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Redox Signaling in Widespread Health Benefits of Exercise

Article in Antioxidants and Redox Signaling · March 2020

DOI: 10.1089/ars.2019.7949

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 ANTIOXIDANTS & REDOX SIGNALING
Volume 00, Number 00, 2020
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2019.7949

Redox Signaling in Widespread Health Benefits of Exercise

Ruy A. Louzada,1,2 Jessica Bouviere,1 Leonardo P. Matta,1 Joao Pedro Werneck-de-Castro,3

Corinne Dupuy,2 Denise P. Carvalho,1 and Rodrigo S. Fortunato1
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Abstract

Significance: Exercise-induced reactive oxygen species (ROS) production activates multiple intracellular
signaling pathways through genomic and nongenomic mechanisms that are responsible for the beneficial effects
of exercise in muscle. Beyond the positive effect of exercise on skeletal muscle cells, other tissues such as white
and brown adipose, liver, central nervous system, endothelial, heart, and endocrine organ tissues are also
responsive to exercise.
Recent Advances: Crosstalk between different cells is essential to achieve homeostasis and to promote the
benefits of exercise through paracrine or endocrine signaling. This crosstalk can be mediated by different
effectors that include the secretion of metabolites of muscle contraction, myokines, and exosomes. During the
past 20 years, it has been demonstrated that contracting muscle cells produce and secrete different classes of
myokines, which functionally link muscle with nearly all other cell types.
Critical Issues: The redox signaling behind this exercise-induced crosstalk is now being decoded. Many of
these widespread beneficial effects of exercise require not only a complex ROS-dependent intramuscular
signaling cascade but simultaneously, an integrated network with many remote tissues.
Future Directions: Strong evidence suggests that the powerful beneficial effect of regular physical activity for
preventing (or treating) a large range of disorders might also rely on ROS-mediated signaling. Within a
contracting muscle, ROS signaling may control exosomes and myokines secretion. In remote tissues, exercise
generates regular and synchronized ROS waves, creating a transient pro-oxidative environment in many cells.
These new concepts integrate exercise, ROS-mediated signaling, and the widespread health benefits of exercise.
Antioxid. Redox Signal. 00, 000–000.

Keywords: ROS, exercise, myokines, redox signaling

Introduction physical exercise (64, 133). A more detailed description of

how physical exercise increases skeletal muscle ROS gen-

R eactive oxygen species (ROS), such as superoxide

(O2-), hydroxyl (OH), and the nonfree radical species,
H2O2, are small radical or nonradical molecules derived from
molecular oxygen (O2) (142). Superoxide and nitric oxide
seem to be the primary free radicals generated by different
sources in contracting skeletal muscle during and immedi-
ately after exercise (84, 129, 130). ROS have a critical role as
signaling molecules that are necessary for skeletal muscle
force development and to achieve the adaptive responses to
eration and the role of ROS-mediated signaling for muscular
exercise adaptations has been extensively reviewed by others
(63, 84, 129, 130).
In 1961, the term ‘‘exercise factors’’ was used to define a
group of factors delivered by contracting muscles that were
able to lower glycemia (62). Nowadays, metabolites, lipid
peroxidation products, extracellular ROS, myokines, and
exosomes are the main suggested mechanisms through which
muscle cells communicate with remote cells. These factors

1
Institut of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
2
Université Paris-Sud, Orsay, UMR 8200 CNRS and Institut Gustave Roussy, Villejuif, France.
3
Division of Endocrinology, Diabetes and Metabolism, Miller School of Medicine, University of Miami, Miami, Florida, USA.

1
 2 LOUZADA ET AL.

that are modulated by exercise, in a chronic perspective, are

directly related to the benefits of physical exercise under
different physiological and pathophysiological conditions. In
this review, we will focus on the effects of physical exercise
beyond skeletal muscle, providing an overview about the
communication between contracting muscles and remote
tissues through ROS-mediated mechanisms.

Hypothetical Model of ROS Waves

The ROS waves hypothesis is based on the ability of
physical exercise to increase ROS production in contracting
muscles and its later spread to remote tissues. A hypothetical
model is proposed in Figure 1 to summarize the probable
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mechanisms through which contracting skeletal muscle

communicates with remote tissues, leading to changes in
redox-mediated signaling pathways that operate the wide-
spread health beneficial effects of exercise. A first phase of
ROS waves after exercise occurs in contracting muscle and
promotes multiple post-translational modifications of ami-
noacids in many kinases and phosphatases (e.g., AMPK,
CaMK, PTEN, PPIA, etc.) that are crucial for the local
stimulus of muscular glucose uptake, mitochondrial biogen-
esis, and antioxidant capacity. Further, skeletal muscles also
produce ROS in the extracellular milieu that leads to macro-
molecules oxidation, such as lipid peroxidation. Interestingly,
lipid peroxidation products can act as signaling molecules.
Myokines and exosomes might be released from skeletal
muscle in response to intracellular ROS stimulated by phys-
ical exercise. Once in blood circulation, these mediators
change the function of remote tissues during and after exercise
periods. Further, the second phase of ROS waves might
stimulate many similar redox-sensible signaling pathways in
noncontracting tissues that promote widespread responses to
physical exercise (Fig. 1B).
In 1978, Dillard et al. found that expired pentane, an index
of lipid peroxidation, increased during aerobic physical ex-
ercise (42). One year later, Brady et al. demonstrated that
lipid peroxidation levels were higher in the skeletal muscle
and liver of rats immediately after swimming (22). Probably,
this was the first indirect demonstration of the ability of
physical exercise to increase ROS generation, but the tissue
involved in their production was only revealed 3 years later.
Davies et al. showed by using electron paramagnetic resonance
that skeletal muscle and liver ROS production are increased
immediately after exhaustive exercise (36). Nowadays, it is
well known that skeletal muscle contractile activity leads to
ROS generation, which is related to biomolecule oxidation, not
only in skeletal muscle but also in other tissues (114, 130).
Electrical stimulation of the hind limb muscle in adult mice
was found to increase malonaldehyde levels in serum and liver
and decrease liver glutathione and protein thiol content (34),
suggesting that the ROS generated by skeletal muscle could
oxidize extracellular molecules also in noncontracting tissues,
such as the liver. In humans, the venous concentration of ox-
idized glutathione was found to increase after physical exercise
in an intensity-dependent manner (35, 114).
The redox communication between contracting muscle
and remote tissues can be mediated by several mechanisms:
(i) lactate conversion to pyruvate in almost every cell, which
increases NADH levels; (ii) purines released by contracting
muscle that serve as substrates for xanthine oxidase; (iii)
FIG. 1. Exercise-induced ROS waves illustration. (A)
Contracting muscle creates an oxidative environment through
consecutive ROS spikes. The first ROS wave is produced
mainly by skeletal muscle Nox2, induces activation of ROS-
mediated signaling pathways, and promotes correct folding of
newly born proteins. Skeletal muscle releases signals to re-
mote tissues in many ways during muscle contraction. For
example, metabolites released by skeletal muscle can be ta-
ken up and metabolized in remote tissues, an event that can
stimulate ROS production. Moreover, myokines can also
control the production of ROS in remote tissues. Products of
lipid peroxidation (HODE and HNE) produced during skel-
etal muscle contraction are capable of binding to nuclear
receptors in remote cells in which these compounds can then
regulate gene expression. Finally, exosomes released by the
muscles undergoing contraction might stimulate signaling
pathways in remote tissues. ROS production in remote tissues
after contraction is proposed as a second ROS wave. (B) First
and second ROS waves induced by physical exercise in a
temporal perspective. During skeletal muscle contraction, the
first ROS wave within skeletal muscle occurs during exercise
and can spread to remote tissues. At the end of exercise,
contracting muscle causes a decrease in ROS production,
whereas remote tissues present the second ROS wave, leading
to activation of ROS-mediated signaling pathways. 13-
HODE, 13-hydroxyoctadecadienoic acid; 4-HNE, 4-hydro-
xynonenal; Nox2, NADPH oxidase 2; ROS, reactive oxygen
species. Color images are available online.
myokines; (iv) ROS gradient reaching adjacent cells; (v) lipid
peroxidation products; and (vi) exosomes secretion. The
oxidative environment induced in remote tissues by physical
exercise is probably related to protein modification in their
cysteine residues, which cause structural and functional
 REDOX SIGNALING IN WIDESPREAD EFFECTS OF EXERCISE
modifications (Fig. 1) and increased oxidation markers that
are detected after exercise in noncontracting tissues. More-
over, several myokines released during physical exercise
control the expression of genes in an ROS-dependent man-
ner, as well as the activity of transcription factors, such as
peroxisome proliferator-activated receptor gamma coacti-
vator 1-alpha (PGC-1a), nuclear factor erythroid 2-related
factor 2 (Nrf2), and nuclear factor-kappa B (33, 90, 123, 142).
Adaptations and Benefits of Redox Signaling
Due to Exercise
The expression of PGC-1a during physical exercise is
dependent on a tightly, fine-tuned redox-mediated signal
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(87), and boosting PGC-1a has been related to protection
against several pathological conditions, such as sarcopenia,
insulin resistance, and cancer-induced cachexia (68). PGC-
1a is responsible for many of the beneficial effects of
exercise in muscle due to its ability to orchestrate the tran-
scription of both nuclear and mitochondrial DNA, promoting
a balanced expression of mitochondrial proteins and anti-
oxidant defenses against exacerbated ROS production. Im-
portantly, an accumulation of PGC-1a after each bout of
exercise is indispensable to achieve these beneficial effects
(47, 53, 70).
Many of the intracellular actors involved in obtaining the
beneficial effects of exercise contain redox sensible regions
that are directly affected by ROS. The crucial requirement of
optimal ROS signaling is clearly observed by the effect of
antioxidants in hampering many important adaptations that
are related to endurance training, such as an increase in ex-
ercise capacity (21, 64), mitochondrial biogenesis (153),
muscular glucose uptake, improvement in insulin sensitivity
(133), and the increase of lean mass after resistance training
(17).
Several chronic exercise adaptations are dependent on
ROS produced by skeletal muscle in response to an acute
exercise session. Transient increases in ROS induced through
bouts of acute exercise stimulate Nrf2 activation. Nrf2 is the
master regulator of antioxidant defenses, a transcription
factor that regulates the expression of more than 200 cyto-
protective genes (44). Regular aerobic exercise in rodent
models has consistently been shown to activate Nrf2 sig-
naling in multiple tissues, including skeletal muscle (111),
kidney (122), brain (162), liver (135), and myocardium (145),
which leads to the upregulation of endogenous antioxidant
defenses and an overall greater ability to counteract the
damaging effects of oxidative stress.
Regarding protein quality control, exercise might be im-
portant to the correct folding of proteins, due to the creation
of an oxidative redox potential needed to oxidize the free
sulfhydryl groups of cysteine leading to disulfide bonds for-
mation that stabilize the protein conformation (Fig. 1) (166).
Antioxidant administration before exercise (64, 133) pre-
vents some of the most important exercise responses, prob-
ably by maintaining the intracellular environment in an
inappropriate reduced state. This reduced state may impair
the correct folding of born proteins (166). Loss of protein
folding regulation leads to an accumulation of unfolded or
misfolded proteins inside the endoplasmic reticulum (ER)
lumen and drives ER stress (ERS), which accentuates the
progression of many diseases, including Alzheimer’s. Unlike
3
almost all other cellular locations that have reducing redox
potentials, the typical ER has the oxidative redox potential
that is necessary to form disulfide bonds. It has been reported
that the ER of insulin-resistant rodents contains much higher
proportions of unfolded polypeptides and fewer disulphide
bonds than normal endoplasmic reticula, which is associated
with the loss of protein folding regulation (116).
Interestingly, exercise reduces negative outcomes in Alz-
heimer’s disease, including the accumulation of misfolded
proteins (159). The unfolded protein response (UPR) is in-
duced within contracting muscle cells and plays an important
role in cell protection from stress, mainly through an essential
chaperoning system (50). Exercise seems to improve many
ERS-related pathologies, such as diabetes mellitus, neurode-
generative diseases, sarcopenia, or cardiovascular alterations
(81, 91, 169) and PGC-1a, the redox sensible master gene
involved in the beneficial effects of exercise, interacts with
ATF6a to regulate the UPR in muscle (169). Many proteins
included in the axis that regulates UPR activation (e.g.,
PERK, eIF2a, ATF4, CHOP, IRE1, XBP1, BiP, and ATF-6)
are regulated in both muscle and remote tissues after ex-
ercise training. For example, exercise training upregulates
ER chaperones, such as binding immunoglobulin protein
(BiP) in mouse muscle (39, 110), liver (31), and brain (89,
96). In summary, both acute and chronic exercise seems to
activate UPR signaling to alleviate ERS-related patholo-
gies (50).
A tight regulation of UPR is also crucial to the correct
folding of tumor suppressor proteins (79, 166). An exercise-
mediated control of tumor suppressor proteins has been
proposed to be one plausible mechanism for the lower inci-
dence of cancer in regular physically active individuals (113,
166). Indeed, a tumor suppressor protein might be delivered
from contracting muscle or synthetized directly in different
tissues by ROS-mediated signaling that is induced by phys-
ical exercise (Fig. 1A). Another possible mechanism is re-
lated to decreased insulin resistance in trained individuals,
since insulin resistance seem to be positively related to tumor
development (3). Once again, notably, such hypotheses fit
well with the association of being actively healthy and the
lower incidence of cancer (83).
Lipid Peroxidation as a Mediator of Crosstalk
Between Different Tissues
Over the past three decades, an extensive body of literature
has shown the role of lipid peroxidation, not only in the
pathophysiology of diseases but also in cell biology and
human health (9). Lipid peroxidation is a process in which
oxidants attack lipids containing carbon–carbon double
bond(s) and involve hydrogen abstraction from a carbon,
with oxygen insertion resulting in lipid peroxyl radicals and
hydroperoxides (27). Increased ROS production can lead to
membrane lipid peroxidation and different cell fate, de-
pending on the cellular metabolic state and repair ability (9).
Products of lipid peroxidation (at low rates) can mediate
cell to cell communication during exercise. NADPH oxidase
2 (NOX2) is expressed at the sarcolemma of skeletal muscle,
and it seems to be the main source of ROS during contractile
activities that produce superoxide in the extracellular me-
dium (74, 77, 138). It is believed that superoxide is rapidly
converted into H2O2, which is more stable and membrane
 4 LOUZADA ET AL.
permeable, acting as a signaling molecule. However, super-
oxide production near the sarcolema might increase the
oxidation of extracellular lipids/lipoproteins, converting
linonelic acid into 13-hydroxyoctadecadienoic acid (13-
HODE) and cholesterol into oxysterol, which is able to
activate transcription factors such as peroxisome proliferator-
activated receptor gamma and liver X receptor alpha,
respectively (41). Apart from this, 4-Hydroxynonenal (4-HNE)
is another product of lipid peroxidation that can activate
transcription factors. It was demonstrated that 4-HNE acti-
vates Nrf2, increasing mitochondriogenesis and antioxidant
defense (95). Interestingly, Nrf2 is activated after physical
exercise in several tissues, including the brain, kidney, and
testis (44). Once products of lipid peroxidation are increased
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in the blood after physical exercise (66), it is possible that
they can communicate with skeletal muscle and with other
distant cells in the body (Fig. 2).
Metabolites Acting as Communicating Molecules
Through ROS-Dependent Mechanisms
A growing body of evidence suggests that different me-
tabolites produced during muscle contraction are secreted
during physical exercise. Interestingly, these metabolic
products can act like chemical mediators between cells (20).
Lactate is a very well-known metabolite produced by glucose
oxidation during muscle contraction that has been suggested
as a candidate molecule to communicate muscle with remote
cells. Formerly, it was believed that lactate was a waste
product of glycolysis, even referred to be the cause of fa-
tiguing muscle during physical exercise (24). The role of
lactate has changed from a possibly bad intermediate mole-
cule in muscle physiology to an important messenger mole-
cule during physical exercise (1). Nowadays, many
physiological functions are attributed to lactate, such as
slowing muscular fatigue (117), increasing force production
(92), and serving as a high-energy rich carbon skeleton for
many crucial biochemical processes within cells (24, 140).
The biochemical aspect of lactate production and its
physiological role were extensively reviewed by Brooks (24).
In brief, there are three main proposed functions for lactate,
as follows: (i) as an energy source, (ii) a major gluconeogenic
precursor after exercise, mainly due to a shuttle between the
skeletal muscle and the liver (the Cori cycle), and (iii) a
‘‘lactormone,’’ able to mediate autocrine, paracrine, and
endocrine communication (24). New information regarding a
shuttle between contracting muscle and microbiota has just
become available, which suggests that intestinal microbiota
are able to metabolize lactate and, thus, influence the exercise
capacity (143). Besides, a potential mechanism showing that
lactate might be able to stimulate ROS-mediated signaling
has also been proposed (Fig. 3).
FIG. 2. Lipid peroxidation
as a mediator of skeletal
muscle-remote tissue cross-
talk. NADPH oxidase is pro-
posed as the main source of
ROS during contractile activity
and due to its transmembrane
location, ROS production is
very close to the phospholipids
of the membrane. Circulating
lipids and membrane phos-
pholipids can be attacked by
extracellular-produced ROS to
form lipid peroxidation inter-
mediates that can interact with
nuclear receptors in remote
tissues and modulate gene ex-
pression. Color images are
available online.
 REDOX SIGNALING IN WIDESPREAD EFFECTS OF EXERCISE 5
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FIG. 3. Crosstalk between muscle and other tissues. Skeletal muscle contraction increases glucose oxidation rates in an
exercise intensity-dependent manner and stimulates the conversion of pyruvate into lactate via LDH. Lactate is mainly
produced by fast-twitch muscles during exercise and is released in muscles undergoing contraction to the blood circulation
through specific transporters (such as MCT). Once in circulation, lactate can be taken up by many tissues, such as liver,
WAT, brain, heart, and kidney. In the liver, lactate is converted into pyruvate by PDH, causes an increase in the levels of
NADH+, and stimulates several intracellular metabolic pathways. Robust ATP hydrolysis produces ADP, which is con-
verted into ATP and AMP by AK. AMP deamination yields hypoxanthine, which is released into the circulation. Xanthine
oxidase converts hypoxanthine into urate and also produces superoxide, leading to redox-mediated signaling in the liver.
Urate is a potent antioxidant molecule that is released into the circulation. The proposed crosstalk between muscle and liver
involves spatially and temporally synchronized ROS production and ROS scavenging to ensure that correct ROS-mediated
signaling occurs within the whole body during and after exercise. In the left panel, intestinal NADH-dependent ROS
production is proposed as a mechanism that may use NADH derived from lactate oxidation by NAD(P)H oxidase to produce
ROS in a microbiota-dependent way. ADP, adenosine diphosphate; AK, adenylate kinase; AMP, adenosine monophosphate;
ATP, adenosine triphosphate; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; PDH, pyruvate dehydro-
genase; WAT, white adipose tissue. Color images are available online.

Intracellular lactate levels increase during physical ex-
ercise in an intensity dependent way. Lactate can cross the
sarcolemma and reach blood circulation through mono-
carboxylate transporters (MCTs). Serum lactate can be
uptaken by a wide range of cells such as contracting skel-
etal muscles themselves (16), heart (59), brain (132), liver
(48), kidney (112), astrocytes, or neurons (126). It is be-
lieved that slow-twitch skeletal muscle fibers and liver are
the most important tissues for the removal of lactate from
blood (Fig. 3) (60), and the expression of MCTs is crucial
for the uptake of lactate from circulation. Moreover,
functional mitochondria are fundamental for lactate to be
oxidized (12, 24).
As proposed by Hashimoto and Brooks, lactate may act as
a communicating mediator called ‘‘lactormone’’ that couples
contracting muscle to other tissues (69). Supporting this hy-
pothesis, intraperitoneal l-lactate administration was found
to increase mitochondrial content in diverse mouse tissues
such as liver and brain (46), as well as PGC-1a and un-
coupling protein 3 expression in gastrocnemius (90). Because
these events are also observed after physical exercise, the
transitory increase in blood lactate during physical exercise
can contribute to the widespread health adaptations that occur
in remote tissues. Recently, it was demonstrated that lactate
was able to upregulate transforming growth factor beta2
(TGF-b2) in white adipose tissue. Further, TGF-b2 was se-
creted by adipose tissue in response to physical exercise,
which is related to the improvement of glucose tolerance.
Interestingly, the administration of a lactate-lowering agent
dichloroacetate during exercise training decreased circulat-
ing TGF-b2 levels and reduced exercise-stimulated im-
provements in glucose tolerance (154).
A hypothetical model is proposed in Figure 1, which shows
the probable redox-mediated mechanisms through which
contracting skeletal muscle communicates with remote tis-
sues. ROS might be involved in lactate action in different
tissues. Indeed, lactate increases the expression of genes in-
volved in lactate transport and mitochondrial oxidation in an
ROS-dependent manner (69), although the source of ROS has
not been defined. Recently, it has been shown that lactate-
mediated increase in PGC1-a expression was blunted by
N-acetyl cysteine pretreatment in skeletal muscle (90, 115).
Figure 3 illustrates the hypothetical sources of ROS that can
be activated by lactate: mitochondria, xanthine oxidase, and
NAD(P)H oxidase. During lactate oxidation, NADH is gen-
erated, increasing mitochondrial oxidative phosphorylation,
 6 LOUZADA ET AL.

which can increase ROS production. Another potential signaling can lead to impaired function in many tissues and
mechanism may be the use of NADH derived from lactate blunt the widespread health benefits of exercise.
oxidation by NAD(P)H oxidase to produce ROS. This hy-
pothesis is supported by experiments in Drosophila in which H2O2 Gradient as a Chemoattracting Agent
lactate stimulates intestinal NADH-dependent ROS produc- Supporting Skeletal Muscle Regeneration
tion (82). A dysbiosis marked by overgrowth of Lactobacillus
In skeletal muscle, cooperation between immune and
plantarum in the fly gut increases lactate production and
muscular cells is observed during muscular regeneration. The
promotes over-proliferation of intestinal stem cells. Using
recruitment of neutrophils and monocytes from blood cir-
this model, the authors proposed that lactate is a key element
culation is mediated by chemoattracting molecules that are
for communication between microbes and intestinal cells,
secreted by many cells (e.g., endothelial cells, satellites cells,
and that NADH produced by oxidation of lactate derived
and immune-resident cells) at the lesion site. Multiple factors
from bacteria could be a substrate for NAD(P)H oxidase to
such as C-C motif ligand 2/monocyte chemoattractant
increase ROS production (82). In this context, it would be
protein-1 (MCP1) and tumor necrosis factor alpha are known
interesting to test whether the same process occurs after ex-
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to attract monocytes and to differentiate them into macro-

ercise.
phages, respectively (128), orchestrating the process of
Lactate also seems to exert a free radical scavenger func-
myogenesis (32, 94, 137). H2O2 gradient also serves as a
tion, able to act as an antioxidant in some specific conditions
chemoattracting agent. In the zebrafish model, an H2O2
as heart ischemia, since the perfusion with a lactate solution
gradient generated by the DUOX enzyme (after tissue injury)
protects the tissue from oxidative damage (65). These newly
recruits leucocytes to the site of the lesion to promote wound
proposed functions of lactate, acting as a free radical scav-
healing (118). Moreover, H2O2 production seems to be cru-
enger and as a signaling molecule, are illustrated in Figure 3.
cial to promote progenitor cell proliferation during acute
A purine cycle also intermediates the crosstalk between
kidney lesions (33). In these two examples, the inhibition of
contracting muscle and liver. Mostly in the liver, inosine and
H2O2 generation abolished the regenerative process. In
hypoxanthine derived from the muscle can be converted into
mammals, the role of DUOX1 in the recruitment of immune
uric acid by xanthine oxidase (71). This cycle might be a
cells was demonstrated in lung inflammatory response, which
potential mechanism by which exercise promotes (paradox-
was found to be crucial to the regenerative process (30, 67).
ically) a pro-oxidant effect due to xanthine oxidase in the
Thus, one can speculate that exercise-induced H2O2 gener-
liver, and an increase of urate, a powerful antioxidant in
ation in skeletal muscle cells could create a gradient to attract
circulation (114). In fact, increased plasma total antioxidant
supportive cells to participate in exercise responses.
capacity has been observed after high-intensity exercise (71).
After eccentric exercise, there is a peak in pro-
The circulating urate can be taken up by different cells and,
inflammatory CD68+ macrophages infiltration at 48 h (100,
after a recovery period after exercise, converted into allantoin
102) that switches to anti-inflammatory CD163+ macro-
(73). During this process, there is a scavenger of the hydroxyl
phages after 72 h (57, 131). Given that ROS are produced and
radical, hydroperoxide, and lipid peroxidation (37). Inter-
released into the blood from skeletal muscle (11, 34, 84), one
estingly, urate is taken up preferentially by the contracting
can hypothesize that these molecules participate in the re-
muscle, and not by the resting ones (72). Although the
cruitment of immune cells to support cellular regeneration
mechanism is not well understood, urate might be useful for
after damaging exercise. In fact, DUOX1 is expressed in
protecting the contracting muscle from oxidative damage
muscular progenitor cells and its expression declines during
once its exogenous administration reduces exercise-induced
myogenesis (139), suggesting that DUOX1 could be a source
oxidative stress (165).
of ROS from satellite muscular progenitor cells to commu-
Based on this intimate co-operation between muscle and
nicate to other cells through an H2O2 gradient, as observed
liver, we propose synchronized and tightly regulated ROS-
during zebrafish and mammalian lung and kidney injury.
mediated signaling in remote tissues that might be dependent
on the products of muscle contraction and liver processing.
Myokines and Its ROS-Dependent Regulation:
This crosstalk is a well-organized network that synchronizes
Communicating the Contracting Muscle
contracting muscle and other remote tissues to ensure optimal
to the Whole Body
redox-mediated signaling during exercise and recovery (as
illustrated in the hypothetical schema in Fig. 1). For example, After contractile activity, skeletal muscle cells increase the
lactate is produced and released into blood circulation, production and release of several molecules such as cytokines
reaching multiple different tissues, then altering the metab- or other small proteins (*5–20 kDa) and proteoglycan pep-
olism, and finally promoting an ROS-mediated signaling. tides that show autocrine, paracrine, and/or endocrine effects.
Simultaneously, intense ATP hydrolyses, mostly found dur- Several well-known cytokines (e.g., IL-6, IL-8, IL-10, IL-15,
ing intense exercise, promote the release of inositol and etc.) have been shown to be secreted by skeletal muscle
hypoxanthine, which mostly reaches its peak after exercise. during physical exercise and, as a result, they are referred to
Once in the liver, urate is produced from hypoxanthine and as myokines (123). Myokines are involved in exercise-
released into circulating blood, delivering a potent antioxi- associated metabolic changes and not only in muscle but also
dant and probably counteracting the peaks of ROS produc- in other tissues such as heart, adipose tissue, liver, pancreas
tion. This hypothetical possibility elaborates the fine-tuning bone, and brain. Myokines play important roles in metabolic
process required for the exchange of purines and lactates that changes that are induced by physical exercise. They also
are derived from contracting muscle and is dictated by the participate in tissue regeneration and repair, immunomodula-
intensity of exercise. When this fascinating and natural tion. and cell differentiation (124). The interconnections be-
communication is perturbed, a disaccord in redox-mediated tween contracting muscle and practically all cells are complex
 REDOX SIGNALING IN WIDESPREAD EFFECTS OF EXERCISE
and not a completely deciphered system. Currently, several
myokines have been described to be stimulated by physical
exercise, including IL-6 (121), Irisin (19), IL-15 (146), FGF21
(156), LIF (23), and MCP1 (28). Interestingly, the PGC-1a and
Akt/mTOR pathways seem to be involved in myokine secre-
tion, and both of them are redox-sensitive intracellular path-
ways (Fig. 4). The most well-studied myokines are IL-6 and
Irisin (121).
Skeletal muscle IL-6 secretion increases after a single bout
of physical exercise (124), which induces liver glucose out-
put (52). IL-6 secretion during physical exercise is not related
to tissue damage or pro-inflammatory processes (124, 142,
150), and it seems to be regulated by intracellular calcium
levels (167). It was demonstrated that the administration of a
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cocktail of antioxidants containing Vitamins A, C, and E
(161) or Vitamin C and E (54) before a single bout of physical
exercise attenuated IL-6 secretion in humans. L-NAME, a
nitric oxide synthase (NOS) inhibitor, also attenuated the
increase in IL-6 mRNA levels in response to exercise (151).
In addition, it has been suggested that NOX enzymes are
involved in IL-6 secretion by skeletal muscle during physical
exercise, since apocynin, an NOX inhibitor, attenuated this
phenomenon, which also occurred in a gp91phox knockout
animal (75).
IL-6, secreted by contracting skeletal muscle during re-
sistance training, seems to activate satellite cells through
the activation of STAT3, which is crucial for hypertrophy
(144). It has not been investigated whether this action is
dependent on ROS-mediated signaling; however, ROS can
stimulate STAT3 via a modification on its Tyr 7055 and
Cys 253 (147). Further, IL-6 stimulates ROS production in
plenty of different cell types (163), so it is possible that
ROS can act as an intermediate in the effect of IL-6 in
remote tissues during exercise. For instance, increased in-
sulin sensitivity and fatty acid oxidation that is stimulated
by IL-6 (26) might, in part, be dependent on the activation
of AMP-activated protein kinase (AMPK) by ROS (157).
Also, the stimulus of muscle glucose uptake after exercise
might be through a similar mechanism (61). The interplay
between IL-6 and AMPK has also been confirmed in dif-
ferent tissues (88).
Irisin was first described as a myokine with the ability to
induce a browning phenotype in white adipose tissue, raising
basal metabolic rates (Fig. 5) (19). Several studies showed
that irisin promotes widespread effects in remote tissues,
FIG. 4. Myokines and ROS dependence. The most studied myokines are illustrated and their ROS dependence is
highlighted. IL-6, IL-8, and IL-15 are regulated by ROS. The control of several myokines relies on the activation of human
PGC-1a and AKT/mTOR signaling, but their dependence on redox signaling has not yet been completely investigated.
AKT/mTOR, protein kinase B/mechanistic target of rapamycin; IL, interleukin; PGC-1a, peroxisome proliferator-activated
receptor gamma coactivator 1-alpha. Color images are available online.
7
preventing oxidative stress and tissue damage. Nrf2 activates
a well-known antioxidant pathway that protects against var-
ious pathological conditions (43, 97, 99), and its activation
seems to correlate with the antifibrotic and antioxidative ef-
fects of irisin (33, 45, 171). For instance, irisin treatment
protects hepatocytes in vitro from overproduction of ROS
that is induced by H2O2 (13). Moreover, irisin mediates part
of the protective effect of dexmedetomidine in livers under
intestinal ischemia/reperfusion damages via decreasing in-
flammasome markers and ROS production (51). In addition,
irisin is known to decrease several pro-inflammatory cyto-
kines and increase antioxidant defenses (e.g., SOD and GPX)
in hepatocytes through Nrf2 upregulation (45). In endothelial
cells, irisin stimulates angiogenesis via AKT/mTOR/S6K1/
Nrf2 signaling, which reduces the higher ROS content in-
duced by oxidized LDL (Fig. 5) (171).
A major role of oxidative stress in fibrosis development is
caused by over-activated Smad-3 signaling (18) that leads to
exacerbated ROS production (14). Mechanistically, irisin-
induced Nrf2 activation in cardiac cells suppresses the ROS
generation that is induced by angiotensin II, which is triggered
by TGF-b1 in fibroblasts (33). Moreover, irisin was found to
prevent heart remodeling (98) and reduce blood pressure of
spontaneously hypertensive rats through endothelial cells
oxidative stress reduction via AMPK and NO signaling (56).
In a type 2 diabetes mice model, irisin ameliorated endothelial
function by reducing the overproduction of superoxide and
peroxynitrite (172). However, in endothelial cells, irisin also
reverted the oxidative stress induced by advanced glycation
end products by decreasing endothelial NOS activity and
therefore, preventing the endothelial damage that is secondary
to inflammasome overactivation (40).
In macrophages, irisin was found to increase proliferation
and phagocytosis ability (105) and protected these cells
against oxidative stress damage via upregulation of antioxi-
dant defenses, even under LPS stimulation (106). Given the
undeniable implication of macrophages in the development
and progression of many diseases, a direct action of exercise-
induced irisin on macrophages could contribute to the
widespread beneficial effects of exercise. Some of these ef-
fects are illustrated in Figure 5.
Previous studies have suggested that myokines mediate the
beneficial effects of exercise in brain function (Fig. 5). Under
an ischemia/reperfusion stroke, brain cells experiment a
glucose/oxygen deprivation that triggers an intense ROS
 8 LOUZADA ET AL.
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production that can lead to an irreversible injury. Using
in vitro model, irisin protected neuron cells from oxidative
stress via decreasing the higher ROS production and in-
flammation through increased SOD expression, consequently
protecting neurons from stroke-induced apoptosis (127).
In 1944, one of the first reports suggested that exercise
reduced tumor growth and progression in mice (136). Since
then, there are several evidences showing that a healthy
lifestyle hinders the development of several types of cancer
and regular exercise can protect from a large range of dis-
eases (49, 119, 134). However, the precise mechanisms are
still unclear. Regarding the effect of exercise on tumor
growth, there are many conflicting results that have been
obtained from rodent experiments. Recent studies have
shown that exercise may decrease tumor growth if tumor-
bearing mice were trained 4 weeks before inoculation with
B16 melanoma cells (125), whereas another study suggested
avoiding free access to exercise because they observed in-
creased tumor growth and decreased lifespan in a model of
intramuscular tumor inoculation (8). Despite the gap of
knowledge about the mechanisms of actions involved in this
process, regular exercise has been recommended as a sup-
plementary therapy for cancer patients (83). Based on exist-
ing evidence, some public health organizations have issued
physical activity guidelines also for cancer prevention. Re-
garding the large number of studies conducted on physical
FIG. 5. Crosstalk between
skeletal muscle and other
tissues. The regular practice
of physical exercise promotes
widespread health benefits
that can prevent and treat
many diseases. Most of the
desirable effects of physical
exercise are mediated by
myokines that can facilitate
communication of contract-
ing muscles with remote tis-
sues. The mechanism of
action of many myokines re-
lies directly or indirectly on
redox signaling. Color ima-
ges are available online.
activity and cancer prevention, most of them have an in-
complete description of physical activity (such as type, in-
tensity, duration, and metabolic responses).
Mechanistically, an antiproliferative effect of serum col-
lected from mice after moderate exercise intensity (50% of
maximal exercise capacity), but not from mice submitted to
high-intensity swimming exercise (50% and 80% of maximal
capacity, respectively), confirms the idea that a mix of
myokines are secreted that are able to affect tumor cells,
reducing tumor growth (2). However, another study showed
that high-intensity training (corresponding to 85% of VO2
max) was able to promote a reduction in tumor mass (10). By
screening 28 potential candidate molecules upregulated in
serum after swimming exercise, Oncostatin M has emerged
as a candidate for reducing mammary cancer cells growth
(78). Another candidate able to prevent the initiation of a
tumor was the ‘‘Secreted Protein Acidic and Rich in Cy-
steine’’ (SPARC). The level of circulating SPARC increased
in mice and humans after a single bout of exercise. Low-
intensity exercise induces SPARC expression and secretion
that reduces the formation of aberrant crypt foci and prevents
the onset of colon cancer (6). SPARC expression seems to be
induced by low and moderate exercise programs (149) and is
mainly secreted by slow-twitch fibers instead of fast-twitch
fibers after resistance exercise (Fig. 5) (148). Apart from its
possible direct action as an antitumor molecule, SPARC also
 REDOX SIGNALING IN WIDESPREAD EFFECTS OF EXERCISE
seems to mimic some of the beneficial effects of exercise on
glucose metabolism via AMPK activation (5).
Therefore, myokines secreted by the muscles may mediate
the beneficial effects of exercise on tumor development.
Although it is a promising and innovative theme, the effects
of contracting muscle myokines (e.g., IL-6, Irisin, FGF21,
Oncostatin M, SPARC, etc.) on tumor growth and progres-
sion using xenograph and inoculated rodent models are still
relatively unknown.
Extracellular Vesicles Released After Exercise
and Physiological Perspectives
Other important products secreted by skeletal muscle
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during contraction are extracellular vesicles that include
exosomes and microvesicles (also known as shedding vesi-
cles, ectosome). Exosomes are complex 20–100-nm vesicles
formed by the inward budding of endosomal membranes to
form large multivesicular bodies. These vesicles are released
out of the cell when the multivesicular body fuses with the
plasma membrane. The exosome is characterized by soluble
and membrane-bound proteins, lipids, metabolites, DNA,
and RNA (mRNA, miRNAs, and other small regulatory
RNAs) that are involved in a protective lipid bilayer that can
modify signaling pathways and protein expression in remote
tissues (160).
Physical exercise considerably increases the metabolic
demands of the body. and the vesicle trafficking of metabolic
mediators might be a tool through which tissues can share
resources during this physiological situation. Using quanti-
tative proteomic techniques and intravital imaging experi-
ments, Whitham et al. demonstrated that skeletal muscle
contraction stimulates extracellular vesicle release and pro-
vides the means for tissue crosstalk during exercise (168).
Although the temporal aspects of exosome and small vesicle
biogenesis and transport in exercise are unknown, it has been
demonstrated that the release of exosomes is associated with
increases in the levels of intracellular calcium (141). Because
motor neuron stimulation of skeletal muscle fibers leads to a
rapid release of Ca2+ from the sarcoplasmic reticulum (109),
it is plausible that a transient Ca2+ increase during skeletal
muscle contraction is likely involved in exosome release.
ROS seems to be involved in the secretion of extracellular
vesicles since pro-oxidant conditions seem to induce their
release (15). In addition, NADPH oxidase and NOS-2 in-
hibitors reduced the production and release of neutrophil
microvesicles (158). Further, tumor and senescent cells have
altered redox balances with elevated ROS levels, which is
related to the higher number of microvesicles secreted by
these cells (29, 80). From an exercise perspective, more re-
search is necessary to elucidate the relationship between in-
creases in ROS production during skeletal muscle contraction
and microvesicle release (Fig. 1). However, some evidence
suggests that disrupted ROS-mediated signaling caused by
NAD(P)H oxidase inhibition (75), or aging, through exac-
erbated ROS generation (107), can affect the nature of exo-
somes (e.g., heat shock proteins [HSPs] expression),
delivering and potentially hampering the widespread bene-
ficial effect of exercise.
HSPs, for example, can be transferred between two cells
(170) and participate in the maintenance of protein homeo-
stasis (155). Previous studies suggested that Hsp70 secretion
9
is mediated by ATP-binding cassette transporter (ABCA1)
transmembrane transporters (4). Further, three other mecha-
nisms can also be related to Hsp70 release, such as: (i) cell
lysis with consequent release of cytoplasmic content; (ii) cell
surface blebbing and Hsp70 release in microvesicles to the
extracellular fluid; and (iii) through endolysosomes (58, 103,
104). HSPs are molecular chaperones that facilitate protein
folding and maintain protein structure and function during
cellular stress. HSPs are involved in a wide range of cellular
processes that are perturbed by oxidative stress, such as
protein folding, apoptosis, and inflammation (85, 86). As
recently discussed (108), the intercellular communication via
exosomes might compensate for the imbalance in HSP levels
in almost every cell (155). For example, differentiated neu-
rons seem to be dependent on a transfer of HSP from other
cells to maintain their cytoprotection against stress (101). It
has been demonstrated that intercellular HSP transfer occurs
by exosomes after exercise (93, 164), raising the idea that this
intercellular communication is critical to ensure an integrated
stress response across different tissues (160a). The exosomes
secreted after acute exercise have higher levels of HSPs (55,
168). On the contrary, muscle denervation alters the content
of exosomes (38), suggesting a crucial role for contractile
activity on exosome content and secretion.
Alzheimer’s is a neurodegenerative disease in which there
is a prevalent protein folding disorder in the brain, and the
HSP response in some neurons becomes reduced (25). It has
been extensively demonstrated that HSP is involved in pro-
tein homeostasis in the brain, and an accumulation of mis-
folded proteins (b-amyloid peptides) contributes to the
progression of this neurodegenerative disease (7). Interest-
ingly, an increase in HSP70 expression improves cognition
function and reduces amyloid-b peptide levels in Alzhei-
mer’s model mice (152). Based on the mechanisms men-
tioned earlier, it is tempting to suggest that HSP transfer
through exosomes, which follows exercise, could retard or
even prevent the development of brain diseases; and this
hypothesis is illustrated in Figure 5.
Conclusions
The widespread beneficial effects of physical exercise are
mediated by multiple soluble factors that interfere with a
variety of signaling pathways, and the decoding of these
pathways is a challenge for basic and translational research.
Fascinatingly, optimal ROS-mediated signaling is necessary
to achieve the beneficial effects of exercise. In a common
way, exercise-secreted mediators seem to operate via PGC-
1a and Nrf2, two redox sensible pathways that promote
healthier conditions globally.
Recently, attention has focused on the mechanisms of
action of new myokines. Many exercise responses and ad-
aptations may be dictated by the (un)balance of ‘‘cytokines
cocktail,’’ defined as a net balance between deleterious
hampering molecules and benefit enhancing molecules. Un-
derstanding how aging and obesity blunt some of these ex-
ercise adaptations and proposing a multiple and combined
therapeutic strategy that will optimize the beneficial effect of
exercise in many diseases is a critical issue in exercise bi-
ology. In addition, the ROS-rich environment that is created
after exercise is not exclusively restricted to contracting
muscle. Understanding the processes by which the beneficial
 10
effects of exercise occurs is, therefore, a prerequisite to de-
fining how lack of exercise contributes to a poor quality of
life and high incidence of disease. Therefore, further studies
designed to decipher the mechanism of action of myokines
are indispensable to understand the redox code that underlies
the beneficial effects of exercise and, thus, will expand the
therapeutic strategies for many diseases.
Authors’ Contributions
All authors listed have made a substantial, direct, and in-
tellectual contribution to the work, and approved it for pub-
lication. R.A.L., J.B., and L.P.M. wrote the article; D.P.C.,
C.D., and J.P.W-.d.-C. revised the article; and R.A.L. and
Downloaded by UNIV OF MIAMI MILLER SCH MED Package NERL from www.liebertpub.com at 04/27/20. For personal use only.
R.S.F. designed the outline of the article and wrote the article.
Acknowledgment
The authors would like to thank Sapiens scientific illus-
trations for the design of the figures.
Funding Information
The study was supported by research grants from CNPq,
Fundaçao Carlos Chagas Filho de Amparo a Pesquisa do
Estado do Rio de Janeiro (FAPERJ), and Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES).
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 16 LOUZADA ET AL.

171. Zhang M, Xu Y, and Jiang L. Irisin attenuates oxidized AMP ¼ adenosine monophosphate
low-density lipoprotein impaired angiogenesis through AMPK ¼ AMP-activated protein kinase
AKT/mTOR/S6K1/Nrf2 pathway. J Cell Physiol 234: ATP ¼ adenosine triphosphate
18951–18962, 2019. BiP ¼ binding immunoglobulin protein
172. Zhu D, Wang H, Zhang J, Zhang X, Xin C, Zhang F, Lee CAMK ¼ Ca2+/calmodulin-dependent protein kinases
Y, Zhang L, Lian K, Yan W, Ma X, Liu Y, and Tao DUOX ¼ dual oxidase
L. Irisin improves endothelial function in type 2 diabetes ER ¼ endoplasmic reticulum
through reducing oxidative/nitrative stresses. J Mol Cell ERS ¼ endoplasmic reticulum stress
Cardiol 87: 138–147, 2015. FGF21 ¼ fibroblast growth factor 21
GLUT4 ¼ glucose transporter 4
Address correspondence to: H2O2 ¼ hydrogen peroxide
Dr. Ruy A. Louzada HSPs ¼ heat shock proteins
Institut of Biophysics Carlos Chagas Filho IL ¼ interleukin
Federal University of Rio de Janeiro LIF ¼ leukemia inhibitory factor
Downloaded by UNIV OF MIAMI MILLER SCH MED Package NERL from www.liebertpub.com at 04/27/20. For personal use only.

LDH ¼ lactate dehydrogenase

Rio de Janeiro 21941-590
L-NAME ¼ N(G)-nitro-l-arginine methyl ester
Brazil
MAPK ¼ mitogen-activated protein kinase
MCP1 ¼ monocyte chemoattractant protein 1
E-mail: andraderuy@hotmail.com MCT ¼ monocarboxylate transporter
NADH ¼ nicotinamide adenine dinucleotides
Date of first submission to ARS Central, November 11, 2019; NOX ¼ NADPH oxidase
date of final revised submission, February 29, 2020; date of Nrf2 ¼ nuclear factor erythroid 2-related factor 2
acceptance, March 9, 2020. O2 ¼ oxygen
O2- ¼ superoxide
OH ¼ hydroxyl
PDK4 ¼ pyruvate dehydrogenase kinase 4
Abbreviations Used PGC-1a ¼ peroxisome proliferator-activated receptor
13-HODE ¼ 13-hydroxyoctadecadienoic acid gamma coactivator 1-alpha
4-HNE ¼ 4-hydroxynonenal ROS ¼ reactive oxygen species
AK ¼ adenylate kinase SPARC ¼ secreted protein acidic and rich
AKT/mTOR ¼ protein kinase B/mechanistic target in cysteine
of rapamycin TGF-b ¼ transforming growth factor beta

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