Many secreted proteins have been suggested to be involved in the regulation of metabolic function of skeletal muscle itself and other metabolic organs. Interleukin (IL)-6 is a representative myokine that is transiently elevated in muscle following one bout of exercise [8]. IL-6 may act locally within the contracting skeletal muscle in a paracrine manner or be released into the circulation and may increase over tenfold, thus inducing systemic effects [9, 10]. IL-6 directly increases glucose metabolism of skeletal muscle in resting state [11] and can lead to further improvement of insulin sensitivity in response to exercise [12]. In addition, previous studies also showed that recombinant IL-6 infusion at normal physiological levels selectively stimulates lipid metabolism in skeletal muscle in healthy subjects [13] and in subjects with type 2 diabetes [14]. In addition, muscle-derived IL-6 has been suggested to play a role in increased lipolysis in adipose tissue through an endocrine mechanism [15]. In fact, recombinant IL-6 intra-lipid infusion elevates plasma fatty acid levels, which probably caused by adipose tissue lipolysis [12]. Furthermore, injection of IL-6 results in hepatic glycogen catabolism and accelerated circulatory glucose output in rats [16]. This may contribute to the maintenance of blood glucose and supply the required energy substrate during exercise. In addition, physiological elevation of IL-6 levels stimulates production of an insulin secretagogue, glucagon-like peptide-1, from intestinal L cells and pancreatic alpha cells, which ultimately promotes insulin secretion from pancreatic β cells [17].

In addition to IL-6, other muscle-secreted proteins including brain-derived neurotrophic factor, fibroblast growth factor (FGF) 21, and IL-15 have been shown to be produced in skeletal muscle in response to acute or chronic exercise and have been suggested to increase nutrient metabolism [18–21]. FGF21 is secreted from muscle cells with activation of Akt signaling and can stimulate lipolysis, glucose transporter (GLUT) 1-mediated glucose uptake in adipocytes [22], fatty acid utilization for energy, and ketone body production in the liver [23]. In muscle cells, FGF21 exposure increases glucose uptake in both absence and presence of insulin via GLUT-1 and GLUT-4 [19]. Additionally, FGF21 prevents palmitate-induced insulin resistance in skeletal muscle [24]. These observations suggest that FGF21 plays a role in lipolysis and glucose uptake in the liver, adipose tissue, and skeletal muscle. IL-15 has been shown to be increased in skeletal muscle and plasma by acute exercise [20, 25], although this remains controversial. IL-15 overexpression in muscle resulted in increased expression of oxidative metabolic factors including sirtuin 1, peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ coactivator (PGC)-1α, and PGC-1β in skeletal muscle and adipose tissue [26, 27], which would contribute exercise-induced activation of glucose and lipid metabolism. The chemokine CXC motif ligand-1 (CXCL-1), referred to as keratinocyte-derived chemokine (KC) in mice and IL-8 in humans, is also elevated by one bout of exercise in skeletal muscle locally [28, 29]. CXCL-1 overexpression induces aerobic metabolism in skeletal muscle and suppresses diet-induced fat accumulation in adipose tissue. Myonectin has been also identified as a regulatory factor of lipid metabolism in the liver and adipose tissue [30].

A recent study showed that PGC-1α1 expression in muscle stimulates expression of FNDC5, a membrane protein that is cleaved and secreted as irisin [31]. PGC-1α1 has been shown to play a central role as part of transcriptional coactivators involved in aerobic metabolism; thus, a considerable amount of attention has been focused on it as a target for the prevention or treatment of metabolic syndrome through activation of lipid metabolism. Acute and habitual exercise elevates PGC-1α1 expression in skeletal muscle [32, 33] and, consequently, the secretion of irisin from muscle into the circulation. Secreted irisin acts on white adipose cells and facilitates brown fatlike development, which may account for metabolic elevation and body fat reduction induced by exercise although it remains controversial how this factor actually contributes. More recently, the same group reported that another PGC-1α1-dependent small molecule myokine, ß-aminoisobutyric acid (BAIBA), is also increased by habitual exercise and stimulates browning of white adipose tissue along with hepatic fat oxidation via PPARα-mediated signaling [34]. Habitual exercise has been shown to increase circulating BAIBA along with a reduction of metabolic risk factors in mice. Additionally, meteorin-like (Metrnl) is also secreted from skeletal muscle with overexpression PGC-1α4 [35] which is known as a regulator of muscle hypertrophy but not aerobic metabolic capacity [36]. The elevation of circulating Metrnl induces oxygen uptake along with thermogenesis, which is caused by increasing IL-4 expression via eosinophils and then induces the browning of white adipose tissue indirectly via modulation of macrophage activity [35]. In contrast to BAIBA, the level of circulating Metrnl is increased in response to a single bout of exercise.

It has been suggested that muscle-secreted proteins have additional functions, and some proteins contribute to muscle hypertrophy via autocrine or paracrine effects. Insulin-like growth factor-1 (IGF-1) is a well-known secreted protein contributing to muscle mass [37, 38]. It has been shown that overexpression of IGF-1 caused by transgenic and adeno-associated virus injection results in hypertrophy and higher strength and accelerates muscle regeneration in normal and dystrophic mice [39–41]. The hypertrophic effect is mainly mediated by protein synthesis through increased Akt-mTOR-p70S6 K signaling. The insulin signaling pathways, PI3K/Akt and mTOR/p70S6 K, play important roles in glucose uptake as well as protein synthesis in muscle cells. In the synthesis cascade, Akt signals to p70S6 K via mTOR activation, resulting in phosphorylation of S6-rebosomal protein and increased translation of various proteins related to hypertrophy [42]. IGF-I overexpression can also prevent age-induced muscle atrophy in mice [43]; thus, upregulation of IGF-I would be an appropriate method for inhibiting sarcopenia. However, there are controversial findings regarding the effect of IGF-I administration to the elderly on muscle mass and function because decrease in sensitivity to IGF-I occurs in aged muscle [44]. Leukemia inhibitory factor (LIF) is another secreted protein that accelerates muscle mass. The expression level is regulated by intracellular Ca²⁺ induced by exercise in skeletal muscle, and LIF is released into extracellular fluid but not seem to reach the circulation [45]; thus, it acts locally in muscle tissues. LIF stimulates satellite cell proliferation via mainly activation of the JAK2-STAT3 signaling pathway and promotes muscle hypertrophy and regeneration [46].

In contrast, growth and differentiation factor 8, known as myostatin, which belongs to the transforming growth factor-b (TGF-b) superfamily, is an established inhibitor of muscle hypertrophy. Myostatin is secreted and circulates in blood in an inactive complex with several proteins and peptides [47]. After binding activin receptor IIB on the cell membrane, myostatin stimulates Smad signals and decreases the level of myogenic regulatory factors including MyD and Pax3, leading to inhibition of myoblastic proliferation and differentiation during developmental myogenesis [48, 49]. In addition, it suppresses protein synthesis via Akt-mTOR signaling. As a result, myostatin leads to inhibition of cell cycle progression and reduction of both myogenesis and protein synthesis.

Follistatin-related factors directly bind to myostatin and inhibit its anti-hypertrophic effect [50]. Follistatin-like protein 1 (Fstl1), a muscle-secreted protein, is known as a representative inhibitor of myostatin. It has been shown that Fstl1 is secreted from muscle cells into circulation through activation of Akt-mTOR signal in response to IGF-I [51]. In addition, inflammatory cytokines such as interferon gamma and IL-1β stimulate secretion of Fstl1. Because the level of Fstl1 in circulation is increased by a single bout of exercise in humans [51], the effect of this protein would spread to the entire body in an endocrine manner. Decorin, a leucine-rich proteoglycan, is secreted from contracting muscle into the circulation in response to a bout of exercise and can inhibit function of myostatin via not only direct binding but also stimulating expression of Mighty, a downstream factor of the myostatin cascade [52]. In decorin overexpression muscles, Myod1 and follistatin were increased, whereas ubiquitin ligases, atrogin1 and MuRF1, were decreased [52]. Thus, decorin secreted from muscle cells is involved in hypertrophy and atrophy of skeletal muscle. The expression of decorin has been shown to be increased by strength training in humans and mice, which could contribute adaptation to muscle mass.

More myokines have been reported to induce muscle proliferation and differentiation of satellite cells. Chitinase-3-like protein 1 is elevated in the circulation and muscle tissues by acute exercise and activates myoblast proliferation via protease-activated receptor 2 [53], which is involved in restructuring of skeletal muscle in response to exercise training. IL-7, identified in media from primary cultures of human myotubes differentiated from satellite cells, stimulates satellite cell migration [54]. Interestingly, strength training markedly increases IL-7 expression in skeletal muscle, which suggests that IL-7 is associated with muscle hypertrophic adaptation induced by training. IL-6 also has an aspect that contributes exercise-induced proliferation of satellite cells after muscle damaging exercise [55]. STAT3 signaling induced by IL-6 has been suggested to act as a regulator of the proliferation.

Several proteins secreted by skeletal muscle can regulate bone metabolism; namely, there is a cross talk system between muscle and bone. Conditioned medium from cultured muscle cells stimulates differentiation of bone marrow stromal cells, promotes bone healing, and results in osteoblast formation [56, 57]. It has been suggested that candidate effectors among secreted proteins include the growth factors IGF-I and TGF-β and cytokines such as IL-6 and IL-15 [58–60]. Inaddition, irisin also serves to increase formation of osteoblasts [61]. This effect is mainly exerted through Wnt/β-catenin signaling that can protect osteocytes from glucocorticoid-induced apoptosis [62] and through MAP kinase signaling that promotes osteoblast proliferation and differentiation [63]. Recently, connective tissue growth factor was found as a novel osteogenic factor which secreted from skeletal muscle in bioinformatics analysis [64], and its expression has been shown to be increased in response to exercise [65].

Muscle-secreted proteins may also have anti-inflammatory properties, and muscle-derived IL-6 likely contributes to reduced inflammation when in circulation [66]. IL-6 can increase the levels if anti-inflammatory factors including IL-10, IL-1 receptor agonist, and C-reactive protein in neutrophiles and the liver [67]. Indeed, recombinant IL-6 infusion inhibits the endotoxin-induced increase in circulating levels of tumor necrosis factor (TNF-α), a representative pro-inflammatory cytokine [68]. IL-6 is also recognized as a pro-inflammatory cytokine. In severe systemic infection, circulating IL-6 is drastically elevated and may reach over 10,000-fold the level in resting healthy state. In contrast, chronic low-grade elevation of IL-6 (below tenfold of that in resting healthy state) is induced by sedentary life, obesity, and dietary habits, which are associated with the development of metabolic diseases, although regular physical activity reduces the elevation of circulating IL-6 in the resting state along with metabolic improvement [69, 70]. Therefore, it is necessary to consider separately the exercise-induced secretion of IL-6, which is a transient/moderate elevation, and the pathological states, which are transient/high or chronic/low elevations. Other myokines such as Fstl1 also exert anti-inflammatory activity as a systemic effect [71].

A number of epidemiological studies have showed the average individual’s level of physical activity and its relationship to the incidence of cancer in Europe, the United States, and Japan. The general consensus among the authors of these studies is that physical activity can prevent cancer in the colon, breast, uterus, pancreas, and lungs [72–75]. In particular, almost all studies clearly demonstrate that physical activity significantly reduces the incidence of colon cancer. A review of these epidemiological studies by the World Cancer Research Fund/United States Cancer Research (WCRF/AICR) showed that physical activity was the only lifestyle change that would convincingly reduce an individual’s risk of colon cancer [76]. Although the exact mechanism underlying the beneficial results obtained in epidemiological studies remains unclear, various potential mechanisms such as activation of the immune system and antioxidant status, anti-inflammatory signals, improved insulin sensitivity, proportion of different bile acids, and exercise-induced increases in gastrointestinal transit have been suggested [77–81]. Previously, we reported that regular exercise prevents the formation of aberrant crypt foci (ACF), which are the precursor lesions of colon adenocarcinoma, associated with anti-inflammatory activity on the mucosal surface of the mouse colon [82]. However, the endogenous defense system, such as antioxidants and chaperone proteins remained unchanged, suggesting that the anti-tumorigenesis effect of habitual exercise is affected by the levels of circulating factors such as myokine, rather than endogenous proteins in the colon.

Secreted protein acidic and rich in cysteine (SPARC) was identified as a muscle-derived protein that suppresses tumorigenesis, using comprehensive transcriptome approach of muscle tissue in sedentary and exercised young and old mice [83]. SPARC in the muscle tissue was elevated in response to one bout of exercise. However, circulating SPARC does not adaptively elevate by habitual exercise in the resting state, suggesting that increased SPARC levels in muscle tissues due to regular exercise do not contribute substantially to the circulating concentration while at rest. However, habitual exercise significantly promoted the transient exercise-induced increase in the circulating SPARC levels.

SPARC belongs to the matricellular protein family, which is primarily involved in development, remodeling, and tissue repair through modulation of cell-cell and cell-matrix interactions [84–86]. In addition, SPARC has been reported to have more unique functions such as angiogenesis regulation, collagen production/fibrillogenesis, chaperoning, inhibiting adipogenesis, and further exerting anti-tumorigenic effects [87–90]. Previous studies have revealed that a lack of SPARC promotes pancreatic and ovarian tumorigenesis in vivo [91, 92]. In addition, the presence of exogenous SPARC in cancer cell lines reduces cell proliferation in vitro [93]. Furthermore, epigenetic silencing of the SPARC gene via promoter hypermethylation is frequently identified in colon cancers and is linked to rapid progression of the tumor [94, 95]. Moreover, modulation of SPARC expression affects the sensitivity of colorectal tumors to radiation and chemotherapy [96, 97]. Intriguingly, a clinical study showed that the 5-year survival of patients with tumors that expressed high levels of SPARC was significantly better than that of those with tumors that did not express SPARC [95]. Based on these studies, we examined the effect of the myokine SPARC on the onset of colon tumors by using SPARC-null mice. In azoxymethane (AOM)-induced colon cancer model in mice, habitual low-intensity exercise for 6 weeks reduced the formation of ACF in the colons of wild-type mice [83]. In contrast, more ACF were found in AOM-treated SPARC-null mice than in wild-type mice, and exercise did not have an inhibitory effect. We also examined the effect of exogenous SPARC on ACF formation in the colon by injecting AOM-treated mice with recombinant SPARC. Furthermore, in a cell culture experiment, addition of recombinant SPARC to colon carcinoma cells inhibited cell proliferation in a dose-dependent manner. However, addition of conditioned medium from short interfering RNA (siRNA)-treated muscle cells to the colon carcinoma cells accelerated their proliferation. These results indicated that secreted SPARC suppresses colon tumorigenesis.

A cause of the precursor lesions of tumor formation is dysregulation of apoptosis [98]. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that regular exercise increased the number of apoptotic colon cells in wild-type mice; however, the number did not differ between sedentary and exercised SPARC-null mice [83]. Furthermore, the levels of cleaved caspase-3 and caspase-8 were higher in wild-type mice than in SPARC-null mice, and regular exercise further increased the levels of these apoptosis markers in wild-type mice but not in SPARC-null mice. These findings indicated that SPARC mediates exercise-induced colon reduction via caspase-3- and caspase-8-dependent apoptosis. In addition, we identified an effect of exogenous SPARC on colon tumor by using colon carcinoma cells and found that apoptosis of these cells was elevated by addition of recombinant SPARC in a dose-dependent manner. This in vitro result supports the hypothesis that SPARC prevents proliferation of colon tumor cells via increased apoptosis.

Myokines might also mediate suppression of mammary cancer. Accumulated evidence in a review from WCRF/AICR has shown that a lifestyle of habitual physical activity probably reduces the risk of developing mammary cancer in both premenopausal and menopausal stages [76]. In addition, conditioned medium with serum obtained from mice immediately after exercise suppressed cell growth and enhanced caspase activity in the mammary cancer cell line MCF-7, which was reduced in the serum obtained at 2 h after exercise [99]. Thus, Hojman et al. tried to identify tumor suppressive myokines from candidates in contracting muscle from exercised mice, and identified the secreted protein oncostatin M, which is increased in both muscle tissues and serum in response to one bout of exercise. This myokine inhibits proliferation of MCF-7 cells by activating caspase, suggesting it may inhibit tumor [99].

As discussed above, the novel concept that skeletal muscle is a secretory organ has recently been developed. Currently, over 20 proteins and peptides have been identified and one secreted protein has multiple functions (Table 9.1), for example, IL-6 induces nutrient metabolism in metabolic tissues, insulin secretion from pancreas, and anti-inflammatory activities in blood vessels. This shows that exercise exerts physiological changes and adaptations through complex mechanism. In addition, multiple studies have suggested that several other proteins secreted from muscle have not been identified. For example, a bioinformatics study showed that the secretome of human muscle cells includes over 300 proteins [100]. In addition, an in vitro study demonstrated that myocytes secrete many proteins into the medium during differentiation [101, 102]. Furthermore, transcriptome and proteome studies of human and rodent muscle tissue have demonstrated that the expression of many genes and proteins increases in response to exercise [103–106]. Except proteins and peptides, exercise also releases various metabolic factors from skeletal muscle into circulation. For example, lactate is generated from carbohydrates via glycolytic metabolism and the amount is linked to the intensity of exercise. After its release into blood, lactate is carried to other tissues and is utilized as a substrate for aerobic metabolism or gluconeogenesis. Recently, studies into further roles of such muscle-mediated metabolites, including mitochondria biogenesis and as energy substrates in the brain [107, 108], have suggested that lactate and other substances such as amino acids and ions should be reconsidered as endocrine bioactive factors. In addition, microRNAs (miRNAs) may be secreted from muscle into the circulation and function in an endocrine manner. Some miRNAs are taken into intracellular vesicles (e.g., exosomes) and released into circulation without being degraded by RNase [109, 110]. In addition, the circulating miRNAs (c-miRNAs) can move from the circulation into other cells and regulate their function. This occurs via regulation of gene expression at the posttranscriptional level through translational inhibition or mRNA degradation. Several miRNAs are highly enriched in skeletal muscle [111–113] and may be secreted from muscle into circulation [114]. In the future, many other muscle-secreted bioactive factors including metabolites and microRNA could be identified, which may accelerate the understanding of the effect of exercise on improvement of physical performance and prevention of diseases.