Ferulic acid regulates muscle fiber type formation through the Sirt1/AMPK signaling pathway

Abstract

Ferulic acid (FA) is a polyphenolic compound of plant cell walls. Several recent studies demonstrated that polyphenolic compounds could regulate muscle fiber type formation. In this study, we investigated the effect of FA on muscle fiber type formation and also explored its molecular mechanism in mouse C2C12 myotubes. Real-time quantitative PCR showed that FA significantly increased the mRNA abundances of myosin heavy chain (MyHC) I, MyHC IIa, sirtuin1 (Sirt1), peroxisome proliferator-activated receptor gamma coactivator-1-α (PGC-1α) and myocyte enhancer factor 2C (MEF2C), but decreased the mRNA abundance of MyHC IIb. FA increased succinate dehydrogenase (SDH) activity and decreased lactate dehydrogenase (LDH) activity. Western blot analysis showed that FA significantly increased the protein level of slow-MyHC, but significantly decreased the protein level of fast-MyHC, which were attenuated by AMP-activated protein kinase (AMPK) inhibitor Compound C, AMPKα2 siRNA or Sirt1 inhibitor EX527. We also showed that EX527 attenuated FA-induced increase in the phosphorylation levels of AMPK and liver kinase B1 (LKB1), indicating that AMPK serves as a downstream target of Sirt1. Our present study provides the first in vitro evidence that FA regulates muscle fiber type formation via the Sirt1/AMPK signal pathway.

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Introduction

Skeletal muscle is a heterogeneous tissue composed of different types of fibers, which can be distinguished according to the myosin heavy chain (MyHC) isoform expression. There are four types of muscle fibers in mammals including slow oxidative type I with MyHC I (slow-MyHC), fast oxidative type IIa with MyHC IIa, intermediate type IIx with MyHC IIx and fast glycolytic type IIb with MyHC IIb.^1,2 It has been reported that slow oxidative muscle fiber proportion is positively correlated to insulin sensitivity³ and high-quality meat of livestock and poultry.⁴ Therefore, more and more attention has been paid to promoting slow muscle fiber formation and increasing slow muscle fiber content through nutrition strategies.

Ferulic acid (FA, 3-methoxy-4-hydroxycinnamic acid) is a common polyphenolic compound present in barley grain, wheat bran, rice endosperm, maize bran and other plant cell walls.⁵ It is covalently linked to polysaccharides by ester bonds.^6,7 FA can be released from plant cell walls by the action of feruloyl esterases.⁸ Numerous studies have shown that FA has a powerful antioxidant effect.^5,9,10 FA has also been shown to have an effect of improving mitochondrial function.^11,12 It has been proved that improvement of mitochondrial function is beneficial for the functioning of oxidative myofibers.^13,14 In addition, several recent studies have reported that polyphenolic compounds such as resveratrol and apple polyphenols can regulate muscle fiber type formation.^15,16 However, the effect of FA on muscle fiber type formation is not clear.

In the present study, we investigated the effect of FA on muscle fiber type formation in mouse C2C12 myotubes. We also investigated its molecular mechanism.

Materials and methods

Reagents

The slow-MyHC (1 : 500) and fast-MyHC (1 : 250) antibodies were obtained from Sigma (St Louis, MO, USA). The troponin I slow type (Troponin I-SS) (1 : 1000) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PGC-1α (1 : 500), Sirt1 (1 : 1000), total AMPK (1 : 1000), p-AMPK (1 : 1000), total LKB1 (1 : 1000) and p-LKB1 (1 : 1000) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The β-actin (1 : 3000) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

C2C12 myoblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotics (100 U ml⁻¹ penicillin and 100 μg L⁻¹ streptomycin) at 37 °C under a 5% CO₂ atmosphere. When the cells were grown to 80–90% confluence, cell differentiation was induced using a differentiation medium containing DMEM with 2% horse serum (Hyclone, Logan, UT, USA). After 1 d of differentiation, the cells were treated with FA (dissolved in DMSO). Compound C (AMPK inhibitor) or EX527 (Sirt1 inhibitor) was added 1 h before FA treatment. The medium was then replaced with a fresh differentiation medium daily for 5 days before analysis.

Small RNA interference (siRNA)

Once C2C12 myoblasts reached 80–90% confluence, they were transfected with 50 nM of negative control (NC) siRNA or AMPKα2-specific siRNA (GenePharm, Shanghai, China) using Lipofectamine 3000 (Invitrogen, California, USA) according to the manufacturer's protocol. The siRNA sequences used were 5′-GCAUACCAUCUUCGAGUAATT-3′ (forward) and 5′-UUACUCGAAGAUGG UAUGCTT-3′ (reverse) for AMPKα2, and 5′-UUCUCCGAACGUGUCACGUTT-3′ (forward) and 5′-ACGUGACACGUUCGGAGAATT-3′ (reverse) for NC.

RNA extraction and reverse transcription

Total RNA from the C2C12 myotubes was isolated using RNAiso Plus reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol. The concentration of RNA was determined using a Beckman Coulter DU 800 system (Beckman Coulter, Fullerton, CA, USA). One microgram of total RNA was transcribed into single-stranded cDNA using the PrimeScript® RT reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer's instructions. The cDNA was subsequently used as a template for PCR amplification.

Real-time quantitative PCR

Real-time quantitative PCR was performed in triplicate using the SYBR Select Master Mix (Applied Biosystems, Foster, CA, USA) and an ABI 7900HT real-time PCR detection system. The gene specific primers used are listed in Table 1. The cycling conditions used in real-time quantitative PCR were 45 cycles at 95 °C for 15 s and 60 °C for 30 s. The relative expression of each gene was calculated according to the comparative Ct value method¹⁷ and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same sample.

Table 1 List of genes, primer sequences, GenBank accession numbers, and product sizes in this study

Gene name	Primer	Sequence	GenBank accession no.	Product size (bp)
MyHC I	Forward	5′-CTTCTACAGGCCTGGGCTTAC-3′	NM_080728	128
	Reverse	5′-CTCCTTCTCAGACTTCCGCAG-3′
MyHC IIb	Forward	5′-CTTGTCTGACTCAAGCCTGCC-3′	NM_010855	158
	Reverse	5′-TCGCTCCTTTTCAGACTTCCG-3′
MHC IIa	Forward	5′-TTCCAGAAGCCTAAGGTGGTC-3′	NM_001039545	94
	Reverse	5′-GCCAGCCAGTGATGTTGTAAT-3′
MEF2C	Forward	5′-GATCTCCGCGTTCTTATCCC-3′	L13171	91
	Reverse	5′-CCAATGACTGAGCCGACTG-3′
PGC-1α	Forward	5′-CCAGTACAACAATGAGCCTGC-3′	NM_008904	118
	Reverse	5′-CAATCCGTCTTCATCCACG-3′
Sirt1	Forward	5′-ACAGTGACAGTGGCACATGC-3′	NM_019812	130
	Reverse	5′-AATCCAGATCCTCCAGCACA-3′
AMPKα2	Forward	5′-TCTGGAGGTGAATTGTTCGAC-3′	NM_178143	146
	Reverse	5′-ACATTCTCTGGCTTCAGGTCC-3′
GAPDH	Forward	5-AGGGCATCTTGGGCTACAC-3′	NM_008084	211
	Reverse	5′-TGGTCCAGGGTTTCTTACTCC-3′

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Western blotting analysis

Cells were lysed in RIPA lysis buffer (Pierce, Rockford, IL, USA) containing the protease inhibitor cocktail (Sigma). After centrifugation at 12 000g for 10 min at 4 °C, the supernatant obtained was used for western blot analysis. The protein content was determined using the BCA protein assay kit (Pierce). The proteins were then electrophoresed, transferred, immunoblotted, and visualized as previously described.¹⁸ β-Actin was used as a housekeeping protein to control equal loading. The density of the protein bands was determined with Gel-Pro Analyzer Version 4.2 (Media Cybernetics, Rockville, MD, USA).

Metabolic enzyme activity assay

Metabolic enzyme activity assays were performed on cell lysates. The activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All data were normalized for the corresponding protein concentration of cell lysates.

Statistical analysis

Data are presented as mean ± SE (standard error). The data of Fig. 4B, C, 5 and 6 were analyzed by one-way ANOVA followed by Tukey's tests using SPSS11.5 software. Student's t test was performed to assess the statistical significance between groups in Fig. 1, 2, 3 and 4A. P values < 0.05 were considered significant.

Fig. 1 Effect of FA on the protein expression level of slow-MyHC. C2C12 myoblasts were treated with different concentrations of FA after 1 d of differentiation, and then cultured in the differentiation medium for 5 d. Western blotting analysis was used to analyze the slow-MyHC protein expression normalized to β-actin. Results were the mean and standard errors from three independent experiments. **P < 0.01 and ***P < 0.001 as compared with the control.

Fig. 2 FA regulates muscle fiber type formation. C2C12 myoblasts were treated with 0.5 μM FA or an equal volume of DMSO (control) after 1 d of differentiation, and then cultured in the differentiation medium for 5 d. (A) Effect of FA on the protein expressions of slow-MyHC, fast-MyHC, Troponin I-SS, Sirt1 and PGC-1α. Equal loading was monitored with the anti-β-actin antibody. (B) Effect of FA on the activity of SDH. (C) Effect of FA on the activity of LDH. (D) Effect of FA on mRNA abundances of MyHC I, MyHC IIa, MyHC IIb, MEF2C, PGC-1α, Sirt1 and AMPKα2. The amount of target gene mRNA was normalized to the amount of GAPDH mRNA. Results were the mean and standard errors from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the control.

Fig. 3 Effect of FA on p-AMPK protein expression. Samples are prepared as described in Fig. 2. Western blotting analysis was used to analyze the protein expressions of p-AMPK and AMPK. Results were the mean and standard errors from three independent experiments. ***P < 0.001 as compared with the control.

Fig. 4 AMPK signaling pathway contributes to the regulation of muscle fiber type formation by FA. About 90% confluent C2C12 myoblasts cultured in the differentiation medium were transfected with AMPKα2 siRNA (50 nM) or treated with Compound C (50 μM), followed by treatment with 0.5 μM FA or an equal volume of DMSO (control) for 5 d. (A) Effect of AMPKα2 siRNA on AMPKα2 mRNA expression. (B) AMPKα2 siRNA attenuated the effect of FA on the slow-MyHC and fast-MyHC protein expressions. (C) AMPK inhibitor compound C attenuated the effect of FA on the slow-MyHC and fast-MyHC protein expressions. Results were the mean and standard errors from three independent experiments. **P < 0.01 and ***P < 0.001.

Fig. 5 Sirt1 inhibitor EX527 attenuates the effect of FA on muscle fiber type formation. About 90% confluent C2C12 myoblasts were cultured in the differentiation medium. Twenty-four hours later, cells cultured in the differentiation medium were pretreated with Sirt1 inhibitor EX527 (25 μM) for 1 h and then treated with 0.5 μM FA or an equal volume of DMSO (control) for 5 d. Sirt1 (A), PGC-1α (A), slow-MyHC (B) and fast-MyHC (B) protein expressions were analyzed by western blot. Results were the mean and standard errors from three independent experiments. **P < 0.01 and ***P < 0.001.

Fig. 6 Sirt1 is required for FA-induced AMPK activation. Samples were prepared as described in Fig. 5. p-LKB1 (A) and p-AMPK (B) protein expressions were analyzed by western blot. Results were the mean and standard errors from three independent experiments. **P < 0.01 and ***P < 0.001.

Results

Determination of the optimum level of FA

As shown in Fig. 1, the protein level of slow-MyHC was significantly increased when supplementing with 0.5 μM and 1 μM FA, compared with the unsupplemented control. The maximal increase appeared in the 0.5 μM FA treatment group (Fig. 1). No significant difference was observed between 0 μM and 5 μM FA treatment groups (Fig. 1). Then we chose 0.5 μM of FA for use in our subsequent research.

FA regulates muscle fiber type formation

As shown in Fig. 2A, FA treatment significantly increased the protein expressions of slow-MyHC, troponin I-SS, Sirt1 and PGC-1α, and decreased the protein expression of fast-MyHC. FA treatment significantly increased the activity of SDH (Fig. 2B) and decreased the activity of LDH (Fig. 2C). Real-time quantitative PCR analysis showed that FA significantly increased the mRNA abundances of MyHC I and MyHC IIa but decreased the mRNA abundance of MyHC IIb (Fig. 2D).

AMPK signaling pathway contributes to the regulation of muscle fiber type formation by FA

As shown in Fig. 2D, the AMPKα2, PGC-1α, Sirt1 and MEF2C mRNA abundances in the FA treated group were higher than those in the control group. Compared with the control group, FA treatment significantly increased the p-AMPK protein level (Fig. 3), indicating that FA activates the AMPK signaling pathway.

To explore the mechanism by which FA regulates muscle fiber type formation, C2C12 myoblasts were transfected with AMPKα2 siRNA 24 h before the addition of FA or treated with AMPK inhibitor Compound C 1 h before the addition of FA. The results showed that AMPKα2 siRNA significantly decreased the mRNA abundance of AMPKα2 (Fig. 4A). In addition, AMPKα2 siRNA or AMPK inhibitor Compound C abolished FA-induced upregulation of slow-MyHC and downregulation of fast-MyHC (Fig. 4B and C). Taken together, these results suggested that regulation of muscle fiber type formation by FA is dependent on AMPK signaling.

Inhibition of Sirt1 by EX527 disrupts the effect of FA on muscle fiber type formation

Inhibition of Sirt1 by EX527 significantly decreased the Sirt1 protein level (Fig. 5A). As shown in Fig. 5, EX527 treatment attenuated FA-induced increase in the protein expressions of slow-MyHC and PGC-1α and decrease in the protein expression of fast-MyHC.

Sirt1 is required for FA-induced AMPK activation

As shown in Fig. 6, EX527 treatment attenuated FA-induced activation of LKB1, a primary upstream kinase of AMPK, and also attenuated FA-induced AMPK activation.

Discussion

In this study, we showed that FA significantly increased the mRNA abundances of MyHC I and MyHC IIa, decreased the mRNA abundance of MyHC IIb, increased the protein level of slow-MyHC and decreased the protein level of fast-MyHC. We also showed that FA increased the protein level of slow skeletal Troponin I (troponin I-SS). For the metabolic characteristics of muscle fibers, we showed that FA increased the activity of SDH and decreased the activity of LDH (a key glycolytic enzyme). To the best of our knowledge, our present study provides the first evidence that FA promotes slow oxidative muscle fiber formation and inhibits fast glycolytic muscle fiber formation in C2C12 myotubes. However, FA was reported to promote hypertrophic growth of fast skeletal muscle in adult zebrafish.¹⁹ The reason may be that the function of FA is different in mammals and aquatic animals.

The 5′-AMP-activated protein kinase (AMPK) is a heterotrimetric enzyme (α1, α2, β1, β2, γ1, γ2, and γ3) activated by phosphorylation of its α catalytic subunit.²⁰ The AMPK signalling pathway is a well-studied mechanism that regulates myosin heavy chain expression, resulting in a slower, more oxidative phenotype.²¹ Additionally, AMPK may regulate the expression of PGC-1α, a principal factor in regulating muscle fiber type determination.²² PGC-1α can physically interact with myocyte enhancer factor 2C (MEF2C) to promote the formation of slow-twitch muscle fibers.²³ In the present study, we showed that FA not only upregulated the mRNA abundance of AMPKα2, but also increased the protein levels of p-AMPK and PGC-1α. In order to further explore the mechanism by which FA affects myofiber specific gene expression, AMPK signaling was inhibited by AMPKα2 siRNA or AMPK inhibitor compound C. Our data showed that the inhibition of AMPK signalling abolished the effect of FA on muscle fiber type formation, indicating that the regulation of muscle fiber type formation by FA requires AMPK.

In addition, Sirt1 can directly activate LKB1, resulting in the phosphorylation of AMPK.²⁴ It has been reported that overexpression of Sirt1 resulted in more oxidative fibers²⁵ and upregulated PGC-1α expression.²⁶ In the present study, we found that FA-induced increase in the protein expressions of slow-MyHC and PGC-1α and FA-induced decrease in the protein expression of fast-MyHC were attenuated by Sirt1 inhibitor EX527. We also found that EX527 attenuated FA-induced increase in the phosphorylation levels of AMPK and LKB1. These results indicated that the regulation of muscle fiber type formation by FA requires Sirt1 and AMPK serves as a downstream target of Sirt1. Our data support a working model for FA (Fig. 7), that is, FA activates Sirt1, which leads to the activation of LKB1 and AMPK. The activation of Sirt1 and AMPK converges on enhancing the expression of PGC-1α,²⁷ thereby promoting slow oxidative muscle fiber formation.^23,28

Fig. 7 Scheme illustrating the regulation of muscle fiber type formation by ferulic acid.

Conclusion

In summary, we provide the first evidence that FA regulates muscle fiber type formation through the Sirt1/AMPK signaling pathway. Our study not only brings a novel insight into the function of FA in mammals, but also indicates the necessity of further studying the role of FA in skeletal muscle biology in vivo.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Key R&D Program of China (No. 2018YFD0500403) and the National Natural Science Foundation of China (No. 31672432).

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