Eugenol mimics exercise to promote skeletal muscle fiber remodeling and myokine IL-15 expression by activating TRPV1 channel

Reviewer #1 (Public Review):

Summary:
In this manuscript, Huang et al have investigated the exercise mimetic role of Eugenol (a natural product) in skeletal muscle and whole-body fitness. The authors report that Eugenol facilitates skeletal muscle remodeling to a slower/oxidative phenotype typically associated with endurance. Eugenol also remodels the fat driving browning the WAT. In both skeletal muscle and fat Eugenol promotes oxidative capacity and mitochondrial biogenesis markers. Eugenol also improves exercise tolerance in a swimming test. Through a series of in vitro studies the authors demonstrate that eugenol may function through the trpv1 channel, Ca mobilization, and activation of CaN/NFAT signaling in the skeletal muscle to regulate slow-twitch phenotype. In addition, Eugenol also induces several myokines but mainly IL-15 through which it may exert its exercise mimetic effects. Overall, the manuscript is well-written, but there are several mechanistic gaps, physiological characterization is limited, and some data are mostly co-relative without vigorous testing (e.g. link between Eugenol, IL15 induction, and endurance). Specific major concerns are listed below.

Strengths:
A natural product activator of the TRPV1 channel that could elicit exercise-like effects through skeletal muscle remodeling. Potential for discovering other mechanisms of action of Eugenol.

Weaknesses:
(1) Figure 1: Histomorphological analysis using immunostaining for type I, IIA, IIX, and IIB should be performed and quantified across different muscle groups and also in the soleus. Fiber type switch measured based on qPCR and Westerns does not sufficiently indicate the extent of fiber type switch. Better images for Fig. 1c should be provided.

(2) Figure 2: Histomorphological analysis for SDH and NADH-TR should be performed and quantified in different muscle groups. Seahorse or oroborous respirometry experiments should be performed to determine the actually increase in mitochondrial respiratory capacity either in isolated mitochondria or single fibers from vehicle and Eugenol-treated mice. Em for mitochondrial should be added to determine the extent of mitochondrial remodeling. The current data is insufficient to indicate the extent of mitochondrial or oxidative remodeling.

(3) Figure 2: Gene expression analysis is limited to a few transcriptional factors. A thorough analysis of gene expression through RNA-seq should be performed to get an unbiased effect of Eugenol on muscle transcriptome. This is especially important because eugenol is proposed to work through CaN/NFAT signaling, major transcriptional regulators of muscle phenotype.

(4) I suggest the inclusion of additional exercise or performance testing including treadmill running, wheel running, and tensiometry. Quantification with a swimming test and measurement of the exact intensity of exercise, etc. is limited.

(5) In addition to muscle performance, whole-body metabolic/energy homeostatic effects should also be measured to determine a potential increase in aerobic metabolism over anaerobic metabolism.

(6) For the swimming test and other measurements, only 4 weeks of vehicle vs. Eugenol treatment was used. For this type of pharmacological study, a time course should be performed to determine the saturation point of the effect. Does exercise tolerance progressively increase with time?

(7) The authors should also consider measuring adaptation to exercise training with or without Eugenol.

(8) Histomorphological analysis of Wat is also lacking. EchoMRI would give a better picture of lean and fat mass.

(9) The experiments performed to demonstrate that Eugenol functions through trpv1 are mostly correlational. Some experiments are needed with trpv1 KO or KD instead of inhibitor. Similarly, KD for other trpv channels should be tested (at least 1-4 that seem to be expressed in the muscle). Triple KO or trpv null cells should be considered to demonstrate that eugenol does not have another biological target.

(10) Eugenol + trpv1 inhibition studies are performed in c2c12 cells and only looks at myofiber genes expression. This is incomplete. Some studies in mitochondrial and oxsphos genes should be done.

(11) The experiments linking Eugenol to ca handling, and calcineurin/nfat activation are all performed in c2c12 cells. There seems to be a link between Eugenol activation and CaN/NFAT activation and fiber type regulation in cells, however, this needs to be tested in mouse studies at the functional level using some of the parameters measured in aims 1 and 2.

(12) The myokine studies are incomplete. The authors show a link between Eugenol treatment and myokines/IL-15 induction. However, this is purely co-relational, without any experiments performed to show whether IL-15 mediates any of the effects of eugenol in mice.

(13) An additional major concern is that it cannot be ruled out that Engenol is uniquely mediating its effects through trpv1. Ideally, muscle-specific trpv1 mice should be used to perform some experiments with Eugenol to confirm that this ion channel is involved in the physiological effects of eugenol.

Reviewer #2 (Public Review):

Summary:
The authors examined the hypothesis that eugenol enhances the metabolic profiles of skeletal muscles by activating the TRPV1-Ca2+-calcineurin-NFATc1-IL-15 signalling pathway. They first show that eugenol promotes skeletal muscle transformation and metabolic functions in mitochondria and adipose tissues by analysing changes in the expression of mRNA and proteins of relevant representative protein markers. With similar methodologies, they further found that eugenol increases the expression of mRNA and/or proteins of TRPV1, CaN, NFATC1, and IL-15. These processes were, however, prevented by inhibiting TRPV1 and CaN. Similar expression changes were also triggered by increasing intracellular Ca2+ with A23187, suggesting a Ca2+-dependent process.

Strengths:
Different protein markers were used as a readout of the functions of skeletal muscles, mitochondria, and adipose tissues and analysed at both mRNA and protein levels. The results are mostly consistent though it is not always the case. Although the signaling pathway of TRPV1-Ca2+-CaN-NFAT is not new and well documented, they identified IL-15 as a new downstream target of this pathway,

Weaknesses:
Apart from Fig.2A and 2B, they mostly utilised protein expression changes as an index of tissue functional changes. Most of the data supporting the conclusions are thus rather indirect. More direct functional evidence would be more compelling. For example, a lipolysis assay could be used to measure the metabolic function of adipocytes after eugenol treatment in Fig.3. Functional activation of NFAT can be demonstrated by examining the nuclear translocation of NFAT.

To further demonstrate the role of TRPV1 channels in the effects of eugenol, TRPV1-deficient mice and tissues could also be used. Will the improved swimming test in Fig. 2B and increased CaN, NFAT, and IL-15 triggered by eugenol be all prevented in TRPV1-lacking mice and tissues?

Direct evidence of eugenol activation of TRPV1 channels in skeletal muscles is also lacking. The flow cytometry assay was used to measure Ca2+ changes in the C2C12 cell line in Fig. 5A. But this assay is rather indirect. It would be more convincing to monitor real-time activation of TRPV1 channels in skeletal muscles not in cell lines using Ca2+ imaging or electrophysiology.