compared to maximal or resistance exercise (Lee et al. 2014c). When
tested on human preadipocytes undergoing white differentiation, Irisin promoted a
beige phenotype not dissimilar to the one observed following FGF-21 treatment
(Lee et al. 2014a). Collectively these data confirm the role of Irisin as a myokine
that is able to expand beige adipocyte mass, increasing the non-shivering thermo-
genesis capacity and thus promoting a shift from an inefficient (shivering) to a more
efficient and sustainable form of thermogenesis (Fig. 2).
Human Brown Adipose Tissue Plasticity: Hormonal and Environmental Manipulation 9

Fig. 2  Model of interplay between shivering and non-shivering thermogenesis. Exposure to cold
promotes shivering in non-acclimated individuals, with release of Irisin. This response is costly
from an energy-conservation perspective and not sustainable. Irisin promotes the expansion and
differentiation of beige adipocytes, which increase resilience to cold enhancing non-shivering ther-
mogenesis and delay the onset of shivering. The greater beige adipocyte mass self-sustains by the
autocrine-paracrine effects of FGF-21

Conclusions

In conclusion, human integrated physiology observations coupled with translational

studies offer the possibility to explore the potential therapeutic exploitation of the
adaptive thermogenesis response, an evolutionary mechanism that has greatly
expanded the footprint of mammalians on a wide range of climates. The ability to
promote substrate utilization has the obvious appeal of an “effortless diet pill,” but
the capacity of the system is probably insufficient to generate alone a negative
energy balance to induce weight loss. Nonetheless, even a modest increase in sub-
strate utilization has been proven able to positively impact energy and carbohydrate
metabolism. Thus, the search for novel pharmacologic interventions able to expand
the beige adipocytes mass, and thereby increase the capacity for non-shivering ther-
mogenesis, holds the promise of being a successful treatment to improve insulin
resistance and the negative metabolic consequences of obesity.

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The Energy Sensor AMPK: Adaptations
to Exercise, Nutritional and Hormonal
Signals

Benoit Viollet

Abstract To sustain metabolism, intracellular ATP concentration must be regu-

lated within an appropriate range. This coordination is achieved through the func-
tion of the AMP-activated protein kinase (AMPK), a cellular “fuel gauge” that is
expressed in essentially all eukaryotic cells as heterotrimeric complexes containing
catalytic α subunits and regulatory β and γ subunits. When cellular energy status has
been compromised, AMPK is activated by increases in AMP:ATP or ADP:ATP
ratios and acts to restore energy homeostasis by stimulating energy production via
catabolic pathways while decreasing non-essential energy-consuming pathways.
Although the primary function of AMPK is to regulate energy homeostasis at a cell-­
autonomous level, in multicellular organisms, the AMPK system has evolved to
interact with hormones to regulate energy intake and expenditure at the whole body
level. Thus, AMPK functions as a signaling hub, coordinating anabolic and cata-
bolic pathways to balance nutrient supply with energy demand at both the cellular
and whole-body levels. AMPK is activated by various metabolic stresses such as
ischemia or hypoxia or glucose deprivation and has both acute and long-term effects
on metabolic pathways and key cellular functions. In addition, AMPK appears to be
a major sensor of energy demand in exercising muscle and acts both as a multitask
gatekeeper and an energy regulator in skeletal muscle. Acute activation of AMPK
has been shown to promote glucose transport and fatty acid oxidation while sup-
pressing glycogen synthase activity and protein synthesis. Chronic activation of
AMPK induces a shift in muscle fiber type composition, reduces markers of muscle
degeneration and enhances muscle oxidative capacity potentially by stimulating
mitochondrial biogenesis. Furthermore, recent evidence demonstrates that AMPK
may not only regulate metabolism during exercise but also in the recovery phase.
AMPK acts as a molecular transducer between exercise and insulin signaling and is

B. Viollet (*)
Institut Cochin, INSERM, U1016, Paris, France
CNRS, UMR8104, Paris, France
Université Paris Descartes, Sorbonne Paris cité, Paris, France
e-mail: benoit.viollet@inserm.fr

© The Author(s) 2017 13

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_2
14 B. Viollet

necessary for the ability of prior contraction/exercise to increase muscle insulin

sensitivity. Based on these observations, drugs that activate AMPK might be
expected to be useful in the treatment of metabolic disorders and insulin resistance
in various conditions.

Introduction

One fundamental parameter that living cells need to sustain essential cellular func-
tions is the maintenance of sufficiently high level of ATP.  Thus, cell survival is
dependent on a dynamic control of energy metabolism when ATP demand needs to
remain in balance with ATP supply. If ATP consumption exceeds ATP production,
the ADP:ATP ratio rises, but this is converted into an even larger rise in AMP:ATP
ratio due to the reaction catalyzed by adenylate kinase (2ADP ↔ ATP + AMP). If
the reaction is at equilibrium, the AMP:ATP ratio will vary as the square of the
ADP:ATP ratio, making increases in AMP a more sensitive indicator of energy
stress than decreases in ATP or increases in ADP. On this basis, the cell requires an
efficient energy sensory mechanism based on the detection of the ratios of ADP:ATP
or AMP:ATP. Such a system has been identified as the AMP-activated protein kinase
(AMPK), a heterotrimeric serine/threonine kinase conserved throughout eukaryote
evolution (Hardie et al. 2012). The primary function of AMPK is to monitor changes
in the intracellular level of ATP and maintain energy stores by reprogramming
metabolism through an increase in the rate of catabolic ATP-producing pathways
and a decrease in the rate of nonessential anabolic ATP-utilizing pathways. These
regulatory features are initiated by the phosphorylation of key metabolic enzyme as
well as transcription factors for both short-term effects and long-range regulatory
actions for a better response to future challenges. Although the AMPK system origi-
nally evolved to regulate energy homeostasis in a cell-autonomous manner, in mul-
ticellular organisms, its role has adapted to integrate stress responses such as
exercise as well as nutrient and hormonal signals to control food intake, energy
expenditure, and substrate utilization at the whole body level (Hardie 2014).
Activation of AMPK is triggered by a diverse array of external (e.g., exercise, hor-
mones, nutrients) and internal signals (e.g., AMP/ATP and ADP/ATP ratios) and has
been implicated in the regulation of a wide range of biochemical pathways and
physiological processes. As a consequence, AMPK has stimulated much interest
due to its potential impact on metabolic disorders. The aim of this chapter is to dis-
cuss the possible role of AMPK in the adaptations to exercise, nutrient and hor-
monal signals and its potential as a therapeutic drug target, mimicking the beneficial
effects of exercise.
The Energy Sensor AMPK: Adaptations to Exercise, Nutritional and Hormonal Signals 15

Fig. 1  Schematic representation of AMPK subunit. (a) AMPK domain structure. (b) AMPK het-
erotrimer composition in human skeletal muscle

AMPK: Structure and Regulation

AMPK is a heterotrimeric complex composed of one catalytic α-subunit comprising

a typical Ser/Thr kinase domain, in combination with scaffolding β-subunit contain-
ing a carbohydrate binding module (CBM) and γ-subunit containing four
cystathionine-β-synthase (CBS) domains that serve to bind adenine nucleotides
(Fig. 1). Each of these subunits has several isoforms encoded by different genes (α1,
α2, β1, β2, γ1, γ2 and γ3) that can theoretically combine to form 12 possible hetero-
trimeric complexes (Hardie et al. 2012). Differential expression of the AMPK iso-
form in tissues and post-translational modifications that may locate AMPK in
different cellular compartments also contribute to the specialized functions of
AMPK heterotrimeric complexes. Of interest, in contrast to other AMPK isoforms,
expression of the AMPKγ3 isoform is restricted to the fast-twitch glycolytic skeletal
muscle, suggesting a particular role for AMPKγ3-containing complexes to handle
metabolic challenges in skeletal muscle (Barnes et al. 2004). Mutation in the gene
encoding for the AMPKγ3 subunit PRKAG3 has been reported in pigs and humans,
causing increased deposition of glycogen in skeletal muscle (Milan et  al. 2000;
Costford et  al. 2007). In human skeletal muscle, only three heterotrimeric com-
plexes have been detected, α2β2γ1 (65% of the total pool), α2β2γ3 (20%), and
α1β2γ1 (15%) (Birk and Wojtaszewski 2006; Fig. 1). The heterotrimer combination
varies in mouse skeletal muscle, with the detection of five complexes, including α1
and α2-associated AMPK complexes with both β1 and β2 isoforms (Treebak et al.
2009). Interestingly, each heterotrimer combination displays a distinct activation
profile in response to physical exercise, with γ3-containing complexes being pre-
dominantly activated and α2β2γ1 and α1β2γ1 heterotrimers being unchanged or
activated only after prolonged exercise (Birk and Wojtaszewski 2006).
The mechanism of AMPK activation involves two steps, a reversible phosphory-
lation at a conserved residue (Thr174  in α1 and Thr172  in α2 catalytic subunit,
16 B. Viollet

hereafter referred to as Thr172) within the activation loop in the α-subunit, and a
stimulatory allosteric effect upon binding of AMP within the CBS domains of the
γ-subunit (Hardie et al. 2012). Activity of the complex increases more than 100-fold
when AMPK is phosphorylated on Thr172 by identified upstream kinases. The
combined effect of phosphorylation on Thr172 and allosteric regulation causes a
>1000-fold increase in kinase activity, allowing high sensitivity in responses to
small changes in cellular energy status. In addition, AMP and ADP binding r­ egulates
AMPK activity by promoting Thr172 phosphorylation by the upstream kinases and
by protecting Thr172 from dephosphorylation by phosphatases. All the binding
effects of AMP and ADP are antagonized by binding of ATP, providing a very sensi-
tive mechanism for the activation of AMPK in conditions of cellular energy stress.
Recent crystallographic studies of full-length AMPK heterotrimeric complexes
have provided insights into the domain structure and the regulation upon binding of
adenosine nucleotides (Hardie et al. 2016). Because the activating ligand is bound
on the γ subunit and the kinase domain is in the α subunit, intersubunit communica-
tion has to occur when switching to fully active states. Important regulatory features
for this conformational switch are provided by α subunit flexible components
(Fig. 1), the autoinhibitory domain (AID) and the α-regulatory subunit interacting
motif (α-RIM)/α-hook interacting with the exchangeable nucleotide-binding sites
on the γ subunit, offering a signaling mechanism for nucleotide allosteric regulation
and protection against dephosphorylation of AMPK heterotrimeric complex.
In mammals, the major upstream kinases are the liver kinase B1 (LKB1) and
Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2; Hardie et al. 2012).
Interestingly, CaMKK2 has been shown to phosphorylate and activate AMPK in
response to an increase in intracellular Ca2+ concentration, independent of any
change in cellular AMP:ATP or ADP:ATP ratios. In skeletal muscle, the major
upstream kinase phosphorylating α subunit Thr172 is liver kinase B1 (LKB1), as
exercise-induced AMPK phosphorylation is prevented in mouse models lacking
LKB1 (Sakamoto et al. 2005; Thomson et al. 2007). However, CaMKKβ has been
shown to activate AMPK during mild tetanic skeletal muscle contraction (Jensen
et al. 2007) and to increase AMPKα1 activity in response to skeletal muscle over-
load in LKB1-deficient mice (McGee et al. 2008).

AMPK: Regulation by Hormones and Nutrients

Although AMPK was originally identified as a sensor of cellular energy status by

coordinating anabolic and catabolic pathways to balance nutrient supply with energy
demand, it is now clear that it also participates in controlling whole-body energy
homeostasis by integrating hormonal and nutritional signals from the cellular envi-
ronment and the whole organism. Hypothalamic AMPK has been suggested to be a
key mediator in the regulation of neuronal regulation of feeding behaviour and
energy balance (Fig.  2). Inhibition of AMPK by expressing a dominant negative
isoform in the arcuate nucleus (ARC) of the hypothalamus decreases mRNA
The Energy Sensor AMPK: Adaptations to Exercise, Nutritional and Hormonal Signals 17

Fig. 2 Hypothalamic
AMPK in the regulation of
energy balance

expression of the orexigenic neuropeptides agouti-related peptide (AgRP) and neu-

ropeptide Y (NPY; Minokoshi et al. 2004). Conversely, activation of AMPK in the
ARC by expressing a constitutively active AMPK form can further increase the
fasting-­induced expression of AgRP and NPY and then elevated feeding (Minokoshi
et al. 2004). Similarly, starvation induces AMPK activation and food intake. In con-
trast, refeeding and glucose administration promote AMPK inactivation, accompa-
nied by increased levels of anorexigenic neuropeptides proopiomelanocortin
(POMC) and reduced levels of orexigenic neuropeptides AgRP mRNA in the ARC,
highlighting the sensing levels of nutrients in the hypothalamus (Andersson et al.
2004; Minokoshi et al. 2004). There are also a number of hormones involved in the
regulation of appetite that alter AMPK activity in the hypothalamus. For example,
the orexigenic hormones, such as ghrelin and adiponectin, activate AMPK in the
ARC and promote food intake (Andersson et al. 2004; Kubota et al. 2007); in con-
trast, anorectic hormones, such as leptin and oestradiol, inhibit AMPK in the ARC
and inhibit food intake (Yang et al. 2011; Martinez de Morentin et al. 2014). Another
layer of signal integration for the regulation of whole-body energy balance happens
at the level of the ventromedial nucleus (VMH) of the hypothalamus, where AMPK
regulates energy expenditure by controlling brown adipose tissue (BAT) thermogen-
esis (López et al. 2010; Whittle et al. 2012; Beiroa et al. 2014; Martinez de Morentin
et al. 2014). Importantly, expression of a constitutively active AMPK form in the
VMH is associated with a specific reduction in the expression of BAT thermogenic
markers. In contrast, inhibition of AMPK by administration of thyroid hormone T3
to the VMH promotes whole body energy expenditure by triggering BAT
18 B. Viollet

thermogenesis via activation of the sympathetic nervous system (López et al. 2010).
More recently, it was found that injection of glucagon-like peptide-1 (GLP-­1) recep-
tor agonist liraglutide into VMH decreased AMPK activity, stimulated expression of
thermogenic markers in BAT, and promoted weight loss without affecting food
intake (Beiroa et al. 2014). Overall these findings demonstrate a key role for central
AMPK in the regulation of energy balance by influencing food intake and energy
expenditure in response to peripheral signals, such as hormones and nutrients.

AMPK: Regulation by Exercise

Lifestyle intervention such as regular physical exercise is widely recognized to

improve whole-body performance and metabolism in health and disease. An
increase in daily physical activity is an effective approach to combat many disease
symptoms associated with metabolic syndrome. Endurance exercise can improve
insulin sensitivity and metabolic homeostasis. However, our understanding of how
exercise exerts these beneficial effects is incomplete. In response to exercise, ATP
turnover is increased by more than 100-fold (Gaitanos et  al. 1993), resulting in
increased ATP consumption and a rise in intracellular AMP levels due to the ade-
nylate kinase reaction. These changes in the adenylate energy charge lead to the
activation of AMPK in an intensity- and time-dependent manner, as shown in
rodents (Winder and Hardie 1996) and in human muscle (Wojtaszewski et al. 2000).
Once activated, AMPK regulates multiple signaling pathways whose overall effects
are to increase ATP production, including fatty acid oxidation and glucose uptake.
Given that AMPK is at the nexus of metabolic signaling pathways, a great deal of
interest has focused on the role of AMPK in the adaptation of skeletal muscle to
exercise as well as its use as a possible therapeutic target for the treatment of type
2 diabetes.

Control of Exercise-Induced Glucose Transport

During exercise, contracting skeletal muscle rapidly increases glucose uptake in an

intensity-dependent manner to sustain the energy demand caused by increased ATP
turnover. The first compelling evidence for a role for AMPK in regulating glucose
uptake in skeletal muscle has been obtained with pharmacological activation of
AMPK by AICAR (Merrill et al. 1997). This finding was further supported by the
observation of impaired response to AICAR stimulation in mice expressing a domi-
nant negative AMPK form in skeletal muscle (Mu et al. 2001). However, the role for
AMPK in regulating glucose uptake during muscle contractile activity remains con-
troversial and no solid genetic evidence has been put forward. Exercise-induced
muscle glucose uptake is impaired in AMPK β1β2M-KO mice but remains intact in
AMPKα mdKO (O’Neill et  al. 2011; Lantier et  al. 2014). The reason for the
The Energy Sensor AMPK: Adaptations to Exercise, Nutritional and Hormonal Signals 19

Fig. 3  AMPK-mediated regulation of skeletal muscle adaptation to exercise

difference between studies is not clear and future studies using inducible mouse
models to study the role of AMPK in developed adult skeletal muscle are
warranted.
It has been suggested that AMPK enhances glucose uptake by increasing the
translocation of glucose transporter type 4 (GLUT4) to the plasma membrane
(Fig. 3). Recent findings using AMPK-deficient mouse models have shown a con-
vergence to the phosphorylation of the downstream target of TBC1D1, Rab-GTPase
activating protein, which is emerging as an essential player in contraction-­stimulated
GLUT4 translocation (Stockli et al. 2015). In support of this finding, mice express-
ing TBC1D1 that mutated at predicted AMPK phosphorylation sites showed reduced
contraction-stimulated glucose uptake (Vichaiwong et  al. 2010). Interestingly, in
exercised human skeletal muscle, TBC1D1 phosphorylation was significantly
­correlated with the activity of the α2β2γ3 heterotrimer, supporting the idea that
AMPK is a direct upstream TBC1D1 kinase (Treebak et al. 2014).
Recent studies using compound 991, a cyclic benzimidazole derivative and
potent direct AMPK activator, have shown that pharmacological activation of
AMPK is sufficient to elicit metabolic effects in muscle appropriate for treating type
2 diabetes (Lai et al. 2014). It is also important to note that AMPK-mediated ­glucose
uptake is not impaired in type 2 diabetes during exercise (Musi et al. 2001); there-
fore, activation of AMPK represents an attractive target for intervention.
20 B. Viollet

Control of Training-Induced Muscle Adaptations

In response to repeated metabolic stress, AMPK orchestrates a coordinated response

to enhance mitochondrial biogenesis to match substrate utilization to demand
(Fig. 3; Zong et al. 2002). This response is mediated through the regulation of per-
oxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), a transcrip-
tional co-activator that promotes the expression of mitochondrial genes encoded in
both nuclear and mitochondrial DNA. AMPK activation causes the stimulation of
PGC-1α expression by direct phosphorylation, which drives expression from its
own promoter but also involves deacetylation of PGC-1α via the silent mating-type
information regulator 2 homolog 1 (SIRT1; McGee and Hargreaves 2010). This
finding was further supported by the increase seen in PGC-1α and mitochondrial
function in mice expressing a constitutively active AMPK form in skeletal muscle
(Garcia-Roves et al. 2008). Moreover, deletion of AMPK greatly reduced exercise-­
induced SIRT1-dependent activation of PGC-1α signaling in skeletal muscle (Canto
et  al. 2010). Consistent with these findings, reduced muscle AMPK activity has
been associated with decreased mitochondrial content during aging (Reznick et al.
2007) and in skeletal muscle from AMPKβ1β2- and LKB1-deficient mice (O’Neill
et al. 2011; Tanner et al. 2013).

Control of Muscle Insulin Sensitivity

Skeletal muscle demonstrates increased insulin-stimulated glucose uptake in the

period after exercise (Richter et al. 1982). It may involve an increased translocation
of GLUT4 at the plasma membrane in response to insulin. The underlying mecha-
nism appears to involve AMPK-dependent phosphorylation of the Rab-GTPase
TBC1D4 regulating GLUT4 translocation (Kjobsted et al. 2016). These results are
fully in line with previous findings showing that prior pharmacological AMPK acti-
vation by AICAR enhances insulin sensitivity in rat skeletal muscle with increased
phosphorylation of TBC1D4 (Kjobsted et al. 2015). Thus, activation of AMPK in
the recovery after exercise may be important for exercise-induced adaptations and
may serve to enhance muscle insulin sensitivity (Fig.  3). These findings may be
highly relevant for pharmacological interventions in the treatment of muscle insulin
resistance.

Conclusions and Therapeutic Perspectives

In mammals, AMPK has emerged as a major energy sensor that integrates multiple
extracellular and intracellular input signals to coordinate cellular energy balance.
Targeted by nutritional and hormone signals, AMPK has a crucial role in the
The Energy Sensor AMPK: Adaptations to Exercise, Nutritional and Hormonal Signals 21

hypothalamus to regulate energy intake and energy expenditure. In skeletal muscle,

AMPK has been identified as an important integrator of the metabolic changes that
occur during physical exercise. Given these roles, AMPK is an obvious target for
treatment of metabolic disorders such as obesity and diabetes. However, although
pharmacological activation in peripheral organs is expected to provide therapeutic
benefits, activating central AMPK could cause deleterious consequences, specifi-
cally on body weight control. Recent studies have highlighted the adverse metabolic
consequence of AMPK activation throughout all tissues (Yavari et al. 2016). Thus,
a better understanding of the precise role of AMPK in the cell and its effects at the
whole-body level will be essential for delineating therapeutic strategies aimed at
targeting AMPK.

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Plasma Steroids and Cardiorespiratory
Fitness Response to Regular Exercise

Zihong He, Tuomo Rankinen, Arthur S. Leon, James S. Skinner,

André Tchernof, and Claude Bouchard

Abstract  The aim of this report is to evaluate the relationships between baseline
levels of adrenal, gonadal and conjugated steroids and baseline cardiorespiratory
fitness, as assessed by maximal oxygen uptake (VO2max), as well as its response to
a standardized exercise program. To address this aim we used a subset of the
HERITAGE Family Study (N = 448). In men, significant positive associations were
found between baseline VO2max/kg weight and plasma levels of androsterone gluc-
uronide (ADTG), dihydrotesterone (DHT), 17 hydroxy progesterone (OHPROG),
sex hormone binding globulin (SHBG), and testosterone (TESTO), and negative
association with aldosterone (ALDO). In women, only the free androgen index
(FAI) was negatively associated with baseline VO2max/kg weight. Neither baseline
plasma steroid levels nor SHBG concentrations were associated with the gains in
VO2max resulting from exposure to the 20-week aerobic exercise program after
adjustment for baseline values, age and ethnicity (white or black). We conclude that
baseline plasma steroid levels are only weakly associated with individual differ-
ences in cardiorespiratory fitness in the sedentary state in men but not in women,
whereas no association could be detected with trainability, as defined by the change
in VO2max with the exercise program.

Z. He
Human Genomics Laboratory, Pennington Biomedical Research Center,
Baton Rouge, LA, USA
Department of Biology, China Institute of Sport Science, Beijing, China
T. Rankinen • C. Bouchard (*)
Human Genomics Laboratory, Pennington Biomedical Research Center,
Baton Rouge, LA, USA
e-mail: claude.bouchard@pbrc.edu
A. S. Leon
School of Kinesiology, University of Minnesota, Minneapolis, MN, USA
J. S. Skinner
Department of Kinesiology, Indiana University, Bloomington, IN, USA
A. Tchernof
School of Nutrition, Laval University, Quebec City, QC, Canada

© The Author(s) 2017 25

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_3
26 Z. He et al.

Introduction

The ongoing global epidemic of chronic non-communicable diseases (NCDs) is

related to changes in lifestyle, including low physical activity (PA) levels. Physical
inactivity is a major public health challenge (Engberg et  al. 2012) and has been
defined as the fourth leading cause of death worldwide (Kohl et  al. 2012). The
health benefits of regular PA are well established, and elimination of physical inac-
tivity would remove between 6 and 10% of NCDs and increase life expectancy (Lee
et  al. 2012). Moreover, cardiorespiratory fitness (CRF) is a strong predictor of
health and longevity (United States Department of Health and Human Services
2008). There are large individual differences in CRF among adults who are seden-
tary and who have a history of not engaging in regular exercise (Bouchard et al.
2015). The response of CRF to regular exercise is also highly variable, even when
the exercise prescription is highly standardized and when compliance is not an issue
(Skinner et al. 2001). As shown by the findings of the HERITAGE Family Study,
there are many factors contributing to variation in CRF exercise training response
(Sarzynski et al. 2016). However, little is known about the relationships between
plasma levels of steroid hormones and CRF in the sedentary state or in response to
regular exercise in men or women.
Steroid hormones are mainly synthesized from cholesterol in the gonads and
adrenal glands but some are also produced in other tissues such as skeletal muscle
(Sato and Lemitsu 2015), adipose tissue, liver and others (Luu-The and Labrie
2010; Labrie 2015). Steroid hormones can be grouped into two classes, corticoste-
roids and sex steroids. Within those two classes, there are five types of hormones
depending on the receptors to which they bind: glucocorticoids, mineralocorti-
coids, androgens, estrogens, and progestogens. Steroid hormones are generally car-
ried in the blood, bound to specific carrier proteins such as sex hormone-binding
globulin (SHBG; Hammond 2016) or corticosteroid-binding globulin (Meyer et al.
2016).
Steroid hormones exert a wide variety of effects on cardiorespiratory function. In
brief, corticosteroids help mediate the stress response and promote energy replen-
ishment and efficient cardiovascular function (Nicolaides et  al. 2015). They also
regulate muscle function by acting on muscle mass and strength (Hasan et al. 2012).
Mineralocorticoids contribute to the regulation of blood pressure and participate in
the regulation of cardiac and vascular function. The mineralocorticoid aldosterone
(ALDO) is a key regulator of water and electrolyte homeostasis. Clinical trials have
shown the beneficial effects of ALDO antagonists in chronic heart failure and post-­
myocardial infarction treatment (Lother et al. 2015). Long-term infusion of miner-
alocorticoid (ALDO) in mice resulted in elevation of plasma interleukin-1β levels
and vascular abnormalities (Bruder-Nascimento et  al. 2016). Androgens enhance
skeletal muscle mass by promoting the enlargement of skeletal muscle cells (Sinha-­
Hikim et  al. 2004). Muscle steroid hormone levels were shown to be positively
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise 27

c­ orrelated with muscle strength and cross-sectional area (Sato et al. 2014). Also,
androgens inhibit the ability of adipocytes to store lipids by blocking a signal trans-
duction pathway (Singh et al. 2006). Androgen deficiency has been shown to be a
major risk factor for several disorders, including obesity, metabolic syndrome, and
ischemic heart disease (Pongkan et al. 2016). The CARDIA Male Hormone Study
showed that there are associations of androgens with PA and fitness in young black
and white men (Wolin et al. 2007). Estrogens have vasculo-protective actions that
are thought to prevent atherosclerosis (Reslan and Khalil 2012). Estrogens contrib-
ute to the regulation of the delicate balance between fighting infections and protect-
ing arteries from damage, thus lowering the risk of cardiovascular disease (Meyer
and Barton 2016). Some cardiovascular effects of estrogens may be counteracted by
progestogens (Haddock et  al. 2000; Group 2006). Progestogens have favorable
effects on lipid, glucose and insulin profiles (Group 2006). Higher sex hormone
binding globulin (SHBG) concentration was shown to be associated with a more
favorable cardiovascular disease (CVD) risk profile, independent of total testoster-
one (Canoy et al. 2014).
A significant positive correlation has been observed between the changes in
serum testosterone levels and the number of steps executed on a daily basis; this
finding suggests that the level of PA affects serum testosterone concentrations in
overweight and obese men (Kumagai et al. 2016). Steroid hormones are responsive
to acute and chronic endurance exercise. Both acute and chronic exercise influence
circulating steroid hormone levels (Sato and Lemitsu 2015) and SHBG (Kim and
Kim 2012; Ennour-Idrissi et al. 2015) and activate local steroidogenesis in skeletal
muscle (Sato and Lemitsu 2015).
In the present report, we focus on the relationships between plasma levels of
adrenal, gonadal and conjugated steroids in the sedentary state and baseline (intrin-
sic) CRF, as assessed by maximal oxygen uptake (VO2max), as well as its response
to a standardized exercise program taking into account factors such as age, gender,
ethnicity, and baseline characteristics when appropriate.

Methods

Subjects

The HERITAGE Family Study cohort has been previously described (Bouchard
et al. 1995). A total of 488 adults with complete steroid hormones and VO2max data,
who were fully compliant with the 20-week HERITAGE aerobic exercise program,
were available for the present study. The study protocol had been previously
approved by the institutional review board at each of the four clinical centers of
HERITAGE. Informed written consent was obtained from each subject.
28 Z. He et al.

Anthropometric and Body Composition Measurements

Body weight and height were measured following standardized procedures. Body
density was measured using the hydrostatic weighing technique. The mean of the
highest three (of 10) measurements was used in the calculation of percent body fat
from body density using the equation of Siri (1956). Fat mass was obtained by mul-
tiplying body weight by percent body fat. These measurements have been shown to
be highly reproducible, with no difference between clinical centers or drift over
time in the course of data collection (Wilmore et al. 1997).

Aerobic Exercise Training Program

Each subject in the HERITAGE study exercised three times per week for 20 weeks
on cycle ergometers. The intensity of the training was customized for each individ-
ual on the basis of heart rate (HR) and VO2max measurements taken at a baseline
maximal exercise test. Details of the exercise training protocol can be found else-
where (Bouchard et al. 1995). Briefly, subjects trained at the HR associated with
55% of baseline VO2max for 30 min per session for the first 2 weeks. The duration
and intensity were gradually increased every 2 weeks, until reaching 50 min and
75% of the HR associated with baseline VO2max. This level was maintained for the
final six weeks of training. All training was performed on Universal Aerobicycles
(Cedar Rapids, IA) and power output was controlled by direct HR monitoring using
the Universal Gym Mednet (Cedar Rapids, IA) computerized system. The protocol
was standardized across all four clinical centers and supervised to ensure that the
equipment was working properly and that participants were compliant with the
protocol.

VO2max Measurement

Two maximal exercise tests to measure VO2max were performed on two separate
days at baseline and again on two separate days after training using a SensorMedics
800S (Yorba Linda, CA) cycle ergometer and a SensorMedics 2900 metabolic mea-
surement cart (Skinner et al. 2000). The tests were conducted at about the same time
of day, with at least 48 h between the two tests. In the first test, subjects exercised at
a power output of 50 W for 3 min, followed by increases of 25 W every 2 min until
volitional exhaustion. For older, smaller, or less fit individuals, the test was started
at 40 W, with increases of 10–20 W every 2 min thereafter. In the second test, sub-
jects exercised for 10  min at an absolute (50 W) and at a relative power output
equivalent to 60% VO2max. They then exercised for 3 min at a relative power output
that was 80% of their VO2max, after which resistance was increased to the highest
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise 29

power output attained in the first maximal test. If the subjects were able to pedal
after 2 min, power output was increased every 2 min thereafter until they reached
volitional fatigue. The average VO2max from these two sets was taken as the
VO2max for that subject and used in analyses if both values were within 5% of each
other. If they differed by >5%, the higher VO2max value was used.

Plasma Steroid Hormone and SHBG Concentrations

The hormonal assays have been previously described (Couillard et al. 2000; Ukkola
et al. 2001). Fourteen steroid hormones or their derivatives and SHBG concentra-
tions were assayed. Table 1 provides the full name of each hormone, the abbrevia-
tions used in the present report and a brief comment on the source of production or

Table 1  Description of steroid hormones assayed in the HERITAGE Family Study

Name (unit) Notes and sources
I: Steroids in the 19-carbon family (androstanes) and their derivatives
Adrenal androgen precursors
DHEA Dehydroepiandrosterone (nmol/L) Source: Adrenals
DELTA4 Androstenedione (nmol/L) Source: Adrenals
Intermediate in synthesis of TESTO, DHT, E2
DHEAS DHEA sulfate (nmol/L) Sulfated form of DHEA
Most abundant steroid in circulation
DHEAE DHEA fatty acid ester (nmol/L) A second form of DHEA ester
Active androgens
TESTO Testosterone (nmol/L) Source: Mainly by testicles
DHT Dihydrotesterone (nmol/L) Derived from TESTO or adrenal androgens
precursors in multiple tissues
Androgen metabolites
DIOLG Androstane 3α17βdiol glucuronide Metabolite of TESTO and DHT
(nmol/L)
ADTG Androsterone glucuronide (nmol/L) Metabolite of TESTO and DHT
II: Pregnanes, Progestanes, and Estradiol
PREGE Pregnenolone fatty acid ester Derived from PREG in HDL particles and
(nmol/L) other sites
PROG Progesterone (nmol/L) Source: Mainly in the ovaries
OHPROG 17 hydroxy progesterone (nmol/L) Derived from PROG in adrenals and gonads
E2 Estradiol (pmol/L) Source: ovaries and other tissues
III: Aldosterone, Cortisol, SHBG and FAI
ALDO Aldosterone (nmol/L) Source: adrenal cortex
CORT Cortisol (nmol/L) Source: adrenal cortex
SHBG Sex hormone binding globulin Peptide transporting TESTO and E2
(nmol/L) Source: mainly liver
FAI Free androgen index Calculated as TESTO/SHBG * 100
30 Z. He et al.

transformation of the hormones and their precursors. On 2 consecutive days, two

blood samples were obtained after a 12-h fast pre- and post- exercise program. For
eumenorrheic women, all samples were obtained in the early follicular phase of the
menstrual cycle. None of the women of reproductive age had dramatically irregular
menstrual cycles, as determined by questionnaire and during an interview. After
centrifugation, aliquots were frozen at −80  °C.  For nonconjugated steroids (see
Table 1 for abbreviations), ALDO, dehydroepiandrosterone (DHEA) and testoster-
one (TESTO) were differentially extracted with hexane-ethyl acetate. Petroleum
ether was used for the extraction of androstenedione (DELTA4) and dihydrotester-
one (DHT). In-house RIAs were used to measure the levels of these four steroids.
Progesterone, 17 hydroxy progesterone (OHPROG), cortisol (CORT), estradiol
(E2), and DHEA sulfate (DHEAS) were assayed using commercial kits (Diagnostics
Systems Lab, Webster, TX, USA). For glucuronide [androsterone glucuronide
(ADTG) and androstane 3α17βdiol glucuronide (DIOLG)] and ester [DHEA fatty
acid ester (DHEAE) and prenenolone fatty acid ester (PREGE)] conjugated ste-
roids, ethanol extraction was performed, followed by C18 column chromatography.
Glucuronide conjugates were submitted to hydrolysis with β-glucuronidase (Sigma
Co., St. Louis, MO, USA). Fatty acid derivatives were submitted to saponification.
Steroids were then separated by elution on LH-20 columns and measured by
RIA. SHBG was determined with an IRMA-Count solid phase immunoradiometric
assay using 125I (Diagnostics Systems Laboratories, Inc.). All assays were per-
formed in the laboratory of Dr. Alain Belanger, Molecular Endocrinology, Laval
University Medical Center (CHUL), Québec, Canada.

Statistical Analysis

The present study is based on sedentary adults from the HERITAGE Family Study
who had complete baseline hormonal data. The mean of two measurements both
before and after the exercise program has been used for all hormones and SHBG
levels. Data of men and women were analyzed separately. Pearson product-
moment correlation coefficients were used to quantify the relationships between
baseline VO2max (after adjustment for age and ethnicity) and its exercise training
response (after adjustment for baseline, age and ethnicity) with fasting plasma
steroid hormone, SHBG concentrations and the free androgen index (FAI).
ANCOVA was used to compare each steroid, SHBG and FAI across quartiles of
baseline VO2max or its training response, with age and ethnicity as covariates.
ANCOVA was used to compare the difference between the lowest 5% of the
TESTO distribution and the men in the fourth quartile and between the highest 5%
of the E2 distribution versus the women in the first quartile, with age and ethnicity
as covariates. All analyses were performed using the SAS statistical package (SAS
Institute, Inc., Cary, NC). Significance level was set at a more conservative p-value
threshold of <0.01.
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise 31

Table 2  Reproducibility of fasting steroid hormone levels in three HERITAGE ancillary studies
Intraclass correlation coefficient
Three-day quality
Reliability of assaya HERITAGE test-retestb controlc
Males Females Males Females Males Females
(N = 35) (N = 25) (N = 325) (N = 420) (N = 35) (N = 25)
ADTG 0.81 0.95 0.91 0.93 0.77 0.96
ALDO 0.95 0.97 0.70 0.75 0.79 0.40
CORT 0.98 0.96 0.52 0.88 0.55 0.70
DELTA4 0.97 0.98 0.91 0.94 0.95 0.93
DHEA 0.97 0.97 0.76 0.80 0.86 0.71
DHEAE 0.77 0.94 0.79 0.85 0.79 0.82
DHEAS 0.97 0.98 0.96 0.97 0.94 0.92
DHT 0.96 0.86 0.93 0.83 0.95 0.86
DIOLG 0.80 0.97 0.96 0.93 0.73 0.98
E2 0.91 0.98 0.59 0.84 0.86 N/A
OHPROG 0.98 0.96 0.86 0.80 0.90 0.74
PREGE 0.89 0.87 0.83 0.86 0.69 0.79
PROG 0.92 0.99 0.90 0.84 0.79 N/A
SHBG 0.98 0.99 0.97 0.97 0.98 0.98
TESTO 0.97 0.95 0.95 0.90 0.95 0.92
a
Repeated assays from the same aliquot. Samples from women obtained in the early follicular
phase
b
Test-retest from sample drawn on two different days. Samples from women obtained in the early
follicular phase
c
From samples tested three times in 3 weeks; menstrual cycle phase was not controlled in the
female sample

Results

In the HERITAGE Family Study research program, three ancillary studies focusing
on the within-subject variance and the reproducibility of test data were executed.
Table 2 summarizes the main findings from these three sources for the steroid hor-
mones and SHBG concentrations using the intraclass coefficients for repeated mea-
sures. In study 1, each assay was repeated from samples drawn from the same
aliquot. Blood samples were drawn in the fasted state and those from eumenorrheic
women were obtained in the early follicular phase. In study 2, the test-retest data
were from samples drawn on two different days in the fasted state. Again, samples
from eumenorrheic women were obtained in the early follicular phase. In study 3,
samples were drawn in the fasted state three times in 3 weeks. However, the men-
strual cycle phase was not controlled for in the female sample of the latter study.
Globally, the results of these reproducibility studies indicate that the within-subject
variation in hormone and SHBG levels was quite small compared to the between-­
subject fluctuations. The assays were quite reliable, as revealed by the very high
intraclass coefficients for repeated assays on samples drawn from the same aliquot.
32 Z. He et al.

Table 3  Basic characteristics of sedentary males and females

Men Women
N Age BMI (kg/m2) N Age BMI (kg/m2)
All 244 36 ± 14 26.5 ± 4.4 244 35 ± 14 25.1 ± 25.0
Blacks 61 33 ± 11 26.7 ± 4.1 59 33 ± 12 27.2 ± 5.8
Whites 183 37 ± 15 26.4 ± 4.5 185 35 ± 14 24.5 ± 4.6
17–29 years 114 23 ± 4 24.7 ± 4.3 121 22 ± 3 23.5 ± 4.8
30–49 years 74 40 ± 7 28.3 ± 3.7 75 41 ± 6 25.8 ± 4.4
50–65 years 56 56 ± 4 27.8 ± 4.3 48 55 ± 4 28.0 ± 5.0
Mean ± SD

This finding is particularly true for the steroids of high interest in the present paper,
such as DHEA and DHEAS with coefficients >0.97, DHT and TESTO >0.86, E2
and PROG >0.91 and SHBG >0.95, all in both genders. When the day-to-day varia-
tion (plus the technical error of the assays) was considered in studies 2 and 3, all
coefficients were high (>0.70), with the notable exception of CORT in males (~0.50).
Table 3 shows the physical characteristics of subjects stratified on the basis of
age, gender and ethnicity. There were 244 adult subjects for each gender. About
25% of the subjects were blacks, with no difference between men and women. The
male cohort was slightly overweight and heavier than the females, with the excep-
tion of black women.
Table 4 shows the baseline value of steroid hormones and SHBG in men and
women. Mean and median values plus 95% confidence intervals and intervals
between the first and fourth quartiles of the distributions are presented. Testing for
skewness of distributions revealed that there was no indication of a major skewness
problem with any of the variables.
Table 5 describes the VO2max at baseline and training response. Shown are base-
line VO2max per kg of body weight and kg of fat-free mass. The most important
inclusion criterion of the HERITAGE participants was that they needed to be con-
firmed sedentary. The low levels of CRF measured in males and females of this
HERITAGE subsample confirm that they were indeed very sedentary as a group:
with a mean age of about 35 years, VO2max/kg weight reached only 36 ml O2/kg in
males and 28 in females.
Overall, steroid hormone levels were weakly associated with CRF in men but
almost not at all in women. Table 6 summarizes the associations in men. Shown are
partial correlations (controlling for age and ethnicity) between baseline plasma ste-
roid hormone levels and baseline VO2max/kg body weight, as well as hormonal
levels (adjusted for age and ethnicity) by quartiles of fitness in men. There were
substantial differences across the four quartiles of fitness, with Q1 having a mean
VO2max/kg weight value of 26.5 (SD = 2.3) and Q4 a mean of 48.4 (SD = 3.5).
Significant partial correlations and significant differences across the four quartiles
of VO2max/kg weight were consistently found for ADTG, ALDO, DHT, OHPROG,
SHBG, and TESTO.  The patterns of differences across the fitness quartiles are
depicted for DHT, OHPROG, TESTO and SHBG in Fig. 1, with indications of spe-
cific group comparison differences. Males with the lowest TESTO levels (lowest
Table 4  Steroid hormone levels at baseline in adult males and females
Men (N = 244) Women (N = 244)
Mean ± SD 95%CI Median Interquartile range Mean ± SD 95%CI Median Interquartile range
ADTG 158 ± 87 −12–329 140 80 91 ± 54 −15–197 76 63
ALDO 0.3 ± 0.2 −0.0–0.6 0.2 0.2 0.3 ± 0.2 −0.1–0.6 0.2 0.2
CORT 376 ± 111 159–594 355 156 470 ± 241 −2–942 414 259
DELTA4 2.8 ± 1.5 0.1–5.6 2.7 1.8 2.4 ± 1.5 −0.5–5.3 2.1 2.1
DHEA 15.8 ± 10.3 −4.4–20.1 13.9 11.4 14.3 ± 10.2 −5.7–34.4 11.8 12.0
DHEAE 9.4 ± 5.8 −1.8–20.5 7.7 6.7 7.9 ± 5.4 −2.8–18.5 6.5 5.3
DHEAS 5696 ± 3172 −521–11913 5128 4538 3730 ± 2277 −734–8193 3202 2610
DHT 2.8 ± 1.2 0.3–5.2 2.7 1.8 0.5 ± 0.3 0.0–1.0 0.5 0.4
DIOLG 28.7 ± 14.0 1.3–56.0 26.7 13.1 14.1 ± 7.7 1.1–29.2 12.2 8.3
E2 72 ± 45 −17–161 66 47 136 ± 195 −246–518 82 108.2
OHPRG 5.5 ± 2.9 −0.2–11.1 5.1 3.5 2.1 ± 1.4 −0.7–4.8 1.8 1.4
PREGE 16.8 ± 9.6 −2.1–35.6 14.0 12.1 13.9 ± 9.1 −3.9–31.7 10.9 11.0
PROG 1.7 ± 0.8 0.1–3.3 1.6 0.7 1.6 ± 3.0 −4.3–7.5 1.2 1.2
SHBG 39.5 ± 16.6 7.2–71.7 36.9 25.0 86.4 ± 49.8 −11.2–184.0 70.7 53.3
TESTO 15.1 ± 6.1 3.2–27.0 14.8 9.0 1.3 ± 0.7 0.0–2.6 1.2 0.7
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise

FAI 43.1 ± 21.3 1.4–84.8 39.1 25.3 2.0 ± 1.4 −0.7–4.7 1.6 1.6

Units of steroid hormone and SHBG were nmol/L except E2 (pmol/L). Unit of FAI is in %
33
 34

Table 5 VO2max at baseline and its training response in adult males and females of HERITAGE study
Men (N = 244) Women (N = 244)
X ± SD 95%CI Median Interquartile range X ± SD 95%CI Median Interquartile range
VO2max (ml/min/kg BW) 36.3 ± 8.6 19.4–53.2 35.3 13.4 28.3 ± 6.9 14.8–41.8 28.7 10.3
VO2max (ml/min/kg FFM) 46.5 ± 7.3 32.2–60.8 46.1 10.6 40.7 ± 6.4 28.2–53.2 41.3 9.2
△VO2max (ml/min)a 453 ± 234 −5–911 448 321 347 ± 177 −1–694 338 233
BW body weight, FFM fat-free mass
a
Unadjusted data
Z. He et al.
Table 6  Partial correlations between baseline plasma steroid hormone levels, baseline VO2max/kg body weight, and hormonal levels by quartiles of fitness
in men
Quartilesb
Partial correlation coefficientsa Q1 Q2 Q3 Q4 F, P for trendc
VO2max/kg BW 26.5 ± 2.3 32.0 ± 1.7 38.5 ± 2.4 48.4 ± 3.5
Ages 49 ± 11 41 ± 13 31 ± 11 23 ± 6
Ethnicity 67% (white) 75% (white) 64% (white) 93% (white)
ADTG −0.18* 181.1 ± 12.3 172.7 ± 10.5 155.0 ± 10.5 123.4 ± 12.7 3.11, p = 0.0272
ALDO 0.19* 0.24 ± 0.02 0.25 ± 0.02 0.24 ± 0.02 0.33 ± 0.02 3.83, P = 0.0105
CORT 0.14 355 ± 16 361 ± 14 383 ± 14 407 ± 17 1.40, p = 0.2435
DELT4 0.12 2.5 ± 0.2 2.7 ± 0.2 2.8 ± 0.2 3.1 ± 0.2 1.25, p = 0.2906
DHEA 0.00 15.2 ± 1.4 15.1 ± 1.2 16.9 ± 1.2 15.9 ± 1.4 0.42, p = 0.7386
DHEAE −0.12 10.2 ± 0.8 9.5 ± 0.7 9.2 ± 0.7 8.5 ± 0.9 0.51, p = 0.6725
DHEAS −0.01 5299 ± 424 5877 ± 359 5655 ± 359 5551 ± 437 0.48, p = 0.6951
DHT 0.26*** 2.2 ± 0.2 2.6 ± 0.1 3.0 ± 0.1 3.2 ± 0.2 14.93, P < 0.0001
DIOLG −0.12 30.3 ± 2.1 29.6 ± 1.8 29.9 ± 1.8 24.9 ± 2.1 1.32, p = 0.2677
E2 0.03 72.7 ± 7.0 66.3 ± 5.9 73.1 ± 5.9 76.5 ± 7.2 0.82, p = 0.4817
OHPROG 0.25*** 4.5 ± 0.4 4.8 ± 0.3 6.1 ± 0.3 6.5 ± 0.4 15.47, P < 0.0001
PREGE −0.01 15.9 ± 1.4 17.1 ± 1.2 18.4 ± 1.2 15.6 ± 1.5 1.15, p = 0.3299
PROG 0.11 1.4 ± 0.1 1.7 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 2.37, p = 0.0713
SHBG 0.30*** 32.6 ± 2.4 37.0 ± 2.0 39.3 ± 2.0 49.1 ± 2.4 6.46, p = 0.0003
TESTO 0.34*** 11.5 ± 0.9 13.4 ± 0.7 16.9 ± 0.7 18.6 ± 0.9 9.94, P < 0.0001
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise

FAI −0.08 43.3 ± 2.9 41.6 ± 2.5 48.3 ± 2.5 39.2 ± 3.0 2.45, p = 0.0645

Units of steroid hormone and SHBG are nmol/L except E2 (pmol/L). Unit of FAI is in %
The bold results have the highest significance levels
*P < 0.01; ***P < 0.0001
a
Age and ethnicity were controlled for in the partial correlations between baseline plasma steroid hormone levels and baseline VO2max/kg body weight
b
Adjusted data for steroid hormone, SHBG, and FAI, mean ± SEM
c
35

Age and ethnicity as covariates when comparing hormonal levels by quartiles of fitness in men; F = F ratio, P = p-value
36 Z. He et al.

Fig. 1  Hormone levels adjusted for age and ethnicity by quartiles of baseline VO2max/kg weight
in men. Mean and SEM are shown. Dihydrotesterone (DHT), 17 hydroxy progesterone (OHPROG),
sex hormone binding globulin (SHBG), testosterone (TESTO) *P  <  0.01; **P  <  0.001;
***P < 0.0001

5% of the distribution; N  =  13; TESTO concentration <6.16 nmol/L) were com-

pared to the subjects in the upper quartile of the TESTO distribution (N  =  61;
TESTO concentration >19.56  nmol/L) for potential differences in VO2max.
Significant gaps in CRF were observed, with the low TESTO subjects exhibiting
lower VO2max/kg BW (33.7 ± 1.4 vs. 39.9 ± 0.6, F = 15.2, P = 0.0002) and VO2max/
kg FFM (43.9 ± 1.3 vs. 48.3 ± 0.6, F = 9.3, P = 0.0032).
The same analyses were performed in the female sample and the results are
shown in Table 7. Only FAI was significantly correlated (partial correlation) with
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise 37

Table 7  Partial correlations between baseline plasma steroid hormone levels, baseline VO2max/kg
body weight and hormonal levels by quartiles of fitness in women
Partial Quartilesb
correlation F, P for
coefficientsa Q1 Q2 Q3 Q4 trendc
VO2max/kg BW 19.7 ± 2.2 25.4 ± 2.0 30.7 ± 1.3 37.4 ± 2.8
Ages 47 ± 11 38 ± 13 30 ± 10 23 ± 6
Ethnicity 61% 62% 85% 95%
(white) (white) (white) (white)
ADTG −0.08 98.3 ± 8.1 90.2 ± 6.9 91.6 ± 6.9 83.7 ± 8.1
0.48,
p = 0.6992
ALDO 0.15 0.23 ± 0.03 0.1 ± 0.02 0.22 ± 0.02 0.32 ± 0.03 3.68,
p = 0.0128
CORT 0.14 409.3 ± 35 442 ± 29 525 ± 29 505 ± 34 1.95,
p = 0.1215
DELT4 0.05 2.2 ± 0.2 2.5 ± 0.2 2.3 ± 0.2 2.5 ± 0.2 0.48,
p = 0.6975
DHEA 0.01 14.9 ± 1.4 14.6 ± 1.2 13.3 ± 1.2 14.6 ± 1.4 0.36,
p = 0.7841
DHEAE −0.06 9.3 ± 0.8 8.5 ± 0.7 6.2 ± 0.7 7.5 ± 0.8 2.72,
p = 0.0452
DHEAS −0.04 3954 ± 314 3843 ± 265 3455 ± 265 3667 ± 312 0.49,
P = 0.6919
DHT −0.04 0.54 ± 0.04 0.59 ± 0.03 0.48 ± 0.03 0.48 ± 0.04 2.19,
p = 0.0897
DIOLG −0.08 15.1 ± 1.2 14.9 ± 1.0 13.6 ± 1.0 12.6 ± 1.2 1.25,
p = 0.2922
E2 −0.04 155 ± 31 132 ± 26 150 ± 26 107 ± 30 2.46,
p = 0.0636
OHPROG 0.02 2.4 ± 0.2 1.8 ± 0.2 1.8 ± 0.2 2.1 ± 0.2 2.06,
p = 0.1064
PREGE −0.05 16.1 ± 1.4 14.1 ± 1.2 12.1 ± 1.2 13.4 ± 1.4 1.43,
p = 0.2353
PROG −0.02 2.2 ± 0.5 1.5 ± 0.4 1.2 ± 0.4 1.4 ± 0.5 0.79,
p = 0.5002
SHBG 0.14 66.7 ± 7.8 86.0 ± 6.6 101.3 ± 6.6 91.7 ± 7.7 3.41,
p = 0.0183
TESTO −0.04 1.3 ± 0.1 1.4 ± 0.1 1.3 ± 0.1 1.2 ± 0.1 1.01,
p = 0.3889
FAI −0.20* 2.6 ± 0.2 2.2 ± 0.2 1.5 ± 0.2 1.5 ± 0.2 4.45,
p = 0.0046
Units of steroid hormone and SHBG are nmol/L except E2 (pmol/L). Unit of FAI is in %
*P < 0.01
a
Age and ethnicity as adjustment factors for partial correlation between baseline plasma steroid
hormone levels and baseline VO2max/kg body weight
b
Adjusted data for steroid hormone, SHBG, and FAI, mean ± SEM
c
Age and ethnicity as covariance when comparing hormonal levels by quartiles of fitness in
women; F = F ratio, P = p-value
38 Z. He et al.

Fig. 2  Free androgen

index (FAI) adjusted for
age and ethnicity by
quartiles of baseline
VO2max/kg weight in
women. Mean and SEM
are shown. F = 4.5;
*P = 0.05

baseline VO2max/kg weight and differed across the four fitness groups. None of the
hormones was consistently associated with fitness in women. The FAI values across
the four fitness quartiles are depicted in Fig. 2.
Baseline plasma steroid levels, SHBG concentrations and FAI were not associ-
ated with the gains in VO2max resulting from exposure to the 20-week aerobic exer-
cise program in men or in women. For the latter analyses, the gains in VO2max in
mlO2 were adjusted for baseline level, age and ethnicity. Notably, the men in the low
TESTO group (lowest 5% of the distribution) did not show a deficit in CRF respon-
siveness to exercise training (results not shown).

Discussion

The present study is the most comprehensive report published to date on the rela-
tionships between a large panel of plasma steroid hormones and CRF in sedentary
men and women at baseline and in response to a fully standardized and monitored
20-week exercise program. Of particular significance, the plasma concentrations of
steroid hormones were measured twice in the morning in a fasted state (samples
from two different days) before and again twice (from samples obtained on 2 days)
after the exercise training program. Also of significance, all blood samples were
drawn in the early follicular phase of the menstrual cycle for eumenorrheic women.
Furthermore, three quality-control studies were implemented to quantify the magni-
tude of the assay variance and the within-individual day-to-day variation in hor-
mone concentrations. As a large number of tests of significance were performed, an
alpha threshold of p < 0.01 was used to reduce the risk of false positive results.
Significant associations were found by partial correlation analyses between base-
line VO2max/kg weight and plasma levels of ADTG, ALDO, DHT, OHPROG,
SHBG, and TESTO in men. In contrast, in women, only the FAI was significantly
associated with baseline VO2max/kg weight. When comparing steroids, SHBG and
Plasma Steroids and Cardiorespiratory Fitness Response to Regular Exercise 39

FAI across quartiles of baseline VO2max ml/kg weight, the plasma concentration of
DHT, OHPROG, SHBG, and TESTO increased gradually from Q1 to Q4 in men
whereas FAI progressively decreased as fitness increased in women, indicating that
a higher level of TESTO was associated with better CRF. Indeed, once the effects of
age and ethnicity were taken into account, TESTO explained about 3% of the vari-
ance in baseline VO2max/kg weight in men. In females, FAI also accounted for
about 3% of the baseline VO2max/kg weight, with the same statistical control over
age and ethnicity. Baseline plasma steroid levels, SHBG concentrations or FAI were
not associated with the gains in VO2max resulting from exposure to the 20-week
aerobic exercise program after adjustment for baseline values, age and ethnicity.
The present results reinforce the idea that SHBG3 and TESTO (Storer et al. 2016)
are weakly correlated with baseline fitness. In the CARDIA Male Hormone Study
(391 blacks and 604 whites, aged 24–32 years), SHBG was significantly (p = 0.05)
associated with estimated CRF among white men, with quartiles four and two having
the highest (33.5 nmol/L) and lowest (27.8 nmol/L) SHBG concentrations, respec-
tively (Wolin et  al. 2007). TESTO supplementation in mobility-limited older men
(n = 64) increased hemoglobin and attenuated the age-related declines in VO2peak
(Storer et  al. 2016). However, our results also contradict those of other reports.
Lucertini et al. (2015) found basal CORT levels were lower in the high-fit (VO2max,
37.9 ± 0.9 ml/kg/min) than the low-fit group (VO2max, 26.4 ± 0.7 ml/kg/min). We
could not herein confirm the relationship between CORT and fitness. This discrep-
ancy may be explained by the basic characteristics of the study designs: for instance,
VO2max was estimated using the Rockport Walking Test in the study of Lucertini
et al. (2015), the sample size of their study was quite small and subjects were older
(n = 22, mean age 68 years), and CORT was assayed from saliva samples obtained at
18:00, late afternoon. Huang et al. also did not find a difference in CORT between
high-fitness (VO2max, 51.0  ±  0.8, ml/kg/min, ages: 22.3  ±  1.97, n  =  7) and low-­
fitness groups (VO2max, 36.3 ± 1.3, ml/kg/min, ages, 20.9 ± 0.8, n = 7) in males
(Huang et al. 2014).
The World Anti-Doping Agency (WADA) has developed a specific steroid mod-
ule as a part of their Athlete Biological Passport (ABP; Alladio et al. 2016), which
was implemented internationally in January 2014. Anabolic steroids represent the
most abundant class of substance used to enhance performance in elite sports.
Anabolic steroids are generally used to enhance muscle mass and muscular strength
or power. Their use for improvement of endurance performance or recovery from
training is less frequent. Our three quality control studies of plasma levels of ste-
roids revealed that their blood concentrations were quite stable within an individual.
We observed that TESTO and DHT were weakly but positively associated with CRF
in men only. These associations were lost when VO2max was adjusted for fat-free
mass (results not shown here), suggesting that the associations with fitness may
have been mediated by the muscle mass and not by the ability to deliver oxygen
(cardiac output and hemoglobin content of blood) to the working muscle.
Importantly, there was no relation between plasma levels of steroids and the VO2max
training response to the HERITAGE exercise program. Globally, our results provide
little support for a substantial role of high plasma steroids content in CRF and
endurance performance.
40 Z. He et al.

We conclude from our detailed observations that baseline plasma steroid levels
are only weakly associated with individual differences in CRF in the sedentary state
in men, whereas no association could be detected with trainability, as defined by the
change in VO2max with the exercise program. The associations are particularly con-
sistent for the plasma concentrations of testosterone and SHBG. In women, the most
consistent association pattern was seen with the FAI, potentially reflecting the fact
that high levels of free testosterone in females could be associated with slightly
elevated CRF.

Acknowledgments  The HERITAGE Family Study has been supported over the years by multiple
grants from the National Institute for Heart, Lung and Blood Diseases of the National Institutes of
Health (HL45670, C.  Bouchard and T.  Rankinen; HL47323, A.S.  Leon; HL47317, D.C.  Rao;
HL47327, J.S. Skinner; HL47321, J.H. Wilmore, deceased). CB is partially funded by the John
W.  Barton Sr. Chair in Genetics and Nutrition. Zihong HE is funded by the China Scholarship
Council (File No. 201603620001) and China Institute of Sport Science (2015-01, 2016-01). We
would like to express our gratitude to Dr. Alain Belanger (retired) and his staff from the Molecular
Endocrinology Laboratory of the Laval University Medical Center in Quebec City, Canada, for the
assays of the steroids and their dedication to the HERITAGE Family Study.

Disclosures  AT receives research funding from Johnson & Johnson Medical

Companies for studies unrelated to the present paper.

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Sending the Signal: Muscle Glycogen
Availability as a Regulator of Training
Adaptation

John A. Hawley

Abstract Exercise training-induced adaptations in human skeletal muscle are

largely determined by the mode, volume, intensity and frequency of the training
stimulus. However, a growing body of evidence demonstrates that the availability of
endogenous and exogenous macronutrients can modify multiple intramuscular
responses to both endurance- and resistance-based exercise. Acutely manipulating
substrate availability (by altering diet composition and/or timing of meals) rapidly
alters the concentration of blood-borne substrates and hormones that modulate sev-
eral receptor-mediated signaling pathways. The release of cytokines and growth
factors from contracting skeletal muscle also stimulates cell surface receptors and
activates many intracellular signaling cascades. These local and systemic factors
cause marked perturbations in the storage profile of skeletal muscle (and other
insulin-­sensitive tissues) that, in turn, exert pronounced effects on resting fuel
metabolism and patterns of fuel utilization during exercise. When repeated over
weeks and months, such nutrient-exercise interactions have the potential to alter
numerous adaptive processes in skeletal muscle that ultimately drive the phenotype-­
specific variability observed between individuals. One strategy that augments
endurance-training adaptation is commencing exercise with low muscle glycogen
concentration (“train-low”). The amplified training response observed with low
endogenous carbohydrate availability is likely regulated by enhanced activation of
key cell signalling kinases (e.g., AMPK, p38MAPK), transcription factors (e.g.,
p53, PPARδ) and transcriptional co-activators (e.g., PGC-1α), such that a coordi-
nated up-regulation of both the nuclear and mitochondrial genomes occurs. This
chapter provides a contemporary perspective of our understanding of the molecular
and cellular events that take place in skeletal muscle in response to exercise com-
menced after alterations in nutrient availability and discusses how the ensuing hor-

J. A. Hawley (*)
Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne,
VIC, Australia
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University,
Liverpool, UK
e-mail: john.hawley@acu.edu.au

© The Author(s) 2017 43

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_4
44 J. A. Hawley

monal milieu interacts with specific contractile stimulus to modulate many of the
acute responses to exercise, thereby potentially promoting or inhibiting subsequent
training adaptation.

Introduction

Classic studies conducted in the late nineteenth and early twentieth century deter-
mined that the fuels that supported contracting skeletal muscle during continuous
exercise lasting longer than several minutes were intra- and extra-muscular carbohy-
drate and lipid substrates, with only a minor contribution from amino acids (for
review, see Hawley et  al. 2015). During this time, it was also demonstrated that
manipulation of the macronutrient content of the preceding diet resulted in marked
changes in the time for which exercise at a fixed, submaximal power output could be
sustained; furthermore, subjects perceived exercise to be easier after several days of
a high carbohydrate diet than when the preceding diet was low in carbohydrate and
high in fat (Hawley et al. 2015). In the mid twentieth century, a large body of work,
mainly undertaken in laboratories in Europe, extended the earlier observations of
the importance of carbohydrate-based fuels for prolonged, intense exercise.
The period from 1960 up until the late 1980s has been termed the “classical
period” of exercise biochemistry (Brooks and Mercier 1994). With remarkable fore-
sight, Goldstein (1961) proposed that a “humoral factor” was liberated by muscle
during contraction that “activates the glucose transport system” and helps “regulate
carbohydrate metabolism” in what was later to become known as “muscle cross
talk.” At the beginning of the 1960s, little was known about the regulation of mito-
chondrial biogenesis in skeletal muscle in response to exercise training. However,
seminal work by Holloszy (1967) heralded a major advance in unravelling some of
the cellular events controlling muscle bioenergetics in response to exercise. It was
during this time that the percutaneous needle biopsy technique was introduced into
exercise biochemistry (Bergström and Hultman 1966), making it possible to con-
duct invasive studies and determine the impact of training, diet, and other manipula-
tions on selected metabolic characteristics. Studies conducted in labs in Europe and
North America advanced our understanding of the impact of training and diet
manipulations on muscle substrate stores, the interactive effects of exercise and
orally administered carbohydrate on muscle glucose kinetics and exercise capacity,
and the protein synthetic response to exercise (Hawley et  al. 2015; Brooks and
Mercier 1994). New insights into the influence of substrate availability on the hor-
monal response to prolonged exercise were also advanced in this period (Galbo
et al. 1979). At the same time, major progress in our knowledge of how exercise
activates cellular, molecular, and biochemical pathways with regulatory roles in
training response adaptation took place, and skeletal muscle was confirmed to have
endocrine-like effects, releasing cytokines and other peptides to orchestrate inter-­
organ “cross talk” (Pedersen et al. 2003).
Sending the Signal: Muscle Glycogen Availability as a Regulator of Training Adaptation 45

During the past two decades, exercise training-induced adaptations in skeletal

muscle have been shown to be the result of the cumulative effects of the acute
responses to repeated, single exercise bouts (Perry et al. 2010), with the rapid and
transient changes in gene transcription following a single bout of exercise leading to
chronic effects on protein expression when repeated over time. However, only
recently have we begun to appreciate the impact that alterations in substrate avail-
ability have on modifying training responses-adaptations. While substrates like car-
bohydrate and fat were viewed merely as energy stores for contracting muscles, we
are now beginning to understand that fuel availability per se is a crucial regulator of
many biochemical and cellular signaling events with roles in mitochondrial biogen-
esis, autophagy, health and performance. This chapter will provide a contemporary
perspective of our understanding of the regulation of substrate metabolism; the
molecular and cellular events that take place in skeletal muscle in response to exer-
cise commenced after alterations in nutrient availability; and how the ensuing hor-
monal milieu interacts with specific contractile stimulus to modulate many of the
acute responses to exercise, thereby potentially promoting or inhibiting subsequent
training adaptation.

Fuels for the Fire: Patterns of Skeletal Muscle Fuel Utilization

Both carbohydrate- (muscle and liver glycogen, blood glucose, muscle and liver
lactate) and fat- (adipose and intramuscular triglycerides, blood-borne free fatty
acids and triglycerides) based fuels are important substrates for oxidative phos-
phorylation and energy production in skeletal muscle (Brooks and Mercier 1994).
Compared to the finite stores of carbohydrate, endogenous lipid stores in humans
are plentiful and represent a potentially unlimited source of fuel for skeletal muscle
metabolism during aerobic exercise. However, fatty acid (FA) oxidation by muscle
is limited, at least during the power outputs sustained by athletes during training and
competition (Hawley and Leckey 2015). Unlike the oxidation of carbohydrate,
which is closely geared to the energy requirements of the working muscle, there are
no mechanisms for matching the availability and utilization of FA to the rate of
energy expenditure (Holloszy et al. 1988). However, the oxidation of any one fuel
or another at rest or during exercise does not occur in isolation and is typically the
result of the integration of many metabolic signals involving exogenous and endog-
enous substrate availability, the prevailing hormonal milieu as well as muscle and
whole-body energetic demands.
Both carbohydrate- and fat-based fuels are oxidized at rest to provide the energy
required for basal metabolic processes in skeletal muscle, with a reciprocal rela-
tionship between the utilization of carbohydrate and fat: fuel shifts occurring at
rest are largely driven by the availability of substrates and the concomitant hor-
monal environment (i.e., plasma glucose, insulin, FFA and glucagon concentra-
tions) in the face of a generally unchanged metabolic demand. For example,
increasing blood glucose availability by feeding of a carbohydrate-rich meal
46 J. A. Hawley

increases the uptake and o­ xidation of glucose into skeletal muscle while concomi-
tantly decreasing the oxidation of fat; but overall, there is little change in resting
metabolic rate. The decreased rate of FA oxidation at rest is largely attributable to
a carbohydrate feeding-induced rise in insulin concentration that impairs lipolysis
(i.e., the rate of appearance [Ra] of FFA into the systemic circulation), an effect
that can persist for up to four hours after a carbohydrate-rich meal (Bonadonna
et al. 1990).
The concept of a reciprocal relationship between fat and carbohydrate oxidation,
at least in resting muscle, stemmed from the work of Randle and colleagues in the
1960s (Randle et  al. 1964). Their experiments demonstrated that increased lipid
availability [i.e., circulating plasma free fatty acids (FFA)] increased FA oxidation
and decreased carbohydrate oxidation in muscle and that it was the increase in fat
availability that resulted in key intracellular perturbations, including increased con-
tents of muscle acetyl-coenzyme A (CoA), citrate, and glucose-6-phosphate: lipid-­
induced changes at these key regulatory sites decreased muscle carbohydrate
metabolism. The results of these early studies, which subsequently formed the bases
of the “glucose-fatty acid cycle” hypothesis (Randle 1986), were obtained by com-
paring supra-physiological levels of FFA while glucose concentration was
“clamped.” The prevailing paradigm was that carbohydrate-based substrates were
the muscle’s “default” fuel but that increased FA availability could “switch” muscle
fuel use and down-regulate carbohydrate metabolism. While the “glucose-fatty acid
cycle” proposed by Randle and coworkers (Randle et al. 1964) explained the mech-
anisms responsible for the down regulation of carbohydrate metabolism in the face
of high FA availability, these researchers failed to perform the control or “cross-
over” experiments, in which FA availability was held constant but glucose levels
were increased. This, apparently, was because they steadfastly believed that there
was little regulation of fat metabolism in skeletal muscle at rest (or presumably dur-
ing exercise) and that the key determinant of the rate of FA oxidation was simply a
function of the availability of fat and its regulation at the level of the mitochondrial
membranes through the carnitine-palmitoyl transferase (CPT) complex (Randle
et al. 1964).
While there are situations in which the glucose-fatty acid cycle operates, a series
of elegant studies conducted in the late 1990s clearly demonstrated that carbohy-
drate availability directly regulates fat oxidation at rest and during exercise (Coyle
et al. 1997; Horowitz et al. 1997; Romijn et al. 1995). Coyle et al. (1997) demon-
strated that a carbohydrate-rich meal that induced hyperglycemia and hyperinsu-
linemia increased glycolytic flux and directly reduced rates of FA oxidation during
low-intensity exercise. Carbohydrate feeding reduced the Ra of plasma FFA by
~35%, resulting in a reduction in both plasma FFA and intramuscular triglyceride
oxidation, suggesting a coordinated effect of increased glucose availability on adi-
pose tissue and muscle. These observations indicate that glucose can directly regu-
late fat oxidation during exercise and that the classic “glucose-fatty acid cycle”
proposed by Randle and colleagues (Randle 1986; Randle et al. 1964), in which fat
availability alone regulates rates of FA oxidation, is an overly simplistic view of fuel
regulation.
Sending the Signal: Muscle Glycogen Availability as a Regulator of Training Adaptation 47

The concept that altering substrate availability can modify the proportions of
carbohydrate and fat utilized by working muscle is certainly not new (Hawley et al.
2015). However, during the past decade, data from several independent laboratories
has provided evidence that commencing selected exercise sessions under conditions
of reduced carbohydrate availability can promote training-induced adaptations in
human skeletal muscle to a greater magnitude than if the same sessions were com-
menced with normal or high glycogen levels.

Sending the Signal: The Training Response-Adaptation

The goals of endurance exercise training are to induce an array of physiological and
metabolic adaptations that enable an individual to increase the rate of energy pro-
duction from both aerobic and oxygen-independent pathways, maintain tighter
metabolic control (i.e., match ATP production with ATP hydrolysis), minimize cel-
lular perturbations, increase efficiency of motion, and improve the capacity of the
trained musculature to resist fatigue (Hawley 2002). The mechanisms and meta-
bolic signals by which active muscle senses homeostatic perturbations and then
translates them into improved function has been a topic of intense research for sev-
eral decades (for review, see Perry and Hawley 2017). It is now accepted that a
variety of cellular disruptions takes place at the onset of exercise, including (but not
limited to) increased cytoplasmic free [Ca2+], increased free AMP (AMPf) and an
increased ADP/ATP ratio, reduced creatine phosphate and glycogen levels, increased
FA concentrations and reactive oxygen/nitrogen species (ROS/RNS), acidosis and
altered redox state, including [NAD/NADH] (Hawley et al. 2014; Perry and Hawley
2017). Within the context of metabolic homeostasis, an array of regulatory networks
is stimulated that sustain rates of ATP synthesis over time through the activation of
rate-limiting enzymes controlling carbohydrate and fat catabolism. A long-standing
question in exercise biology is how do these acute disruptions in cellular signals that
maintain energy supply also stimulate long-term adaptive processes that improve
the ability of muscle to sustain a future contractile challenge?
A key component of improved “muscle fitness” following exercise training is
biogenesis of mitochondria in skeletal muscle. A comprehensive discussion of this
topic is beyond the scope of this chapter and the reader is referred to recent reviews
(Hood et al. 2016; Perry and Hawley 2017). In brief, the molecular bases of skeletal
muscle adaptations to an endurance exercise stimulus requires increased expression
and/or activity of key mitochondrial proteins mediated by an array of intra-cellular
signaling events, pre- and post-transcriptional processes, regulation of translation
and protein expression, and modulation of protein (enzyme) activities and/or intra-
cellular localization. There are multiple (and often redundant) stimuli associated
with endurance exercise adaptations and numerous signaling kinases that respond to
contractile stimuli, with numerous downstream pathways that are targets of these
kinases (Egan et al. 2016; Hawley et al. 2014). A major advance in unraveling the
cellular events that promote mitochondrial biogenesis was the discovery of the
48 J. A. Hawley

peroxisome-­proliferator-activated receptor γ coactivator α (PGC-1α), an inducible

coactivator that regulates the coordinated expression of mitochondrial proteins
encoded in the nuclear and mitochondrial genomes (Lin et al. 2002). A critical fea-
ture of the PGC-1α coactivators is that they interact with many different transcrip-
tion factors to activate distinct biological programs in several tissues (Lin et  al.
2002). Certainly in skeletal muscle, PGC-1α has emerged as a key regulator of
mitochondrial biogenesis responsive to the prevailing contractile activity.
The AMPK and p38 MAPK are two other important signaling cascades that con-
verge upon the regulation of PGC-1α and consequently the regulation of mitochon-
drial biogenesis. The AMPK induces mitochondrial biogenesis partly by directly
phosphorylating and activating PGC-1α (Jager et al. 2007) but also by phosphory-
lating the transcriptional repressor HDAC5, which relieves inhibition of the tran-
scription factor myocyte enhancer factor 2 (MEF2), a known regulator of PGC-1α
(McGee and Hargreaves 2010). p38 MAPK phosphorylates and activates PGC-1α
(Puigserver et al. 2001) and also increases PGC-1α expression by phosphorylating
the transcription factor ATF-2, which in turn increases PGC-1α protein abundance
by binding to and activating the CREB site on the PGC-1α promoter (Akimoto et al.
2005). Another transcription factor involved in exercise-induced mitochondrial bio-
genesis in skeletal muscle and presumably activated by AMPK and/or p38 MAPK
is the tumor suppressor protein p53. p53 knockout mice display reduced endurance
exercise capacity compared with wild-type mice, along with reduced subsarcolem-
mal and inter-myofibrillar mitochondrial content and PGC-1α expression. p53 may
also regulate exercise-induced mitochondrial biogenesis through interactions with
TFAM in the mitochondria, where it functions to co-ordinate regulation of the mito-
chondrial genome (Saleem et al. 2011).

 rain-Low, Compete High: A Novel Strategy to Augment

T
Training Adaptation

Skeletal muscle glycogen availability exerts a regulatory effect on many cellular pro-
cesses. As discussed, one protein with a fundamental role in monitoring cellular energy
status is the AMPK. The recent discovery of glycogen-binding sites on the AMPK
β-subunits (McBride et al. 2009) has led to the hypothesis that this regulatory domain
may also allow AMPK to act as a sensor of endogenous glycogen stores (McBride and
Hardie 2009). In this scenario, the glycogen-binding domains act as sensors, enabling
AMPK to gauge the state of cellular glycogen, increasing AMPK activity when stores
are low and decreasing the signal when stores are replete or elevated. The first experi-
mental evidence from human skeletal muscle to provide indirect support for this
hypothesis was undertaken by Wojtaszewski et al. (2003). These workers measured
muscle-signaling responses and substrate utilization in well-trained males under con-
ditions in which a standardized bout of exercise (1 h at 70% peak oxygen consump-
tion) was commenced with either low (∼160 ­mmol/kg dry wt) or high (∼900 mmol/kg
dry wt) muscle glycogen concentration. At rest, AMPK activity and acetyl-CoA
Sending the Signal: Muscle Glycogen Availability as a Regulator of Training Adaptation 49

carboxylase-β (ACC-β) Ser221phosphorylation were lower in glycogen-loaded com-

pared with glycogen-depleted muscles. Of note was the finding that concentrations of
creatine phosphate and adenine nucleotides in resting muscles were similar despite
large differences in muscle glycogen status, suggesting that fuel-dependent mecha-
nisms independent of energy status might regulate AMPK signaling. AMPK-α1 and
α2 activity and ACC-β Ser221 phosphorylation were activated to a greater extent when
exercise was commenced with low compared to high glycogen levels. Subsequently,
Steinberg et al. (2006) reported that commencing exercise with low muscle glycogen
availability increased AMPK-α2 nuclear protein abundance. The results of these stud-
ies make it tempting to ascribe a direct mechanistic link between glycogen availability
and AMPK activation. However, manipulating resting muscle glycogen content in
humans also alters the concentrations of circulating blood metabolites (FFAs, cate-
cholamines). These systemic factors are likely to “fine-tune” the signaling responses
observed when exercise is commenced with low glycogen stores.
The early observations of increased AMPK activation when exercise is com-
menced with low muscle glycogen availability coupled with the putative role of the
AMPK in many of the metabolic adaptations of skeletal muscle to exercise-training
(Jørgensen et al. 2006; Pilegaard et al. 2002) were the bases of a hypothesis formu-
lated by Hansen et al. (2005), namely that chronic exercise training undertaken with
low muscle glycogen concentration would improve training adaptation to a greater
extent compared to when all sessions were commenced with high glycogen avail-
ability. This training protocol was termed “train-low, compete-high.” In a pioneering
study, Hansen et al. (2005) used an experimental design in which the right and left
legs of the same (untrained) subject performed the same amount of total work during
a 10-week intervention period, albeit with different pre-exercise muscle glycogen
content for half of the workouts. In agreement with their original hypothesis, they
reported that markers of “training adaptation” (i.e., resting muscle glycogen content,
the maximal activity of citrate synthase and performance time to exhaustion) were
all enhanced to a greater extent in the leg that commenced selected training sessions
with low compared with high muscle glycogen content (Hansen et al. 2005). The
results of this study were groundbreaking, and the term “train-low, compete-­high”
rapidly spread among the athletic and scientific communities to describe this novel
approach to training. However, the question of whether athletes with a history of
endurance training would benefit to the same extent as previously untrained indi-
viduals when replacing a portion of their training program with “low glycogen”
workouts could not be answered from the original study of Hansen et al. (2005).
In a follow-up investigation, Yeo et al. (2008) investigated the “train-low” para-
digm during a 3-week intervention in well-trained endurance subjects: these
researchers hypothesized that competitive athletes would have maximized their
training adaptation, and further gains, even in the face of training-low, would be
trivial. Yeo et  al. (2008) imposed a training protocol in which nine intense
­interval-­training sessions during the 3-week training block were commenced when
glycogen stores were lowered by ∼50% (through prior exercise) or were replete.
Importantly, during these sessions, the work rate (power output, W) was not
“clamped” as in the study of Hansen et  al. (2005); rather athletes were asked to
50 J. A. Hawley

exercise as hard as they could and produce as much total work as possible. As
expected, when athletes commenced training with low compared with normal
­glycogen availability, their maximal self-selected power output was significantly
lower (an average of 7%). The remarkable finding, however, was that, despite a
lower “training impulse” to the exercising musculature, resting muscle glycogen
concentration, the maximal activities of citrate synthase and β-hydroxyacyl-CoA-
dehydrogenase and the total protein content of cytochrome c oxidase subunit IV
were higher (compared to pre-training values) in individuals who commenced
­interval training sessions with low muscle glycogen content. Despite these augmented
training adaptations, which would be expected to enhance athletic performance, and
in contrast to the findings of Hansen et al. (2005), there was no difference between
the two treatment conditions in a sport-specific cycling time-trial (Yeo et al. (2008).
The precise mechanisms underlying an enhanced adaptive skeletal muscle
response when training sessions were commenced with lowered glycogen availabil-
ity were not investigated during these investigations (Hansen et al. 2005; Yeo et al.
2008). However, from earlier work, MAPK pathways have been implicated as pos-
sible signaling mechanisms involved in the regulation of exercise/nutrient adapta-
tions. Chan et  al. (2004) were the first to demonstrate that alterations in nutrient
availability that reduced muscle glycogen content led to an increased phosphoryla-
tion of nuclear p38 MAPK in human skeletal muscle in response to moderate-­
intensity exercise. However, others have found little change in either the
phosphorylation state of this kinase or one of its downstream targets (activating
transcription factor 2) after a bout of intense cycling commenced with either low or
normal muscle glycogen content (Yeo et al. 2010).
It is important to note that altering carbohydrate availability (i.e., lowering mus-
cle glycogen content) has reciprocal and pronounced effects on lipid availability.
Indeed it seems entirely plausible that increases in skeletal muscle mitochondria
capacity could be further enhanced if training were performed under conditions that
further elevated the levels of circulating FFAs. To test this hypothesis, Fillmore
et al. (2010) determined whether a combination of chronic chemical activation of
AMPK and high-fat availability (a low-CHO, high-fat diet) would have an additive
effect on skeletal muscle mitochondria markers in a rodent model. They treated
male Wistar rats with a high-fat diet, with injections of AICAR (an AMPK activa-
tor), or with a high-fat diet combined with AICAR injections for 6 weeks. Compared
to either AICAR or high-fat feeding alone, AICAR treatment combined with high
fat availability had an additive effect on markers of oxidative capacity (i.e., the citric
acid cycle and electron transport chain) as well as transcriptional regulation.
Specifically, long-chain acyl-CoA dehydrogenase, cytochrome c, PGC-1α protein,
as well citrate synthase and β-hydroxyacyl-CoA dehydrogenase activity were all
increased by a greater magnitude in the AICAR plus high-fat treatment. While the
mechanism(s) responsible for the additive increase in mitochondrial content
observed with chronic activation of AMPK and elevating circulating FFAs could not
be directly determined, Fillmore et  al. (2010) observed an synergistic effect of
chronic AMPK activation and high-fat feeding on PGC-1α protein abundance and
elevations in PGC-1α mRNA in response to chronic AMPK activation (but not by
Sending the Signal: Muscle Glycogen Availability as a Regulator of Training Adaptation 51

h­ igh-­fat feeding). They concluded that the additive increase in PGC-1α protein
abundance was a combined effect of high-fat feeding-induced posttranscriptional
and AMPK-­ dependent transcriptional increases in PGC-1α protein expression.
However, it should be noted that, when post- exercise increases in FA availability
are abolished by administration of acipimox, a pharmacological inhibitor of lipoly-
sis, the normal exercise-induced increase in mRNA abundance of selected meta-
bolic genes (i.e., PDK4 and PGC-1α) persists (Tunstall et  al. 2007). These data
clearly demonstrate that the increase in circulating FFA concentrations observed
during the later stages of exercise and subsequent recovery are not essential to
induce skeletal muscle mRNA expression of several proteins involved in regulating
muscle metabolism and training adaptation.

Evolution of a Paradigm: Train-High, Sleep-Low

The original “train low” protocol proposed by Hansen et al. (2005) and subsequently
investigated by others (Hulston et al. 2010; Yeo et al. 2008) employed a twice-a-day
training protocol in which the second exercise session was undertaken with low
glycogen availability. As noted, a direct consequence of this strategy was that the
maximal self-selected training intensity of the second session was substantially
reduced when it was commenced with low compared with normal glycogen levels.
Such an outcome is counter-intuitive for the preparation of competitive athletes for
whom high-intensity workouts are a critical component of any periodized training
program (Hawley and Burke 2010). Furthermore, it could be argued that training
twice each day with workouts undertaken in close proximity underpinned part of the
augmented training adaptation compared to once-a-day workouts. Against this
background, we formulated a novel approach in which we can prolong the duration
of low carbohydrate availability, thereby potentially enhancing and extending the
time course of transcriptional activation of metabolic genes and their target proteins,
while simultaneously conserving the training intensity of the initial session and
hence the “training impulse” to the working muscles (Lane et al. 2015). We have
termed this strategy “train-high, sleep-low.” In this protocol, we periodized the tim-
ing of nutrient intake such that athletes performed an evening bout of high-intensity
training with high carbohydrate availability, then restricted carbohydrate intake so
that they slept with low carbohydrate availability before undertaking a prolonged
exercise bout (120  min) in the fasted state the following morning. In our acute
model (Lane et al. 2015), we found that, when feeding was withheld overnight and
subjects slept with reduced carbohydrate availability, AMPKThr172, p38MAPK-
Thr180/Tyr182, and p-ACCSer79 were upregulated to a greater extent the following
morning, compared with when subjects were fed a high- carbohydrate meal the
prior evening. We also showed that, when a second, prolonged steady-state training
session was commenced after “sleeping low,” the expression of selected genes and
abundance of phosphorylated signaling proteins with putative roles in lipid oxida-
tion and transport were higher compared with when a post-exercise meal was
52 J. A. Hawley

consumed and glycogen availability was partially restored (Lane et  al. 2015).
Importantly, subsequent chronic interventions utilizing the “train-high, sleep-low”
approach over 1–3 weeks showed clear benefits to performance (Marquet et  al.
2016a, b).

Summary and Directions for Future Research

Independent of prior training status, short-term (3- to 10-week) training programs in

which some of the workouts are commenced with either low muscle glycogen avail-
ability (or low exogenous carbohydrate availability) augment training adaptation
(i.e., they increase the maximal activities of selected enzymes involved in carbohy-
drate and/or lipid metabolism and promote mitochondrial biogenesis) to a greater
extent than when all workouts are undertaken with normal or elevated glycogen
stores. One aspect that is unclear from the present literature is the absolute levels of
glycogen depletion needed to potentiate the effect of the training stimulus on out-
comes such as mitochondrial biogenesis, or the length of time periodic low-glyco-
gen training needs to be undertaken to demonstrate functional changes to training
and/or performance outcomes. To answer such questions, a complex series of stud-
ies would need to be undertaken that systematically ‘titrate’ muscle glycogen levels
and determine subsequent cellular (and performance) response to a standardized
training regimen. A major problem for the basic scientist trying to unravel potential
mechanism(s) underlying the benefit to training adaptation with reduced muscle
glycogen availability is the fact that carbohydrate restriction has reciprocal and pro-
nounced effects on lipid availability. To address this question, it will be necessary to
study human subjects while they are consuming isoenergetic low carbohydrate diets
in combination with either high protein or fat while undertaking a supervised exer-
cise training protocol. Such experiments are currently being undertaken in our lab.
Invasive measures of metabolism (i.e., markers of skeletal muscle mitochondrial
biogenesis and respiration) are necessary to determine if it is high fat or low carbo-
hydrate availability that drives the augmented training response. What is clear, how-
ever, is that in addition to their role as energy substrates, endogenous fuel stores and
the concomitant changes in circulating hormones and metabolites can act as potent
signaling intermediates to modify the normal responses to contractile activity,
thereby augmenting training adaptation and ultimately driving some of the pheno-
typical changes observed with chronic exercise.

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Optimized Engagement of Macrophages
and Satellite Cells in the Repair
and Regeneration of Exercised Muscle

Regula Furrer and Christoph Handschin

Abstract  Recurring contraction-relaxation cycles exert a massive mechanical load

on muscle fibers. Training adaptation therefore entails the promotion of a series of
biological programs aimed at inducing a better stress response but also at optimiz-
ing repair processes. Muscle regeneration is controlled by an intricate, tightly coor-
dinated engagement of muscle fibers, satellite cells, macrophages and other cell
types. In this review, we discuss some of the recent insights into the regulation of
muscle repair and regeneration in exercised muscle, elucidate the role of the peroxi-
some proliferator-activated receptor γ coactivator 1α (PGC-1α) in this context, and
speculate about potential implications for the treatment of muscle diseases.

Introduction

Skeletal muscle is a highly plastic organ that adapts its properties depending on
contractile demand. These adaptations are not only affected by mechanical loading,
e.g., as seen after a period of inactivity or training, but also by the availability of
nutrients and hormones, temperature or oxygen levels, which collectively determine
the balance between protein synthesis and degradation, mitochondrial activity, con-
tractile function, a shift in fiber type distribution and other biological programs. For
example, endurance training promotes mitochondrial function and oxidative metab-
olism, a shift towards high endurance muscle fibers and tissue vascularization,
among other changes. The peroxisome proliferator-activated receptor γ coactivator
1α (PGC-1α) is an important driver of endurance training adaptation (Lin et  al.
2002, 2005; Handschin 2010). Accordingly, PGC-1α strongly boosts oxidative
metabolism, a fiber type shift, glucose uptake, vascularization and other properties
of endurance-trained fibers by co-activating a variety of transcription factors in a
complex transcriptional network (Handschin 2010; Kupr and Handschin 2015).

R. Furrer • C. Handschin (*)

Biozentrum, University of Basel, Basel, Switzerland
e-mail: christoph.handschin@unibas.ch

© The Author(s) 2017 57

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_5
58 R. Furrer and C. Handschin

Exercise is one of the best interventions to preserve muscle mass through its
strong anti-atrophic effects. As one of the main effectors of exercise adaptation,
PGC-1α also reduces the pathological consequences of muscle wasting. For exam-
ple, elevation of PGC-1α in skeletal muscle reduces fiber damage and atrophy and
improves muscle functionality in etiologically diverse muscle wasting contexts such
as hind limb unloading (Cannavino et al. 2014), denervation or fasting (Sandri et al.
2006), or even Duchenne muscular dystrophy (DMD; Handschin et al. 2007; Selsby
et al. 2012; Hollinger et al. 2013). Several mechanisms have been proposed to be
involved in the therapeutic effect of PGC-1α in muscle pathologies, for example, the
inhibition of the transcriptional activity of forkhead box O3 (FoxO3) and thereby
the induction of E3 ubiquitin ligases muscle ring finger 1 (MuRF-1) and muscle
atrophy f-box (MAFbx) that promote protein degradation and fiber atrophy (Sandri
et al. 2006). However, other consequences of increased PGC-1α activity have also
been implicated; therefore, the exact mechanisms that underlie the beneficial effect
of elevation of PGC-1α remain unclear. More recently, the involvement of inflam-
mation, both in muscle fibers and through activation of resident macrophages and
satellite cells (SCs), the lineage-committed adult muscle stem cells, has been stud-
ied in more detail in this context.

Repair and Regeneration After Muscle Damage

For proper muscle regeneration, a series of highly coordinated events take place that
entail a tightly orchestrated cross-talk between different cell types to ensure proper
initiation, activation, cell type transition and, ultimately, termination of various cel-
lular programs. Initially, in response to injury, muscle cells and tissue-resident mac-
rophages secrete cytokines and chemokines such as tumor necrosis factor α (TNFα)
and C-C motif ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) to
attract additional immune cells (Pillon et al. 2013). Neutrophils are among the first
leukocytes infiltrating the damaged area, and they subsequently release chemotactic
signals to promote the tissue infiltration by circulating monocytes (Saclier et  al.
2013a). During the initial inflammatory response, monocytes polarize into classical
M1-type-activated macrophages and remove tissue debris by phagocytosis. The
concomitantly secreted cytokine and chemokine cocktail not only further attracts
monocytes but also activates SC proliferation and commitment (Saclier et  al.
2013b). The SCs are located in the niche between the sarcolemma and the basal
lamina and express different factors depending on the state of the myogenic process.
Quiescent SCs are characterized by high levels of paired-box 7 (Pax7). Upon activa-
tion, e.g., by muscle injury, proliferation and commitment are initiated by a switch
to myogenic regulatory factor 5 (Myf5) and MyoD expression, ultimately resulting
in the differentiation into myoblasts and subsequent fusion to myofibers with ele-
vated myogenin and MRF4 (Charge and Rudnicki 2004). This differentiation pro-
cess is promoted by the shift in macrophage polarization into the alternatively
Optimized Engagement of Macrophages and Satellite Cells in the Repair… 59

activated M2 types that release anti-inflammatory factors (Saclier et al. 2013b). The
requirement for a tight orchestration of these different phases is illustrated, for
example, by the fibrotic tissue accumulation in the case of a disruption of the regen-
eration process by a prolongation of the initial pro-inflammatory context, thereby
resulting in impaired muscle function. Inversely, a reduced phase involving M1
macrophages may impair regeneration by increased formation of necrotic tissue and
inhibition of proliferation of myogenic cells. Therefore, the tight coordination of the
transition from M1 to M2 macrophages is critical for proper regeneration.

Effects of Exercise and PGC-1α on SCs

In several pathological conditions such as DMD, cancer cachexia and aging, oxida-
tive muscle fibers are preferentially spared, suggesting that these muscle fibers are
more protected from muscle damage and degradation. Interestingly, the SC pool in
highly oxidative muscle fibers is larger (Gibson and Schultz 1982; Dinulovic et al.
2016b), which could contribute to an improved regeneration after muscle damage.
Therefore, inducing a shift towards an oxidative phenotype of the muscle by, for
example, exercise (Rowe et al. 2014a), which is also accompanied by a higher SC
content (Shefer et al. 2010; Kurosaka et al. 2012; Fry et al. 2014; Abreu et al. 2017),
could be a strategy to ameliorate the progression of these pathologies. Even in old
muscle, the aging-induced loss of SCs and differentiation potential can be preserved
by voluntary wheel running or forced treadmill exercise by increased Wnt signaling
(Fujimaki et al. 2014; Cisterna et al. 2016). The rejuvenating effect of exercise on
SC function was demonstrated in aged trained mice that recovered to a similar
extent as young sedentary animals after muscle injury (Joanisse et al. 2016), indicat-
ing that the reduced regenerative capacity of aging muscles could be restored by
exercise.
Curiously, even though many of the exercise-induced adaptations in muscle,
such as the fiber type shift and oxidative metabolism, are regulated by PGC-1α (Lin
et  al. 2002, 2005; Handschin 2010); the number of SCs in mice overexpressing
PGC-1α specifically in muscle (MCKα) is lower than in wild-type animals and thus
diametrically opposite to the higher number seen after exercise or in oxidative mus-
cles (Dinulovic et al. 2016b). Importantly however, despite the reduced SC pool,
regeneration is not impaired and the proliferative potential of the SCs is even
enhanced in these mice (Dinulovic et al. 2016b). These observations suggest that
PGC-1α indirectly affects SC function by co-activating factors that activate SCs or
induce a change in the SC niche (Fig. 1).
The composition of the extracellular matrix (ECM) and the basal lamina deter-
mines the efficacy of the niche to modulate SC proliferation and differentiation. For
example, fibronectin (FN) is an important niche protein involved in the activation
and proliferation of SCs (Bentzinger et al. 2013). Accordingly, the impaired regen-
erative capacity in aging muscle is associated with reduced levels of FN and can be
restored by treating mice with FN (Lukjanenko et  al. 2016). It is conceivable,
60 R. Furrer and C. Handschin

Fig. 1  Pre-conditioning of the muscle for faster regeneration. Schematic representation of the
exercise- and PGC-1α-induced muscle cross-talk to macrophages and satellite cells (SC) that cre-
ates an environment that could prime the tissue for improved regeneration in response to damage.
ECM extracellular matrix, BL basal lamina, Mɸ Macrophage

t­herefore, that the enhanced proliferative potential of SCs of MCKα mice is linked
to FN function, since higher levels of FN are observed in the muscles of these ani-
mals (Dinulovic et al. 2016b). The increase in matrix metalloproteinases 2 (MMP2)
and MMP9 induced by exercise further underscores the importance of niche remod-
eling, since the ensuing ECM degradation could subsequently facilitate the migra-
tion of SCs (Garg and Boppart 2016) whereas MMP-controlled vascularization
enhances the delivery of nutrients and growth factors to the niche (Gustafsson
2011). Accordingly, differentiating SCs are closer to capillaries compared to quies-
cent SCs (Christov et al. 2007). Inversely, SCs are more distantly located from cap-
illaries in old compared to young men (Nederveen et al. 2016), suggesting that the
reduced transport of signaling molecules to the niche could contribute to the reduced
regenerating potential observed in aging muscles. The angiogenesis induced by
exercise or PGC-1α (Gustafsson 2011; Rowe et al. 2014b) results in a closer prox-
imity of SCs to capillaries; hence there is improved transport of circulating factors
to the niche and ultimately enhanced regenerative processes.

 ffects of Exercise and PGC-1α on Macrophages

E
and Inflammation

In contrast to the numerous studies on the effects of exercise on SCs, literature on

exercise-induced changes on macrophages is scarce. Most studies investigating
macrophage infiltration in the context of regeneration post-exercise were performed
in humans after damaging eccentric exercise (Przybyla et al. 2006; Mahoney et al.
2008; MacNeil et al. 2011), and less is known about the effects of endurance exer-
cise on macrophage infiltration. As infiltration of immune cells is tightly coordi-
nated, the timing of tissue collection is crucial and complicates the experimental
Optimized Engagement of Macrophages and Satellite Cells in the Repair… 61

setup. In response to resistance exercise, infiltration of CD68+ macrophages is

increased after 48 h (Mahoney et al. 2008; MacNeil et al. 2011) and unchanged in
the early (3 h) and late (72 h) phases of recovery (Przybyla et al. 2006; Mahoney
et al. 2008; MacNeil et al. 2011). The expected switch to CD163+ M2 macrophages
is observed after 72 h, i.e., during the later stage of regeneration (Przybyla et al.
2006). In contrast to the infiltration of macrophages seen 48 h post-resistance exer-
cise in humans, endurance exercise had no effect on the number of macrophages at
that time point in rats (Kurosaka et al. 2012). Therefore, the timing of macrophage
activation, infiltration and polarization depends on the type of exercise, training
intensity and most likely other factors that remain to be elucidated.
In skeletal muscle fibers, PGC-1α reduces the expression of pro-inflammatory
gene expression by inhibiting the transcriptional activity of the nuclear factor κB
(NF-κB; Eisele et al. 2013). Nevertheless, somewhat unexpectedly, sustained over-
expression of PGC-1α in muscle increases the number of tissue-resident macro-
phages in uninjured muscle (Rowe et al. 2014b; Dinulovic et al. 2016a). However,
these macrophages have a predominant M2 phenotype (Dinulovic et  al. 2016a),
indicating that PGC-1α induces an anti-inflammatory environment and may thereby
modulate the response to muscle damage. Interestingly, in contrast to the glycolytic
phenotype of M1 macrophages, M2 macrophages have a more oxidative phenotype
(Galvan-Pena and O’Neill 2014; Kelly and O’Neill 2015), analogous to the meta-
bolic phenotype of muscle overexpressing PGC-1α or PGC-1β. The metabolic shift
required for the anti-inflammatory phenotype of macrophages is mediated by
PGC-1β (Vats et al. 2006).
Although the mechanisms by which PGC-1α affects macrophage polarization
leading to the higher number of M2 macrophages in muscle is unknown, mediators
of PGC-1α-dependent macrophage attraction and/or activation have been described
(Fig.  1). PGC-1α induces the transcription of secreted phosphoprotein 1 (SPP1),
which in turn is involved in the recruitment of macrophages and activation of epi-
thelial cells to ensure functional neovascularization (Rowe et al. 2014b). In the con-
text of acute exercise, PGC-1α controls the expression of the B-type natriuretic
peptide (BNP), which promotes macrophage activation in a paracrine manner
(Furrer et al. 2017). While M1 macrophage activation is resolved more rapidly in
damaged PGC-1α-overexpressing muscles (Dinulovic et  al. 2016a), the mecha-
nisms by which muscle PGC-1α affects infiltration and polarization in this context
are still unclear.

 xercise-Induced Pre-conditioning for Faster Muscle

E
Regeneration

As described above, exercise and muscle PGC-1α induce several adaptations such
as an increase in the SC pool, remodeling of the SC niche, enhancement of the pro-
liferation and differentiation potential of SCs as well as a higher number of
62 R. Furrer and C. Handschin

tissue-­resident macrophages that could all contribute to an improved regenerative

capacity of the muscle. It is conceivable that exercise creates an environment within
the muscle and the niche that could prime the muscle for improved regeneration in
response to muscle damage. This hypothesis of exercise-induced pre-conditioning
is supported by various observations. For example, the altered tissue-resident mac-
rophage population in muscles overexpressing PGC-1α could contribute to the
improved SC proliferation and commitment in damaged muscles of these mice,
since a variety of cytokines and growth factors that activate SCs are secreted by both
muscle and macrophages. M1 macrophages release interleukin 6 (IL-6), insulin-like
growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), whereas
M2 macrophages mainly secrete IGF-1 and VEGF (Wu et al. 2010; Lu et al. 2011;
Tonkin et al. 2015). IL-6 and IGF-1 are important for the activation and prolifera-
tion of SC (Charge and Rudnicki 2004; Serrano et al. 2008) and may thereby con-
tribute not only to the repair and regeneration but, in the case of resistance training,
also the hypertrophic response to exercise. Besides the direct effect of IL-6 on SCs,
changes in IL-6 levels may also be involved in the remodeling of the niche, as IL-6
KO mice show lower levels of FN (Serrano et al. 2008). These reduced FN levels
contribute to impaired regeneration, which is in line with the FN-dependent decrease
in regeneration observed in aging (Lukjanenko et al. 2016). Therefore, the number
of macrophages as well as the proportion of M1 and M2 macrophages in a trained
muscle could play a role in the pre-conditioning of the muscle.
Of note, several of these factors, such as IL-6, IGF-1 and VEGF, are also myo-
kines that are produced and secreted from muscle after exercise (Schnyder and
Handschin 2015). Collectively, the local levels of such signaling molecules could,
therefore, affect the activation of both macrophages and SCs. For example, eleva-
tion of VEGF stimulates angiogenesis and thereby increases vascularization
(Gustafsson 2011; Rowe et al. 2014b). The increase in VEGF is directly regulated
by PGC-1α in muscle fibers (Arany et al. 2008; Baresic et al. 2014; Rowe et al.
2014b) and potentially also indirectly through the stimulation of M2 macrophage
accumulation (Dinulovic et al. 2016a). The resulting increase in tissue vasculariza-
tion improves activation of SCs and facilitates infiltration of immune cells.
Besides paracrine effects of muscle and macrophages, exercise-regulated modu-
lation of intrinsic SC properties also influences the proliferation and differentiation
potential. For example, isolated SCs of muscles exposed to functional overload fuse
better than control SCs (Fujimaki et al. 2016), suggesting that the differentiation
potential of SCs is improved by exercise in a manner that is maintained even after
removal from the niche. Inactivation of Notch signaling and stimulation of Wnt
signaling contribute to improved fusion of trained SCs (Fujimaki et al. 2016), but
the exact mechanisms of this priming of SC memory is unclear. Recently, a G0alert
state of SCs in the contralateral, non-damaged leg of mice with a pharmacologically
induced muscle injury has been proposed to be linked to mammalian target of
rapamycin complex 1 (mTORC1) activity (Rodgers et  al. 2014). This SC pool
exhibits an accelerated proliferative response upon activation. Similar to these
observations in SCs, muscles may also have a memory in terms of the post-exercise
inflammatory response: in human volunteers, MCP-1 levels as well as CD68+
Optimized Engagement of Macrophages and Satellite Cells in the Repair… 63

macrophages were higher two days after a repeated bout compared to the first bout
of exercise (Deyhle et  al. 2015). While the mechanisms underlying this finding
remain enigmatic, similar exacerbated macrophage activation is observed upon
elevation of BNP by PGC-1α in exercised muscle (Furrer et al. 2017). Moreover, in
muscles overexpressing PGC-1α, a larger area of cardiotoxin-damaged muscle is
covered by macrophages and thus undergoing regeneration (Dinulovic et al. 2016a).
In addition, after chronic muscle damage by multiple cardiotoxin injections, fiber
size was recovered more efficiently and fibrotic tissue was reduced by PGC-1α
overexpression (Dinulovic et al. 2016a, b), indicating that exercise- and PGC-1α-
induced intrinsic and extrinsic adaptations contributed to enhanced regenerative
capacity. Importantly, improved regeneration upon cardiotoxin-induced muscle
damage occurred despite strong downregulation of PGC-1α expression (Dinulovic
et al. 2016a, b). These data strongly imply a pre-conditioning effect to help trained
muscle to cope with damage that includes a broad spectrum of functional modula-
tion of SCs, endothelial cells and macrophages as well as cellular cross-talk that
together create an environment that primes the muscle for faster regeneration.

Conclusion

Overexpression of PGC-1α in skeletal muscle ameliorates the symptoms of many

different muscle diseases. Similarly, in those pathologies in which patients are exer-
cise tolerant, training confers a beneficial effect on muscle functionality. While
many different properties of exercise and PGC-1α most likely contribute to this
therapeutic effect, the modulation and orchestration of macrophages and SCs could
directly improve repair and regeneration in these diseases. Therefore, a more careful
elucidation of the cell autonomous changes and cellular cross-talk that optimize
repair and regeneration of damaged muscle tissue after exercise could provide novel
targets and avenues to treat a wide variety of different muscle diseases.

Acknowledgments  The work in our group related to the topic of this review is supported by the
Swiss National Science Foundation, the European Research Council (ERC) Consolidator grant
616830-MUSCLE_NET, Swiss Cancer Research grant KFS-3733-08-2015, the Swiss Society for
Research on Muscle Diseases (SSEM), SystemsX.ch and the University of Basel.

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Skeletal Muscle microRNAs: Roles
in Differentiation, Disease and Exercise

Rasmus J. O. Sjögren, Magnus H. L. Lindgren Niss, and Anna Krook

Abstract MicroRNAs (miRNA) are a noncoding RNA species that play important

roles in the regulation of gene expression. Since miRNAs are able to target multiple
genes simultaneously, miRNAs provide a mechanism for efficiently modulating a
whole pathway to change or alter the functional properties in a particular target tissue.
Ablation of miRNA processing specifically in skeletal muscle results in muscle abnor-
malities and perinatal death, underscoring that miRNAs control essential processes in
skeletal muscle development and function. In this chapter we summarise current knowl-
edge on miRNAs involved in skeletal muscle differentiation, disease and exercise.

Discovery and Biological Role of miRNAs

Precise control of gene expression and hence transcriptional regulation is a prerequi-

site for maintaining cellular functions, including growth and metabolic homeostasis.
Several epigenetic factors, including DNA methylation, histone modification and
miRNAs play important roles in gene expression in multicellular organisms. miR-
NAs were first described at the beginning of the 1990s, when it was appreciated that
these short single-stranded non-coding RNAs function to post-transcriptionally regu-
late mRNA abundance and protein translation. Lin-4 and let-7 were the first miRNAs
to be identified and found to be involved in the regulation of larval development of C.
elegans (Lee et al. 1993; Wightman et al. 1993; Reinhart et al. 2000). miRNAs have
since been observed in viruses, plants and mammals (Bartel 2009) and found to regu-
late a wide variety of cellular functions, including proliferation, differentiation and
metabolism (He et al. 2007b; Williams et al. 2009a; Massart et al. 2016).

R. J. O. Sjögren
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
M. H. L. Lindgren Niss • A. Krook (*)
Department of Physiology and Pharmacology, Integrative Physiology, Karolinska Institutet,
Stockholm, Sweden
e-mail: anna.krook@ki.se

© The Author(s) 2017 67

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_6
68 R. J. O. Sjögren et al.

miRNA Biogenesis, mRNA Interaction and Target Prediction

miRNAs are expressed in the nucleus as primary-miRNA (pri-miRNA) transcripts,

which require processing before becoming functional miRNAs. Pri-miRNAs con-
tain stem-loop structures that are recognized and cleaved by the RNase III endonu-
clease Drosha to form precursor miRNAs (pre-miRNAs; Lee et  al. 2003). The
pre-miRNA is then transported to the cytoplasm, where it is subsequently processed
by RNase III Dicer into a mature miRNA of approximately 21 nucleotides (Hutvagner
et al. 2001). The mature miRNA is then incorporated into the RNA-­induced silenc-
ing complex (RISC), where mRNA target interaction takes place (Hammond et al.
2000). The canonical model of miRNA-mRNA interaction occurs by perfect or par-
tial pairing at the 5′-end of the miRNA to a complementary site of the 3′ UTR of the
target. A sequence of 6–8 nucleotides at the 5′ end of the miRNA, called the seed
region, guides the RISC complex to targeted mRNAs. Repression of mRNAs may
then occur by inhibition of translation or by degradation of the transcript, ultimately
reducing protein abundance of targeted genes (Melo and Melo 2014).
A key challenge in miRNA research to date involves identifying the target genes
for each miRNA. The seed is a dominant motif in miRNAs and is, therefore, used in
prediction algorithms to identify potential targets. The algorithms are centred on find-
ing complementary sequences in the 3′ UTR of mRNAs. Evolutionary conservation
of the complementary sequence is also taken into consideration, and whether there is
a supplementary pairing site in the 3′ part of the miRNA (for review, see Bartel 2009).
However, at present the prediction algorithms have been only partially accurate in
identifying true target sites, yielding both false positive and false negative predictions.
When comparing results from silencing and over-expression experiments, only a
small percentage of mRNA targets were found to be affected by both (Nicolas et al.
2008). Of these, approximately half had a sequence in the 3′-UTR matching the seed,
but none was found by prediction programs (Nicolas et  al. 2008). Since miRNAs
exert their function both at the level of mRNA abundance and at protein abundance,
transcriptomic analysis will miss targets that are regulated only at the protein level. To
further add to the complexity of miRNA regulation, one gene may be targeted by
several different miRNAs. Additionally, the same miRNA may have several target
sites on the same gene (Bartel 2009). One miRNA may even have opposing effects on
a target gene in different tissues (Lu et al. 2010; Chuang et al. 2015).

 ole of miRNAs in the Regulation of Skeletal Muscle

R
Development

Skeletal Muscle Development and Regeneration

Skeletal muscle has been shown to express several RNA species (Gallagher et  al.
2010), and dysregulation of skeletal muscle miRNAs has been implicated in a num-
ber of different disease states (Williams et al. 2009a; Massart et al. 2016). Skeletal
Skeletal Muscle microRNAs: Roles in Differentiation, Disease and Exercise 69

muscle has a large adaptive potential in response to contractile activity. This plastic-
ity is necessary to modify contractile features and adapt metabolic capacity to new
requirements following endurance and/or resistance exercise. Skeletal muscle also
has a high regenerative capacity in response to injury or damage. Critical to this plas-
ticity and the regenerative potential of skeletal muscle are myogenic progenitor cells,
termed satellite cells (Dumont et al. 2015). In adults, satellite cells are quiescent but
they can, in response to injury or increased contractile activity, re-enter the cell cycle
and proliferate and thereafter differentiate, fuse, and regenerate myofibres.
Skeletal muscle development has been studied extensively in vitro through isola-
tion, growth and differentiation of satellite cells and in vivo during embryogenesis
(see Fig. 1). Molecular regulation of cell determination to the myogenic lineage and
proliferation of these cells involves the paired box transcription factors PAX3 and
PAX7 (Braun and Gautel 2011). Pax3 loss-of-function during embryogenesis in
mice results in the absence of musculature in the trunk region due to reduced migra-
tion of satellite cells (Bober et al. 1994; Tremblay et al. 1998). While Pax7 deletion
in animals has little effect on embryogenic muscle formation (Oustanina et  al.
2004), it is essential for satellite cell formation and adult myogenesis (Sambasivan
et al. 2011). When activated, satellite cells can migrate and re-enter the cell cycle to
start proliferation. The processes of satellite cell commitment and differentiation
into multinucleated myotubes are controlled by a group of basic helix-loop-helix
transcription factors known as myogenic regulatory factors (MRFs). The MRFs
consist of four members: myogenic factor 5 (MYF5), myogenic differentiation 1
(MYOD1), myogenin (MYOG), and myogenic regulatory factor 4 (MRF4). These
transcription factors promote terminal differentiation by regulating the expression
of several myogenic genes (Braun and Gautel 2011). Satellite cells committed to the
myogenic program and myogenesis are termed myoblasts, which typically express
PAX7 and early MRFs such as MYOD and MYF5. Expression of these early MRFs
is crucial for myoblast maturation because a combined Myod and Myf5 loss-of-­
function results in the absence of skeletal muscle formation during embryogenesis
(Rudnicki et  al. 1993). MYF5 and MYOD are considered determination factors
required for establishment of myogenic identity since they are upstream transcrip-
tional regulators of late MRFs, MYOG and MRF4 (Braun and Gautel 2011).
Myoblasts will leave the cell cycle for terminal differentiation by lowering PAX7
and MYF5 expression while inducing the late MRFs. The late MRFs are important
for terminal differentiation, and lack of, for example, Myog in mice leads to postna-
tal lethality due to muscle deficiency (Hasty et al. 1993).

miRNA Regulation of Myogenesis In vitro and In vivo

miRNAs are important regulators of cell proliferation and differentiation, processes

that are critical to proper tissue development and maintenance. The relative expres-
sion of miRNAs is tissue dependent, with several miRNAs showing enrichment in
skeletal muscle (and heart muscle). These muscle-enriched miRNAs are termed
 70

Fig. 1  mRNA and miRNA expression during human skeletal muscle cell differentiation in vitro. Human skeletal muscle cells were cultured until approxi-
mately 80% confluence (Day 0; white bars) and induced to differentiate for 10 days (Day 10; black bars). RNA was collected and mRNA and miRNA expres-
sion was assessed by RT qPCR. (a) Expression of the myogenic markers desmin (DES), myogenic factor 5 (MYF5) and myogenin (MYOG) before inducing
myoblasts to differentiate and following differentiation into myotubes. (b) Expression of a muscle-enriched (miR-1) and a non-muscle-enriched (miR-30c)
miRNA before and after differentiation. (c) Representative microscope bright-field pictures of cells prior to inducing differentiation (top) and following myo-
R. J. O. Sjögren et al.

tube formation after differentiation (bottom). All data represent average ± SEM, *p < 0.05 [paired student’s t-test (normal distribution of data) or Wilcoxon
matched-pairs singed rank test (non-normal distribution of data)]
Skeletal Muscle microRNAs: Roles in Differentiation, Disease and Exercise 71

“myomiRs” (Sempere et al. 2004). Some members of the myomiRs are organized
in bicistronic clusters with genomic colocalisation of miR-1-1/miR-133a-2, miR-­
1-­2/miR-133a-1 and miR-133b/206. Thus, these miRNAs share similar expression
patterns and are induced during skeletal muscle cell differentiation by upstream
transcriptional regulation of different MRF members, including MYOD and MYOG
(Rao et  al. 2006; Sweetman et  al. 2008). Besides sharing similar regulation of
expression, they also share similar sequences. For example, miR-133a-1/2 (identi-
cal sequences) and miR-133b differ by only one single nucleotide outside of the
seed region, and miR-1-1/2 (identical sequences) and miR-206 differ in four nucle-
otides at the 3′ end but share identical seed sequences in humans. Thus, miR-1 and
miR-206, and miR-133a and miR-133b, respectively, regulate a set of similar, but
not identical, target genes.
The aforementioned myomiRs are all potent regulators of satellite cell differen-
tiation and proliferation. miR-1 and miR-206 are induced upon satellite cell com-
mitment and differentiation, and increased expression promotes differentiation of
these cells (Fig. 1; Chen et al. 2006; Kim et al. 2006). miR-1 and miR-206 induce
differentiation by targeting several repressors of skeletal muscle cell differentiation,
including histone deacetylase 4 (Hdac4); Chen et al. 2006; Williams et al. 2009b),
Pax3 (Goljanek-Whysall et al. 2011) and Pax7 (Chen et al. 2010). The effects of
miR-133a/b on myogenesis are still debated. It is clear that miR-133, like several
other myomiRs, is induced during skeletal muscle differentiation (Chen et al. 2006;
Kim et  al. 2006), whereas different effects of miR-133 on the myogenic process
have been reported. Initial observations have indicated that miR-133 promotes the
proliferative state of myoblasts by repressing Serum Response Factor (Srf; Chen
et  al. 2006). More recent evidence suggests that miR-133 instead functions by
inhibiting myoblast proliferation to promote muscle cell differentiation (Zhang
et al. 2012; Feng et al. 2013). These effects of miR-133 are due to targeting of pro-­
proliferative target genes. Such targets include SP1 transcription factor (Sp1), an
upstream regulator of Cyclin D1 expression, and Fibroblast Growth Factor Receptor
1 (Fgfr1) and Protein Phosphatase 2 Catalytic Subunit Alpha (Pp2ac), which are
important for regulation of ERK1/2 phosphorylation status (Zhang et al. 2012; Feng
et al. 2013). The differences in results noted could indicate that miR-133 can influ-
ence both proliferation and differentiation in a context-dependent manner.
While the effects of miR-1 and miR-206 on skeletal muscle cell differentiation
in vitro are clear, ablation of miR-1-2 or miR-206 in mice did not result in disturbed
skeletal muscle development during embryogenesis in  vivo (Zhao et  al. 2007;
Williams et  al. 2009b). Nevertheless, genetic deletion of miR-206 negatively
affected post-embryonic regeneration of muscle and neuromuscular connections
following injury (Williams et  al. 2009b; Liu et  al. 2012). Furthermore, miR-206
deletion exacerbated the dystrophic phenotype in a mouse model of muscular dys-
trophy (Liu et al. 2012). Contrary to these results, deletion of the miR-206/miR-­
133b cluster was found not to be required for proper muscle regeneration (Boettger
et  al. 2014). These differences, and the absence of a robust phenotype following
myomiR loss-of-function during embryogenesis, could be explained by the large
redundancy in this group of myomiRs. Not only are there several genomic copies of
72 R. J. O. Sjögren et al.

miR-1 and miR-133a, but they also share similar sequences with miR-206 and miR-­
133b, respectively. Thus, miR-1 and miR-133a could compensate for the loss of
miR-206/133b expression (Boettger et al. 2014). In contrast, D. melanogaster has
only one genomic copy of miR-1 and, in this context, the absence of miR-206 is
coincident with severely deformed musculature in miR-1 mutant larvae (Sokol and
Ambros 2005), implicating that the presence of several genomic copies, as found in
other species, provides system redundancy. Furthermore, a double knockout of
miR-133a-1 and miR-133a-2 in mice resulted in cardiomyopathy and lethal defects
in approximately 50% of offspring, whereas mice lacking only miR-133a-1 or miR-­
133a-­2 did not show this phenotype (Liu et al. 2008). The miR-133a double knock-
out mice that survived until adulthood also showed skeletal muscle defects, including
mitochondrial dysfunction and centronuclear myopathy of type II fibres (Liu et al.
2011).
Skeletal muscle-specific Dicer ablation in mice results in reduced muscle-­
specific miRNA expression and perinatal death, abnormal muscle morphology and
reduced skeletal muscle mass (O'Rourke et al. 2007), indicating that proper miRNA
maturation is essential for overall muscle function and development. Conditional
knockout of Dicer in satellite cells in adult mice results in satellite cells exiting
quiescence and entering the cell cycle, leading to severely impaired regeneration
following skeletal muscle injury (Cheung et al. 2012). Collectively, evidence points
towards a central function of miRNAs, not only for normal embryonic skeletal mus-
cle biogenesis but also for adult muscle function and repair.
Not only is the expression of several myomiRs altered in response to satellite cell
expansion and differentiation, but also several non-muscle enriched miRNAs are
temporally regulated in expression during muscle cell differentiation (see Fig. 1).
Several studies have determined miRNA expression profiles during differentiation
in  vitro and during myogenesis in  vivo (Chen et  al. 2011; Cheung et  al. 2012;
Koning et al. 2012; Dmitriev et al. 2013; Sjögren et al. 2015). We identified 44 miR-
NAs, including the canonical myomiRs, to be altered during primary human skele-
tal muscle cell differentiation (Sjögren et al. 2015), comparable to the 60 miRNAs
altered in a similar study (Dmitriev et al. 2013). Nevertheless, these miRNAs might
not influence either the proliferation or differentiation process but may be involved
in other phenotypic changes associated with muscle cell differentiation, such as
metabolism, hypertrophy or mitochondrial function. Examples of non-muscle-­
enriched miRNAs identified to have altered expression and directly affecting prolif-
eration or differentiation of skeletal muscle cells include, for example, miR-26a
(Dey et al. 2012), miR-30 (Fig. 1; Guess et al. 2015) and miR-199a-5p (Alexander
et al. 2013). Future studies are required to determine the targets and effects on skel-
etal muscle cell proliferation and differentiation, both in vivo and in vitro, of several
of the miRNAs that have altered expression during satellite cell activation and
differentiation.
Skeletal Muscle microRNAs: Roles in Differentiation, Disease and Exercise 73

Skeletal Muscle miRNA in Metabolic Disease

Approximately half of the total body mass in healthy individuals consists of skeletal
muscle, representing a substantial part of whole-body metabolism (DeFronzo and
Tripathy 2009). Skeletal muscle also represents a primary target for insulin-­mediated
glucose disposal, and skeletal muscle insulin resistance is a characteristic feature in
type 2 diabetes and in conditions of impaired glucose metabolism transport activity
(Krook et  al. 2000; Ryder et  al. 2000). Furthermore, skeletal muscle function is
crucial for proper posturing and locomotion patterns, and disruption in metabolic or
motile functionality may greatly decrease an individual’s quality of life. While there
is good evidence that miRNAs play a key role in the regulation of muscle growth
and differentiation, dysregulation of skeletal muscle miRNA expression in disease
states has been less well studied, in particular in human cohorts.
Several miRNAs were found to be downregulated in response to a 3-h
euglycaemic-­hyperinsulinaemic clamp in human skeletal muscle, including miR-1
and miR-133a (Granjon et al. 2009). Expression profiles of skeletal muscle miR-
NAs have been reported in a number of different rodent models of obesity and dia-
betes, including Goto-Kakizaki (GK) rats, a non-obese spontaneous T2D model (He
et al. 2007a; Huang et al. 2009; Herrera et al. 2010), rats rendered diabetic by a
combination of high fat diet and a low dose of streptozotocin (Karolina et al. 2011),
as well as mice fed a high fat diet (Chen et al. 2012; Mohamed et al. 2014). These
different models share skeletal muscle insulin resistance as a key characteristic.
When miRNA profiles from patients with type 2 diabetes were compared against
these different animal models, eight upregulated miRNAs found in type 2 diabetic
patients were also found to be upregulated in at least one of the rodent arrays (for
review and analysis, see Massart et al. 2016). Similarly, 16 of the downregulated
miRNAs were recapitulated in rodent studies. Interestingly, two thirds of the com-
monly regulated miRNAs have been identified in mice rendered insulin resistant in
response to a high fat diet (Massart et al. 2016). However, different miRNAs showed
altered expression depending on the model. For example, miR-99a and miR-100,
which were downregulated in skeletal muscle from patients with type 2 diabetes and
in mice fed a high fat diet, were upregulated in rats on a high fat diet rendered dia-
betic with streptozotocin, suggesting that the aetiology of the insulin resistance
influenced the miRNA expression signature. As yet, only a few of these miRNAs
have been studied in vitro and/or in vivo in relation to metabolic disease, and their
targets remain poorly characterized.

Skeletal Muscle miRNAs in Response to Exercise

Skeletal muscle responds rapidly to exercise, and even a single bout of acute exer-
cise is sufficient to induce changes in gene expression. In response to many bouts of
acute exercise, i.e., exercise training, skeletal muscle adapts by altering protein
74 R. J. O. Sjögren et al.

production, which impacts the functional as well as the metabolic properties of the
exercised muscle. The inter-individual variability in skeletal muscle hypertrophic
response to resistance exercise has been attributed to variations in the ability of
satellite cell mobilization to proliferate and subsequent fuse with existing muscle
fibres (Petrella et al. 2006, 2008). As detailed above, several miRNAs regulate skel-
etal muscle satellite cell quiescence, activation and differentiation, indicating poten-
tial roles for these miRNAs in skeletal muscle hypertrophy.
Identification of pathways and molecular mechanisms regulating skeletal muscle
exercise adaptation has been a key focus from a number of different angles; from
probing understanding of athletic performance to alterations relevant for metabolic
disease. Different modes of exercise training lead to different adaptive responses;
for example, strength training leads to more hypertrophic responses whereas aero-
bic exercise increases endurance. These differences are also mirrored in the differ-
ent intracellular signalling pathways that are activated (for review, see Egan and
Zierath 2013). Several lines of evidence suggest an important role for miRNAs in
skeletal muscle development and hypertrophy (for review see Zacharewicz et  al.
2013; Kovanda et al. 2014).
Several human studies have reported that short exercise bouts/training duration
lead to increased skeletal muscle expression of miR-1 (Nielsen et al. 2010; Russell
et al. 2013), whereas longer endurance training protocols lead to miR-1 downregu-
lation (Nielsen et al. 2010; Drummond et al. 2011; Keller et al. 2011). Rodent stud-
ies are in general agreement regarding regulation of myomiRs in response to
exercise (for review see Zacharewicz et al. 2013). In contrast, although several of
the classical myomiRs have been noted to respond to exercise, a clear consensus has
yet to emerge regarding which other miRNAs are regulated in response to exercise.
These inconsistencies in the literature may be due to species differences as well as
differences in the exercise protocols used, but they may even depend on different
RNA extraction protocols (for more in depth discussion see Zacharewicz et  al.
2013).

 iRNA Regulation of Skeletal Muscle Atrophy and During

m
States of Muscle Mass Loss

Skeletal muscle is the largest organ in adult mammals and muscle function is critical
to metabolic homeostasis and health across the whole life span. Loss of skeletal
muscle mass (atrophy) and function, also referred to as sarcopenia, occurs with
aging (Cartee et al. 2016). Muscle loss is also noted in obese individuals, a phenom-
enon known as sarcopenic obesity, and is associated with lowered skeletal muscle
insulin sensitivity. In addition, individuals with cancer, HIV-AIDS, chronic heart
failure, chronic obstructive pulmonary disease, renal failure, rheumatoid arthritis,
and osteoarthritis can all experience a dramatic loss of muscle mass (Wolfe 2006).
Thus understanding the regulation of skeletal muscle mass and function is of clini-
cal relevance.
Skeletal Muscle microRNAs: Roles in Differentiation, Disease and Exercise 75

Three members of the myomiR family, miR-208a, miR-208b and miR-499, are
encoded and expressed from sequences embedded within heart- and skeletal muscle-­
enriched myosin genes, MYH6, MYH7 and MYH7B, respectively (van Rooij et al.
2009). miR-208a is exclusively expressed in heart muscle, whereas miR-208b and
miR-499 are expressed both in heart muscle and in type I (oxidative) skeletal mus-
cle fibres. Loss of function of both miR-208b and miR-499 in mice results in loss of
type I fibres and is associated with increased expression of miR-208b and miR-499
pro-atrophic target genes, including purine rich element binding protein beta (Purb)
and SRY-box containing gene 6 (Sox6; van Rooij et al. 2009). Furthermore, four
weeks of hind limb suspension, resulting in severe skeletal muscle atrophy, reduces
expression of miR-208b and miR-499 in rat skeletal muscle (McCarthy et al. 2009).
In humans with skeletal muscle loss due to spinal cord injury, expression of miR-­
208b and miR-499-5p is reduced and inversely related to expression of myostatin, a
critical regulator of skeletal muscle mass (Boon et al. 2015). These miRNAs also
participate in a transcriptional network together with estrogen-related receptors
(ERRs) and members of the peroxisome proliferator activated receptor (PPAR)
family to coordinate the regulation of skeletal muscle energy metabolism and fibre
type specification (Gan et al. 2013).
Muscular dystrophy is characterized by muscle atrophy and weakness. A large
microarray-based study found expression of 185 miRNAs to be altered in skeletal
muscle in at least one of 10 common muscular disorders, including Duchenne mus-
cular dystrophy (DMD; Eisenberg et al. 2007). Several miRNAs have been noted to
be dysregulated both in human Duchenne muscular dystrophy and in a mouse model
of the disease (Greco et al. 2009). Examples of dysregulated miRNAs include miR-­
206, which was upregulated in states of muscular dystrophy (McCarthy et al. 2007;
Greco et al. 2009), potentially due to constant regenerative processes in dystrophic
skeletal muscle. However, as mentioned above, mice lacking miR-206 have a mus-
cle dystrophic phenotype (Liu et al. 2012), underscoring the complexity of miRNA
regulation. In the case of muscular dystrophy, a number of miRNAs has been pro-
posed to have possible therapeutic potential. For example, overexpression of miR-­
486, another muscle-enriched miRNA with decreased expression in muscle
dystrophy (Eisenberg et al. 2007), improved muscle function in a mouse model of
muscle dystrophy (Alexander et  al. 2014). Another example is miR-431, where
transgenic miR-431 mice with muscular dystrophy have improved skeletal muscle
fatigability and force generation, potentially due to improved muscle regeneration
and enhanced muscle cell differentiation capacity in vitro (Wu et al. 2015).
miRNAs are also potent regulators of signalling pathways known to regulate
muscle atrophy. Two critical regulators of skeletal muscle atrophy are F-box Protein
32 [FBXO32; also known as Muscle atrophy F-box (MAFbx)] and Tripartite Motif
Containing 63 [TRIM63; also known as Muscle RING finger 1 (MuRF1)], which
regulate protein degradation through regulation of ubiquitination. The family of
Forkhead Box O (FOXO) transcription factors, especially FOXO1 and FOXO3, are
strong upstream regulators of both these genes during skeletal muscle atrophy
(Glass 2010). FOXO1 activity was reduced following miR-486 overexpression in
skeletal muscle cells in  vitro, both through decreased FOXO1 expression and
through increased FOXO1 phosphorylation (inhibitory) through direct miR-486-
76 R. J. O. Sjögren et al.

targeting of Phosphatase and Tensin Homolog (PTEN), a negative regulator of

FOXO1-upstream kinase AKT (Xu et  al. 2012). miR-486 overexpression also
reduced induction of FOXO1 targets MAFbx and MuRF-1 protein expression in
primary mouse muscle cells following treatment with dexamethasone, a potent
inducer of muscle atrophy (Xu et al. 2012). In addition, overexpression of miR-486
protected against skeletal muscle loss in a mouse model of chronic kidney disease
(Xu et al. 2012). Furthermore, miR-486 inhibition in vitro slightly reduced myotube
diameter, and inhibition in vivo reduced the cross-sectional area of muscle fibres,
confirming the effects of miR-486 on regulation of skeletal muscle mass (Hitachi
et al. 2014). MAFbx and MuRF1 are also directly under miRNA-dependent regula-
tion by two different miRNAs, miR-23a and miR-23b (Wada et al. 2011). miR-23a
overexpression either in  vitro or with a transgene in mouse skeletal muscle pro-
tected against dexamethasone-induced muscle atrophy (Wada et al. 2011). Cellular
miR-23a is decreased in dexamethasone-induced skeletal muscle atrophy, poten-
tially through a mechanism including enhanced exosomal release of miR-23a
(Hudson et al. 2014).
miRNA expression has also been assessed following age or sarcopenia-induced
skeletal muscle loss (Drummond et al. 2011; Rivas et al. 2014; Zacharewicz et al.
2014). Nevertheless, the overlap of miRNA signatures is small between these stud-
ies, potentially due to the small number of biological replicates in each study or due
to different platforms to assess miRNA expression. Further studies will be needed
to determine the impact of miRNA expression and regulation of target genes in
human sarcopenia and muscle loss with ageing.

Conclusions

miRNAs are critical regulators of skeletal muscle function and play important roles
in maintaining muscle function and regulating adaptation to different situations,
including muscle use and disuse, as well as in different disease states. While the role
of some of the classical myomiRs is increasingly appreciated and well understood,
the challenge for the field lies in understanding not only the precise regulation of
different miRNA species but also in dissecting and understanding the precise targets
regulated by each miRNA or combination of miRNAs. Unravelling these molecular
signatures could facilitate targeted and tissue-specific miRNA interventions in dif-
ferent disease states affecting skeletal muscle function and metabolism.

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Tryptophan-Kynurenine Metabolites
in Exercise and Mental Health

Paula Valente-Silva and Jorge Lira Ruas

Abstract  In our efforts to identify molecular mediators of the benefits of exercise

to human health, we have uncovered a biochemical pathway in skeletal muscle that
positively impacts mental health. This mechanism is activated by endurance train-
ing and controlled by the transcriptional coactivator PGC-1α1, which induces tran-
scription of several kynurenine aminotransferase (KAT) genes in muscle. KAT
enzymes catabolize the neuroinflammatory tryptophan metabolite kynurenine,
which can accumulate in the brain and lead to alterations associated with stress-­
induced depression (among other psychiatric diseases). Here, we discuss our find-
ings in the context of what is known about the kynurenine pathway of tryptophan
degradation and how its many metabolites can directly affect the brain. These find-
ings provide a mechanism for how physical exercise can improve mental health and
offers potential therapeutic targets for future antidepressant medications.

Introduction

The many benefits of physical exercise to human health are widely recognized.
Depending on their type, intensity, duration, and frequency, different exercise
modalities can be used to improve diverse physiological parameters (Hawley et al.
2014). These include cardiovascular fitness, strength, energy metabolism, and resis-
tance to fatigue, among others. Importantly, exercise training can be used as a pro-
phylactic or therapeutic intervention for a variety of pathologies ranging from
obesity and diabetes to cancer and mental health disorders (Cooper et  al. 2017;
Hawley 2004). Indeed, maintaining an active lifestyle continues to be the best way
to promote healthy longevity. However, the molecular mechanisms that allow the
human body to adapt to exercise challenges are still poorly understood. This is true

P. Valente-Silva • J. L. Ruas (*)

Department of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology,
Karolinska Institutet, Stockholm, Sweden
e-mail: jorge.ruas@ki.se

© The Author(s) 2017 83

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_7
84 P. Valente-Silva and J. L. Ruas

for individual tissues and organs but even more so for the network of inter-organ
communication events that coordinates whole-body adaptation to the multitude of
stimuli that exercise training entails. Skeletal muscle plays a central role in this
process and has been the most studied tissue in this context. From this work, we
have learned valuable information about key players in the regulation of muscle
function, such as the peroxisome proliferator-activated receptor (PPAR)-γ
coactivator-1α (PGC-1α) proteins (Correia et al. 2015). This still expanding family
of transcriptional coactivators is composed of several splicing variants with differ-
ent biological activities and discrete regulation, important for the many effects of
physical exercise (Martínez-Redondo et al. 2015). By understanding these mecha-
nisms, we have also started to elucidate how myokines (muscle-derived factors with
local and/or distal effects) communicate to the rest of the body the changes in skel-
etal muscle elicited by exercise (Giudice and Taylor 2017). For example, exercise
training can reduce the levels of several mediators of chronic low-grade inflamma-
tion (Handschin and Spiegelman 2008), which increase with sedentary habits. This
kind of sustained, unresolved, low-grade sterile inflammation has been linked to the
etiology of many diseases such as diabetes, cancer, and depression. In this context,
the discovery that trained muscle can actively participate in the catabolism of neu-
rotoxic tryptophan metabolites with known deleterious effects on mental health
(Agudelo et al. 2014) has added another layer of complexity to the many functions
of exercised muscle.

The PGC-1α Family of Transcriptional Coactivators

in Skeletal Muscle

Although our understanding of the mechanisms that regulate skeletal muscle adap-
tation to different exercise challenges remains incomplete, PGC-1α coactivators
have been shown to play important roles in this process. These proteins are expressed
in energy-demanding tissues such as heart, skeletal muscle, adipose tissue, and
brain (Correia et al. 2015). Interestingly, the PGC-1α gene can be transcribed from
alternative promoters and its transcripts can be spliced to generate several PGC-1α
variants with different biological activities (Martínez-Redondo et  al. 2015; Ruas
et  al. 2012). The expression of PGC-1α1 [the founding member of the family
(Puigserver et  al. 1998)] is increased by aerobic exercise and regulates genes
involved in mitochondrial biogenesis, adaptive thermogenesis, lipid and glucose
homeostasis, and fiber-type switching, among others (Correia et al. 2015). For these
reasons, reduced PGC-1α1 expression in different tissues has been linked to obesity,
diabetes, sarcopenia, and neurodegeneration. Conversely, it has been shown that
sustained PGC-1α1 expression in mouse skeletal muscle has several beneficial
effects. PGC-1α4 is induced by resistance exercise training and promotes skeletal
muscle growth and strength. Importantly, transgenic animals with elevated PGC-1α4
levels in skeletal muscle show increased exercise performance and resistance to
Tryptophan-Kynurenine Metabolites in Exercise and Mental Health 85

atrophy and to cancer-induced cachexia (Ruas et  al. 2012). Of the many other
PGC-1α isoforms (Martínez-Redondo et  al. 2015), PGC-1α2 and α3 have been
shown to be involved in alternative splicing of their target genes (Martinez-Redondo
et al. 2016), but their main biological roles in skeletal muscle remain unknown.
In addition to helping us understand local adaptations in skeletal muscle, PGC-1α
coactivators have been used as tools to investigate the distal actions of exercise-­
induced myokines. This research has been greatly helped by the use of mouse
genetic models with tissue-specific gain or loss of PGC-1α function developed to
mimic the physiological and pathophysiological situations associated with altered
PGC-1α expression (Rowe and Arany 2014). From these efforts, PGCs have been
found to regulate the expression of several myokines involved in inflammation
(Handschin and Spiegelman 2008), angiogenesis (VEGF; Arany et  al. 2008;
Chinsomboon et  al. 2009), non-shivering thermogenesis (Fndc5/Irisin, Meteorin-­
like 1, and β-aminoisobutyric acid; Boström et al. 2012; Rao et al. 2014; Roberts
et al. 2014), and muscle mass regulation (Myostatin; Ruas et al. 2012). Similarly,
the observation that muscle-specific PGC-1α1 transgenic mice (MKC-PGC-1α; Lin
et al. 2002) are resistant to developing depressive-like behaviors when exposed to
chronic mild stress resulted in the identification of skeletal muscle as an important
site for kynurenine (Kyn) detoxification (Agudelo et al. 2014). Kyn and some of its
metabolites are known neurotoxic compounds linked to mental health disorders,
such as depression and schizophrenia (Cervenka et al. 2017).

The Kyn Pathway of Tryptophan Degradation

Tryptophan (TRP) is an essential amino acid that cannot be synthesized by the

human body and must be acquired from diet. TRP is taken up through the large
neutral amino acid transporter (LAT) isoforms 1 to 4, which are also responsible for
the uptake of other amino acids such as phenylalanine, tyrosine, and cysteine, and
others. However, with the exception of the blood-brain barrier (BBB), LATs have
sufficient capacity to avoid significant competition between TRP and other amino
acids. A large fraction of plasma TRP circulates bound to albumin and is thus not
available for cellular uptake and usage, since only free TRP is taken up by LATs.
Under normal circumstances, only about 1% of absorbed TRP is used in protein
synthesis, 4–5% is used in the production of the neurotransmitter serotonin and the
hormone melatonin, and about 95% is catabolized by the Kyn pathway of TRP deg-
radation (KP). The KP generates NAD+ as an end-product (mainly in liver and kid-
ney; Houtkooper et  al. 2010) and a collection of intermediary metabolites with
diverse biological activities (Fig. 1), which have been mainly studied in the context
of psychiatric disease.
There are two isozymes that catalyze the first step of TRP degradation: trypto-
phan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). TDO is spe-
cific for TRP and has a more restricted expression pattern (highest in the liver; Yu
et al. 2016), whereas IDO is more ubiquitous and metabolizes any compound with
86 P. Valente-Silva and J. L. Ruas

Protein Tryptophan
IDOs
Tph
TDOs
Kynureninase
AA L-Kyn N-Formyl- Serotonin
L-Kyn
KMO KATs
KATs
XA 3-HK Kyna Melatonin
Kynureninase
3HAO
PA
Quin Toxicity

QPRT

Nicotinate NAPRT1 Nicotinic

ribonucleotide Acid

NAD+
Fig. 1 The kynurenine pathway of tryptophan degradation. Tph tryptophan hydroxylase,
IDO  indoleamine 2,3-dioxygenase, TDO tryptophan 2,3-dioxygenase, L-Kyn L-kynurenine,
AA Anthranilic acid, KMO Kynurenine 3-Monooxygenase, KATs Kynurenine amino-transferases,
3HAO 3-Hydroxyanthranilate 3,4-Dioxygenase, PA Picolinic acid, Quin Quinolinic acid,
QPRT Quinolinate phosphoribosyltransferase, NAD+ Nicotinamide adenine dinucleotide, NAPRT1
Nicotinate phosphoribosyltransferase

an indol ring structure. The expression and activity of TDO are regulated by cues
that include tryptophan, glucocorticoid, and estrogen levels (Yu et al. 2016) and are
inhibited under pro-inflammatory conditions. Under those conditions, liver TDO
ceases to be the main isozyme responsible for TRP metabolism (normally account-
ing for 90%), and the extra-hepatic IDO takes on a more prominent role. This shift
in TRP metabolism causes an increase of Kyn produced by immune cells that
express high levels of IDO in an attempt to control the inflammatory environment.
Indeed, as pro-inflammatory cytokines stimulate IDO activity, rising Kyn levels
activate the aryl hydrocarbon receptor (AhR) in discrete immune populations and
reduce the activity of natural killer cells (NKT), dendritic cells (DC), and T-cells
while allowing for Treg proliferation. The net result of this process is an increase in
immune tolerance. In line with this, some tumor cells express high IDO/TDO levels
as a strategy for escaping the immune system (Platten et al. 2015). Importantly, not
all cells possess the complete enzymatic machinery to fully convert TRP to NAD+.
In general, the metabolites generated by the KP depend on which enzymes are
expressed in each cell type. For example, Kyn aminotransferases (KATs), of which
Tryptophan-Kynurenine Metabolites in Exercise and Mental Health 87

there are four genes in the human genome, are responsible for metabolizing Kyn to
kynurenic acid (Kyna) and 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA)
(Fig. 1). This is particularly important in the brain, as both Kyna or XA have neuro-
protective properties as opposed to several of the downstream metabolites of the KP.

KP Metabolites and Mental Health

The link between the KP and central nervous system (CNS) toxicity has been appre-
ciated for a long time (Lapin 1978). However, it was only in the early 1980s that
quinolinic acid (QA) was shown to be a NMDAR agonist (Stone and Perkins 1981),
thus providing the first mechanism for KP-induced CNS toxicity. The majority of
Kyn reaches the brain from the periphery, as it can easily cross the BBB. However,
Kyn can also be produced locally from TRP by astrocytes and microglial cells
(Guillemin et  al. 2001; Stone and Darlington 2002). Interestingly, although Kyn
accumulation in the CNS is consistently associated with neuroinflammation and
negative outcomes, its precise mechanism of action remains elusive. In fact, activa-
tion of astrocytic AhR (for which Kyn is an agonist) has been shown to be anti-­
inflammatory in the context of multiple sclerosis (Rothhammer et al. 2016). To date,
KP-associated toxicity is mostly understood as the balance between the excitotoxic
actions of QA and the neuroprotective effect of Kyna as a NMDAR and α7-nicotinic
acetylcholine receptor (α7-nAChR) antagonist. Microglial cells can produce QA
whereas astrocytes preferentially generate Kyna (although they can produce QA
from extracellular 3-HK, which increases with neuroinflammation). Interestingly,
elevated Kyna levels have been found in the cerebrospinal fluid of schizophrenic
patients (Linderholm et al. 2012). Conversely, reducing brain Kyna levels in pre-­
clinical studies (using a KAT2 inhibitor) resulted in improved cognitive function
during chemically induced challenges to working and spatial memory tasks (Kozak
et al. 2014). In this situation, the pathological consequences of high Kyna concen-
trations are likely to manifest due to hypoglutamatergic function. 3-HK induces
neuronal apoptosis through free-radical generation (Okuda et al. 1998; Polyzos and
Ketelhuth 2015). Several metabolites of the pathway have also been linked to neu-
rodegenerative diseases such as Alzheimer’s disease, Huntington’s disease,
Parkinson’s disease, among others.

Crosstalk Between Physical Exercise and KP Metabolites

The importance of TRP in exercise has been extensively investigated (Newsholme

and Blomstrand 2006), not only due to its critical role in protein synthesis but also
in efforts to understand the development of central fatigue and the interplay between
diet, exercise, and mood disorders. For example, the increase in circulating free
fatty acids that occurs during endurance exercise displaces TRP from albumin and
88 P. Valente-Silva and J. L. Ruas

increases free TRP levels, resulting in higher TRP uptake and metabolism in differ-
ent tissues, with direct impact on the KP.  Accordingly, an increase in free TRP
plasma levels is followed by an increase in TRP and some of its metabolites in
several brain regions associated with the control of mood and fatigue (Blomstrand
et al. 1989; Newsholme and Blomstrand 2006).
High TRP uptake during exercise could lead to higher NAD+ production, which
is indeed increased in tissues such as the liver (which is able to fully catabolize
TRP). However, the risk of accumulating KP metabolites that can cross the BBB
(e.g., Kyn and 3-HK) could have detrimental effects. Recently, it has been shown
that exercise increases the expression of KAT enzymes in skeletal muscle (Agudelo
et  al. 2014; Schlittler et  al. 2016), promoting the conversion of Kyn into Kyna,
which does not cross the BBB (Agudelo et al. 2014). Peripheral Kyn catabolism
prevents its accumulation in the brain and the associated deleterious effects. This is
particularly relevant in the context of stress-induced depression, which is character-
ized by high Kyn levels. This work has provided a mechanism for how physical
exercise can improve mental health. Exercise training activates muscle KAT expres-
sion and Kyn detoxification by inducing the expression of PGC-1α1 and the
PPARα/δ transcription factors, which offers potential therapeutic targets for future
antidepressant medications. Interestingly, elevated NAD+ levels could further pro-
mote this mechanism, as NAD+ is a positive regulator of Sirtuin1, which in turn
activates PGC-1α1 through lysine deacetylation (Rodgers et al. 2005). Sirtuins have
multiple effects on the regulation of cellular energy metabolism, metabolic enzymes
and oxidative stress responses in mitochondria (Cantó et al. 2015). Therefore, alter-
ations in the KP may ultimately affect skeletal muscle metabolism and function
through multiple concurrent mechanisms.

Future Perspectives and Considerations

TRP is without doubt an important player in many critical functions of a living

organism. In addition to being a building block for protein synthesis, TRP plays a
role in mental health, energy homeostasis, and immune regulation. It is exciting to
see research in these different areas becoming more and more interdisciplinary.
Although we have focused here mainly on post-absorptive TRP metabolism, this
amino acid can be sequestered by gut microbiota and converted to tryptamine
(Williams et al. 2014) or other indole compounds such as indole-3-propionic acid
and indole-3-aldehyde (Rothhammer et al. 2016). Indol compounds serve as impor-
tant signals between bacteria and between bacteria and their host. Indols are also
AhR agonists involved in the local immunomodulation of host tolerance responses
and, distally, in the regulation of CNS inflammation (Hubbard et  al. 2015). It is
therefore critical to consider the effect of the microbiome on TRP metabolism and
host physiology. For example, germ-free (GF) mice show reduced anxiety-like
behavior, and this phenotype is reversed after colonization. This observation sug-
gests that gut-derived molecules can modulate mood and possibly cognition, and it
Tryptophan-Kynurenine Metabolites in Exercise and Mental Health 89

opens interesting challenges in the discovery of novel pathways of gut-brain com-

munication (Heijtz et  al. 2011). Physical exercise also has an impact on gut
­microbiome composition, but it remains to be explored if this directly impacts on
gut TRP metabolism.
The KP is highly conserved evolutionarily, which allows for its study in simpler
organisms to dissect its many functions. For example, Kyna has been shown to be
involved in C. elegans feeding behavior in a NMDAR-dependent mechanism
(Lemieux et al. 2015). It will be interesting to continue exploring the role of KP
metabolites as possible mediators of inter-organ communication, which could inte-
grate nutrition, metabolism, and immune responses, with all the physiological and
pathophysiological implications this could have.

Acknowledgments  The authors wish to acknowledge members of the Ruas lab for critical read-
ing of the manuscript and funding from the Swedish Research Council, the Novo Nordisk
Foundation (Denmark), Karolinska Institutet, The Lars Hierta Memorial Foundation, The Strategic
Research Program (SRP) in Diabetes, and The SRP in Regenerative Medicine at Karolinska
Institutet.

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The Role of FNDC5/Irisin in the Nervous
System and as a Mediator for Beneficial
Effects of Exercise on the Brain

Mohammad Rashedul Islam, Michael F. Young, and Christiane D. Wrann

Abstract  Exercise can improve cognitive function and the outcome of neurode-
generative diseases like Alzheimer’s disease. This effect has been linked to the
increased expression of brain-derived neurotrophic factor (BDNF). However, the
underlying molecular mechanisms driving the elevation of this neurotrophin remain
unknown. Recently, we have reported a PGC-1α-FNDC5/irisin pathway that is acti-
vated by exercise in the hippocampus in mice and induces a neuroprotective gene
program, including Bdnf. This review will focus on FNDC5 and its secreted form
“irisin,” a newly discovered myokine, its role in the nervous system and its thera-
peutic potential. In addition, we will briefly discuss the role of other exercise-­
induced myokines in positive brain effects.

Introduction

Exercise, especially endurance exercise, is known to have beneficial effects on brain

health and cognitive function (Cotman et al. 2007; Mattson 2012; Voss et al. 2013).
This improvement in cognitive function with exercise has been most prominently
observed in the aging population (Colcombe and Kramer 2003). Exercise has also
been reported to ameliorate outcomes in neurological diseases such as depression,
epilepsy, stroke, and Alzheimer’s and Parkinson’s disease (Ahlskog 2011; Arida
et al. 2008; Buchman et al. 2012; Russo-Neustadt et al. 1999; Zhang et al. 2012).
The effects of exercise on the brain are most apparent in the hippocampus and the
dentate gyrus, a part of the brain involved in learning and memory. Specific benefi-
cial effects of exercise in the brain have been reported to include increases in the
size of, and blood flow to, the hippocampus in humans and morphological changes
in dendrites and dendritic spines, increased synapse plasticity and, importantly, de
novo neurogenesis in the dentate gyrus in various mouse models of exercise (Cotman

M. R. Islam • M. F. Young • C. D. Wrann (*)

Massachussetts General Hospital and Harvard Medical School, Charlestown, MA, USA
e-mail: cwrann@mgh.harvard.edu

© The Author(s) 2017 93

B. Spiegelman (ed.), Hormones, Metabolism and the Benefits of Exercise,
Research and Perspectives in Endocrine Interactions,
https://doi.org/10.1007/978-3-319-72790-5_8
94 M. R. Islam et al.

et al. 2007; Mattson 2012). De novo neurogenesis in the adult brain is observed in
only two areas; the dentate gyrus of the hippocampus is one of them. Exercise is one
of the few known stimuli of this de novo neurogenesis (Kobilo et al. 2011).
One important molecular mediator of these beneficial responses in the brain to
exercise is the induction of neurotrophins/growth factors, most notably brain-­
derived neurotrophic factor (BDNF). In animal models, BDNF is induced in various
regions of the brain with exercise, most robustly in the hippocampus (Cotman et al.
2007). BDNF promotes many aspects of brain development, including neuronal cell
survival, differentiation, migration, dendritic arborization, synaptogenesis and plas-
ticity (Greenberg et al. 2009; Park and Poo 2013). In addition, BDNF is essential for
synaptic plasticity, hippocampal function, and learning (Kuipers and Bramham
2006). Highlighting the relevance of BDNF in humans, individuals carrying the
Val66Met mutation in the Bdnf gene exhibit decreased secretion of BDNF, decreased
volume of specific brain regions, deficits in episodic memory function and increased
anxiety and depression (Egan et al. 2003; Hariri et al. 2003). Blocking BDNF sig-
naling with anti-TrkB antibodies attenuates the exercise-induced improvement of
acquisition and retention of a spatial learning task, as well as the exercise-induced
expression of synaptic proteins (Vaynman et al. 2004, 2006). However, the underly-
ing mechanism that induces BDNF in exercise remains incompletely understood.
We recently described a role for the newly discovered “exercise-hormone,”
FNDC5 (Bostrom et  al. 2012), and its secreted form, “irisin,” in the protective
effects of exercise on the brain. Fndc5 expression is induced by exercise in the hip-
pocampus in mice; it can, in turn, activate BDNF and other neuroprotective genes
(Wrann et  al. 2013). Importantly, peripheral delivery of FNDC5 to the liver via
adenoviral vectors, resulting in elevated blood irisin, induces expression of Bdnf and
other neuroprotective genes in the hippocampus. These data indicate that either iri-
sin itself can cross the blood-brain-barrier to induce gene expression changes or
irisin induces a “factor x” that can, which has significant implications for irisin as a
novel therapeutic target. This review will examine previous literature about FNDC5/
irisin as well as its therapeutic potential for treating neurodegenerative disease.

Discovery of FNDC5/Irisin

In 2002, two groups independently cloned a novel gene termed either PeP or, alterna-
tively, Frcp2, that contained a fibronectin type III (FNIII) domain; it is now named
FNDC5 (Ferrer-Martinez et al. 2002; Teufel et al. 2002). Recently, our group identi-
fied FNDC5 as a PGC-1α-dependent myokine that is secreted from muscle during
exercise and induces some major metabolic benefits of exercise (Bostrom et al. 2012).
FNDC5 is a glycosylated type I membrane protein. It contains a N-terminal sig-
nal peptide [amino acid (aa) 1-28], a FNIII domain (aa 33–124), a transmembrane
domain (aa 150–170), and a cytoplasmic tail (aa 171–209) (www.uniporot.org)
(Fig. 1). The secreted form of FNDC5 contains 112 amino acids (aa 29-140), named
irisin, and is generated by proteolytic cleavage and is released into the circulation.
The Role of FNDC5/Irisin in the Nervous System and as a Mediator for Beneficial… 95

Fig. 1  Analysis of Irisin Peptides by Mass Spectrometry. (a) Scheme of the murine FNDC5 pro-
tein structure (top) and murine irisin protein structure (bottom). SP signal peptide, H hydrophobic
domain, C cytoplasmic domain. (b) Murine FNDC5 amino acid sequence with corresponding
domains colored. The irisin sequence is underlined

The protease/sheddase responsible for that cleavage has not yet been identified.
Irisin has been crystallized and its structure has been solved (Schumacher et  al.
2013). Interestingly, the FNIII-like domain shows an unusual confirmation, with a
continuous intersubunit beta-sheet dimer, that has not been previously described for
any other FNIII protein. Subsequent biochemical experiments confirmed the exis-
tence of irisin (bacterial recombinant) as a homodimer.

Irisin in Humans

Irisin is a highly conserved polypeptide across mammals and is, in fact, 100% iden-
tical in mice and humans (Bostrom et al. 2012). Such a high degree of conservation
is often the result of evolutionary pressure to conserve function. Interestingly, the
human FNDC5 has an atypical start of translation, ATA in place of ATG, as com-
pared to mouse Fndc5. While it is now known that a few percent of eukaryotic
mRNAs begin translation with non-ATG start codons (Ingolia et al. 2011; Ivanov
et al. 2011; Peabody 1989) and are often associated with regulation on the transla-
tional level (Chang and Wang 2004; Starck et al. 2012), recent reports (Albrecht
et al. 2015; Raschke et al. 2013) have argued that this ATA codon in human FNDC5
was a “null mutation” or a “myth” and, as a result, human irisin would not be pro-
duced. Furthermore, many reports from other groups measuring irisin in humans by
Western blot or ELISA have suggested results to be artefacts of poor antibody speci-
ficity (Albrecht et al. 2015; Erickson 2013; Raschke et al. 2013), even though an
earlier study had detected irisin circulating in human plasma using mass spectrom-
etry, an unbiased method independent of the quality of existing antibodies
(Jedrychowski et al. 2015; Lee et al. 2014). To identify and quantify irisin in human
plasma, we used targeted mass spectrometry with control peptides enriched with
stable isotopes as internal standards. This precise, state-of-the-art method demon-
strated that human irisin was mainly translated from its non-canonical ATA start
96 M. R. Islam et al.

codon (Jedrychowski et al. 2015). In addition, it showed that, in sedentary individu-

als, irisin circulated at ~3.6 ng/ml and that it was significantly increased in individu-
als undergoing aerobic interval training. This study determined that, at the atomic
level, human irisin exists, and is regulated by certain forms of aerobic exercise.

FNDC5/Irisin in Exercise

FNDC5/irisin was first described as an exercise-induced myokine by Bostrom et al.

in 2012, where they observed upregulation of Fndc5 gene expression in skeletal
muscle and increased serum irisin levels after prolonged endurance exercise in both
mice and humans. Increasing the circulating levels of irisin by overexpression of
FNDC5 from adenoviral vectors in the liver led to an increase in “browning” of the
white inguinal adipose tissue, i.e., the upregulation of mitochondrial gene expres-
sion, especially of Ucp1, and an increased glucose tolerance in mice. Both are
among the major metabolic benefits of endurance exercise. By now, there are around
50 published papers that investigate the role of FNDC5 and/or irisin in exercise,
including both rodent studies and clinical trials in humans. Induction of Fndc5
mRNA in skeletal muscle by endurance exercise has been confirmed in several stud-
ies in both mice (Quinn et  al. 2015; Tiano et  al. 2015; Wrann et  al. 2013) and
humans (Albrecht et al. 2015; Lecker et al. 2012; Norheim et al. 2014) using qPCR
or RNA sequencing. As with all clinical studies, there are a lot of variables to con-
sider, such as retrospective studies vs. interventional trials, age and fitness level of
the subjects and, most importantly, the type of exercise protocol used and time point
of sampling. However, a consensus is building, supported by studies that reported
positive associations between irisin plasma level and exercise, performed early sam-
pling and high intensity training protocols levels (Daskalopoulou et al. 2014; Huh
et al. 2014; Kraemer et al. 2014; Malcolm Eaton et al. 2017; Norheim et al. 2014).
Plasma levels of irisin and BDNF have been shown to be positively correlated with
cognitive function in endurance athletes (Belviranli et al. 2016).
The brief rise in circulating irisin levels after exercise is suggestive of an acute
shedding event of irisin during exercise. There is little to no evidence thus far that
FNDC5 or irisin is upregulated by resistance exercise in mouse or human This is not
unexpected, since endurance exercise activates PGC-1α1, which has been shown to
be the upstream regulator of Fndc5 gene expression, whereas resistance exercise
activates a different isoform of PGC-1α1, PGC-1α4 (Ruas et al. 2012).

FNDC5/Irisin in Neuronal Development

Fndc5 is highly expressed in the brain, including the Purkinje cells of the cerebel-
lum (Dun et al. 2013; Ferrer-Martinez et al. 2002; Teufel et al. 2002). Irisin, the shed
form of FNDC5, was identified in human cerebrospinal fluid by Western blot (Piya
The Role of FNDC5/Irisin in the Nervous System and as a Mediator for Beneficial… 97

et  al. 2014). In addition, immunoreactivity against the extracellular domain of

FNDC5/irisin was detected in human hypothalamic sections, especially paraven-
tricular neurons (Piya et al. 2014). Other tissues with high FNDC5 levels include
skeletal muscle and the heart. Fndc5 gene expression increases during differentia-
tion of rat pheochromocytoma-derived PC12 cells into neuron-like cells (Ostadsharif
et al. 2011). FNDC5 levels are enhanced after the differentiation of human embry-
onic stem cell-derived neural cells into neurons (Ghahrizjani et al. 2015) as well as
during the maturation of primary cortical neurons in culture and during brain devel-
opment in vivo (Wrann et al. 2013).
Knockdown of FNDC5 in neuronal precursors impaired their development into
mature neurons (and astrocytes), suggesting a developmental role for FNDC5  in
neurons (Hashemi et al. 2013). Despite this finding, forced expression of FNDC5
during neuronal precursor formation from mouse embryonic stem cells increased
mature neuronal markers (Map2, b-tubulinIII and Neurocan) and an astrocyte
marker (GFAP) and BDNF. However, overexpression of FNDC5 in undifferentiated
mouse embryonic stem cells did not have these effects, indicating that FNDC5 sup-
ports neural differentiation rather than lineage commitment (Forouzanfar et  al.
2015). Pharmacological doses of recombinant irisin increased cell proliferation in
the mouse H19-7 hippocampal cell line (Moon et  al. 2013). Furthermore, forced
expression of FNDC5 in primary cortical neurons increased cell survival in culture,
whereas knockdown of FNDC5 had the opposite effect (Wrann et al. 2013).

FNDC5/Irisin: Other Effects in the CNS

The group of Dr. Mulholland (University of Michigan) had taken in interest in the
central nervous effects of irisin. In a first study, they injected irisin either into third
ventricle of rats or intravenously measured the effects on blood pressure and cardiac
contractibility (Zhang et al. 2015a). Central administration of irisin activated neu-
rons in the paraventricular nuclei of the hypothalamus, as indicated by increased
c-fos immunoreactivity. Central irisin administration also increased both blood
pressure and cardiac contractibility. In contrast, i.v. injection of irisin reduced blood
pressure in both control and spontaneously hypertensive rats. In a second study,
Zhang et al. showed that central treatment of rats with irisin-Fc led to an increase in
physical activity as measured as total travel distance, ambulatory counts and time,
and vertical counts and time compared to control animals receiving IgG Fc peptide
(Zhang et al. 2015b). In addition, the centrally applied irisin also induced significant
increases in oxygen consumption, carbon dioxide production and heat production,
indicating an increase in metabolic activity- possibly through SNS activation.
Similarly, intra-hypothalamic injection of irisin decreased food intake in rats pos-
sibly by stimulating anorexigenic peptides and inhibiting dopamine, norepinephrine
and orexin-A (Ferrante et al. 2016).
A recent study investigated the neuroprotective potential of irisin treatments in
cerebral ischemia. Irisin improved survival of cultured PC12 neuronal cells in
98 M. R. Islam et al.

o­ xygen glucose deprivation. I.v. injection of recombinant irisin reduced brain infarct
and edema volume and improved the neurological score in MCAO stroke model (Li
et  al. 2017). Systemic irisin administration has also been shown to ameliorate
depressive-like behavior in a chronic unpredictable stress model in rats (Wang and
Pan 2016).

 xercise Induces Hippocampal BDNF Through a PGC-1α/

E
FNDC5 Pathway

In a recent study, we have shown that FNDC5 is also elevated in the hippocampus
of mice undergoing endurance exercise regimen of 30-days free-wheel running.
Neuronal Fndc5 gene expression is regulated by PGC-1α and Pgc1a−/− mice show
reduced Fndc5 expression in the brain. Forced expression of FNDC5  in primary
cortical neurons increases Bdnf expression, whereas RNAi-mediated knockdown of
FNDC5 reduces Bdnf. Importantly, peripheral delivery of FNDC5 to the liver via
adenoviral vectors, resulting in elevated blood irisin, induces expression of Bdnf and
other neuroprotective genes in the hippocampus. Taken together, our findings link
endurance exercise and the important metabolic mediators, PGC-1α and FNDC5,
with BDNF expression in the brain. Interestingly, a recent study investigating the
effects of the flavonoid quercetin hypobaric hypoxia, reported that quercetin admin-
istration to hyperbaric hypoxic rats increased expression of PGC-1α, FNDC5, and
BDNF in the hippocampus (Liu et al. 2015). While more research will be required
to determine whether the FNDC5/irisin protein can improve cognitive function in
animals, this study suggests that a hormone administered peripherally could induce
some of the effects of endurance exercise on the brain (Fig. 2).

Other Circulating Factors from the Muscle

While FNDC5/irisin is a very interesting molecule that holds therapeutic promise,

this is not to say that we think that FNDC5/irisin captures all the benefits of exercise
on the brain or that is the only important secreted molecule from muscle in exercise.
In fact, other such molecules have been described, including BDNF, IGF-1, and
VEGF, kynurenic acid, cathepsin B, as well as a variety of cytokines and chemo-
kines, to name a few (Agudelo et al. 2014; Moon et al. 2016; Phillips et al. 2014;
Voss et al. 2013). We expect that in the future, additional molecules will be discov-
ered and some may reach their full therapeutic potential.
The Role of FNDC5/Irisin in the Nervous System and as a Mediator for Beneficial… 99

Neuronal
Exercise
Development

CA1
Maturation
FNDC5
BDNF CA3 Dentate Gyrus
Knockdown
Voluntary
Free Wheel Astrocytes Neurons
Exercise

FNDC5 knockdown in neuronal

Exercise induces hippocampal BDNF
precursors leads to decreased
through a PGC-1alpha/FNDC5 pathway
(Wrann et al., 2013). FNDC5/ maturation
(Hashemi et al., 2013).
Irisin

Induction of FNDC5 mRNA Pharmacoligical doses of

is found in skeletal muscle recombinant irisin lead to
after endurance exercise increased cell proliferation
(Quinn et al., 2015; Tiano et al., 2015; in mouse HC cell line
Wrann et al., 2013) (Moon et al., 2013).

Force expression of FNDC5

Increased levels of circulating
in primary culture neurons
irisin post-exercise
increased cell survivial
(Bostrom et al., 2012). (Wrann et al., 2013).

FNDC5 seems to support cell

differentiation rather than
Central cell lineage commitment
Nervous (Forouzanfar et al., 2015).
System
FNDC5 levels are enhanced
after human embryonic
stem cell differentiation
Central administration of irisin into neurons
leads to neuronal activiation in (Ghahrizjani et al., 2015)
paraventricular nuclei and increases
both blood pressure and cardiac
contractibility
(Zhang et al., 2015)

Fig. 2  Summary of FNDC5/irisin effects on the brain

Acknowledgements  This work was supported by the NIH (NS087096). I also thank Dr. Mark
Jedrychowski for support with the art work.

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