Eugenol promotes the transformation of fast to slow musle fiber.

(A) The body weight of the mice. (B, C), Skeletal muscle weight and representative images of skeletal muscle. (D-F) The protein expression of muscle fiber type in GAS and TA muscle and in C2C12 myotubes. (G-H) The mRNA expression of muscle fiber type in GAS and TA muscle and in C2C12 myotubes. For A-B, N=12 per group. For D-E, N=3 per group. For F and H, N=4 per group. For G, N=6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol promotes oxidative metabolism activity, mitochondrial function, and endurance performance of mice.

(A) The effect of eugenol on exhausting swimming time in mice. (B) The effect of eugenol on metabolism enzymes activity in GAS and TA muscle. (C) The heatmap for the mRNA expression of genes encoding mitochondrial complex components and transcription factors controling mitochondrial biogenesis in GAS muscle. Color gradient represents relative mRNA expression with darker colors indicating higher expression. (D) Protein expression of mitochondrial electron transport complexes in GAS muscle. Complex I (NDUFA1), complex II (SDHA), complex III (UQCRC1), complex IV (MTCO1), and comlex V (ATP5B). (E) mtDNA copy number in muscles and cells. For A, N=15 per group. For B, N=6 per group. For C and F, N=4 per group. For D, N=3 per group. For E, N=6 per group. #P<0.1, *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol enhances lipolysis and thermogenesis.

(A) The ADF and the ration of ADF to ADG. (B) Tissue weight of adipose weight. (C) The level of T-CHO, LDL, and HDL in serum. (D) The mRNA expression of genes related to lipolysis, lipogenesis, and lipid transport in iWAT and gWAT. (E) The mRNA expression of genes related to adipose browning and thermogenesis in iWAT and gWAT. (F) The protein expression of FABP1 and UCP1 in iWAT and gWAT. (G) The expression of protein related to adipose browning and thermogenesis in BAT. (H) The protein expression of mitochondrial electron transport complexes in BAT. For A-B, N=12 per group. For C-E, N=6 per group. For F-H, N=3 per group. #P<0.1, *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol activated TRPV1-mediated CaN/NFATc1 signaling pathway.

(A) The gene expression profile of TRP channels in skeletal muscle was obtained from the GTEx dataset in The Human Protein Atlas (https://www.proteinatlas.org/). (B, C) The mRNA expression of TRP channels in TA muscle and C2C12 myotubes. (D-F) The TRPV1 and CnA protein expression in GAS and TA muscle and in C2C12 myotubes. (G-I) The protein expression of NFATc1 in GAS and TA muscle and in C2C12 myotubes. For B, N=6 per group. For C, F, and I, N=4 per group. For D-E and G-H, N=3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol promotes fast to slow muscle fiber transformation by activating TRPV1-mediated CaN/NFATc1 signaling pathway.

C2C12 myotubes were treated by 25 μM eugenol and 1 μM TRPV1 inhibitor AMG-517 or 0.5 μM CaN inhibitor CsA for 1 day after 4 days of differentiation. (A) The flow cytometry assay was used to detect Ca2+ levels in C2C12 myotubes; FITC means the fluo-4 fluorescence and SSC means side scatter. (B) Western blot was uesd to detect CnA protein expression in C2C12 myotubes. (C) Western blot was uesd to detect mitochondrial electron transport complexes protein expression in C2C12 myotubes. (D) Western blot was uesd to detect slow MyHC and fast MyHC protein expression in C2C12 myotubes. (E) Representative immunofluorescence images of Slow MyHC (Green fluorescence) and relative mean fluorescence intensity quantification. Magnification: × 200. For A-D, N=4 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

The myokines controlled by CaN.

C2C12 myotubes were treated for 16 h with 0, 0.1, 0.5, and 1 μM Ca2+ ionophore after 2 days of differentiation. (A) The mRNA expression of MCIP1. (B) The protein expression of CnA. (C) The enzyme activity of CaN. (D) Fluo-4 was used to stain the Ca2+ and the flow cytometry assay was used to detect Ca2+ fluorescence in C2C12 myotubes in control and 0.5 μM A23187 groups. (E) The heatmap for the myokines mRNA expression in control and 0.5 μM A23187 groups. Color gradient represents relative mRNA expression with darker colors indicating higher expression. (F) Correlation analysis of gene expression values of myokines and MCIP1 gene performed by linear regression with Pearson’s correlation coefficient (r). Color gradient represents correlation coefficient with darker colors indicating higher positive correlation. (G) The number of binding sites for transcription factors NFATc1 were predicted by hTFtarget and JASPAR. For A, N=6 per group. For B-D, N=3 per group. For E, N=4 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Myokines to be tested in our study.

The myokine IL-15 expression depends on CaN/NFATc1 signaling pathway.

(A) C2C12 myotubes were treated for 16 h with 0, 0.1, 0.5, and 1 μM Ca2+ ionophore after 2 days of differentiation. The protein expression of IL-15. (B-D) C2C12 myotubes were treated by 0.5 μM A23187 and 0.5 μM CsA for 16 h after 2 days of differentiation. The protein expression of CnA, NFATc1, and IL-15. (E) Sequence logo of NFATc1 motif and the predicted NFATc1 binding sites in the promoter region of IL-15. (F) NFATc1 and the key sequence of IL-15 (5’-AATGAAAA-3’) docking. Confidence scores above 0.7 indicate high probability of binding, scores between 0.5 and 0.7 suggest possible binding, and scores below 0.5 indicate unlikely binding. (G) The probe sequence of NFATc1. The underline represents the predicted binding site of NFATc1, bio-NFATc1 means oligonucleotide probes that labeled with biotin at the 5’ end, cold-NFATc1 means oligonucleotide probes that did not labeled with biotin, mu-NFATc1 means oligonucleotide probes that was mutated at the binding site. (H) Nuclear protein extracts (NE) with NFATc1 probe were used to EMSA assay. (I) The protein expression of NFATc1 after transfecting 1 μg/mL pcDNA3.1 vector, 0.5 μg/mL or 1 μg/mL pcDNA3.1-NFATc1 in HEK293T cells. (J) The relative luciferase intensity referred to the ratio between firefly luciferase intensity and renilla luciferase intensity. For A-D, N=3 per group. For H, N=6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol promotes the expression and secretion of IL-15 in skeletal muscle of mice.

(A) The IL-15 mRNA expression in GAS and TA muscle. (B, C) The IL-15 protein expression in GAS and TA muscle. (D) Left: The mRNA expression of MyHC I, MyHC IIa, MyHC IIb and IL-15 in EDL and SOL muscle of mice. Right: Correlation analysis of gene expression values performed by linear regression with Pearson’s correlation coefficient (r). Color gradient represents correlation coefficient with darker colors indicating higher positive correlation. (E) The protein expreesion of slow MyHC, fast MyHC, and IL-15. (F) The concertration of IL-15 in serum. For A, D, and F, N=6 per group. For B-C and E, N=3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Eugenol promotes IL-15 level through TRPV1-mediated CaN/NFATc1 signaling pathway.

C2C12 myotubes were treated by 0-200 eugenol for 1 day after 4 days of differentiation. (A) The effect of eugenol on IL-15 mRNA expression in C2C12 myotubes. (B) The effect of eugenol on IL-15 protein expression in the C2C12 cell medium. Coomassie staining as loading control. (C, D) C2C12 myotubes were treated by 25 μM eugenol and 1 μM TRPV1 inhibitor AMG-517 or 0.5 μM CaN inhibitor CsA for 1 day after 4 days of differentiation. The mRNA and protein expression of IL-15. N=4 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Effect of eugenol on C2C12 cell viability.

Cells were treated with 0, 12.5, 25, 50, 100, 200 μM EUG for 1 day after 4 days of differentiation. The C2C12 cell viability was measured using CCK-8 kit. N=6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

TRP channels expression profiles and TRPV1 mRNA eexpression in adipose tissue.

The gene expression profile was obtained from the GTEx dataset in The Human Protein Atlas (https://www.proteinatlas.org/). (A) TRPV1 expression profiles in tissues. (B) TRP channels expression profiles in adipose tissue. (C) The mRNA expression of TRP channels in adipose tissue. For C, N=6 per group. #P<0.1, *P < 0.05, **P < 0.01, and ***P < 0.001.

Molecular docking for eugenol and TRPV1.

The capsaicin binding sites (TYR511, SER512, THR550, and GLU570) were selected as the binding pocket. The figure showed TRPV1 amino acid residues interacting with eugenol and the intermolecular force.

The mRNA expression of MCIP1.

N=6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Ca2+ ionophore A23187 promotes the transformation of fast to slow muscle fiber by CaN signaling pathway.

A-C The mRNA and protein expression of muscle fiber type in C2C12 myotubes. For B, N=4 per group. For A and C, N=3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Representative immunofluorescence images of IL-15.

IL-15 (Green fluorescence) and DAPI (Blue fluorescence). Magnification: × 200.