Review Article

https://doi.org/10.1038/s42255-020-0258-x

Metabolic communication during exercise

Robyn M. Murphy1,2, Matthew J. Watt3 and Mark A. Febbraio   4 ✉

The coordination of nutrient sensing, delivery, uptake and utilization is essential for maintaining cellular, tissue and whole-body
homeostasis. Such synchronization can be achieved only if metabolic information is communicated between the cells and tissues
of the entire organism. During intense exercise, the metabolic demand of the body can increase approximately 100-fold. Thus,
exercise is a physiological state in which intertissue communication is of paramount importance. In this Review, we discuss the
physiological processes governing intertissue communication during exercise and the molecules mediating such cross-talk.

H
ippocrates, born in ~460 bc on the Greek island of Kos, is
credited with being the first person to believe that diseases
are a result of natural causes rather than superstitions or
the will of gods. Acknowledged as the creator of Western medicine,
Hippocrates was also the first documented person to recognize
that an active lifestyle is beneficial to health, stating that “walking
is man’s best medicine,” and “if there is a deficiency in food and
exercise, the body will fall sick.” Inactivity is now well established to
lead to obesity, the accumulation of visceral fat, insulin resistance,
hyperlipidaemia and skeletal muscle atrophy1. Moreover, physical
inactivity is now known to increase the risk of acquiring many life-
style diseases including colon, endometrial and breast cancers, type
2 diabetes, cardiovascular disease, bone degenerative diseases, dis-
eases involving cognitive impairment (such as dementia), and poly-
cystic ovarian syndrome1–3. Much of the benefit of physical activity
in preventing disease has been attributed to several mechanisms
including weight loss, increased cardiorespiratory fitness (maxi-
mum rate of oxygen consumption, VO2 max) and the maintenance
of muscle mass2. However, over the past 20 years, interorgan com-
munication during exercise has emerged as an alternative mecha-
nism through which physical activity can protect the body against
an array of lifestyle diseases.
Metabolic demand during exercise
Metabolic demand during exercise varies greatly, in a manner
dependent on exercise intensity and the requisite ATP supply to
sustain skeletal muscle contraction4. In the most demanding exer-
cise, such as when an athlete competes in an ‘all-out’ capacity, the
exercise duration is typically <60 s, and the metabolic rate in skel-
etal muscle can increase ~100-fold (ref. 5). Carbohydrate, fatty acids
and protein can all be used for ATP production, and the efficiency
is highest when sufficient oxygen (O2) is continually supplied to the
muscle and is not limited to the mitochondria, thus ensuring oxida-
tive phosphorylation via the tricarboxylic acid cycle (also referred
to as the citric acid or Krebs cycle).
During periods of high energy demand, such as whole-body
exercise, O2 availability cannot meet local demands in the contract-
ing muscle6. The rate-limiting factor is the capacity for O2 delivery
to skeletal muscle, because oxygen uptake (VO2) is able to meet
the delivery rate6. This aspect is perhaps best demonstrated by the
observations that mitochondrial oxidative enzymes increase rapidly
with endurance training, whereas the increase in VO2 max occurs
to a smaller extent7. Through a series of steps, glucose moieties
are converted to pyruvate, which has two fates. In the presence of
O2, it is converted to acetyl-CoA and enters the Krebs cycle. In the
absence of sufficient O2, to maintain at least some ATP production,
pyruvate undergoes anaerobic metabolism and is converted to lac-
tate, in a much less efficient pathway that produces only ~6% the
amount of ATP produced by aerobic metabolism.
Fatigue is of paramount concern to competing athletes and is
defined as an inability to maintain force production or a progres-
sive decline in performance8. As their names suggest, central fatigue
occurs through the brain, whereas peripheral fatigue occurs in skel-
etal muscle. The concept of central fatigue is discussed in further
detail below. Until the early 2000s, lactic acid (lactate and hydro-
gen ions) had been blamed as the primary culprit determining
muscle fatigue, although this conclusion was based on correlative
rather than causative, data9. Research using a minimalist prepara-
tion, mechanically skinned individual muscle fibres, has provided
clear evidence that lactate per se does not cause the muscle fibre to
lose the ability to produce force via depolarization-induced calcium
release, also known as fatigue10. Indeed, the same minimalist prepa-
ration has also indicated that lactic acid, specifically the increases
in both hydrogen ions (or reduced pH)11 and lactate12, actually con-
tribute to the maintenance of excitability of the muscle cell mem-
brane and the ability to undergo repeated cycles of contraction via
excitation–contraction coupling, thus completely debunking the
notion that lactic acid causes muscle fatigue.
The precise cause of muscle fatigue is complex and dependent on
several factors including the duration of exercise, the species studied
and the experimental model. During high-intensity exercise and/or
in the single-fibre model, fatigue is thought to be linked to the accu-
mulation of inorganic phosphate as a result of the breakdown of cre-
atine phosphate, which has direct effects on sarcoplasmic reticulum
handling of Ca2+, and a decreased ability of the muscle to produce
force11,13
The functioning of excitable tissues (that is, the brain and the
skeletal and cardiac muscle) is energetically demanding. Each of
these tissues meets some demands with internal stores of glyco-
gen, which can be enzymatically processed to produce glucose.
Systemically, the liver plays an important role in supplying glucose
to the circulation during periods of increased energy demand. The

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia. 2Department of
1

Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University, Melbourne, Victoria, Australia. 3Department of Physiology, University
of Melbourne, Melbourne, Victoria, Australia. 4Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.
✉e-mail: mark.febbraio@monash.edu

Nature Metabolism | www.nature.com/natmetab

Review Article
Box 1 | Lactate, the first identified muscle secretory factor
The relationship between muscle activity and the production of
lactic acid from skeletal muscle was first observed in amphibians
in 1907 (ref. 174). Skeletal muscle is now well known to release
lactate during exercise, in a manner influenced by both exercise
intensity and muscle mass175. Generally, the release of lactate from
contracting muscle is seen as a mere by-product of intense mus-
cle contractions, and lactate must be rapidly cleared from the
contracting muscle for contractions to continue176. However,
lactate has long been known to be an important fuel source for
other tissues9. The finding that lactate can be oxidized by the brain
dates back more than 80 years (refs. 176,177). However, the notion
that lactate can be a fuel source for the brain was not adopted
until much later. With the advent of new in vivo markers, such as
the genetically encoded fluorescence resonance energy transfer
sensor Laconic, in combination with two-photon microscopy,
the concept that lactate in the brain is differentially present in
various compartments and moves among these compartments
via monocarboxylate transporters, with a lactate flux from astro-
cytes to neurons, has been supported178. To trace lactate move-
ments in humans, van Hall and co-workers have analysed blood
samples from the arterial, jugular and femoral veins after the in-
fusion of 13C-labelled lactate in participants cycling at 75% VO2
max179. The authors concluded that lactate contributes 20–25% of
the brain’s total energy demand, probably in preference to glucose
oxidation. The brain is clearly adaptable to substrate provision as
glucose or lactate, and once inside a cell, lactate is oxidized rather
than stored, thus maintaining a constant driving force for lactate
uptake by various brain regions179. Whether the increased deliv-
ery of lactate to the brain with exercise is involved in processes
other than substrate provision, such as through other signalling
events, is unknown but warrants consideration.
extent to which liver glycogenolysis or gluconeogenesis contributes
to hepatic glucose production depends on factors including exercise
intensity, temperature and exogenous carbohydrate availability14,15.
Skeletal muscle is highly adaptable to enhancing glycogen stores,
and consequently, elite athletes are able to build large stores of gly-
cogen, a process sometimes referred to as glycogen supercompen-
sation, as evidenced by the roughly spherical granular structures
seen under electron microscopy16. At least in rodent tissue, liver17,
cardiac18 and brain19 glycogen also increases in response to exer-
cise training. Glycogen stores are finite in all tissues, although the
amount of ATP obtainable is variable and dependent on oxygen
availability, as described above.
Skeletal muscle as the primary metabolic communicator
during exercise
Muscle as a signalling organ. As early as the 1960s, skeletal muscle
was hypothesized to potentially be more than an organ that helps
maintain posture and provide locomotion, when Goldstein postu-
lated the existence of a humoral component in skeletal muscle that
may help regulate glucose homeostasis20. This concept that skeletal
muscle may be a signalling and/or endocrine organ was largely
ignored until the turn of this millennium, but the evidence that skel-
etal muscle, is a signalling organ, particularly during contraction,
has been known for several decades21 (Box 1).
Although research has centred on the release of cytokines and/
or peptides from skeletal muscle, metabolites are also released from
this organ. Roberts and co-workers22,23 have recently identified
candidate metabolites released from skeletal muscle by using liq-
uid chromatography–mass spectrometry metabolic profiling from
muscle cells overexpressing the transcriptional coactivator PGC-1α
NaTuRe MeTaBolISm
IL-6
Lactate BAIBA
Metrnl
Cathepsin B
Irisin
BAIBA
SPARC
Irisin
Metrnl
Fig. 1 | Muscle secretory factors during exercise. During muscle
contraction, an array of molecules are secreted that communicate with
other organs such as the liver, BAT and WAT, colon and brain. These
factors include those that signal to the brain and improve cognition and/or
memory (lactate, Metrnl, cathepsin B and irisin), those that signal to
the liver and improve metabolism (IL-6 and BAIBA), those that signal
to adipose tissue and increase browning or energy expenditure (BAIBA,
irisin and Metrnl) and those that provide protection against colon
cancer (SPARC).
(ref. 7). PGC-1α regulates gene expression in skeletal muscle and has
a major influence on the muscle’s metabolic response to exercise24.
The liquid chromatography–mass spectrometry metabolic screen
identified four metabolites (β-aminoisobutyric acid (BAIBA),
GABA (γ-aminobutyric acid), cystine and 2´-deoxycytidine) that
are released by PGC-1α-overexpressing myocytes, two of which
appear to have biological roles. BAIBA induces browning of
white fat and β-oxidation in the liver7,25, whereas GABA has been
implicated in muscle fibre-type switching8. The earlier observations
with respect to lactate, coupled with these recent findings, high-
light the importance of skeletal muscle as a signalling organ during
exercise, in terms of metabolite cross-talk, a component of muscle
communication during exercise that is somewhat clear but often
overlooked (Fig. 1).
Muscle as an endocrine organ. IL-6 as the prototypical myokine. As
described above, the concept that skeletal muscle is an endocrine
organ gained popularity in the early twenty-first century, when sev-
eral studies demonstrated that the cytokine interleukin-6 (IL-6) is
produced by, and subsequently released from, skeletal muscle dur-
ing exercise1,26. The term myokine, denoting a cytokine or peptide
that is produced and released by skeletal muscle and subsequently
exerts paracrine or endocrine effects, was then coined1,26. The
observation that muscle produces and releases IL-6 during exercise
and the recognition of myokines spawned this new field of research.
In a review published 4 years ago, we suggested that to satisfy the
criteria for being considered a myokine, a protein or peptide must
(1) be derived from skeletal muscle cells (2) be secreted, either via
classical secretory pathways or via extracellular vesicles (EVs), and
(3) induce a biological function in an endocrine and/or paracrine
manner3.
The earliest and most extensively studied bona fide myokine is
IL-6, which was somewhat serendipitously discovered as the pro-
totypical myokine. Plasma IL-6 was shown to increase in response
to strenuous exercise27, and, given that IL-6 is a cytokine, the
blood mononuclear cells (BMNCs) were assumed to be the source.
However, the messenger RNA27 and protein28,29 expression of IL-6
does not increase in BMNCs during exercise in humans. Moreover,
the hepatosplanchnic bed clears, rather than releases, IL-6 during
exercise30.
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NaTuRe MeTaBolISm
Using muscle biopsy and arteriovenous (a-v) balance techniques,
the Pedersen and Febbraio laboratories have demonstrated that
both the mRNA and protein expression of IL-6 increases in skeletal
muscle cells during exercise31–33, and that IL-6 protein net release
from contacting limbs increases up to 100-fold during muscle con-
traction34,35. Importantly, the production of IL-6 via skeletal muscle
during exercise is somewhat unique. Whereas Il6 mRNA expres-
sion in BMNCs during inflammation is under transcriptional con-
trol of the p65 subunit of the transcription factor NF-κB, exercise
increases the phosphorylation of c-jun terminal kinase (JNK), the
reporter activity of the downstream transcription factor AP-1 and
the recruitment of AP-1 to the promoter region of the Il6 gene in
skeletal muscle36. Moreover, IL-6 production during exercise is
entirely dependent on this transcriptional pathway, because mice
with a muscle-specific deletion of JNK do not show increased IL-6
transcription in response to exercise36.
Despite this definitive evidence that IL-6 is transcribed, trans-
lated and released from skeletal muscle during exercise, whether
it serves a biological function was unknown until recombinant
human IL-6 (rhIL-6) was infused in combination with exercise. One
hypothesis for why IL-6 is released during exercise is inhibition of
the production of tumour necrosis factor (TNF), which is known
to cause pathogenesis in sepsis. Both exercise and rhIL-6 infusion
to levels similar to the IL-6 levels during exercise completely sup-
pressed the TNF response to a bolus of Escherichia coli lipopoly-
saccharide endotoxin, which induces low-grade inflammation
in healthy humans37. This study has provided clear evidence that
exercise is anti-inflammatory and that the underlying mechanism
involves the production of IL-6.
As discussed above, the mechanisms that mediate the tightly con-
trolled production and clearance of glucose during exercise were not
fully elucidated until relatively recently, and an unidentified ‘work
factor’ has been suggested to influence the contraction-induced
whole-body glucose production20. Experiments with rhIL-6 have
demonstrated that IL-6 released during exercise contributes to the
increase in whole-body glucose production in humans38. These data
provide potential new insights into factors that mediate glucose
production and disposal, and implicate IL-6 as the first myokine
that results in muscle–liver cross-talk during contraction.
Other interleukins. Beyond IL-6, several other cytokines have been
linked to the skeletal muscle signalling response during exercise.
Soon after our work describing IL-6 as a myokine, we conducted
a cytokine gene expression screen of several immunoregulatory
cytokines. We detected skeletal muscle mRNA expression levels of
IL1Β, IL6, IL8 (official symbol CXCL8), IL18 and TNF from human
biopsy samples. Of note, however, the only cytokine other than IL-6,
influenced by exercise was IL-8 (ref. 39). Interestingly, as with IL-6,
the contraction-induced increase in the mRNA expression of IL8
was exacerbated by low intramuscular glycogen. Critically, unlike
IL-6, plasma IL-8 did not increase during exercise39, thus suggesting
that it is not a contraction-induced myokine.
IL-15 is an interesting cytokine because it increases within skel-
etal muscle during contraction and is linked to skeletal muscle
growth40. IL-15 has also been demonstrated to affect both adipose
tissue mass41 and the ability of adipocytes to secrete adiponec-
tin42, thus suggesting muscle–adipose cross-talk during exercise.
Similarly to IL-8, however, IL-15 has not been found to be released
from the contracting limb during exercise, thereby suggesting that
it is not a contraction-mediated myokine.
A recent study has identified IL-13 as a potential myokine. In a
comprehensive series of experiments, Lee and colleagues have shown
that IL-13 increases in the plasma in exercise trained humans and in
the skeletal muscle in exercise-trained mice43. Moreover, they have
found that IL-13-deficient animals have defective fatty acid metab-
olism after acute exercise and impaired mitochondrial biogenesis
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Review Article
after endurance training43. Intriguingly, however, the authors have
identified that the source of the increase in circulating IL-13 during
exercise is type 2 innate lymphoid cells, in a manner not muscle spe-
cific, and rather than studying animals with muscle-specific IL-13
deficiency, they used animals with whole-body IL-13 deficiency
as their model. Therefore, whether IL-13 is a bona fide myokine
remains to be determined.
Irisin. Apart from the interleukins described above, other candidate
myokines have recently been identified. Similarly to the actions of
the metabolite BAIBA22,25, myokines have been found to result in
adipose tissue browning. Using gene expression array analysis of
skeletal muscle from PGC1α-transgenic mice, which exhibit a simi-
lar phenotype to that of endurance-trained animals, Böstrom et al.44
have identified that irisin, a C-terminally cleaved portion of the pro-
tein FNDC5, increases in plasma in response to exercise in both
mice and humans. The protein is biologically relevant because its
muscle-specific overexpression improves metabolic homeostasis via
browning of white adipose tissue (WAT)44. Although the Spiegelman
group has demonstrated the existence of irisin via tandem mass
spectrometry (MS)45 and has recently identified a receptor for this
ligand46, the data have been challenged, because human FNDC5
has an unusual start codon of ATA rather than ATG, thus causing
critics to suggest that irisin cannot be measured47. Moreover, avail-
able antibodies to irisin have been reported to non-specifically bind
serum proteins, thus prompting questions regarding the widely
used method of measuring irisin by enzyme-linked immunosorbent
assys47–49 and highlighting the possible need to use the unequivocal
tandem MS method to obtain reliable irisin data.
Meteorin-like protein. Another exercise-induced myokine that results
in adipose tissue browning is meteorin-like protein (Metrnl), iden-
tified by the Spiegelman group50. Metrnl is induced in muscle with
exercise, but it also increases in adipose tissue with cold exposure,
thus leading to increased Metrnl levels in the circulation under
these stimuli. Increasing circulating levels of Metrnl increase energy
expenditure and enhance glucose tolerance50 .
Apart from these important myokines, other muscle secretory
factors, such as myostatin, fibroblast growth factor 21, myonectin,
myostatin, brain-derived neurotrophic factor (BDNF), IL-8, IL-15
and mitochondrially encoded peptide-c have all been suggested to
act as myokines. These myokine candidates have been discussed in
previous reviews3,51
Although most of the above myokines were discovered seren-
dipitously, several groups have attempted to uncover the so-called
‘myokinome’. Using a computational approach, Schiaffino and col-
leagues have suggested that more than 300 proteins are potentially
secreted by contracting skeletal muscle52. This number is, however,
likely to be markedly underestimated, because many proteins that
lack a signal-sequence peptide and recognition of transmembrane
regions can be secreted via EVs53. In fact, through a-v balance stud-
ies across the contracting human limb, purification of EVs from
arterial and venous blood during exercise, and label-free quantita-
tive proteomic techniques, we have identified more than 600 poten-
tial muscle secretory factors, most of which lack secretory-sequence
peptides53. The roles of EVs in tissue cross-talk during exercise are
discussed in greater detail below.
Muscle–brain cross-talk during exercise. Many studies have sug-
gested that regular physical activity can modify both cognition
and brain biochemistry via circulating exercise-induced factors
or ‘exerkines’54,55, but identifying whether myokines or exerkines
can affect brain function has been challenging. Apart from irisin’s
reported role in adipose tissue browning, a different role has recently
been uncovered. Irisin levels are diminished in both the hippo-
campus and cerebrospinal fluid of mouse models of Alzheimer’s
Review Article
disease56. Moreover, decreasing irisin in the brain in these mouse
models impairs cognition, but this impairment is rescued if irisin
levels are subsequently increased56. These recent studies highlight
irisin as an important mediator of the beneficial effects of exercise
in Alzheimer’s disease preclinical models.
The notion that muscle secretory factors may affect cognition is
not, however, without precedent. Ruas and colleagues have identi-
fied that exercise training influences kynurenine metabolism and
protects against stress-induced depression57. They have shown that
exercise increases the skeletal muscle expression of kynurenine
aminotransferases, thus enhancing the conversion of kynurenine
into kynurenic acid, a metabolite unable to cross the blood–brain
barrier. Decreasing plasma kynurenine protects the brain from
stress-induced changes associated with depression57. This study has
opened therapeutic avenues for the treatment of depression by tar-
geting skeletal muscle, without the need to cross the blood–brain
barrier.
Shortly thereafter, Moon et al.58 demonstrated that cathepsin B,
a muscle secretory factor, mechanistically underlies the finding that
running is beneficial for neuroregeneration and cognition. Increased
cathepsin B levels have been observed in conditioned medium
derived from skeletal muscle cell cultures treated with the AMP
kinase agonist AICAR, to mimic contraction. In mice, non-human
primates and humans, running increases plasma cathepsin B, which
importantly correlates with fitness and hippocampus-dependent
memory function58. These recent findings56–58 suggest that myo-
kines contribute to the beneficial effects of exercise on cognition.
Myokine effects on cancer. Multiple epidemiological studies have
indicated that physical activity can prevent several cancers includ-
ing colon, breast and endometrial cancer59. Circulating secreted
protein acidic and rich in cysteine (SPARC) increases in the plasma
in both mice and humans during exercise60. The effect of SPARC on
colon tumorigenesis in SPARC-deficient mice has also been exam-
ined. In addition, in an azoxymethane-induced colon cancer mouse
model, regular low-intensity exercise educes the formation of aber-
rant crypt foci in wild-type mice but not in SPARC-null mice60.
These findings suggest that myokines are a molecular mechanism
through which exercise can decrease the incidence and/or progres-
sion of certain cancers (Fig. 1).
Role of the liver in intertissue communication and exercise
metabolism
General considerations. The liver integrates complex signals
derived from the systemic (that is, arterial blood) and portal (that
is, nutrient-rich blood from the intestines, pancreas or spleen) cir-
culation, thereby regulating an array of metabolic functions includ-
ing the metabolism of carbohydrates, fatty acids and amino acids,
and the production and secretion of lipoproteins and hormones. As
mentioned earlier, glucose derived from liver glycogen provides an
essential metabolic substrate to contracting skeletal muscle during
exercise14,15. The liver also produces very-low-density-lipoprotein
triacylglycerol (VLDL-TG), which can contribute as much as
10–15% of the total energy expenditure in the postabsorptive state61.
VLDL-TG secretion decreases during both exercise and recovery,
whereas VLDL-TG clearance remains unchanged; thus, VLDL-TG
oxidation makes a very small contribution to total energy expendi-
ture during exercise62.The extensive vascular network of the liver,
particularly the ‘open-pore’ sinusoids located between hepatocyte
planes, facilitates the free exchange of nutrients and hormones
within the liver, and the hepatokines released in response to meta-
bolic stresses are likely to be prominent in paracrine and autocrine
regulation of hepatocyte function63. Extending the likelihood of sig-
nificant exercise-induced alterations in protein secretion, evidence
from rodent studies has shown that the liver responds to an acute
bout of exercise, at least at the gene level, by upregulating an array
NaTuRe MeTaBolISm
of genes typically associated with energy depletion, whose products
are involved in stress signalling pathways including MAPK, JNK,
p53 and IL-6-like signalling64.
Transcriptomic analyses of the liver have revealed
exercise-induced changes in the mRNAs encoding secreted pro-
teins, thus suggesting the likelihood of changes in liver protein
secretion65. Advances in sample preparation, liquid chromatogra-
phy and MS technology have enabled the deepest coverage of the
liver proteome, and 11,520 proteins have been identified in mouse
liver66. Given that ~7% of these proteins enter the bloodstream
through secretion, there is clearly a large scope for the involvement
of hepatokine interorgan cross-talk. In this regard, our group has
identified 564 hepatocyte-secreted proteins, 30% of which are clas-
sically secreted67. Our recent unpublished work indicates that the
liver secretes ~2,500 proteins, 20% of which are classically secreted
and 80% of which are contained within EV’s.
Hepatokines as exercise-regulated signals that affect metabo-
lism. HSP72. Although much of the research focus on hepato-
kines has been directed at elucidating their roles in the aetiology
of metabolic diseases63, interest has grown in understanding the
roles of hepatokines in regulating systemic metabolism during both
exercise and recovery. We were one of the first groups to identify a
bona fide exercise-induced hepatokine, when we conducted human
hepatosplanchnic a-v balance studies almost two decades ago. We
demonstrated that exercise induces the hepatosplanchnic release of
heat shock protein 72 (HSP72) in healthy humans during prolonged
bicycle exercise68.
FGF21. Probably the best-studied and most physiologically impor-
tant hepatokine is fibroblast growth factor 21 (FGF-21). FGF21 is
expressed in many organs, most prominently the liver, and it has
been demonstrated to regulate an ever-expanding array of biologi-
cal functions69–71. The actions of FGF21 are mediated by binding to
FGF receptors and interaction with a coreceptor, β-Klotho, which is
essential for effective FGF21 binding and signal transduction72. The
target-organ selectivity of FGF21 is determined by the restricted tis-
sue expression of β-Klotho, which is predominantly expressed in
the liver, pancreas and adipose tissue. Hence, these tissues are con-
sidered the major targets of FGF21 (ref. 73).
FGF21 was initially shown to stimulate adipose tissue glucose
uptake, and subsequent studies using transgenic overexpression of
FGF21 or FGF21 infusions in mice have shown that FGF21 improves
whole-body insulin sensitivity and glycaemic control, increases adi-
pose tissue lipolysis and increases energy expenditure74. These effects
are associated with diminished adiposity and hepatosteatosis in
obese and diabetic animals74. More recent studies have demonstrated
roles of FGF21 in regulating adiponectin release from adipose tissue,
adipocyte browning, lifespan extension and protection against vas-
cular inflammation and atherosclerotic-plaque formation75.
FGF21 levels increase modestly during exercise in proportion
to exercise intensity and more dramatically immediately after exer-
cise—effects that are sustained for ~4 hours (refs. 76–78). Although
controversial, there has been recent interest in exercise-induced
‘browning’ of WAT79,80, and FGF-21 is a candidate protein that
may drive this process and decrease adiposity, at least in mice.
Experiments in high-fat-fed FGF21-null mice have reported other
impairments in the adaptation to exercise, and an absence of
improvements in glucose tolerance or decreases in hepatic triglyc-
eride content81. Together, these studies clearly show that FGF21 is an
exercise-regulated hepatokine whose action is necessary to achieve
the full metabolic benefits of exercise training.
Follistatin. Muscle growth is a hallmark of exercise training, particu-
larly resistance exercise, and recent data indicate that a liver-secreted
factor participates in promoting muscle mass. Follistatin, a
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NaTuRe MeTaBolISm
member of the transforming growth factor (TGF)-β superfam-
ily, exerts pleiotropic actions including inhibition of myostatin to
regulate skeletal muscle growth. The liver is now understood to be
a primary source of exercise-induced follistatin, and studies using
hepatosplanchnic a-v balance have demonstrated substantial fol-
listatin release after exercise77,78,82,83. In support of these observations,
a striking increase has been observed in follistatin (Fst) mRNA in
the livers of mice after acute exercise, but not in other tissues includ-
ing skeletal muscle82. Notably, follistatin does not increase during
exercise, thus implicating an important role of follistatin in the
adaptation to exercise.
The likelihood that liver-derived follistatin can induce a
change in muscle mass is highlighted by an innovative study
that developed nanoparticles loaded with Fst mRNA to induce
follistatin translation and secretion from the liver84. With this
approach, serum follistatin levels increased to levels comparable
to those observed after acute exercise (for example, 2.5-fold above
resting levels). Liver-derived follistatin was sufficient to decrease
serum myostatin, and long-term repeated injections increased mus-
cle mass in mice by 10%. Follistatin exerts numerous other effects in
several target tissues, including modulation of glucagon and insulin
secretion from the pancreas77,85, and improved whole-body glycae-
mic control85,86.
ANGPTL4. Regular exercise training induces acute and long-term
adaptations that modulate lipid metabolism, particularly in skel-
etal muscle, adipose tissue and the liver. Angiopoietin-like protein
4 (ANGPTL4) is a circulating protein that is highly expressed in
liver and that regulates lipid metabolism by inhibiting lipoprotein
lipase activity, thereby decreasing uptake of triacylglycerol-derived
fatty acids into tissues and stimulating adipose tissue lipolysis87.
Plasma ANGPT4 levels increase only marginally during exercise
and more prominently in recovery from exercise. These effects
appear to be augmented with increasing intensity and duration
of exercise88,89. Examination of tissue ANGPTL4 mRNA levels has
indicated that liver and adipose tissue contribute more than muscle
to the exercise-induced increase in circulating ANGPTL4 (ref. 89).
These data were confirmed by Ingerslev et al.90, who have dem-
onstrated direct secretion of ANGPTL4 from the liver in humans,
which appears to be mediated by a glucagon–cAMP–protein kinase
A pathway.
Whole-body fatty acid oxidation is well known to be elevated
for several hours after aerobic endurance exercise, partly because
of increased plasma free fatty acid (FFA) availability secondary to
increased adipose tissue lipolysis (~0.5–1.0 mM for 2–6 h)91. The
increase in lipolysis can be mediated by a residual increase in the
catecholamines, increased growth hormone and decreased insulin
levels. ANGPTL4 might contribute to postexercise adipose tissue
lipolysis, particularly as the effects of the other lipolytic regulators
dissipate over time91.
Selenoprotein P. Selenoprotein P (SeP) was originally identified as a
hepatokine that functions as a selenium-transport protein, and later
studies showed that SeP contributes to insulin resistance and hyper-
glycaemia in people with type 2 diabetes92. Intriguingly, recent work
suggests that SeP may explain the individual variations in respon-
siveness to exercise training in people with metabolic diseases93. SeP
appears to signal via LDLR-related protein 1 (LRP1), which func-
tions as an uptake receptor of SeP, thus decreasing ROS production
in skeletal muscle and consequently blunting AMPK activity, as a
result of exercise adaptations including mitochondrial biogenesis
and exercise capacity93.
Although these studies using SeP-deficient or LRP1-deficient
mice provided the first evidence of an intrinsic inhibitory factor
that attenuates adaptation to exercise, the physiological relevance
remains unresolved, because circulating SeP levels are unchanged
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Review Article
FGF21
Follistatin
FGF21
ANGPTL4
Follistatin
HSP72
SeP
Fig. 2 | Hepatokines during exercise. During exercise, hepatokines are
secreted that improve brain function (FGF21), pancreatic function (FGF21
and follistatin), muscle function and metabolism (follistatin, HSP72 and
SeP), and adipocyte biology (ANGPTL4).
during or after exercise in humans94. Nevertheless, these findings
in mice, coupled with a small trial in postmenopausal women, indi-
cate that pretraining plasma SeP concentrations might be useful for
predicting the ineffectiveness of exercise training on aerobic exer-
cise capacity.
Other hepatokines. Given the available evidence, most exercise-
responsive hepatokines appear to increase after rather than dur-
ing exercise, thus indicating roles in modulating metabolism in the
recovery from exercise and/or regulation of the adaptation to exer-
cise (Fig. 2). Other hepatokines including GDF15, inhibin βE, IGF1,
IGFBP1 and LECT2 are likely to be identified as exercise responsive.
However, compelling evidence is currently lacking. These hepato-
kines have been reviewed extensively elsewhere95.
Adipose tissue as a metabolic communicator during
exercise
General considerations. Lipolysis in WAT provides FFAs for ATP
production at rest and during exercise. During moderate-intensity
exercise (~50% VO2 max), whole-body adipose tissue lipoly-
sis increases approximately twofold by 30 min and progressively
increases to approximately fivefold higher than that at rest by 4 h.
Rates of whole-body fatty acid oxidation closely match lipolysis
during the first 90 min of moderate intensity exercise, and the FFA
supply from adipocytes is sufficient to provide all substrates for fatty
acid–oxidation requirements at rest and during moderate-intensity
exercise91. Although the contribution of adipose-derived FFAs to
whole-body ATP production decreases during higher-intensity
exercise (>80% VO2 max), this effect remains substantial in
endurance-trained athletes96. The glycerol released from complete
lipolysis can be used by the liver to sustain gluconeogenesis and
hepatic glucose output, which are most prominent during very pro-
longed exercise97.
Aside from these critical functions, adipose tissue is a major reg-
ulator of whole-body energy homeostasis through communication
with the brain and other organs through the secretion of adipokines,
which include small molecules, lipids and proteins. MS approaches
have identified a large diversity of secreted proteins from adipo-
cytes98,99 and adipose tissue100. The distinction between adipocyte
and adipose tissue secretion is important because of the complex
cellular composition of adipose tissue, which includes adipocytes
and various populations of adipocyte progenitor cells, an array of
immune cells, endothelial cells and neurons.
Moreover, adipose tissue is located at various anatomical sites,
including the visceral and subcutaneous depots, and one study
using comparison of isotope-labelled amino acid incorporation
Review Article
rates MS has reported significant site-specific differences in
secretion from human adipose tissues100. A deeper evaluation and
comparative analysis are required across other adipose tissues and
their constituent cells. Adding to this complexity, we have recently
identified three human adipose progenitor cell subtypes that give
rise to adipocytes with unique endocrine profiles101. The distribu-
tion of adipocyte progenitor cell subtypes varies between visceral
and subcutaneous adipose tissues and is likely to underpin the dif-
ferences in protein secretion from these depots.
Adipokines have important roles in a plethora of biologi-
cal process including but not limited to appetite and satiety,
insulin action, lipid metabolism, blood pressure, coagulation
and reproduction102. The secretion of adipokines—including
leptin, adiponectin, retinol-binding protein 4 (RBP4), FGF21,
pigment-epithelium-derived factor, bone morphogenetic protein
(BMP)-4, BMP-7, vaspin, apelin and progranulin—is altered in obe-
sity and contributes to a spectrum of obesity-associated diseases.
This field has been reviewed in depth by others102.
Adipokines and exercise metabolism. TGF-β2. Although exami-
nation of the effects of adipokines on metabolic regulation, parti­
cularly in the context of obesity, is a burgeoning field, the effects
of acute exercise remain somewhat unclear, primarily because of
differences in the phenotypes of study participants and disparities
in study designs. Overall, a single exercise bout appears to induce
either no changes or extremely modest changes in circulating adi-
pokines, thus indicating that adipokines are unlikely to be impor-
tant regulators of metabolism in response to acute exercise103. A
study by Goodyear and colleagues, however, has recently identi-
fied TGF-β2 as a bona fide exercise-induced adipokine104. Exercise
training increases TGF-β2 expression in subcutaneous WAT, serum
and fat explants. Most importantly, improved glucose tolerance
has been observed after transplantation of subcutaneous WAT
from exercise-trained mice into donor mice, but critically not into
TGF-β2 deficient donor mice104. Importantly, lactate, whose roles
as a signalling molecule have been discussed at length (Box 1),
stimulates TGF-β2 production in adipocytes104, thereby suggesting
a model through which muscle/lactate signals to adipose tissue/
TGF-β2, thereby modulating metabolism.
Adiponectin. The beneficial effects of regular physical exercise have
long been postulated to be partly mediated by changes in factors
that are contained within adipose tissue and cause adipose tissue
browning105 and/or adipokine secretion103, given that adipokine lev-
els change dramatically in response to exercise training, particularly
in obese individuals and people with type 2 diabetes103. Perhaps
the most studied adipokine in this context is adiponectin, which
improves insulin sensitivity and stimulates FFA uptake and/or
oxidation in muscle. Adiponectin is generally lower in obesity but
importantly is increased after exercise training in overweight and
obese individuals106,107. Similarly, long-term exercise training (≥2
weeks) is associated with a decrease in plasma leptin levels regard-
less of age and sex, to an extent is largely dependent on the amount
of fat loss induced by training108.
Other adipokines. Other adipokines known to cause inflammation
and insulin resistance, perturbed lipid metabolism, or fibrosis or vas-
cular dysfunction are consistently decreased with exercise training.
These include apelin109, RBP4 (refs. 110,111), visfatin112,113, chemerin114
and lipocalin-2 (ref. 115). There are several exceptions; for exam-
ple, the insulin-resistance-inducing adipokine PEDF116,117 and the
lipid-mobilizing factor zinc-α2-glycoprotein118 are unresponsive to
exercise training, whereas vaspin, which improves insulin sensitiv-
ity in mice, decreases with exercise training119,120. Nevertheless, the
changes in adipokine secretion after exercise training are predicted
to confer positive metabolic effects.
NaTuRe MeTaBolISm
Adipocyte-derived small molecules. LPA. Aside from proteins,
adipose tissue secretes several lipids that regulate metabolism.
Lysophosphatidic acids (LPAs), consisting of a glycerol backbone, a
phosphate group and an ester-bound acyl chain, are potent bioactive
lipids secreted by adipocytes. Among a multitude of biological roles,
LPA impairs glucose homeostasis and inhibits insulin secretion in
obese mice121,122. Plasma LPA levels are associated with obesity and
insulin resistance in mice, rhesus monkeys and humans122,123, and
this relationship appears to be dependent on dietary constituents
and the activity of autotaxin, which catalyses LPA synthesis. The
effects of exercise on adipocyte autotaxin secretion and LPA synthe-
sis have not been described.
Fatty acid esters of hydroxyl fatty acids. Fatty acid esters of hydroxyl
fatty acids are produced by WAT, and administration of certain spe-
cies, namely palmitic acid esters of hydroxystearic acids (PAHSAs),
increases glucagon-like peptide 1 and insulin secretion, and
improves insulin action and glycaemic control124. Low circulating
PAHSA concentrations have been associated with insulin resis-
tance in humans124, and although the evidence is limited, one study
in older women has reported increased PAHSAs in adipose tissue
and serum with exercise training125. Hence, there is a clear scope for
further work examining the roles of acute exercise and prolonged
training in regulating adipose-tissue-secreted lipids, to understand
their roles in cellular signalling and their effects in resolving meta-
bolic disease.
Effects of exercise on BAT-secreted factors
Batokines. The concept that brown adipose tissue (BAT) is a meta-
bolic communicator has gained much interest over the past decade
and has led to the discovery of so-called ‘batokines’, which carry out
this communication (Box 2). The breadth of batokines in regulating
systemic physiology has been somewhat underappreciated. However,
it is not yet clear whether most these proteins are present in the cir-
culation and thereby are bona fide endocrine regulators; whether
such proteins circulate at effective concentrations, which is a con-
cern given the relatively small mass of BATs in humans; and perhaps
most importantly, the identities of their target tissues and biological
actions. This will be an area of great interest in the coming years.
BAT-derived lipids. The role of exercise in regulating BAT activa-
tion and protein secretion is not well documented. Although ther-
mogenic activation and norepinephrine are known to alter BAT
protein secretion98, and exercise is highly likely to increase sympa-
thetic drive and norepinephrine stimulation to BAT126, definitive
evidence is required to demonstrate exercise-mediated changes in
protein secretion in vivo.
Perhaps the most exciting work in this domain relates to the
effects of the so-called lipokines, which are lipids secreted from
adipose tissue. The lipokine 12,13-diHOME was identified by MS
lipidomics to be released from BAT in response to cold in mice and
humans, and to act in an autocrine–paracrine manner in increas-
ing fatty acid uptake, lipolysis and thermogenesis in BAT; moreover,
prolonged treatment with this lipokine decreases circulating triglyc-
eride levels127. Studies by Stanford and colleagues have shown that
acute physical exercise in humans and mice increases the circulating
levels of 12,13-diHOME, and acute treatment with this lipokine in
mice increase skeletal muscle fatty acid uptake and oxidation, but
has no effect on glucose uptake128. These are the first studies dem-
onstrating an endocrine role of BAT in response to exercise, thus
indicating a novel mechanism for BAT–skeletal muscle cross-talk.
Role of the brain as a metabolic communicator during
exercise
Central fatigue. The ‘central fatigue hypothesis’ during exercise
postulates that an inability to maintain exercise capacity is associated
Nature Metabolism | www.nature.com/natmetab
NaTuRe MeTaBolISm
Box 2 | Batokines as metabolic communicators
BAT and beige adipose tissue are active secretory tissues releas-
ing batokines, which exert local and systemic actions. Brown
adipocytes have long been known to secrete proteins that con-
tribute, in an autocrine or paracrine manner, to the control of
BAT expansion and activity as well as the browning of WAT180.
Effector batokines include the positive regulators prostaglandins,
FGF-21 and CXCL14, and the negative regulators endothelin-1
and MSTN. More recent work has shown that BAT secretes pro-
teins that signal to other cell types and assist in tissue remodelling
during BAT recruitment to thermogenic stimuli, primarily by
targeting innervation and vascularization. Prominent examples
include BMP8b, CXCL14 and NRG4 (ref. 180). Emerging evidence
also indicates that brown adipose acts in an endocrine manner
in influencing metabolism in distant tissues such as the heart181
and skeletal muscle182. Work by Kong et al.182 has described the
first evidence of BAT–skeletal muscle cross-talk, demonstrating
that the BAT in mice lacking IRF4 secretes myostatin at thermo-
neutrality, but activation of BAT lowers myostatin and improves
exercise performance. These findings have potential implications
for muscle growth and exercise capacity; however, further work
is needed to determine whether this signalling axis is relevant
in humans and to ascertain the relevance of such cross-talk in
the context of regular exercise training. Proteomic analyses and
comparative mapping of proteins secreted from cultured hu-
man brown and white adipocytes show that although most of
the secreted proteins are detected in both adipocyte types, ~100
proteins are secreted exclusively from brown adipocytes98, thus
indicating an unappreciated breadth of batokines in regulating
systemic physiology.
with changes in the synaptic concentrations of neurotransmitters
during exercise. This increase in neurotransmitter concentration in
turn affects muscle function in the absence of hindered motor-unit
recruitment within the skeletal muscle itself or substantial deple-
tion of substrates for generation of ATP129–131. Thus, the brain during
exercise is acknowledged as a very important functional organ.
Cerebral blood flow. Through the pioneering work of the Secher
laboratory, cerebral blood flow is now known to increase by approx-
imately 25% during exercise, and a parallel increase in metabolism
is observed132–134. During activation, an increase in cerebral O2 sup-
ply is required, because there is no capillary recruitment within
the brain, and increased metabolism becomes dependent on an
enhanced O2 diffusion gradient135. Moreover, the increase in cere-
bral oxygenation during exercise136 contrasts with the contracting
skeletal muscle, where oxygenation progressively decreases135. This
difference between cerebral and muscle oxygenation is likely to
arise because skeletal muscle can sustain activity even if O2 satura-
tion is decreased by 90% (ref. 137), whereas brain function deterio-
rates when its average O2 saturation is decreased by 10% (refs. 138,139).
Therefore, maintaining cerebral blood flow and brain oxygenation
is clearly paramount for sustaining exercise performance. However,
the extent to which the brain plays a role as a metabolic communi-
cator during exercise is less clear.
Myokines and hepatokines in peripheral-organ and central-organ
cross-talk. In the brain, lactate is produced in astrocytes and pro-
vided as a substrate to nearby neurons. Many of the identified myo-
kines and hepatokines discussed above have also been found to
exhibit flux across the brain during exercise. One such peptide is
BDNF, a member of the neurotrophic family of proteins that facili-
tates neurogenesis, synaptic plasticity and cell survival140. BDNF has
Nature Metabolism | www.nature.com/natmetab
Review Article
also been linked to cognition and memory, because its expression
in the hippocampus is diminished in people with depression and/
or Alzheimer’s disease141. In contrast, studies in both rodents and
humans have indicated that exercise training and/or increased fit-
ness levels are associated with maintenance or improvements in
brain biology and function, and have been suggested to help pre-
vent Alzheimer’s disease142–144. The protective role of exercise may be
due to several mechanisms. Exercise is able to enhance neurogenesis
in the adult mouse brain145,146. It is also able to decrease amyloid-β
pathology146, and animals that are more physically active have
diminished amyloid-β accumulation147. Together, such changes are
associated with improved memory in rodent behavioural testing146.
The peripheral factors that contribute to such exercise-induced
changes in the brain remain largely unknown but are now begin-
ning to be identified and studied.
Circulating BDNF is well known to be elevated during exer-
cise148, and although its expression increases in skeletal muscle
during exercise148, the increase in blood is likely to be derived from
platelets148,149. One potential mechanism through which exercise
training may confer protection against neurodegeneration is the
uptake of BDNF by the brain during exercise. Intriguingly, however,
the brain releases rather than takes up BDNF during exercise150,151,
thus suggesting that the central nervous system is also a source of
the increased circulating levels of BDNF during exercise.
As discussed above, HSP72 was identified as one of the first
hepatokines nearly 20 years ago68. Of note, this protein is also
released by the brain during exercise152, but whether this release
has a physiological role is unclear. Finally, the first identified myo-
kine, IL-6, is released from the brain during prolonged exercise in
humans, although the release is neither rapid nor marked153. The
physiological consequences of the release of these molecules from
the brain during exercise are unclear, but, as discussed above, some
muscle-derived factors appear to have roles in cognitive function
during exercise. Hence, the area of exercise and ‘tissue cross-talk’
and their potential roles in neurodegenerative brain pathology is
of great interest and importance. Experiments are currently being
undertaken to screen the flux of proteins across the human brain
with techniques developed by the Secher laboratory135. This work
may shed light on the role of the brain as a metabolic communicator
during exercise.
Role of extracellular vesicles in metabolic communication
during exercise
General considerations. As briefly discussed above, in recent
years, exercise has been increasingly appreciated to transiently
stimulate the release of a host of systemic factors into circula-
tion via EVs. EVs were first described in detail in the early 1980s
by two research groups154,155, which reported that transferrin
receptors located on small reticulocytes are secreted into the extra-
cellular medium. These studies were largely ignored for many
years, because EVs were dismissed as unimportant cellular waste
organelles used only to dispose of molecules unwanted by cells.
However, emerging research has revealed that EVs play a far more
complex role in the body as a facilitator of cell-to-cell communica-
tion156,157. EVs are now well accepted to be actively secreted from
virtually all cells in the body158, and growing evidence indicates that
EVs have important roles in tissue-to-tissue communication during
exercise3,159,160.
EVs transport and deliver bioactive materials such as enzymes,
proteins and microRNAs (miRNAs) to target cells in a paracrine
fashion or via the circulation161,162, which contains a heterogeneous
mixture of EVs comprising apoptotic bodies, microvesicles shed
from membranes and exosomes. In the absence of markers and
purification strategies to clearly distinguish these subtypes, EVs
of unclear subcellular origin can be grouped according to size,
into large EVs and small EVs161,162. Shotgun label-free proteomic
Review Article
Transmembrane
Phospholipid
proteins bilayer
DNAs
RNAs
Proteins
Exosomes
Fig. 3 | EVs traffic biological signals during exercise. Proteins, miRNAs,
DNAs and enzymes are carried in EVs from contracting muscle to the
liver and possibly other organs, such as the brain, pancreas and adipose
tissue, where they release these molecules, thereby modifying biology and
possibly providing a mechanism for the protective effects of exercise in
disease states.
analyses of plasma samples have increased in precision in the past
decade, thus overcoming the challenge of identifying high dynamic
ranges of protein abundance, without limiting the identification
of lower-abundance proteins99. Unbiased, hypothesis-free and
high-coverage proteomic experiments have recently enabled char-
acterization of the secretome during exercise3,159,160.
Role of exercise in extracellular-vesicle mobilization. During
exercise, the number of EVs in the circulation increases approxi-
mately twofold (ref. 53) to fourfold (ref. 163), and the number returns
to pre-exercise levels within 4 h of recovery53,163. This exercise-
induced increase appears to be highly enriched in small EVs,
because significant increases in the small-EV markers ACTN4
(ref. 164), ADAM10 (ref. 162), ALIX165, ANAX11 (ref. 162) and CD81
(refs. 162,165,166) are elevated in EVs in postexercise samples53. Recently,
platelets, endothelial cells and leukocytes have also been found
to contribute to the exercise-induced release of EVs167, and these
cellular subtypes may contribute to the increased number of EVs
in the circulation observed during exercise. As described above,
EVs contain a mixture of enzymes, proteins and miRNAs, and
recent studies have examined all three of these bioactive materials
to some extent.
The regions to which exercise-induced EVs relocate are crucial
to the understanding of the biological importance of their cargo. For
many years, researchers hypothesized a ‘muscle–liver axis’ during
exercise168, and early research on the role of IL-6 as a myokine sug-
gested that this axis is important for maintaining glucose homeosta-
sis38. To investigate the biodistribution of EVs after their liberation
into circulation during exercise, Whitham et al.53 have isolated EVs
from exercised or non-exercised donor mice, labelled them with
lipophilic carbocyanine, intravenously injected them into recipient
mice and analysed the tissue distribution of labelled EVs by intra-
vital imaging. EVs from exercised, compared with non-exercised,
donors revealed marked localization of labelled EVs in the liver
and to a lesser extent, the spleen and lung53. Experiments are cur-
rently underway using stable-isotope labelling with amino acids in
cell culture (SILAC) mice, originally described by the Mann labo-
ratory169. Through donation of purified EVs from SILAC mice, we
are currently constructing an exercise/EV mouse atlas in which
we can determine not only the tissue distribution of exercise EVs
in the donor mice but also the identity of the proteins delivered by
these EVs.
NaTuRe MeTaBolISm
Effect of exercise on EV cargo. Exercise-induced EVs secreted into
the circulation are enriched in glycolytic enzymes matching the high
energy demands of exercise53. Of note, glycolytic enzymes delivered
by EVs can influence the glycolytic rate in recipient cells170,171, but the
functional relevance of the enrichment of glycolytic enzymes imme-
diately after exercise in vivo remains to be elucidated. Many proteins
have been observed in EVs after exercise, including CD36, Flotillin1,
α-sarcoglycan, HSP72, and amyloid-β (reviewed in ref. 105), but the
biological importance of these observations remains unclear.
Through a proteomic screening approach, Whitham et al.53 have
observed 322 proteins whose abundance differs between rest and
exercise, although again, the biological meaning of these changes
is unknown. EVs are also enriched in miRNAs, and several studies
have demonstrated that exercise results in an increase in an array
of miRNAs in EVs (reviewed in ref. 105). Similarly to the observa-
tions with proteins, the biological meaning of such increases is also
unknown.
One method to decipher the roles of miRNAs in a biological con-
text has been elegantly demonstrated by Kahn and co-workers. To
process miRNAs, cells must express the miRNA-processing enzyme
Dicer. Briefly, these researchers have demonstrated that mice with
a fat-specific deletion of Dicer, as well as humans with lipodystro-
phy, have significantly less circulating EV miRNA than controls172.
Moreover, the authors identified several miRNAs in EVs that partic-
ipate in intracellular communication and tissue cross-talk173. During
exercise, EVs are likely to be liberated by the contracting skeletal
muscle, because studies of a-v balance across the contracting limb in
humans suggest that many bona fide myokines are contained in EVs
in the contracting muscles of the limb53. Therefore, exercising skel-
etal muscle–specific Dicer-deficient animals and adoptively trans-
ferring the purified EVs from these and littermate-control animals
into resting donor mice may provide insight.
In summary, EVs during exercise are clearly an important mecha-
nism through which tissue cross-talk can occur (Fig. 3). Physical
activity is well known to modulate many biological processes in organs
distant from the contracting skeletal muscle. Whether EVs play roles
in these processes as well as in the protective effects of exercise in
many diseases will be a fertile area of research in the coming years.
Conclusions and future directions
Research interest in cell-to-cell and organ-to-organ communication
during exercise has grown extensively since the turn of the millen-
nium. We now know that interorgan communication during exer-
cise contributes to the mechanism through which physical activity
can protect the body against an array of lifestyle diseases. Although
the myokine field has been well studied, the roles of the liver, brain,
and both WAT and BAT beds in mediating metabolism during exer-
cise requires further study.
Clearly the most intriguing and emerging field is that of EVs.
Although several groups have shown that exercise modulates the
number of EVs secreted and the tissues to which EVs are trans-
ported after an exercise bout, very little else is known. In fact, to our
knowledge, no studies have pinpointed the specific cargo sent from
one tissue to another during exercise, or the roles, if any, of EVs
and their cargo in regulating metabolism and/or contributing to the
protective effect of exercise in the progression of diseases, such as
cognitive impairments and prevalent metabolic diseases including
type 2 diabetes and non-alcoholic fatty liver disease. This field is
likely to be a productive area of research in the near future.
Received: 28 April 2020; Accepted: 2 July 2020;
Published: xx xx xxxx
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Acknowledgements
M.A.F. is supported by a Senior Principal Research Fellowship (APP1116936) and an Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
Investigator Award (APP1194141) from the National Health & Medical Research Council published maps and institutional affiliations.
of Australia. © Springer Nature Limited 2020

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