Muscle Hormones

C H A P T E R

26 Muscle Hormones Ana M. Rodrı´guez, M. Luisa Bonet, Joan Ribot Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB), CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBEROBN), and Institut d’Investigacio´ Sanita`ria Illes Balears (IdISBa), Palma de Mallorca, Spain

1. INTRODUCTION Skeletal muscle, as a whole organ, can be considered the largest organ in the body, making up about 40% of the body mass. Apart from its elevated proportion in the body, it plays critical roles that rely in contraction related to postural retention and locomotion. Therefore, it is a very relevant organ in the consumption of energy fuels, needed for contraction, thus playing a main role in energy metabolism and regulation (e.g., it is a main site for fatty acid and glucose oxidation) (de Lange et al., 2007; Huh, 2018; Iizuka et al., 2014). Not surprisingly given the ability of skeletal muscle activity to influence whole body metabolism, physical activity and exercise have been widely demonstrated as primary ways to maintain and improve health (Huh, 2018; Whitham and Febbraio, 2016). Metabolic consequences of muscle contraction, related with physical activity and exercise training, can logically help to explain the relation with health: improvements in cardiorespiratory fitness, better lean/fat mass ratio, reduction of lipemias, decreased inflammation, improved liver fat and glucose metabolism, and maintenance/construction of muscle mass, etc. (Booth et al., 2012; Hawley et al., 2014; Starkie et al., 2003; Whitham and Febbraio, 2016). However, beneficial effects of physical activity cannot be solely attributed to the simple view of muscle contraction as a way to increase fuel consumption and energy expenditure (and thus counteract the development of overweight/obesity and their related metabolic disturbances). Another very relevant function importantly involved in all these beneficial effects is skeletal muscle secretory activity: the view of skeletal muscle as an endocrine organ (with also autocrine and paracrine activities), able to produce and release signaling molecules

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00026-2

that regulate whole body metabolism and help metabolic integration, communicating muscle with other organs in the body. This will be the focus of our chapter. The signal molecules secreted by muscle are denominated myokines. The term myokine is often defined as “a cytokine or peptide that is produced by skeletal muscle cells and subsequently released into the circulation to exert endocrine or paracrine effects in other cells, tissues or organs” (Febbraio et al., 2004; Whitham and Febbraio, 2016), although the term has been widened to include nonprotein signaling molecules, as we will see in this chapter. The idea of muscle as an endocrine organ is not so new because, actually, more than 50 years ago it was already suggested that skeletal muscle could secrete humoral factors, associated to contraction, which would affect the physiology of other organs (Goldstein, 1961). Nonetheless, the important turning point was approximately with the entrance of the new millennium and the description of interleukin-6 (IL-6) as a skeletal muscle secreted protein, in response to contraction/exercise, with effects in different tissues/organs, now considered the prototypical myokine (Febbraio and Pedersen, 2002; Pal et al., 2014; Pedersen and Febbraio, 2008; Whitham and Febbraio, 2016). Since then, other putative, or even already demonstrated as true, myokines have been discovered and studied. In the present chapter, we will focus in depth on two very relevant myokines, both related to muscle contraction and both with suggested and proven health benefits: IL-6, as the prototypical one, and Irisin, more recently discovered but with specific characteristics that make it to be considered a true myokine. We will also briefly revise other myokines, together with the present state of the art and future directions in the field.

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2. INTERLEUKIN-6 IL-6 is nowadays considered a main insignia of the paradigm of skeletal muscle as an endocrine organ. The groups of B.K. Pedersen and M.A. Febbraio and colleagues pioneered and deeply influenced this paradigm, with a main focus on IL-6 produced in association with muscle contraction and exercise or physical activity and its antiinflammatory and metabolic effects, important to explain health benefits of exercise (Benatti and Pedersen, 2015; Karstoft and Pedersen, 2016; Pedersen and Febbraio, 2012). Since the recognition of skeletal muscle as an important producer of IL-6, numerous groups and wide research evidence have supported endocrine and metabolic roles of this cytokine, as it will be explained later in this chapter. Nevertheless, historically, the concept of muscle-derived IL-6 as a myokine has had to coexist and get along with the paradoxical fact that IL-6 was previously described and widely studied as a typical proinflammatory cytokine, mainly produced by the cells of the immune system (and also secreted by many other cell types), with pleiotropic effects, and involved in inflammatory processes and diseases (Benatti and Pedersen, 2015; Garbers et al., 2018; Karstoft and Pedersen, 2016). This paradox has started to be unraveled in recent years, and the myokine role of IL-6 has gained substantial relevance, reflected in an extensive number of original papers and reviews, thus becoming an important focus for the study of the beneficial effects of physical activity and exercise.

2.1 Structure of IL-6 and Its Receptor Complex The human IL-6 preprotein has 212 amino acids and, upon maturation, a 28 amino acid signal peptide is cleaved, rendering the mature 184 amino acid IL-6 protein (Yasukawa et al., 1987). The three-dimensional structure of mature IL-6 (Somers et al., 1997) consists of a bundle of four antiparallel helixes (AeD) connected by loops and an extra smaller (mini) helix (E), enclosing a core of hydrophobic amino acidic residues. In fact, IL-6 belongs to a family of four-helix-bundle cytokines denominated after itself: the IL-6-type cytokine family, which also includes other important cytokines such as cardiotrophin 1 (CT-1), cardiotrophin-like cytokine, ciliary neurotrophic factor (CNTF), IL-11, IL-27, leukemia inhibitory factor, and oncostatin M (OSM), with pro- and antiinflammatory actions (Rose-John, 2018). The IL-6-type cytokines form a family since they share the property to form complexes with receptors containing one or two molecules of the signaling receptor subunit glycoprotein 130 (gp130, 130 kDa) (Rose-John, 2018), which is a cell plasma membrane ubiquitous

protein and the common transducer of this family. IL-6 has three epitopes, denominated “sites,” important for the formation of the receptor complex: sites I, II, and III, which are conserved epitopes that bind the gp130 subunit, with site III being exclusive to this family of cytokines (Bravo et al., 1998; Chow et al., 2002; Kalai et al., 1997; Simpson et al., 1997). The IL-6 receptor complex also entails interactions of sites I, II, and III with the IL-6-specific receptor (IL-6R), besides gp130, to form composite sites (for more information, see Boulanger et al., 2003). Structure-based sequence alignment studies of the sites for interaction with IL-6R and gp130 in mouse vs. human IL-6 suggest that site III interactions might be of greater importance, mediating the assembly of the IL-6/IL-6R/gp130 ternary complex (Veverka et al., 2012). Signal transduction through the IL-6 receptor system is intricate, and three types of signaling have been described so far: classic signaling, trans-signaling, and trans-presentation (Garbers et al., 2018). The classic signaling depends on the initial interaction of IL-6 with its membrane receptor IL-6R (also referred as IL6Ra). The initially translated protein from IL-6R mRNA has 468 amino acids that include a signal peptide (for the secretory pathway), an extracellular region with different domains, a transmembrane domain, and a final short cytoplasmic region. Moreover, the receptor is glycosylated in the secretory pathway, to give rise to a mature IL-6R of 80 kDa (Hirata et al., 1989; Varghese et al., 2002). The interaction of IL-6 with its membrane receptor IL6R is not enough to transduce the signal; instead, after binding, IL-6/IL-6R needs to form a ternary complex with gp130; therefore gp130 (also referred as the subunit b of the complex) is necessary for IL-6 signal transduction to take place (Garbers et al., 2018). The structure and assembly of the IL-6/IL-6R/gp130 complex was significantly clarified in 2003, by crystallography analysis by Boulanger et al. (2003), who described a hexameric complex where two molecules of each component (IL-6, IL-6R, and gp130) are present, and it was further clarified by electron microscopy (Skiniotis et al., 2005). In the hexameric model (see Fig. 26.1), the molecules of IL-6, IL-6R, and gp130 assemble in a sequential manner and cooperatively (Boulanger et al., 2003). The extracellular domains of both the IL-6R and gp130 receptors contain three b-sheet sandwich domains (D1, D2, and D3 from farthest to nearest to the plasma membrane), and gp130 contains three more domains proximal to the membrane (D4, D5, and D6) (Skiniotis et al., 2005). Neither IL-6 nor IL-6R alone are able to significantly bind gp130 (very low affinity), but first, IL-6 interacts (through site I) with an epitope formed by D2-D3 domains in IL-6R, establishing a nonsignaling binary complex. Thereafter, this binary

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FIGURE 26.1 Molecular model of IL-6 signaling transduction “classic” pathway. IL-6 binding to its membrane-bound receptor IL-6Ra and to the signal transducing glycoprotein gp130 (1) causes the dimerization of the ligated IL-6/IL-6R/gp130 receptor complex, leading to the formation of the hexameric complex and the activation of the constitutively gp130-bound JAK kinases by transphosphorylation (2). Active JAK phosphorylates specific tyrosine residues in the cytosolic part of each gp130 (3) enabling the STAT transcription factor to bind the phosphotyrosine residues, where it becomes substrate of active JAK (4a). Phosphorylated STATs dimerize and, as dimers, translocate into the nucleus to activate the transcription of multiple genes (4b), including SOCS 3, which binds to phosphotyrosine-759 of gp130 (4c), thereby inhibiting JAK and causing the negative feedback regulation of the JAK-STAT intracellular signaling cascade. Phosphotyrosine-759 also acts a docking site for the adaptor molecule SHP2, which is important for initiation of the PI3K-Akt/PKB and the MAPK intracellular signaling pathways, as well as for negative feedback via dephosphorylating gp130 (5). Active JAKs seem to also activate AMPK intracellular signaling (6). See also main text for more complete information.

complex interacts with the D2eD3 domains of the gp130 receptor, allowing it to dimerize with another gp130 receptor, previously bound to another IL-6/IL-6R complex by the same sequential and cooperative way, thus forming the final, signaling-competent, hexameric complex (Boulanger et al., 2003; Skiniotis et al., 2005). The latter contains two copies of each of the three molecules involved, IL-6, IL-6R, and gp130, organized in two trimolecular complexes that dimerize (Boulanger et al., 2003; Skiniotis et al., 2005). In the dimerized structure, the structural model describes the bending of the legs (by the membrane proximal domains) of the two gp130 receptors involved, thus allowing forming gp130-gp130 contact before entering the cell membrane (Skiniotis et al., 2005). This conformation forces the close

apposition of the receptor dimer and thus allows JAK trans-phosphorylation in the gp130 intracellular domains and the initiation of the transduction of signal (Boulanger et al., 2003; Skiniotis et al., 2005; Varghese et al., 2002) (see next section and Fig. 26.1). Whereas gp130 is ubiquitous, IL-6R is significantly expressed only in certain types of cells or tissues including blood cells (macrophages, neutrophils, and some types of T cells), liver, skeletal muscle, adipose tissue, and the brain (including hypothalamus and hippocampus) (Cron et al., 2016; Scheller et al., 2011; Uhle´n et al., 2015) (see also IL6R in Human Protein Atlas, available from www.proteinatlas.org). Thus, tissues not expressing IL-6R cannot respond to IL-6 via classic signaling.

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A second type of IL-6 receptor is the soluble form, sIL6R, which can be generated by two ways: alternative splicing of the IL-6R mRNA (which accounts for a smalldabout 10%dfraction of total sIL-6R) (Lust et al., 1992; Wolf et al., 2014) and proteolytic scission of the IL6-R by the protease ADAM Metallopeptidase Domain 17 (ADAM17, of the metalloproteinase and disintegrin families) (Riethmueller et al., 2017). Main cells shedding sIL-6R are activated neutrophils and other immune cells (Cron et al., 2016). The affinity of the IL-6 binding to sIL6R is similar to the affinity for the membrane IL-6R, and IL-6/sIL-6R interaction also has the potential to recruit and induce dimerization of gp130 on the cell membrane and therefore start signaling, with the additional property of the relatively elevated circulating levels of sIL-6R; therefore, cells not expressing the membrane receptor can also respond to IL-6 by this second type of signaling, denominated trans-signaling (Calabrese and Rose-John, 2014; Cron et al., 2016; Garbers et al., 2018). The biology of IL-6 signaling has additional levels of complexity. Alternative splicing or polyadenylation of gp130 mRNA, and to a lesser extent proteolytic cleavage of the gp130 protein, can originate several different forms of soluble gp130 (sgp130), which can block the IL-6/sIL-6R (trans-)signaling (Wolf et al., 2014, 2016). Another additional point of complexity is given by the blood concentration of the different components of the system. The circulating levels of IL-6 are variable depending on the physiologic situation: in basal nonpathologic and nonexercise-stimulated conditions, levels are about 1e5 pg IL-6/mL, and they can increase up to the range of ng/mL in pathologic conditions (Calabrese and Rose-John, 2014; Garbers et al., 2018). sIL-6R levels oscillate at about 40e80 ng/mL, and sgp130 at approximately 400 ng/mL (Jones et al., 2011). Thus, environmental/physiologic- or pathologic-induced changes in these concentrations, with possible differences in local/paracrine concentrations, and the presence or absence of membrane IL-6R, will determine the final integrated signaling response of a cell/tissue type, together with the presence of other signaling molecules. It is important to note that IL-6 trans-signaling is the specific signaling type that has been more effectively associated with proinflammatory effects, while the classic pathway would not be associated with proinflammation (Calabrese and Rose-John, 2014). The third type of IL-6 signaling, i.e., transpresentation, is a recent discovery and is much more specific. It takes place in the framework of IL-6 provided by dendritic cells (which would act as the signalproducing cells) signaling to T cells (target cells) to commit the T cells to a highly efficiently tissuedestructive phenotype. The peculiarity of this signaling is that trans-presentation of IL-6 is done from dendritic cells to T cells via the dendritic cell membrane IL-6R

(Garbers et al., 2018; Heink et al., 2017). Although this third type of signaling is a recent, specific, discovery and will not be further discussed in this chapter, it must be noted that the three types of signaling (i.e., classic, trans-signaling, and trans-presentation) converge in the modus operandi of receptor complex dimerization (which includes the necessary interaction with gp130) to activate the intracellular signaling corresponding cascades (Garbers et al., 2018).

2.2 Biochemical Reactions of IL-6: Signaling and Secretion We have explained in the previous section the chemical and structural interactions of IL-6 with its specific receptors and the formation of the hexameric complex that includes gp130, as the receptor complex necessary to initiate signal transduction. Now, we are going to deepen discussion on the signaling cascades after receptor activation. 2.2.1 Mechanisms of Signal Transduction Induced by IL-6 Following hexameric complex formation, the general mechanisms of IL-6 signal transduction rely on typical cytokine transduction intracellular pathways (Fig. 26.1). Thus, the homodimerization of the gp130 receptors activates several signaling cascades/pathways in the cytoplasm: the JAK/STAT (Janus kinase/signal transducer and activator of transcription), PI3K (phosphoinositide 3-kinase), MAPK (mitogen-activated protein kinase), and YAP (YES associated protein 1) pathways (Calabrese and Rose-John, 2014; Garbers et al., 2018; Mauer et al., 2015). JAKs are very important for IL-6 signaling. In fact, JAK proteins are very common signal transducers for cytokines in general. Cytokine receptors do not have intrinsic tyrosine protein kinase activity, but they have JAKs constitutively associated; thus a JAK molecule is associated to the intracellular domain of each gp130 of the dimer (different JAK isoforms can be involved, as we will discuss subsequently) (Babon et al., 2014a; Bla¨tke et al., 2013; Calabrese and RoseJohn, 2014; Garbers et al., 2018; Munoz-Canoves et al., 2013). The association of JAKs to gp130 seems to be mediated by two conserved regions denominated box1 and box2 (Heinrich et al., 1998). Dimerization of gp130 upon hexameric complex formation determines the approximation of the intracellular domains of the two gp130 molecules, allowing the JAK bound to each one to be activated by trans-phosphorylation, thus firing the cascade. Afterward, the activated JAKs phosphorylate five tyrosine residues of the cytoplasmic domain of gp130; these phosphorylations cause a

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conformational change that creates docking sites for proteins (which can be adapters or effectors) that contain an SH2 domain (phosphotyrosine recognition domains), such as STAT (with docking sites at the four carboxy-terminal phosphotyrosines of the gp130), or the phosphatase SHP2 (with a docking site in Tyr-759) (Babon et al., 2014a; Bla¨tke et al., 2013; Calabrese and Rose-John, 2014; Garbers et al., 2018). The docked STAT proteins, such as STAT1 and 3, are phosphorylated by JAKs; then, the STAT proteins form homo- or heterodimers and translocate to the nucleus, where they act as transcription factors of key genes for the cellular response (Bla¨tke et al., 2013; Garbers et al., 2018). Docking of SHP2 (also known as PTPN11 and ubiquitously expressed), in its turn, initiates two other important signaling pathways activated by the cytokine: the PI3K-AKT (AKT is also known as protein kinase B) pathway and the MAPK pathway (Babon et al., 2014a; Garbers et al., 2018). The general functioning of the signaling cascades of PI3K-AKT and MAP kinases are widely known and reviewed elsewhere (for a recent revision, see Nelson and Cox, 2017); these cascades regulate diverse intracellular functions and gene expression in the nucleus of specific target genes. The activated SHP2 phosphatase also serves to negate feedback regulation, since it counteracts JAK by dephosphorylating phosphotyrosines of gp130 (Bla¨tke et al., 2013). In the YAP pathway, after IL-6 activation of the receptor complex, two members of the SRC family of protein kinases (Parsons and Parsons, 2004), YES and SRC, which are intracellular protein kinases, interact with the cytoplasmic domain of gp130 and phosphorylate the transcriptional coactivator YAP1. Phosphorylated YAP1 translocates to the nucleus, where it regulates gene expression by interacting with specific transcription factors, such as for genes involved in the regulation of proliferation and cell growth (Garbers et al., 2018). IL-6 signaling is short-lived. SOCS (suppressors of cytokine signaling) proteins are prominent actors in a negative feedback loop to suppress signaling by IL-6 and other cytokines. Within the SOCS family, SOCS3 is especially relevant in the control of the signaling by the IL-6 family of cytokines (Babon et al., 2014b) (Fig. 26.1). Importantly, SOCS genes are among the genes whose expression is induced by STAT proteins, typically activated by IL-6, as explained earlier. SOCS3 proteins bind phosphorylated Tyr-759 in gp130 receptors and directly interact with JAK proteins there, inhibiting them, therefore shutting down the JAK/STAT cascade (Babon et al., 2014b; Bla¨tke et al., 2013). Moreover, as explained before, phospho-Tyr-759 also recruits the phosphatase SHP2 activating the PI3K and MAPK pathways, so SOCS3 also inhibits these other branches

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of the signaling cascades associated to IL-6 activity (Babon et al., 2014b). The families of JAK/STAT proteins have different members, and differences among them might be important when distinguishing the IL-6 effects in different cell types and depending on the physiologic environment. Moreover, the use of the different signaling pathways seen, such as PI3K and MAPK, together with other possible pathways related to IL-6 (such as AMP activated protein kinase (AMPK) signaling, see later), can be highly dependent on the cell type and metabolic context (Carey et al., 2006; Lai et al., 1999; TakahashiTezuka et al., 1998). 2.2.2 IL-6 Signaling in Skeletal Muscle Skeletal muscle is not only an important producer of IL-6: it is also a key target for the myokine, which has autocrine and paracrine effects. Skeletal muscle shows significant expression of the membrane-bound IL-6R (Pedersen and Febbraio, 2012; Uhle´n et al., 2015), thus allowing classic signaling. It must be noted that muscle activities and the myogenic process can be affected not only by muscle-secreted IL-6 but also by IL-6 released by inflammatory cells infiltrated in the tissue (MunozCanoves et al., 2013). The JAK/STAT signaling pathway appears to be a major pathway in muscle for response to IL-6, governing basic functions such as proliferation and differentiation. This pathway has been linked with both the growth and differentiation of myoblasts, and it is involved in satellite cell-dependent myogenesis (Munoz-Canoves et al., 2013). It has been described that JAK1, STAT1, and STAT3 are activated early in the process of rapid proliferation of satellite cells, and they are involved in myoblast proliferation by inducing the expression of cell cycle proteins (Munoz-Canoves et al., 2013; Sun et al., 2007). Other studies showed that the JAK1/ STAT1/3 pathway sets a checkpoint for differentiation of myoblasts, which can only start when a sufficient cell number has been achieved after proliferation (Munoz-Canoves et al., 2013; Sun et al., 2007). Nonetheless, JAK1, STAT1, and STAT3 are not the only JAK/ STAT family members with key functions in muscle. JAK2 is also involved together with STAT2 and 3 in the myogenic differentiation pathway, with opposite actions to JAK1/STAT1/3 (Munoz-Canoves et al., 2013), possibly allowing a fine-tuning of the process. In general, different members of the JAK/STAT family have a role in the proliferation and differentiation of muscle cells, in coordination with other factors and different signals. IL-6 has been proposed as one of the potential signals controlling these actions on muscle growth and differentiation. This is because, apart from being able to activate JAK/STAT pathways, IL-6 is expressed during differentiation in myoblasts and is able to

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stimulate their differentiation and fusion (Baeza-Raja and Mun˜oz-Ca´noves, 2004; Hoene et al., 2013; MunozCanoves et al., 2013). Of course, in the complex process of myogenesis, IL-6 probably does not act alone, and several data (both from cell and in vivo models) suggest that different members of the IL-6 family of cytokines participate in myogenesis, acting at different stages and combining different signaling pathways (MunozCanoves et al., 2013). Another pathway that has been suggested to be activated by IL-6 in skeletal muscle is the AMPK signaling pathway, although its specific activation and role here is less known so far. Experimental evidence links IL-6 with the enhancement of lipolysis and fat oxidation, as well as GLUT-4-mediated glucose uptake in skeletal muscle, via AMPK activation, and it seems that this AMPK activation might be a downstream effect of JAK activation (Fig. 26.1) (Al-Khalili et al., 2006; Benatti and Pedersen, 2015; Carey et al., 2006; Kelly et al., 2009; Mauer et al., 2015; Sarvas et al., 2013). 2.2.3 IL-6 Production and Secretion by Muscle Cells Stimuli that induce IL-6 expression and secretion can significantly vary depending on the physiologic situation and the producing cell type. As already stated, along its history, IL-6 has been discussed both as a pro- and antiinflammatory cytokine; however, the skeletal muscle/physical activity associated IL-6 production, which is the focus of this chapter, seems to be related to antiinflammatory and metabolic effects. We will now concentrate on the present knowledge about the stimuli that promote muscle IL-6 production and secretion related to physical activity or exercise. Accordingly, one key stimulus for muscle IL-6 production is contraction: IL-6 is the first cytokine secreted to blood from muscle during exercise (Benatti and Pedersen, 2015; Pedersen and Febbraio, 2008), exponentially increasing in blood up to 100-fold or even 120-fold after exercise (depending on the type and duration of it) and thereafter (during recovery) decreasing steadily (Benatti and Pedersen, 2015; Peake et al., 2015; Pedersen and Febbraio, 2008). In inflammatory cells such as macrophages, the induction of IL-6 expression depends on the previous induction and activation of the transcription factor NF-kB (nuclear factor kappaB), but this is not the case of myocytes in an exercise/noninflammatory context, where the experimental evidence indicates that IL-6 production depends on downstream events after the intracellular Ca2þ increase triggered by contraction, probably involving a crosstalk between Ca2þ, the nuclear factor of activated T cells (NFAT), and p38 MAPK (Benatti and Pedersen, 2015). Ca2þ activates p38

MAPK and calcineurin, which in turn activate specific transcription factors and coactivators (CREBP/p300/ CBP and AP-1/NFAT) that upregulate IL-6 expression (Benatti and Pedersen, 2015) (CREBP: cyclic AMPresponse element binding protein, CBP: CREB binding protein; AP-1: activator protein 1). Another stimulus affecting the efflux of IL-6 from muscle might be the carbohydrate availability. For instance, it has been reported that the ingestion of carbohydrates during exercise causes a decrease in IL-6 blood levels (without affecting mRNA levels), therefore suggesting that carbohydrate availability is important in the regulation of IL-6 levels during exercise (Lutoslawska, 2012); this makes sense since IL-6 induces fat use as fuel, so the presence of different substrates and their use (carbohydrates and fatty acids) can be finely regulated. See also later (in the physiologic actions Section 2.3.1) the relationship of muscle IL-6 with glycogen levels. IL-6 secretion by skeletal muscle can be affected by other tissues functioning also as endocrine organs. For instance, a recent work (Mera et al., 2017) has reported that bone, acting in an endocrine way, can release osteocalcin in response to exercise. Osteocalcin exerts interesting activities in skeletal muscle. It increases glucose uptake, but also the production and secretion of muscle IL-6, whose activity in turn would provide fuels for exercise, by increasing fatty acids from adipose tissue lipolysis and glucose from liver gluconeogenesis (Febbraio, 2017; Mera et al., 2017). It must be noted that skeletal muscle is a complex tissue that not only comprises myocytes (muscle fibers). Apart from myoblasts and myocytes, and the presence of satellite cells, other cell types can be found in the tissue: fibroblasts, myeloid cells, and pericytes (also able to secrete cytokines) (Peake et al., 2015). IL-6 has been located both in muscle fibers (both type I and II), but also in satellite cells and fibroblasts (Peake et al., 2015). Regarding the type of muscle fibers, it has been reported that IL-6 production in response to exercise is higher in fast-twitch (type I) than slow-twitch (type II) fibers (Lutoslawska, 2012). At any rate, it is clear that skeletal muscle produces IL-6 highly significantly in response to exercise, increasing IL-6 expression and secretion. Although the activity of skeletal muscle as a secretory organ is well established, little is known so far about the secretion pathways in muscle cells and their regulatory mechanisms (Giudice and Taylor, 2017). In the case of IL-6, a study by Lauritzen and colleagues gave some clues (Lauritzen et al., 2013). The study was performed in murine fixed and living muscle fibers with transfected green fluorescent protein tagged to IL-6. At rest, IL-6positive vesicles were localized intracellularly in the muscle fibers (at the sarcolemma and T-tubule regions).

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After inducing contraction, the vesicles with the fluorescent tag decreased both at the surface and interior of the fibers, thus suggesting that the secretory function of the muscle fibers would act in a typical way as classic endocrine cells: the muscle cells would contain depots of IL-6 in intracellular vesicles that would be secreted upon contraction stimulation (Lauritzen et al., 2013).

2.3 Physiological Functions of IL-6: Muscle IL6 in Health and Disease As highlighted, our focus in this chapter is on muscle IL-6, its role as a myokine and, consequently, its metabolic actions and its relation with physical activity or exercise. Therefore, this section will be mainly focused on such a metabolic role, and not the more classical roles of IL-6 related with immune function. Nevertheless, since, as we have seen when explaining the receptor system and its associated signaling cascades, IL-6 is a very complex cytokine, with a very complex biology and pleiotropic effects, some concepts must be clarified first. As stated earlier, IL-6 was first discovered and studied in relation with its immune functions. More than 30 years ago, in 1986, its complementary DNA was isolated, and it was first described as a novel human interleukin able to induce immunoglobulin production by B lymphocytes, termed BSF-2 (B-cell stimulatory factor 2), also receiving other names in the literature, although now it is well established as IL-6 (Hirano et al., 1986; Kishimoto, 2010). It was only after the beginning of the new millennium that IL-6 started to receive attention related to its metabolic actions as a myokine (Whitham and Febbraio, 2016). In the vast literature, IL-6 has often been classified as a “good” or “bad” molecule, since (increased) circulating levels were associated with obesity and inflammation, and both as an anti- and proinflammatory cytokine (with the proinflammatory role as the “bad” one). However, this view is too simplistic. To begin with, proinflammatory responses are important in the function of the immune system, although, of course, an excess of IL-6 production in a proinflammatory environment can be considered a detrimental phenotype, such as happens in diseases as rheumatoid arthritis (RA) and other inflammatory diseases, which could include obesity, certain autoimmune diseases, inflammatory colon cancer, etc. (Garbers et al., 2018; Scheller et al., 2011). In fact, in diseases as RA, blocking IL-6 signaling has been proven as a therapeutically effective treatment, though with some detrimental metabolic side effects. In particular, treatment with the antibody tocilizumab (anti-IL-6R, which prevents all types of signaling: classic, trans-signaling, and transpresentation), approved in multiple countries, is effective for RA and other inflammatory diseases, but it has

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been associated with nondesirable metabolic effects such as increase in serum triglycerides, total cholesterol, and body weight, thus suggesting that IL-6 signaling actually is beneficial for glucose and lipid metabolism (Cron et al., 2016; Garbers et al., 2018). Furthermore, antiinflammatory effects have been attributed to IL-6 in a wide number of studies and publications, and particularly when considering the muscle-derived IL-6 in connection to exercise (Benatti and Pedersen, 2015). Moreover, muscle IL-6 has been associated to different metabolic effects, far beyond its role in inflammation (discussed in more detail subsequently). These antiinflammatory and metabolic effects have been frequently associated to muscle IL-6 as a myokine, and muscle IL6 has been suggested as one of the main actors to explain the health benefits of exercise, as discussed in this chapter. Nonetheless, IL-6 cannot be classified as “good” or “bad” either depending on its antiinflammatory (or even metabolic) or proinflammatory actions nor on the tissue or cell type of origin. For instance, increased IL6 (together with TNF, tumor necrosis factor) was one of the first adipokines associated to obesity and has thus been frequently recognized (even as a marker) in the context of metabolic inflammation or metainflammation related to the obese phenotype, and one of the molecules responsible for insulin resistance in such condition (Mauer et al., 2015). Strikingly, recent findings seem to indicate that even IL-6 associated to obesity (not only the “muscle-exercise” IL-6) might have homeostatic and antiinflammatory roles in obesity meta-inflammation and metabolic disorders (Mauer et al., 2015). In particular, findings suggest that the IL-6 produced in the adipose tissue in the course of obesity would favor the maintenance of one specific type of macrophages (M2), with an antiinflammatory role, in adipose tissue and limit endotoxemia and insulin resistance in obesity (Mauer et al., 2015), thus challenging the general idea of the proinflammatory role of adipose tissue macrophage-produced IL-6. Of course, we are not suggesting that chronically elevated levels of IL-6, in general, would turn out to have positive effects on health, since the literature has widely shown examples of the contrary, for instance in inflammatory diseases (as RA), and other examples such as the role of IL-6 in hepatocellular carcinoma (IL-6 overproduction is common in multiple cancer cells, and increased IL-6 levels in blood correlate with poor outcomes in these patients) (Mauer et al., 2015). What we want to highlight is the high complexity of IL-6 biology and the fact that more research is needed to understand it in different metabolic and disease contexts. If we focus on IL-6 as a myokine, even here, it is important to clarify some aspects of the complex biology of this cytokine that will be important to understand the actions of muscle-secreted IL-6, especially in response to

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contraction. As already stated, the effects of IL-6 are very context-dependent, and some key facts can give us clues about the behavior of IL-6 as a pro- or antiinflammatory molecule. First, the proinflammatory effects of IL-6 have been linked to trans-signaling (involving sIL-R), whereas classic signaling (involving membrane IL-R) would rather be involved in antiinflammatory and regenerative/protective effects in metabolism (Karstoft and Pedersen, 2016; Scheller et al., 2011). Second, the soluble form of gp130 (sgp130), can block IL-6/sIL-6R complex functions as a buffering system in steady-state conditions (Garbers et al., 2018; Rose-John, 2018). Therefore, the relative concentrations (both in general circulation and locally, in a paracrine context) of all these molecules will be relevant. Third, the molecules that accompany IL-6 induction are also important, such as TNF. For instance, one theory states that elevated IL-6 would be a result of TNF increase, and that TNF coming from adipose and other inflamed tissues would be the main culprit of detrimental effects (Benatti and Pedersen, 2015). Fourth, it seems also important if IL-6 is chronically elevated (usually associated with inflammatory conditions) or acutely elevated (as happens after

exercise) (Cron et al., 2016). Finally, five and very important to our chapter, it seems key the type of cells/tissues where IL-6 is produced. In fact, during exercise, the main source (almost exclusive) of the rise in blood IL-6 is skeletal muscle, while up to 35% of blood IL-6 can arise from adipose tissue in obesity (Cron et al., 2016). Bearing in mind all these considerations, we will next concentrate on the physiologic, generally healthpromoting, effects of IL-6 as a myokine, produced by a specific tissue, i.e., skeletal muscle acting as an endocrine organ, associated to contraction/exercise (and the specific stimuli involved), and targeting tissues that express the membrane-bound IL-6R, which can then undergo classic (antiinflammatory, metabolismregulating) signaling (see also Fig. 26.2). 2.3.1 Muscle IL6 Physiology and Effects in Skeletal Muscle As stated, the signals for IL-6 expression in muscle cells are not inflammation-related and are strictly different from the signals involved in its expression in macrophages (see Section 2.2.3 IL-6 production and secretion by muscle cells). Therefore, IL-6 production and

FIGURE 26.2 Roles of IL-6 and irisin produced by active muscle in different organs and cell types. Some of the suggested effects represented in the figure have more support in the literature than others, where the evidence is more recent. See main text for more about each specific proposed role as well as other roles of these two myokines.

2. INTERLEUKIN-6

signaling in working muscle seems to be independent of both NF-kB activation and TNF induction. Induction of IL-6 production by muscle contraction usually is not associated to the production of TNF or other proinflammatory cytokines; only when the exercise is prolonged and strenuous is a small circulating TNF increase observed (Benatti and Pedersen, 2015). As already discussed, these facts are in line with the idea that muscle IL-6 is more related to metabolic roles than to inflammation. Moreover, muscle IL-6 production is also related to the levels of intramuscular glycogen, and carbohydrate availability in general. IL-6 mRNA expression and protein secretion are significantly enhanced when glycogen levels inside muscles are low and muscle glycogen stores are depleted; because of that, IL-6 has been considered an energy sensor in exercise (Lutoslawska, 2012; Benatti and Pedersen, 2015). Moreover, ingestion of carbohydrates during exercise causes a decrease in IL-6 blood levels: therefore, carbohydrate (not only glycogen) availability seems also relevant in the regulation of muscle IL-6 levels and secretion (Lutoslawska, 2012). As a myokine, IL-6 has auto- and paracrine actions in the same muscle, besides endocrine effects mediating crosstalk with other organs. IL-6 has been shown to increase muscle glucose uptake (related with GLUT4 translocation to the plasma membrane), as well as lipolysis and fat oxidation in muscle (this last effect probably via AMPK activation) (Benatti and Pedersen, 2015; Cron et al., 2016; Pedersen and Febbraio, 2012). The rationale behind these effects is clear considering the need of fuel substrate regulation during exercise to burn glucose and fatty acids to fulfill the demands of the working muscle. IL-6 might be also important for the regulation of muscle metabolism during recovery. A recent work using specific muscle IL-6 KO mice (Knudsen et al., 2017) highlights the role of muscle-derived IL-6 in orchestrating the coordination of muscle with adipose tissue to regulate fuel utilization in the recovery phase, concluding that muscle IL-6 is important in the restoration of homeostasis after exercise (Knudsen et al., 2017). These results, together with other findings (see the next Section 2.3.2), outline the relevance of muscle IL-6 as an endocrine regulator in the crosstalk between two relevant tissues in energy homeostasis. IL-6, together with other myokines, has been involved in the promotion of muscle hypertrophy (Pedersen and Febbraio, 2012). It has been reported that IL-6 is an important regulator of hypertrophic muscle growth mediated by satellite cells (Serrano et al., 2008). It is also relevant in the regeneration of muscle, where IL-6 can come from infiltrating macrophages and neutrophils, fibroadipogenic progenitors, and satellite muscle cells, probably helping the myogenic process from satellite cells (Munoz-Canoves et al., 2013).

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The IL-6 system is also important in the resting but trained skeletal muscle. Different studies have outlined the inverse relationship between basal (resting) IL-6 circulating levels and exercise practiced regularly (Fischer et al., 2004; Pedersen and Febbraio, 2012), probably associated to increased IL-6 sensitivity. For instance, endurance training has been shown to significantly upregulate the membrane receptor (IL-6R) expression (classic signaling) (Keller et al., 2005). 2.3.2 Effects of Muscle IL-6 in Adipose Tissue One clear idea in the literature regarding the metabolic roles of skeletal muscle IL-6 is that this myokine is an important mediator of muscle crosstalk with other tissues, and white adipose tissue is one of the main IL-6 targets in this sense (Huh, 2018; Pedersen and Febbraio, 2012). There is evidence including evidence from human studies that IL-6 increases lipid mobilization in adipocytes, i.e., lipolysis, thus having a relevant role in fat metabolism (Huh, 2018). IL-6 also favors the oxidation of the released fatty acids, mainly in other organs, such as skeletal muscle. Interestingly, the more recent paradigm in energy metabolism research, the browning of adipose tissue (also known as beiging or brightening) (Cannon and Nedergaard, 2012), also seems to be a target of IL-6 action. It has been suggested that IL-6 induction associated to exercise training is involved in the induction of UCP1 (uncoupling protein 1, key mediator of adaptive thermogenesis) expression in white adipose tissue depots (browning) (Knudsen et al., 2014). Thus, in mice, exercise training increased UCP1 mRNA levels in inguinal white adipose tissue, and this effect was lost in IL-6 KO mice. Fat browning stimulated by exercise-induced muscle IL-6 might be another metabolic molecular mechanism potentially involved in the promotion of a more healthy/lean phenotype associated with regular exercise. The crosstalk between skeletal muscle and adipose tissue is important in terms of providing energy fuels (by adipose tissue releasing free fatty acids by lipolysis) to muscle when its energy demands are increased, as during physical activity. In this sense, it has also been described that both leptin (a prototypical adipokine with key functions in metabolism) and certain free fatty acids induce IL-6 expression and/or secretion in skeletal muscle cells (Nozhenko et al., 2015; Sanchez et al., 2013). These works suggest a role of leptin and free fatty acids released by adipose tissue in crosstalk with skeletal muscle, regulating muscle IL-6 production and thereby closing a metabolic circle where muscle IL-6 regulates secretion of adipose tissue, which in turn secretes molecules that regulate muscle IL-6 production.

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2.3.3 Effects of Muscle IL-6 in Brain The possible functions of exercise-induced muscle IL6 on brain function and signaling are barely known. In fact, IL-6 is released from the brain itself after exercise, and central, rather than peripheral, IL-6 might be responsible for observed effects (Nybo et al., 2002). Different effects on important neurotransmitters have been associated to exercise, but little is known about how contracting muscles communicate with different parts of the brain and affect such neurotransmitters (Pedersen, 2013). Some clues are given subsequently in this chapter (see Irisin effects on brain). Nonetheless, it is worth studying the possible roles of peripheral muscle-produced IL-6 on brain, since critical regions for the control of feeding behavior/energy metabolism (hypothalamus) and cognitive function (hippocampus) express the IL-6R. In this sense, Ferrer et al. (2014) have shown, using a muscle-specific IL-6 KO murine model, that muscle IL-6 might have a role modulating the expression of key neuropeptides that regulate energy homeostasis in the hypothalamus. 2.3.4 Effects of Muscle IL-6 on Liver Glucose Homeostasis Liver is another main target of IL-6, which has welldocumented effects on liver metabolism, such as in the acute phase response (Gauldie et al., 1987) (important for protection against infections), and the regulation of glucose tolerance (Mauer et al., 2015). Whereas these roles do not specifically refer to muscle IL-6, it is known that muscle maintains a crosstalk with the liver, involving IL-6. In particular, it is known that muscle IL-6 contributes to the production of glucose in the liver during exercise, but the relationship between muscle IL6 and the control of hepatic glucose metabolism has not been completely unraveled (Giudice and Taylor, 2017; Pedersen and Febbraio, 2012). One study proposed that IL-6 does have a role in hepatic glucose production during exercise in humans, but it suggested that its action would be dependent on another, still unidentified, factor, also induced by muscle contraction (Febbraio et al., 2004). 2.3.5 Effects of Muscle IL-6 in Pancreas: More About Glucose Homeostasis The pancreas is another of the key targets of IL-6. Acute IL-6 signaling (as happens with contraction/exercise) promotes insulin secretion by the b-cells, their proliferation, prevents their apoptosis (induced by metabolic stress), and generally increases their viability and survival, regulating the b-cell mass (Ellingsgaard et al., 2011; Herder et al., 2015; Karstoft and Pedersen, 2016; Paula et al., 2015). It is therefore clear that IL-6 has a positive impact on the endocrine pancreas

responsible of producing insulin, so IL-6 might be important in glycemia regulation and protection against diabetes. In this sense, the capacity of the exercisedependent increases in IL-6 to promote the secretion of glucagon-like peptide-1 (GLP-1), both by b-cells in the pancreas and by intestinal L-cells, is of interest (Karstoft and Pedersen, 2016). This combination is positive for improving insulin secretion and the control of blood glucose levels, since GLP-1 is an incretin. Hence, the muscleepancreas axis (and also involving intestinal Lcells), through IL-6 action, can create an endocrine loop with effects in glycemia regulation and insulin function, beneficial for type 2 diabetes patients (Karstoft and Pedersen, 2016) but also for healthy individuals. These data, together with other data given in preceding sections, outlines the importance of muscle IL-6 as an endocrine signal able to communicate muscle with tissues critical for the control of glucose homeostasis. 2.3.6 IL-6 as an Antiinflammatory Myokine Many of the data and references already revised in this chapter have outlined the multiple antiinflammatory roles of IL-6, especially as a myokine. Specific antiinflammatory effects reported for this cytokine are summarized next. IL-6 has been suggested to induce classic antiinflammatory cytokines, such as IL-1ra and IL-10 (Steensberg et al., 2003; Trayhurn et al., 2011). Another antiinflammatory effect of IL-6 relies on its capacity to inhibit lipopolysaccharide-induced TNF production by monocytes (as studied in a cell model) (Schindler et al., 1990). Moreover, other experimental works have suggested that, actually, circulating IL-6 sets up a negative feedback to restrain TNF production (we have to keep in mind that TNF stimulates IL-6 production), thus supporting IL-6 antiinflammatory roles (Benatti and Pedersen, 2015; Pedersen and Febbraio, 2012). There are other multiple experimental data that support antiinflammatory roles of exercise and musclederived IL-6 by regulating leukocyte homeostasis and trafficking, limiting the expression of other inflammatory cytokines (not only TNF), augmenting the macrophage responsiveness to the antiinflammatory cytokine IL-4, etc. (Benatti and Pedersen, 2015). Therefore, IL-6 can be considered a crucial muscle hormone mediating the antiinflammatory effects of exercise. 2.3.7 Muscle IL-6 in Health and Disease, Cancer, and Beyond Available evidence positions IL-6 as a central regulator of physiology, but also involved in different diseases, and with both anti- and proinflammatory actions depending on the context. As already stated, patients with certain inflammatory-related serious diseases can benefit from treatments based on blocking IL-6/IL-6R (for more information, please see the excellent review by Garbers et al.,

3. IRISIN

2018). Nevertheless, completely blocking all types of IL-6 signaling brings about nondesirable metabolic effects (Benatti and Pedersen, 2015; Cron et al., 2016). It also associates to increased risk of bacterial infections. Therefore, new strategies focused on specifically blocking the IL-6/sIL-6R complex (and thus trans-signaling) are being developed (Garbers et al., 2018). Taking this into account, and the many diseases associated with increased IL-6 levels, it is clear that exogenously administrating IL-6 could never be suggested as a treatment for obesity or other metabolic diseases, even considering the multiple beneficial and antiinflammatory effects of IL-6 explained in this chapter. Despite the impossibility of directly using IL-6 as treatment, the multiple beneficial roles of IL-6 make it a very interesting molecule to be induced by means of physiologic ways, i.e., by regular exercise or physical activity, thus boosting muscle-derived IL-6 and its roles as a myokine. This is in line with the concept of “exercise is medicine” (which can also be applied when talking about irisin and other myokines), and all the wide literature about muscle IL-6, together with current advances, supports this concept (Febbraio, 2017; Huh, 2018; Karstoft and Pedersen, 2016). Regarding recent advances, the work of Pedersen et al. (Pedersen et al., 2016) using a murine model of cancer should be outlined, where they demonstrate that exercise induces a complex immunological and hormonal response able to inhibit tumor growth, with a critical role of IL-6. In their work, the authors demonstrate that an increase of IL-6 (exercise associated) and adrenaline secretion reduces tumor growth, by mobilizing and redistributing a specific subset of NK (natural killer) cells (Febbraio, 2017; Pedersen et al., 2016).

3. IRISIN Among the myokines, irisin is attracting a lot of interest as a potential mediator for beneficial effects of exercise on metabolism and brain functions. Irisin was discovered in 2012 as a protein secreted by skeletal muscle that stimulates the appearance of brown adipose tissue (BAT) features in white adipose tissue (WAT) (Bostrom et al., 2012), a phenomenon known as WAT browning. It was shown that transgenic mice with muscle-specific overexpression of peroxisome proliferator-activated receptor-g coactivator 1a (PGC1a), a transcriptional regulator that is activated by exercise, displayed WAT browning and an increased gene and protein expression in muscle of fibronectin type III domain containing 5 (FNDC5), a membrane 1

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protein that is cleaved and secreted as irisin (Bostrom et al., 2012). Subsequent research established that irisin is produced and secreted by other tissues besides skeletal muscle, which implicated irisin in the control of biologic processes and functions other than WAT browning. Irisin has been linked to favorable effects on metabolism and has gained much attention as a potential therapeutic agent for metabolic diseases such as obesity and type 2 diabetes. However, there have been controversial results on irisin significance, especially in humans, boosted by peculiarities of the human FNDC5 gene, technical issues regarding the specificity of available anti-FNDC5/irisin antibodies, and the fact that irisin receptor(s) remain to be identified.1

3.1 Structure of Irisin FNDC5, the precursor of irisin, is a glycosylated type I membrane protein (212 amino acids in humans; 209 in rodents) that contains an N-terminal signal peptide, a fibronectin type III domain, a transmembrane domain, and a cytoplasmic tail. Irisin contains 112 amino acids, most of them mapping to the fibronectin type III domain (in human, amino acids 32e143 of the full-length protein; in mouse and rat, amino acids 29e140). Irisin is generated from FNDC5 by proteolytic cleavage and is released into the circulation. The protease/sheddase responsible for that cleavage remains to be identified. Strikingly, antibodies against the intracellular portion of FNDC5 (such as Abcam 149e178C terminal, no longer available) recognized the secreted form of the protein (Bostrom et al., 2012). The possibility that the full-length FNDC5 is secreted has been put forward (Crujeiras et al., 2015). The predicted size of the shed irisin polypeptide is w12 kDa, and a 12-kDa peptide sequenced as irisin was identified in deglycosylated human plasma samples, using a gold-standard method, namely tandem mass spectrometry (Jedrychowski et al., 2015; PerezSotelo et al., 2017). However, the detection of irisin by more conventional methods employing commercial antibodies, such as immunoblotting or ELISA, has proven problematic. Many publications, including the seminal paper by Bostrom et al. (Bostrom et al., 2012), detected in western blots of serum multiple bands of molecular weight higher than 12 kDa; some of these bands are thought to represent glycosylation or dimerization forms, while other reflect poor antibody specificity (Perez-Sotelo et al., 2017). Recombinant irisin (corresponding to residues 30e140 of the human ˚ FNDC5 protein) has been crystallized, and its 2.28 A structure has been solved; structural and biochemical

By the publication of the present book, Irisin has been described to interact with aV Integrin Receptors, to mediate effects in bone and adipose tissue. For more information you can consult the references of Kim et al., 2018 (https://doi:10.1016/j.cell.2018.10.025) and Farmer, 2019 (https://doi:10.1056/NEJMcibr1900041).

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data have suggested that irisin exists as a homodimer (Schumacher et al., 2013). Regarding its evolution, irisin is a highly conserved polypeptide across mammals, being 100% identical in mice and humans (Bostrom et al., 2012). Such a high degree of conservation is usually considered to reflect evolutionary pressure to conserve function. The human FNDC5 gene has an atypical start of translation, ATA in place of the more typical ATG, as compared to mouse Fndc5. Because of that, it has been claimed that human FNDC5 and, subsequently, irisin would not be produced (the first ATG in the human gene is rather downstream of this ATA codon, so its use would result in a truncated protein) (Raschke et al., 2013b; Albrecht et al., 2015). However, some cellular proteins are translated from non-ATG start codons, and Jedrychowski et al. (2015) using tandem mass spectrometry have unequivocally shown that human irisin exists in circulation (Jedrychowski et al., 2015). Besides in skeletal muscle, FNDC5/irisin is produced in other tissues, notably, cardiac muscle (to even higher levels than in skeletal muscle) (Aydin et al., 2014), adipose tissues (Roca-Rivada et al., 2013; Moreno-Navarrete et al., 2013), the liver (Mo et al., 2016), and parts of the brain including the hypothalamus (Wrann et al., 2013; VarelaRodriguez et al., 2016). FNDC5 expression in adipose tissues is higher in BAT than in WAT depots in rodents (Varela-Rodriguez et al., 2016; Amengual et al., 2018).

3.2 Biochemical Reactions of Irisin: Signaling and Secretion Binding of exogenous irisin to the plasma membrane of 3T3-L1 adipocytes has been demonstrated, suggesting the existence of an irisin receptor on the adipocyte cell membrane (Zhang et al., 2014). However, irisin receptor(s) in humans and animals are yet to be identified, which represents a strong limitation to the understanding of irisin biologic significance, signaling pathways, and mechanisms of action.1 Stimuli known to modulate FNDC5/irisin production are summarized next. Exercise: Irisin was originally identified as a PGC1a-dependent myokine (Bostrom et al., 2012). PGC1a is a transcriptional coactivator closely related to energy metabolism and induced by exercise in muscle, where its activity favors mitochondrial biogenesis, angiogenesis, and fiber-type switching (Handschin and Spiegelman, 2008). In fact, circulating levels of irisin are significantly increased after exercise in mice and human (Bostrom et al., 2012; Lee et al., 2014; Jedrychowski et al., 2015) (from w3.6 ng/mL in sedentary individuals to w4.3 ng/mL in individuals undergoing aerobic interval training, as indicated by mass spectrometry measurements (Jedrychowski et al., 2015)). However, the

relationship of irisin and exercise has been the subject of controversy. This relationship may depend on the type of exercise, its intensity, and the duration of the exercising sessions (Buscemi et al., 2017). Cold exposure: Cold exposure increases circulating irisin in humans proportionally to shivering intensity, suggesting skeletal muscle is the main contributor to the cold-induced irisin rise (Lee et al., 2014). Since FNDC5 treatment induces brown fat gene/protein expression and thermogenesis in human neck adipocytes (see Section 3.3.2 on irisin physiologic functions), it has been suggested that shivering may link exercise- and cold-induced thermogenesis, with irisin-mediated muscleeadipose communication being a key part of this link (Lee et al., 2014). Constitutive androstane receptor (CAR) activation: Ligand-dependent activation of the nuclear receptor CAR upregulates the hepatic production of FNDC5/irisin and circulating irisin levels, without changing skeletal muscle Fndc5 mRNA levels (Mo et al., 2016). Furthermore, the Fndc5 gene was proven to be a direct transcriptional target of CAR (Mo et al., 2016). CAR was originally identified as a xenobiotic receptor predominantly expressed in liver and intestine, yet CAR activity was later shown to impact metabolism by suppressing hepatic lipogenesis and gluconeogenesis, and to protect against hepatic steatosis (Dong et al., 2009; Gao et al., 2009). These are among the effects of irisin in liver cells (see Section 3.3.5 on irisin physiologic functions). Thus, induction of hepatic irisin may mediate, at least in part, the beneficial effects of CAR activation in the liver. Nutritional conditions/dietary factors: Recent findings suggest that in both rodents and humans, FNDC5/irisin production is sensitive to nutritional conditions or specific dietary factors. In rats, circulating irisin levels were not affected by long-term caloric restriction or high-fat diet, but were decreased after a 48-h fast, along with Fndc5 expression in skeletal muscle, BAT and WAT depots (Varela-Rodriguez et al., 2016). Hypothalamic Fndc5 expression did not change with any of the tested diets/nutritional conditions, suggesting that, unlike peripheral FNDC5 expression, central regulation of FNDC5 is independent of alimentation/nutrition (Varela-Rodriguez et al., 2016). Increased serum levels of irisin were reported in mice fed a high-fat diet rich in saturated fat, and irisin is released by cultured skeletal muscle cells upon exposure to saturated fatty acids (palmitate) (Natalicchio et al., 2017), but not monounsaturated fatty acids (oleate) (Natalicchio et al., 2017) or an equimolar mixture of oleate and linoleic acid (Sanchez et al., 2013). Irisin produced in response to saturated fatty acids may serve to protect against saturated fatinduced pancreatic injury (Natalicchio et al., 2017) (see Section 3.3.6 on irisin physiologic functions). In humans,

3. IRISIN

supplementation with n-3 polyunsaturated fatty acids (PUFA) has been reported to increase circulating irisin in patients with type 2 diabetes (Ansari et al., 2017) and patients with coronary artery disease (Agh et al., 2017). In these human studies, the increase in serum irisin paralleled beneficial effects of n-3 PUFA supplementation on metabolic parameters (e.g., HOMA-IR, diastolic blood pressure, or blood lipids) but were independent of changes in body weight/adiposity-related parameters (Ansari et al., 2017; Agh et al., 2017). Another human intervention study compared the effect of three different diets on irisin levels in metabolic syndrome patients: low glycemic index (LGID), Mediterranean (MD), or low glycemic index Mediterranean (LGIMD) diet. Mean irisin levels increased in all diet groups, but the increase reached statistical significance only with the LGID intervention. Positive associations between vegetable proteins and saturated fatty acids consumption and serum irisin were reported (Osella et al., 2018). Retinoic acid: Cell and animal studies indicate that retinoic acid, the acid form of vitamin A, induces FNDC5/ irisin production in skeletal muscle cells, liver, and adipose tissues, elevates serum irisin levels, and favors FNDC5/irisin mobilization from skeletal muscle and the liver toward BAT and WAT depots (Amengual et al., 2018). Stimulation of FNDC5/irisin may contribute to the leaning effects and metabolic benefits observed in mice upon retinoic acid treatment, which include the mobilization and consumption of stored lipids and the induction of WAT browning (Bonet et al., 2015). Although irisin signaling transduction pathways are essentially unknown, molecular targets that appear to mediate irisin effects in biologic systems are being unveiled (see also next Section 3.3). Such targets may functionally be downstream of the irisin-dependent activation of the putative irisin receptor(s). For instance, studies have shown that irisin promotes fat browning by activating p38 MAPK and extracellular signalerelated kinase (ERK) signaling pathways (Zhang et al., 2014). These two pathways also appear to mediate osteogenic effects of irisin (Qiao et al., 2016). Additionally, metabolic effects of irisin in muscle and liver have been related to the activation of AMPK, a cellular energy sensor and a key controller of cellular energy metabolism (Lee et al., 2015; Mo et al., 2016; So and Leung, 2016; Tang et al., 2016). Interestingly, there is evidence that AMPK activation may be involved in the stimulation of irisin production promoted by retinoic acid in skeletal muscle cells (Amengual et al., 2018). Regarding the mechanisms of hormone inactivation, very little is known about irisin inactivation. The recombinant human irisin dimer appears to be highly stable due to interactions between monomers (Schumacher et al., 2013). Accordingly, Huh et al. (2012) observed no significant differences in detectable circulating levels of

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irisin in sera that had undergone multiple freezing and thawing cycles (Huh et al., 2012). In seeming contrast, however, a negative association of irisin concentration with the storage duration of frozen human serum samples has been reported (Hecksteden et al., 2013). A study on the distribution and elimination of 125I-irisin in vivo in mice concluded that metabolic clearance is achieved primarily through both the hepatobiliary and renal systems (Lv et al., 2015).

3.3 Physiological Functions of Irisin and Their Relation With Health and Disease Animal models of irisin loss of function have yet to be characterized, so the physiologic significance of irisin remains to be firmly established. Despite this, FNDC5/irisin has been related to many biologic processes/ functions on the basis of the actions exerted by exogenous irisin in animal and cell models, and the accumulated knowledge on the control of endogenous FNDC5/irisin production (see previous discussion). Biologic processes and functions affected by FNDC5/irisin are summarized next (see also Fig. 26.2). 3.3.1 Effects of Irisin in Skeletal Muscle Treatment with irisin increases mitochondrial oxidative metabolism, mitochondrial uncoupling and fatty acid uptake, and oxidation in skeletal muscle cells (Huh et al., 2014b; Vaughan et al., 2015; Xin et al., 2016). Irisin also favors glucose uptake in human and murine skeletal muscle cells (Huh et al., 2014b; Lee et al., 2015). The latter effect was traced to the enhancement of GLUT4 translocation to the plasma membrane, following reactive oxygen species production and subsequent activation of AMPK and p38 MAPK (Lee et al., 2015). Besides impacting muscle metabolism, recombinant irisin behaves as a promyogenic factor. Thus, irisin induces skeletal muscle hypertrophy and rescues from denervation-induced muscle atrophy in mice (Reza et al., 2017), and it stimulates muscle growth-related genes in human myocytes (Huh et al., 2014a). 3.3.2 Effects of Irisin in Adipose Tissues White adipose tissue browning/beiging: Irisin was discovered as a browning-inducing myokine (Bostrom et al., 2012). Similar to BAT activation, WAT browning may favor leanness and metabolic health by increasing energy expenditure as heat and the oxidation of excess glucose and lipid molecules, and it is therefore of clinical interest in obesity and related metabolic derangements (Bartelt and Heeren, 2014). Recombinant irisin-induced browning has been demonstrated in rodents and confirmed in human primary mature

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26. MUSCLE HORMONES

adipocytes and human subcutaneous WAT explants (Bostrom et al., 2012; Lee et al., 2014; Zhang et al., 2014, 2016). There is also evidence that upregulation of endogenous Fndc5/irisin expression in primary white adipocytes (through the knockdown of the myostatin gene) promotes browning (Ge et al., 2017). The browning effect of irisin appears to be dependent on the activation of p38 MAPK and ERK signaling pathways, as blocking either pathway with specific inhibitors abolished irisin-induced UCP1 upregulation in human subcutaneous WAT ex vivo (Zhang et al., 2014). The browning effect of irisin has also been linked to the induction of peroxisome proliferator-activated receptor a (PPARa) expression in white adipocytes (Bostrom et al., 2012). Interestingly, white (3T3-L1) adipocytes in which browning has been induced secrete increased levels of irisin, suggesting a positive feedback loop between fat browning and irisin production as an autocrine/paracrine signal by the adipose tissue itself (Moreno-Navarrete et al., 2013). Adipogenesis: Besides favoring the browning of mature adipocytes, exogenous irisin inhibits the differentiation of human preadipocytes into mature adipocytes (Huh et al., 2014a; Zhang et al., 2016). In keeping with an inhibitory effect of irisin on adipogenesis, stable silencing of FNDC5 expression in pluripotent stem cells (C3H10T1/2 cells) enhances adipose differentiation and decreases UCP1 expression (Perez-Sotelo et al., 2017). FNDC5/irisin expression and secretion are induced during adipogenic differentiation of C3H10T1/2 cells (Perez-Sotelo et al., 2017). 3.3.3 Effects of Irisin in Bone Exogenous irisin promotes bone formation in mice (Colaianni et al., 2015; Zhang et al., 2017), and it favors osteoblast differentiation and inhibits osteoclast differentiation in culture (Qiao et al., 2016; Zhang et al., 2017). Thus, irisin may represent a factor in musclee bone connectivity, promoting changes in bone, enabling it to support greater loads due to an increased physical activity sensed in muscles (Buscemi et al., 2017). Furthermore, in mice, voluntary exercise increases irisin production in bone, together with increased expression of osteogenic markers (Zhang et al., 2017). 3.3.4 Effects of Irisin in Brain and Cognitive Function De novo neurogenesis and cognitive function: A PGC-1aFNDC5/irisin pathway, which is activated by exercise in the hippocampus and induces a neuroprotective gene program including increased expression of brainderived neurotrophic factor (BDNF), has been described in mice (Wrann et al., 2013). BDNF is a neurotrophin that promotes many aspects of brain development and is essential for synaptic plasticity, hippocampal function,

and learning. Induction of the central PGC-1aFNDC5/irisin-BDNF pathway may contribute to the known positive effects of exercise in the improvement of cognitive function. Peripheral irisin may also favor hippocampal BDNF expression acting as an endocrine signal, as elevation of blood irisin (through delivery of FNDC5 to the liver via adenoviral vectors) induces Bdnf and other neuroprotective genes in the hippocampus in mice (Wrann et al., 2013). Trained human athletes have higher circulating BDNF and irisin levels, and positive correlations between circulating levels of both of these proteins and performance in cognitive function tests have been reported (Belviranli et al., 2016). Thus, irisin could be a mediator for beneficial effects of exercise on the brain (Wrann, 2015). Feeding regulation: Both inhibitory (Ferrante et al., 2016) and stimulatory effects (Tekin et al., 2018) on feeding have been described in rats after central injection of irisin. In goldfish, an anorexigenic effect of exogeneous irisin was reported (Butt et al., 2017). Whereas in zebrafish, intraperitoneal injection of irisin did not affect feeding, but its knockdown using siRNA reduced food intake, suggesting that irisin is required to maintain food intake in this species (Sundarrajan and Unniappan, 2017). All in all, irisin has been connected to feeding control, but its role to this respect remains unclear and controversial. 3.3.5 Effects of Irisin in Liver In hepatic cell models (primary human hepatocytes and human HepG2 hepatoma cells), exposure to exogenous irisin inhibits gluconeogenesis and lipogenesis by activating the AMPK pathway, thus decreasing glucose output and triglyceride content (Mo et al., 2016; So and Leung, 2016). Adenovirus-mediated overexpression of irisin in the liver improves hepatic steatosis and insulin resistance in obese and diabetic (ob/ob) mice while decreasing hepatic lipogenic gene expression (Mo et al., 2016). Further, transgenic mice overexpressing irisin in the liver are protected against dietinduced obesity and insulin resistance (Mo et al., 2016). Thus, hepatic-derived irisin may exert paracrine/autocrine effects as well as endocrine effects of metabolic benefit. Beneficial effects of irisin on hepatic cholesterol metabolism have also been described. Thus, long-term infusion of irisin into obese mice ameliorates hypercholesterolemia and suppresses hepatic cholesterol production via a mechanism dependent on AMPK activation and subsequent inhibition of sterol regulatory element-binding transcription factor 2 (SREBP2) transcription and nuclear translocation (Tang et al., 2016). Further, a study on the distribution of 125I-irisin in vivo showed high radioactivity in the mouse liver, implying the liver as a target for irisin (Lv et al., 2015).

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4. OTHER MYOKINES

3.3.6 Effects of Irisin in Pancreas Studies suggest a capacity of irisin to improve pancreatic b cell survival and function. Exogenous irisin favors proliferation of pancreatic beta cells and protects these cells from high-glucose-induced apoptosis (Liu et al., 2017). Furthermore, the irisin-rich sera from high-fat diet-fed mice and the irisin-rich secretome of palmitate-exposed myotubes protect pancreatic islets and insulin-secreting cells from apoptosis, an effect that was abrogated in the presence of an irisinneutralizing antibody (Natalicchio et al., 2017). Antiapoptotic effects of irisin in pancreatic beta cells are dependent on irisin-mediated protein kinase B (AKT) activation and subsequent induction of antiapoptotic BCL2 protein expression (Natalicchio et al., 2017). Additionally, recombinant irisin stimulates insulin biosynthesis and glucose-stimulated insulin secretion in pancreatic beta cells in a PKA-dependent manner (Natalicchio et al., 2017). In vivo administration of irisin improved glucose-stimulated insulin secretion and increased b-cell proliferation (Natalicchio et al., 2017). 3.3.7 Antiinflammatory Effects of Irisin Treatment with exogenous irisin has been shown to exert antiinflammatory effects in different biologic systems including macrophages, adipocytes, endothelial cells, and in aortic tissues in vivo, as reviewed elsewhere (Buscemi et al., 2017). The antiinflammatory action of irisin translates into decreased levels of proinflammatory cytokines, and it has been linked to decreased production of reactive oxygen species and decreased activity of proinflammatory MAPK and NF-kB pathways (Buscemi et al., 2017). 3.3.8 Irisin Effects on Disease and Aging Work in experimental models suggests positive effects of irisin on metabolic diseases including obesity, type 2 diabetes, dyslipidemia, and nonalcoholic fatty liver disease (NAFLD). However, there have been controversial results in humans regarding the relationship of serum levels of irisin with these various disorders, perhaps partly due to the use of different ELISA kits to measure these levels (Buscemi et al., 2017; Polyzos et al., 2018). Most, but not all observational studies show a direct association between circulating irisin and indexes of adiposity. To explain this association, and knowing that FNDC5/irisin expression and secretion are induced during adipogenesis, it has been hypothesized that in the obese condition, WAT would overexpress and oversecrete FNDC5/irisin in an attempt to counterbalance the metabolic deregulation by increasing energy expenditure (through irisin-induced WAT browning); however, in established obesity, adipose and other peripheral tissues may suffer from FNDC5/

irisin resistance (Perez-Sotelo et al., 2017). High serum concentrations of irisin have been observed in patients with type 2 diabetes in certain studies, although most studies have reported the opposite, i.e., lower levels of irisin in type 2 diabetes patients than in controls (Buscemi et al., 2017; Polyzos et al., 2018). Both positive and negative associations of circulating irisin with insulin sensitivity indexes have been reported (Buscemi et al., 2017). There have also been conflicting reports regarding irisin and NAFLD, with some human studies associating higher circulating irisin levels with the severity of this condition (Buscemi et al., 2017; Polyzos et al., 2018). Regarding cardiovascular diseases, most observational studies reported lower levels in patients than controls; it has been proposed that reduced irisin after myocardial infarction may represent a mechanism against energy depletion in critical situations (Buscemi et al., 2017; Polyzos et al., 2018). Regarding metabolic bone diseases, circulating irisin is positively associated with bone mineral density and strength in athletes, and inversely associated with osteoporotic fractures in postmenopausal osteoporosis (Polyzos et al., 2018). The antiinflammatory, antiapoptotic, and antioxidative properties of irisin have suggested the potential application of irisin therapy in diseases (other than those already mentioned here) whose initiation, progression, and/or prognosis also involve these pathogenic processes, such as kidney diseases, cancer, lung injury, and inflammatory bowel diseases (Askari et al., 2018). In summary, irisin remains a promising and appealing therapeutic target for many diseases, but much research is still needed before recombinant irisin therapy can be tested in humans.

4. OTHER MYOKINES Skeletal muscle produces and secretes a number of other important molecules that are also considered, or are candidates to be, myokines. It is worth shortly reviewing some of them to further understand the great potential of skeletal muscle as an endocrine organ. Interleukin-15 (IL-15) is a purported myokine that belongs to a different family of cytokines compared to IL-6. It belongs to the IL-2 superfamily of cytokines and, like IL-6, is expressed by different cell types and has pleiotropic effects (Huh, 2018; Karstoft and Pedersen, 2016). Its role as a bona fide myokine is still under certain discussion since there is still conflicting data about how exercise affects its expression and secretion, and especially because whether muscle is, actually, a significant source of IL-15 remains greatly unknown (Huh, 2018; Karstoft and Pedersen, 2016). Therefore, there are still some doubts about IL-15 having a true endocrine role

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when produced by muscle. Nevertheless, several experimental data point into the direction of IL-15 as a myokine, responsive to exercise and with possible endocrine effects. First, it is highly expressed in muscle cells and its mRNA levels significantly increase in skeletal muscle after exercise (Benatti and Pedersen, 2015; Grabstein et al., 1994; Karstoft and Pedersen, 2016). The latter effect is not only shown in animal models, but also in humans, where chronic endurance training significantly increases IL-15 expression in skeletal muscle (Rinnov et al., 2014). Second, IL-15 shows direct specific effects on skeletal muscle itself, like its well-known capacity to exert anabolic actions on muscle: IL-15 promotes the accumulation of contractile proteins, such as myosin heavy chain, in differentiated myocytes, as well as myogenic differentiation (Quinn et al., 1995, 1997). There have also been described a variety of other effects on skeletal muscle cells related to glucose uptake regulation, enhancement of mitochondrial activity, and protection against H2O2associated oxidative stress (Huh, 2018). Third, as happens with IL-6, IL-15 also seems to be involved in the skeletal muscle-adipose tissue crosstalk, regulating adipose metabolism. Muscle IL-15 has been involved in decreases of fat deposition in adipose cells and, generally, the reduction of adipose tissue mass (Benatti and Pedersen, 2015; Carbo´ et al., 2001; Quinn et al., 2005), with some experimental data (in a murine model of IL-15 overexpression) outlining a particular effect reducing the accumulation of visceral fat (vs. subcutaneous) specifically (Nielsen et al., 2008). Fourth, recent experimental data suggest a novel endocrine role for muscle IL-15 connecting exercise with skin health, and thus suggesting a skeletal musclee skin crosstalk. A study performed by Crane and colleagues (Crane et al., 2015), with data both in mice and humans, suggests that muscle IL-15, induced by exercise and via AMPK activation in muscle, can affect skin mitochondrial function, attenuating aging-associated changes in skin. Other putative myokines of relatively recent discovery, or with relatively recent functions attributed to their production by skeletal muscle, include molecules such as myonectin, b-aminoisobutyric acid (BAIBA), meteorinlike protein (METRNL), and secreted protein acidic and rich in cysteine (SPARC), among others. Myonectin (also known as CTRP15) was discovered as a new member of the C1q/TNF-related protein family, while characterizing the functions of CTRP proteins in metabolism, and it was described that skeletal muscle secretes it in response to exercise (and also nutrients) (Seldin et al., 2012; Whitham and Febbraio, 2016). Experimental data suggest that myonectin has effects on liver and adipose tissue regulating lipid homeostasis (Giudice and Taylor, 2017; Seldin et al., 2012). BAIBA is not a protein, but it can be classified as a putative myokine. It is a metabolite of the catabolism of thymine, and its circulating levels

have been reported to be increased by exercise training both in mice and humans (Roberts et al., 2014). Different beneficial effects have been attributed to BAIBA in muscle itself, such as promotion of mitochondria free fatty acid oxidation and protection against inflammation (Jung et al., 2015; Roberts et al., 2014). Moreover, it can be potentially considered a true myokine since endocrine effects have also been described for this molecule. In this line, BAIBA upregulates free fatty acid oxidation also in adipocytes, in liver reduces hepatic de novo lipogenesis, and has been shown to induce browning of WAT in mice (Begriche et al., 2008; Huh, 2018; Maisonneuve et al., 2004; Roberts et al., 2014). Meteorin-like protein (or METRNL) has also been related with induction of browning of adipose tissue and is, of course, induced in skeletal muscle by exercise (Rao et al., 2014; Whitham and Febbraio, 2016). Curiously, the effects of meteorin-like on browning seem to be indirect, through regulation of immune cells. Increased levels of meteorin-like protein have also been associated with improvement of glucose tolerance and stimulation of energy expenditure in mice (Rao et al., 2014). Regarding SPARC, it was identified as a contraction-induced myokine by microarray and bioinformatics tools, and it has been suggested to reduce precursor lesions of adenocarcinoma in the colon via regular exercise (Aoi et al., 2013; Whitham and Febbraio, 2016).

5. CONCLUSIONS AND FUTURE DIRECTIONS Nowadays, there is little doubt that muscle is a very relevant endocrine organ, able to communicate with many (if not all) the tissues of the body and regulate/ integrate general metabolism, with an important impact in key metabolic aspects such as glucose homeostasis, lipid metabolism, cognitive function, immunology, etc. The concept of myokine is already generally assumed, and an important number of true and potential myokines have been described. Some of them have been widely studied, especially IL-6, the prototypical myokine, and also irisin, since its discovery and postulated functions generated important expectations (and controversy), which will probably increase (dissipate) after the expected characterization of its receptor/s and system/s of signaling. When talking about muscle and myokines, it is unavoidable to also talk about physical activity and exercise, as the physiologic drivers of myokine induction, useful for the prevention of prevalent diseases and even their treatment, integrating the concept, defended by several authors, of “exercise is medicine.” The future directions in this field will probably go into deepening the knowledge of the most important or already demonstrated as bona fide myokines, but also in the discovery, already very active at present, of

REFERENCES

new myokines. New tools are becoming very relevant in this field, provided by the omics techniques, such as transcriptomics, proteomics, metabolomics, and lipidomics, and also techniques as mass spectrometry, cytokine arrays, and importantly, bioinformatics (Karstoft and Pedersen, 2016; Whitham and Febbraio, 2016). Different studies have already started to apply these new techniques to characterize the myokinome (the conjunct of signaling molecules expressed and secreted by muscle) or the muscle secretome in general (as a first step to identify potential true myokines), some of them putting special emphasis in the myokines regulated and secreted in association with contractile activity (Hartwig et al., 2014; Karstoft and Pedersen, 2016; Raschke et al., 2013a). It has to be outlined that the research on new myokines is not only focused on proteins, but also in other types of molecules such as lipids and specific metabolites (Karstoft and Pedersen, 2016). Another new exciting field includes the study of extracellular vesicles to discover novel putative myokines secreted in nonclassical ways (Whitham et al., 2018). All these high throughput techniques are rendering and will render a high amount of information and putative myokine candidates, which will have to be validated as true muscle-derived hormones by different types of studies including the analysis of plasma levels and their dynamics regarding exercise (exercise timecourse data), the use of gene-manipulated animal models, etc. (Whitham and Febbraio, 2016). In summary, the high relevance of the endocrine functions of skeletal muscle is clear, so this is an active field in metabolic research, especially related with exercise and health, which in the coming years will probably render many new interesting results, with the description of more myokines and the understanding of their involvement in integrated metabolic regulation. It is expected that these discoveries will help to discover new therapeutic targets, as well as paving the way to a more personalized medicine, including the “prescription” of individual-adapted exercise to prevent and combat prevalent diseases such as obesity and its metabolic disorders, cognitive decline, immunological diseases, etc.

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