Due to the lack of regular exercise among many individuals (Carlson et al., 2010), metabolic diseases are highly prevalent in modern society (Hoehn et al., 2010). To address this issue, pharmacological interventions have been considered as potential treatments. Exercise mimetics are pharmacological compounds that produce fitness benefits (Fan and Evans, 2017). Although some synthetic exercise mimetics such as AICAR (AMPK activator), GW501516 (PPARδ activator), SRT1720 (SIRT1 activator), and GSK4716 (ERRγ activator) have been used to improve fitness (Feige et al., 2008; Narkar et al., 2008; Rangwala et al., 2010), the safety and health of each drug must be considered. In recent years, plant extracts such as resveratrol, lycium barbarum, and epicatechin have demonstrated potential as a new class of low-toxin exercise mimetics (Meng et al., 2020; Nogueira et al., 2011; Wen et al., 2020). Therefore, it is of great significance to discover natural medicinal and edible plants that have mimetic effects of exercise.

Based on their metabolism and contractile properties, muscle fibers are commonly divided into slow oxidative fibers (muscle fibers that express myosin heavy chain (MyHC) I) and fast glycolytic/oxidative fibers (muscle fibers that express MyHC IIa, MyHC IIx, or MyHC IIb) (Schiaffino and Reggiani, 2011). Different muscle fibers exhibit a high degree of plasticity and are regulated by exercise (Egan and Zierath, 2013). The increased proportion of slow muscle fibers contributes to the enhancement of mitochondrial biogenesis and lipid metabolism (Carlson et al., 2010). Therefore, one of the benefits of exercise is to treat metabolic disorders by promoting slow muscle fiber remodeling (Duan et al., 2017). In addition, it has been increasingly recognized that myokines released by skeletal muscle play an essential role in mediating the benefits of exercise. Myokines are defined as cytokines or peptides that are released from skeletal muscle cells, which exert autocrine, paracrine, or endocrine effects (Pedersen and Febbraio, 2012). As a medium of crosstalk between muscles and other organs, myokines exert systemic regulation of exercise by acting on the muscle, fat, liver, pancreas, and other organs, effectively improving insulin resistance, obesity, and metabolic disorders of type 2 diabetes (Severinsen and Pedersen, 2020). Therefore, another potential effect of exercise is to induce the release of myokines (Fan and Evans, 2017). Although many previous studies have discovered plant extracts as exercise mimetics by promoting slow oxidative muscle fiber, few studies have investigated the effect of exercise mimetics on the release of myokines.

Eugenol is a safe natural compound extracted from clove oil and various plant spices such as basil, bay leaves, and cinnamon (Kaur et al., 2010). Eugenol exhibits a variety of biological activities, including antioxidative (Magalhães et al., 2019), antibacterial (Devi et al., 2010), anti-inflammatory (Magalhães et al., 2019), and anti-cancer activities (Lesgards et al., 2014). Recent studies have also suggested that eugenol could be a promising therapeutic drug for preventing diabetes and obesity (Al Trad et al., 2019; Jung et al., 2012; Mnafgui et al., 2013; Srinivasan et al., 2014), indicating its potential as an exercise mimetic. In addition, as a member of the transient receptor potential (TRP) channel family, TRP vanilloid 1 (TRPV1), also known as the capsaicin receptor, has been reported to improve endurance capacity and energy metabolism (Luo et al., 2012), counter obesity (Baskaran et al., 2016), and intervene diabetes (Wang et al., 2012), making it a potential target protein for discovering exercise mimetics.

Eugenol contains a vanilloyl fragment that may bind to TRPV1 similarly to capsaicin, implying that eugenol might exert its biological activity via TRPV1 (Harb et al., 2019). It has been found that eugenol activates TRPV1 in a heterologous expression system and rat trigeminal ganglion neurons (Xu et al., 2006; Yang et al., 2003). Furthermore, a recent study demonstrated that eugenol activated TRPV1 in skeletal muscle (Jiang et al., 2022). TRPV1 is an important Ca²⁺ entry pathway contributing to increase intracellular Ca²⁺ (Nilius et al., 2007), suggesting that TRPV1 may regulate many biological processes through Ca²⁺-dependent signaling pathways. As a Ca²⁺-dependent signaling, calcineurin (CaN) is a core signaling to promote fast-to-slow muscle fiber transformation (Sakuma and Yamaguchi, 2010). Additionally, CaN signaling plays an essential role in regulating the expression of myokine IL-6 (Banzet et al., 2005; Banzet et al., 2007). Therefore, we hypothesize that eugenol, as an exercise mimetic, promotes the release of myokines and fast-to-slow muscle fiber transformation via the TRPV1-mediated CaN signaling pathway. The primary objective of this study is to investigate this hypothesis.

Our study reveals that eugenol can serve as an exercise mimetic that promotes remodeling of skeletal muscle fibers and expression of the myokine IL-15 through the TRPV1-mediated CaN/NFATc1 signaling pathway. This expands the traditional biological functions of eugenol and provides a theoretical basis for its potential applications in the food and drug industry. Moreover, we identify a novel TRPV1-mediated CaN/NFATc1 signaling pathway that promotes the transformation of fast-to-slow muscle fibers. Importantly, we provide the first explanation of the mechanism underlying the release of the myokine IL-15.

TRPV1, like all other TRP channels, assembles as tetramers to form cation-permeable pores (Clapham, 2003). Initially identified as a capsaicin receptor and heat-activated ion channel that modulates pain and neurogenic inflammation (Caterina et al., 1997), subsequent studies have found that TRPV1 is expressed on many non-neural sites and plays roles in immunity, vasculature, obesity, and thermogenesis (Fernandes et al., 2012). Previous studies have also shown that capsaicin activated TRPV1 to improve endurance capacity and energy metabolism (Luo et al., 2012), counter obesity (Baskaran et al., 2016), and intervene diabetes (Wang et al., 2012). Therefore, TRPV1 is a strong target for the discovery of exercise mimetics, which drove us to search for TRPV1 agonists that may act as exercise mimetics. We focused on eugenol, a healthy and edible plant extract that may be a potential TRPV1 agonist. Since eugenol contains a vanilloyl fragment, it is possible to bind TRPV1 through a similar pattern to capsaicin. Indeed, previous studies have found that eugenol activates TRPV1 in a heterologous expression system and rat trigeminal ganglion neurons (Xu et al., 2006; Yang et al., 2003), and a recent study reported that 50 and 100 μM eugenol promoted TRPV1 expression in C2C12 myotubes (Jiang et al., 2022). In our study, we investigated the mRNA expression profile of TRP channels in C2C12 myotubes and skeletal muscle, and found that only TRPV1 mRNA was upregulated in response to eugenol treatment. Moreover, it was observed that the protein expression of TRPV1 was consistent with the mRNA expression. Our molecular docking results also indicated that eugenol bound to the capsaicin binding pockets of TRPV1. Based on these findings, we propose that eugenol specifically activates TRPV1 in skeletal muscle, rather than other TRP channels. This may be due to the relatively higher expression of TRPV1 in skeletal muscle compared to other TRP channels.

One of the benefits of exercise mimetics for body is to promote the skeletal muscle oxidative phenotype and endurance performance (Fan and Evans, 2017). Our studies showed that eugenol improved endurance performance and promoted fast-to-slow muscle fiber transformation. Slow muscle fibers are characterized by the improvement in mitochondrial function and oxidative metabolism (Choi and Kim, 2009). As expected, eugenol also promoted mitochondrial function and oxidative metabolism capacity in skeletal muscle. In a previous study, capsaicin-induced TRPV1 activation was shown to promote exercise endurance and the skeletal muscle oxidative phenotype (Wang et al., 2012). The authors suggested that TRPV1 activated calmodulin-dependent protein kinase (CaMK) by increasing intracellular Ca²⁺, and the activation of CaMK further activated PGC-1α, contributing to these effects (Wang et al., 2012). In skeletal muscle, the CaN signaling pathway is also an important Ca²⁺-mediated signal that promotes the muscle oxidative phenotype (Sakuma and Yamaguchi, 2010). CaN induces the translocation of NFATc1 to the nucleus by dephosphorylating NFATc1, thereby switching fast-to-slow muscle fibers (Calabria et al., 2009). TRPV1 activation has been shown to promote CaN activity in several other cells and tissues (Hou et al., 2019; Ma et al., 2011; Yang et al., 2018), but the link between TRPV1 and CaN signaling in skeletal muscle has not yet been established, nor has it been determined whether TRPV1 promotes muscle oxidative phenotype through the CaN/NFATc1 signaling pathway. Therefore, we investigated the role of TRPV1-mediated CaN signaling pathway in the regulation of muscle fiber types. We found that eugenol increased CaN expression through the activation of TRPV1, and that it promoted fast-to-slow muscle fiber transformation via the TRPV1-mediated CaN/NFATc1 signaling pathway. Recent studies have also found that eugenol increases muscle glucose uptake through the TRPV1/CaMK signaling pathway (Jiang et al., 2022), suggesting that both CaN and CaMK signaling may be involved in TRPV1-facilitated skeletal muscle oxidative phenotype.

Another benefit of exercise mimetics is the alleviation of obesity and improvement of metabolic health. It has been reported that an increase in slow-twitch fiber content is positively associated with reduced obesity and improved metabolic health (Carlson et al., 2010). Previous studies have shown that eugenol can reduce blood lipids in rats with hyperlipidemia (Harb et al., 2019), lower blood glucose and insulin resistance in diabetic mice (Al Trad et al., 2019; Sanae et al., 2014), and reduce hepatic lipid accumulation in high-fat-fed mice (Rodrigues et al., 2022), demonstrating its potential in improving glucose and lipid metabolism. In our study, we found that feeding eugenol under standard diet conditions may promote fat thermogenesis and browning to facilitate fat breakdown. Interestingly, it was found that capsaicin alleviated obesity and promoted white fat browning by activating the TRPV1 (Baskaran et al., 2016). Our study found that eugenol promoted TRPV1 mRNA expression in adipose tissue, indicating that eugenol may promote white fat browning through TRPV1 activation.

Exercise mimetics may exert beneficial effects on the body by promoting the release of myokines (Fan and Evans, 2017). Some myokines, such as IL-13 (Knudsen et al., 2020), IL-15 (Quinn et al., 2013), and neurturin (Correia et al., 2021), have been well documented to improve metabolic homeostasis, promote fast-to-slow muscle fiber transformation, and improve endurance capacity. However, the regulatory mechanisms for myokines expression are largely unclear. Since myokines are usually promoted by muscle contraction, and Ca²⁺ is the main signal in muscle contraction, CaN, downstream of Ca²⁺, has been considered to regulate myokine expression. CaN has been reported to regulate the expression of myokine IL-6 (Banzet et al., 2005; Banzet et al., 2007). Additionally, the transcriptional activation activity of myokines IL-4 and IL-13 is thought to be promoted by transcription factor NFATc2, which is downstream of CaN (Jacquemin et al., 2007; Zádor, 2008). Based on the correlation and TFBS analysis, it was found that IL-15 is most likely to be regulated by the CaN/NFATc1 signaling pathway in our study. IL-15 has been reported as a myokine that improves fatty acid utilization, insulin sensitivity, and endurance capacity, and prevents obesity and diabetes (Nadeau and Aguer, 2019). In different cell models, CsA-mediated inhibition of CaN decreased the release of IL-15 (Cho et al., 2002; Cho et al., 2007). Furthermore, an increase in CaN expression in 3T3-L1 cells was accompanied by an increase in IL-15 expression (Almendro et al., 2009). Our study found that the CaN/NFATc1 signaling pathway contributes to the eugenol-promoted expression of IL-15. Furthermore, we observed that the expression of IL-15 was higher in the slow-twitch SOL muscle than in the fast-twitch EDL muscle, suggesting that an increase in muscle oxidative phenotype may further promote the release of IL-15. However, the mechanism underlying how IL-15 promotes the muscle oxidative phenotype remains unclear and requires further investigation.

Apparently, an interesting finding throughout our in vitro and in vivo study was that the high doses of eugenol (200 mg/kg for mice and 200 μM eugenol for C2C12 myotubes) had no effect on TRPV1-mediated CaN/NFATc1 signaling pathway, IL-15 expression, and muscle fiber type. We suspected that high doses of eugenol may cause desensitization of TRPV1 in skeletal muscle under our study conditions. This indirectly verified that the activation of TRPV1 does promote IL-15 expression and fast-to-slow muscle fiber transformation type. It is well known that continuous activation of TRPV1 by capsaicin causes TRPV1 desensitization, which is considered to underlie the analgesic effects of capsaicin (Koplas et al., 1997). As previously reported, TRPV1 agonists promoted TRPV1 desensitization in a dose- and time-dependent manner by targeting lysosomes to degrade TRPV1 in HEK293 cells (Sanz-Salvador et al., 2012). A hypothetical model suggested that upon TRPV1 opening to allow Ca²⁺ influx, Ca²⁺/CaM binds to the CaM-binding domain of TRPV1, leading to channel inactivation, and CaN dephosphorylates TRPV1 to desensitize TRPV1 (Hasan and Zhang, 2018). However, most results on TRPV1 desensitization were obtained by capsaicin treatment of cells, and it is unknown whether high-dose eugenol promotes TRPV1 desensitization through a similar mechanism. Therefore, it is essential to select the appropriate dose of eugenol for the application of TRPV1 activators.

In conclusion, our findings indicate that eugenol can promote the transformation of fast-to-slow muscle fibers and induce the myokine IL-15 expression through the TRPV1-mediated CaN/NFATc1 signaling pathway. Moreover, our study is the first to demonstrate that the expression of IL-15 is regulated by the CaN/NFATc1 signaling pathway. These results suggest that eugenol may have potential as a novel exercise mimetic and that TRPV1 may represent a promising therapeutic target for metabolic disorders.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Tengteng Huang

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Data curation, Formal analysis, Investigation, Writing - original draft

   Contributed equally with

   Xiaoling Chen

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3583-1835

2. Xiaoling Chen

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Conceptualization, Supervision, Methodology, Project administration

   Contributed equally with

   Tengteng Huang

   Competing interests

   No competing interests declared

3. Jun He

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

4. Ping Zheng

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Yuheng Luo

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Aimin Wu

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

7. Hui Yan

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7476-3914

8. Bing Yu

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

9. Daiwen Chen

   Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

10. Zhiqing Huang

    Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, China

    Contribution

    Conceptualization, Supervision, Funding acquisition, Methodology, Writing - review and editing

    For correspondence

    zqhuang@sicau.edu.cn

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5092-9297

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