A few chronic diseases such as type 2 diabetes, cardiovascular disease, colon cancer, breast cancer, dementia, depression and so on are defined as ‘a diseasome of physical inactivity’. Visceral fat accumulation leading to systemic inflammation caused by lack of exercise is an independent and strong risk factor for many chronic diseases. Given that exercise is recognized as an effective method to reduce visceral adipose tissue (VAT) mass, the protective effect of exercise on disease is attributed to the anti-inflammatory effect[8]. Myokines play a key role in exercise-mediated fat loss[7]. However, not everyone is suitable for exercise. The conclusions of this study have suggested that dietary n-3 PUFAs seem to be an alternative to exercise to reap the metabolic benefits of exercise.
Even though IL-6 is often regarded as a pro-inflammatory factor, as the first myokine discovered[9], the metabolic benefits of IL-6 cannot be ignored. In spite of the role of IL-6 on glucose metabolism is still controversial[10], its effect on lipid metabolism is outstanding. A randomized controlled study has provided direct evidence that the reduction of VAT mass after cycling depends on the body's IL-6 level[11]. Besides, more and more studies have reported that IL-6 is an important endocrine factor stimulating white fat browning[12] which plays the biological effects of improving insulin resistance and reducing body weight by increasing the oxidation of glucose and fatty acids[13]. IL-6 level both in skeletal muscle and serum increase significantly after exercise, which confirmed that exercise is the main driving force for the production of muscle-derived IL-6[6]. The present study showed that n-3 PUFAs intervention promoted the expression and secretion of IL-6 in myotube cells. It is an interesting finding that n-3 PUFAs does not work as anti-inflammatory nutrients but may as positive dietary inducers on muscle-derived IL-6. However, it is still unclear whether n-3 PUFAs also mediate the production of IL-6 through the muscle contraction mechanism[6]. The research results of Watt MJ et al. showed that IL-6 infusion reduced hormone-sensitive lipase (HSL) in the adipose tissue of patients with type 2 diabetes[14]. In this study, the expression of HSL in 3T3-L1 adipocytes was increased, and the expression of browning markers in 3T3-L1 adipocytes was also up-regulated. These all provide strong evidence that muscle-derived IL-6 may be a key myokine regulating the skeletal muscle-adipose tissue axis by n-3 PUFAs.
Irisin, as a recently discovered new muscle factor, is always reported to be beneficial to health and its active role in muscle-adipose tissue crosstalk is particularly prominent. Irisin mainly increases thermogenesis by stimulating the browning of white adipose tissue, thereby increasing energy expenditure. It is considered to be a potential target for the treatment of obesity and related diseases[15]. A double-blind controlled trial has showed that n-3 PUFAs supplementation increases the plasma Irisin level in patients with coronary heart disease[16] and type 2 diabete[17] and pregnant women[18]. However, such studies have not determined whether plasma Irisin is derived from skeletal muscle. Our study found that n-3 PUFAs increased the transcription level of Irisin in myotube cells, which related to the browning of 3T3-L1 adipocytes. Combined with the clinical trial results above-mentioned, it is clear that n-3 PUFAs may activate white adipose tissue browning by stimulating expression and secretion of Irisin in skeletal muscle.
IL-15 expresses in bone marrow, secondary lymphoid tissues, and many non-lymphoid tissues including adipose tissue and skeletal muscle, and its expression in skeletal muscle being particularly high[19]. In rodents, high level of IL-15 is associated with decreased adiposity. Overexpression of IL-15 in skeletal muscle has been found to reduce the mass of VAT[20] rich in a large number of IL-15 receptors[21]. In addition, IL-15 has been reported to stimulate adiponectin secretion by 3T3-L1 adipocytes[22]. All these seem to provide evidence that IL-5 works on skeletal muscle-adipose tissue crosstalk. Nevertheless, instead of in an endocrine fashion, IL-15 may likely act locally in skeletal muscle by increasing fatty acid oxidation, in turn, would increase fatty acid uptake into skeletal muscle and finally limit the availability of fatty acid for adipose tissue in humans[23]. Anyway, there is no doubt that high IL-15 level in skeletal muscle improve fatty acid utilization. Lots of studies have demonstrated that circulating IL-15 levels is increased after an acute exercise in humans[24]. Previous studies have also found that supplementing 2.5g of n-3 PUFAs per day (DHA:EPA = 1:2, for 8 weeks) increase the serum IL-15 level in women with depression[21]. However, there was no study on n-3 PUFAs regulating IL-15 expression in skeletal muscle before this article, even though a decrease of intestinal IL-15 expression in the fish oil group was reported[25]. Therefore, present study is the first to make it clear that n-3 PUFAs can stimulate skeletal muscle to express IL-15, but more research is thus needed to determine whether n-3 PUFAs promote the release of myogenic IL-15. Lebris S Quinn et al. have confirmed that IL-15 can stimulate 3T3-L1 adipocytes to secrete adiponectin[25]. However, the increase in adiponectin expression in 3T3-L1 adipocytes in our study did not seem to be related to skeletal muscle IL-15 expression. Consequently, it is still unclear whether IL-15 is involved in skeletal muscle-adipose tissue crosstalk regulating by n-3 PUFAs.
Myostatin (MSTN) is known as a key protein in the regulation of energy metabolism and muscle insulin resistance. Overexpression of MSTN can inhibit the transcription of GLUT4 and block insulin signaling[26]. Conversely, lacking MSTN presents with enhanced insulin sensitivity[27] and increased browning of the SAT[28] in mice. Marzia Bianchi et al. have discovered that maternal intake of n-3 PUFAs during pregnancy influence DNA methylation levels of MSTN in cord blood white cells of newborns, regrettably, have not done any research on the relationship between n-3 PUFAs and skeletal muscle MSTN[28]. In this study, we have found that both ALA and EPA significantly reduce the expression of MSTN, which suggests that ALA and EPA may participate in maintaining the body's glucose and lipid metabolic homeostasis by regulating muscle-derived MSTN.
Fibroblast growth factor (FGF) 21, a mediator of glucose and lipid metabolism, is produced by the liver, adipose tissues, and skeletal muscle. The use of FGF21 reduces hepatic glucose production and plasma glucose levels, while increasing insulin sensitivity and glucose uptake in adipose tissue. In addition, FGF21 reduces liver and plasma triglycerides and body weight, while activating brown adipose tissue[30]. Although exogenous administration of FGF21 exerts beneficial effects on glucose and lipid metabolism, circulating FGF21 levels are elevated in ob/ob and db/db mice, diet-induced obese mice and obese human[31]. Therefore, obesity may be an FGF21-resistant state. n-3 PUFAs have been found to increase the expression of FGF21 in C2C12 myotubes observed in our study. However, Katsunori Nonogak et al. reported that a diet with EPA over 6 days decreased plasma FGF21 levels of individually-housed KKAy mice, but not affected FGF21 mRNA level of soleus muscle[31]. Although the up-regulation of genes related to the lipid metabolism and browning in 3T3L1 adipocytes treated with supernatant of myotube cell culture medium treated with DHA/EPA was observed, the changes in FGF21 levels in the myotube cell culture supernatant was not be detected in our study. Nevertheless, Xavier Escoté et al. found recently that EPA supplementation increased circulating FGF21 in overweight/obese women following a hypocaloric diet[33]. In consequence, we propose that n-3 PUFAs, at least including EPA, play a role in regulating function and metabolism of adipocytes through muscle-derived FGF21.
Adiponectin and leptin are produced and secreted by adipocytes in most cases and are definite as adipokines. Most of the studies targeting this two proteins have been focused on adipose-tissue secretes adiponectin and leptin which can be found in the circulation. However, it has been described that adiponectin and leptin are also produced by muscle cells. Delaigle et al. reported early the presence of adiponectin mRNA and protein in mouse tibialis anterior muscles[33]. Moreover, the evidence that exercise induces adiponectin expression in skeletal muscle is also clear[35]. It is obvious to all that adiponectin plays the prominent role in anti-inflammatory, anti-atherosclerotic, anti-obesity and improving glucose and lipid metabolism disorder[36]. Earlier studies have proven that n-3 PUFAs are promoters of adiponectin in adipose tissue[37]. Although the secretion level was not analyzed, increased expression of adiponectin in C2C12 myotubes treated with EPA was present in current study. Such a result indicates that EPA may participate in the regulation of myogenic adiponectin. However, it is unclear whether myogenic adiponectin is involved in skeletal muscle-adipose tissue axis, because some researchers regard myogenic adiponectin plays an autocrine/paracrine role[38]. Athough leptin derived from adipocytes is considered to be a risk factor for diseases in most cases, it cannot be ignored that leptin in skeletal muscle palys antiobesity and antidiabetic roles[39]. We found that the combined intervention of DHA and EPA stimulated myotube cells to express leptin, which was contrary to the down-regulation of n-3 PUFAs on adipose tissue and circulating leptin in high-fat diet mice. Similarly, whether the myogenic leptin induced by n-3 PUFAs exerts endocrine function need further research, because its local effect in skeletal muscle may be more important[40].
Although TNF-α considered to be a pro-inflammatory, it is also one of the myokines becauce of its expression induced by exercise and related to muscle damage caused by exercise[41]. Importantly, the promote lipolysis effect of TNF-α has been reported[42]. In this study, DHA and EPA seemed to have different effects on the expression of muscle-derived TNF-α. We believe that DHA inhibits skeletal muscle express TNF-α may be the result of DHA exerting anti-inflammatory effect[43], while further study required to figure out whether EPA-mediated skeletal muscle TNF-α expression is related to lipolysis[44].
AMPK is described as a central regulator of metabolism. In this study, we tried to find out the role of AMPK in the regulation of myokines expression by n-3 PUFAs. Although previous reports have pointed out that the production of myogenic IL-15 is also related to AMPK[45], we only found that the expression of Irisin depends on activation of AMPK signaling completely. Irisin is secreted into the circulation after the proteolytic cleavage of fibronectin type III domain-containing protein 5 (FNDC5)[46] of which production has been determined to be related to peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)[47]. The regulatory effect of DHA/EPA on Irisin expression and PGC-1α was blocked by the AMPK antagonist compound C, suggesting that DHA/EPA increased Irisin via the AMPK pathway. The transcription of other myokines can be activated by Metformin, indicating that part of the production of these myokines should be attributed to AMPK signaling, but more mechanisms may be independent of AMPK.