Epigallocatechin Gallate Reduces Slow-Twitch Muscle Fiber

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Article

EGCG reduces slow-twitch muscle fiber formation and mitochondrial biosynthesis in C2C12 cells by repressing AMPK activity and PGC-1# expression Lina Wang, Zhen Wang, Kelin Yang, Gang Shu, Songbo Wang, Ping Gao, Xiaotong Zhu, Qian-yun Xi, Yongliang Zhang, and Qingyan Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02193 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016

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Journal of Agricultural and Food Chemistry

EGCG reduces slow-twitch muscle fiber formation and mitochondrial biosynthesis in C2C12 cells by repressing AMPK activity and PGC-1α expression WANG lina, Wang zhen, Yang kelin, SHU gang, WANG Songbo, GAO Ping, ZHU xiaotong, Xi qianyun, Zhang Yongliang, JIANG qingyan* College of Animal Science and National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510640, Guangdong, China *Address proofs and correspondence to: Pro. Qingyan Jiang College of Animal Science South China Agricultural University Wushan Avenue, Tianhe District, Guangzhou, 510642 P.R. China. E-mail address: [email protected] Tel./fax: +86 20 85284901.

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Abstract Epigallocatechin gallate (EGCG) is a major active compound in Green Tea Polyphenols. EGCG acts as an antioxidant to prevent the cell damage caused by free radicals and their derivatives. In skeletal muscle, exercise causes the accumulation of intracellular Reactive Oxygen Species (ROS) and promotes the formation of slow-type muscle fiber. To determine whether EGCG, as an ROS scavenger, has any effect on skeletal muscle fiber type, we applied different concentrations (0, 5, 25, and 50 µM) of EGCG in the culture medium of differentiated C2C12 cells for 2 days. The fiber-type composition, mitochondrial biogenesis-related gene expression, antioxidant and glucose metabolism enzyme activity and ROS levels in C2C12 cells were then detected. According to our results, 5 µM EGCG significantly decreased the cellular activity of SDH; 25 µM EGCG significantly downregulated the MyHC I, PGC-1α, NRF-1, p-AMPK levels and SDH activity while enhancing the CAT and GSH-Px activity and decreasing the intracellular ROS levels; and 50 µM EGCG significantly downregulated MyHC I, PGC-1α and NRF-1 expression and HK and SDH activity while increasing LDH activity. Furthermore, 300 µM H2O2 and 0.5 mM AMPK agonist (AICAR) improved the expression of MyHC I, PGC-1α and p-AMPK, which were all reversed by 25 µM EGCG. In conclusion, the effect of EGCG on C2C12 cells may occur through the reduction of the ROS level, thereby decreasing both AMPK activity and PGC-1α expression and eventually reducing slow-twitch muscle fiber formation and mitochondrial biosynthesis. Key word: EGCG; C2C12; Muscle fiber types; Mitochondria biosynthesis 2


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1

Introduction

2

Epigallocatechin gallate (EGCG) is a major active compound in Green Tea

3

Polyphenols and acts as the main antioxidant component of tea polyphenols to prevent

4

the cell damage caused by free radicals and their derivatives.1, 2 Reactive oxygen

5

species (ROS), including oxygen ions, peroxide, and oxygen free radicals, are a

6

by-product of aerobic metabolism in cells. High levels of ROS can cause damage to

7

cellular and genetic structures.3, 4 However, studies have shown that physiological

8

concentrations of ROS are important for maintaining normal cellular functions, such

9

as cell growth, differentiation, proliferation and apoptosis.5-7 In skeletal muscle,

10

exercise can promote the formation of slow-type muscle fibers while causing the

11

accumulation of intracellular ROS. It is therefore possible that a certain concentration

12

of ROS may be beneficial to slow-fiber generation.8, 9

13

Adult skeletal muscle is composed of four muscle fiber types that are classified

14

as either slow twitch (MyHC I) or fast twitch (MyHC IIa, MyHC IIx and MyHC

15

IIb).10 These different muscle-fiber types show different metabolic and contractile

16

properties. MyHC I myofibers exhibit a high oxidative capacity and a high

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mitochondrial content and are resistant to fatigue. In contrast, MyHC IIb myofibers

18

display low oxidative metabolism and low mitochondrial content. The metabolic and

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contractile properties of MyHC IIa and MyHC IIx are between those of MyHC I and

20

MyHC IIb.11, 12 Mature skeletal muscle is highly plastic, and hormones, nutrition and

21

other changes can influence the expression of muscle-fiber-specific proteins, thereby

22

promoting the transformation of muscle-fiber types.13, 14 3



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Irrcher et al (2009) and Kang et al (2009) found that ROS regulate PGC-1α

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transcription through AMPK.15, 16 PGC-1α is an auxiliary transcriptional activator that

25

plays a key role in regulating mitochondrial gene expression. PGC-1α expression is

26

higher in slow muscle than in fast muscle.17, 18 Skeletal muscle in PGC-1α transgenic

27

mice shows increased mitochondrial concentrations and oxidative capacity and

28

contains more MyHC I and MyHC IIa fibers.19 Furthermore, AMPK activation is

29

reported to increase both PGC-1α expression and mitochondrial content.20 AMPK

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activation or inhibition also affects the transformation of muscle fiber types.21

31

However, as an ROS scavenger, the effect of EGCG on skeletal muscle fiber

32

types and the underlying intracellular signaling pathway have not been reported. We

33

hypothesis that EGCG attenuates slow fiber generation in muscle cells, as reduced

34

ROS levels cannot effectively activate AMPK and PGC-1α expression. Hence, the

35

present study was designed to investigate the effects of EGCG on muscle fiber types

36

in C2C12 muscle cells. C2C12 cells were treated with EGCG alone or together with

37

H2O2 or AICAR (AMPK activator), and intracellular ROS levels, antioxidant enzyme

38

activities, mitochondrial biosynthesis, AMPK activation and PGC-1α expression were

39

detected to gain insights into the possible pathways mediating the effects of EGCG.

40

Methods and materials Chemicals and reagents. EGCG and hydrogen peroxide (H2O2) were obtained

41 42

from

Sigma-Aldrich

(St.

Louis,

43

5-aminoimidazole-4-carboxamide-1-β-d-ribo-furanoside (AICAR) was purchased

44

from Beyotime (Haimen, China). All compounds were resuspended in sterile DMEM 4


MO,

USA).

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(GIBCO, Grand Island, NY, USA) before use. Antibodies against β-actin, PGC-1α,

46

mtTFA, NRF-1, AMPKα, p-AMPKα were obtained from Cell Signaling Technology

47

(Beverly, MD). MyHC I antibodies were obtained from Abcam (England). MyHC II

48

antibodies were purchased from Millipore (USA). Kits for measuring hexokinase

49

(HK), lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), total superoxide

50

dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), reactive

51

oxygen species (ROS) were purchased from Nanjing Jiancheng Bioengineering

52

Institute (China). Mito-tracker green was purchased from Beyotime Institute of

53

Biotechnology (Haimen, China).

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Cell Culture and Treatment. C2C12 muscle cells were maintained in DMEM

55

(Invitrogen Life Technologies Inc., Burlington, ON, Canada) containing 10% FBS

56

(GIBCO, Grand Island, NY, USA) and 1% antibiotic/antimycotic (GIBCO, Grand

57

Island, NY, USA) at 37°C and 5% CO2. Upon reaching 90% confluence, cells were

58

switched to differentiation medium (DMEM containing 2% heat-inactivated horse

59

serum (GIBCO, Grand Island, NY, USA) and 1% antibiotic/antimycotic) for 4 days to

60

differentiate. Then, cells were cultured in differentiation medium with 0, 5, 25 and 50

61

µM EGCG in the presence or absence of 300 µM H2O2 or 0.5 mM AICAR for an

62

additional 2 days before being harvested. Fresh medium was provided every 2 days.

63

RNA extraction and qPCR. Total RNA was extracted from C2C12 muscle cells

64

using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the

65

manufacturer's instructions. After treatment with DNase I (Takara Bio Inc., Shiga,

66

Japan), total RNA (2 µg) was reverse transcribed to cDNA in a final 20-µL system 5



67

with M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and random

68

primers (oligo-dT18) according to the manufacturer's instructions. β-actin was used as

69

a candidate housekeeping gene. SYBR Green I Real-time PCR Master Mix (Toyobo

70

Co., Ltd., Osaka, Japan) and both sense and antisense primers (200 nM for each gene)

71

were used for qPCR. PCR reactions were performed in a Mx3005p instrument

72

(Stratagene, La Jolla, CA, USA). The primers are listed in Table.1

73

Western Blot C2C12 cells were lysed in RIPA lysis buffer containing 1 mM

74

PMSF. The total protein concentration was determined using a Pierce BCA protein

75

assays kit (Thermo, USA). After separation via 10% SDS-PAGE, the proteins were

76

transferred to polyvinylidene fluoride (PVDF) membranes and then blocked with 5%

77

(wt/vol) nonfat dry milk in Tris-buffered saline containing Tween-20 for 2 h at room

78

temperature. Subsequently, the PVDF membranes were incubated with the indicated

79

antibodies, including rabbit anti-β-actin (1:2000), mouse anti-MyHC I (1:2000),

80

mouse anti-MyHC II (1:2000), rabbit anti-PGC-1α (1:2000), goat anti-mtTFA

81

(1:1000), rabbit anti-NRF-1 (1:2000), rabbit anti-AMPKα (1:1000), and rabbit

82

anti-phospho-AMPKα (Thr172; 1:1000). Primary antibody incubation was performed

83

overnight at 4°C, followed by incubation with the appropriate secondary antibody

84

(1:1000; Bioss) for 1 h at room temperature. Protein expression was measured with a

85

FluorChem M Fluorescent Imaging System (ProteinSimple, Santa Clara, CA, USA)

86

and normalized to β-actin expression.

87

Enzyme assay. Enzyme assays were performed on cell lysates. Hexokinase (HK),

88

lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), total superoxide 6


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dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activity

90

were measured using commercial assay kits. All metabolite data were normalized to

91

the corresponding cellular protein content.

92

Dichlorofluorescein Assay. The intracellular production of reactive oxygen

93

species (ROS) was assayed using DCFH-DA. After 2 days of treatment with 25 µM

94

EGCG, C2C12 cells were washed once with PBS and then incubated for 45 min with

95

10 µM DCFH-DA at 37°C in PBS, followed by an additional PBS wash. Cells were

96

then suspended in PBS, and DCF fluorescence was measured using a Synergy HT

97

reader (BioTek, USA) at 485/20 (excitation) and 528/20 (emission). Data are

98

presented as relative fluorescence units/well.

99

Mito Tracker staining. After 2 days of treatment with 25 µM EGCG, C2C12

100

cells were washed twice with pre-warmed fresh PBS and incubated in medium

101

containing 100 nmol/L of ﬂuorescent mitochondrial probe (Mito-Tracker Green,

102

emission at 490 nm) for 30 min. After two washes with PBS, cells were photographed

103

on a fluorescence Microscope (TI-U, Nikon, Japan) and analyzed with NIS-Elements

104

software.

105

Statistical analysis. All data are expressed as the means ± SEM. Significant

106

differences between the control and the treated groups were determined by one-way

107

ANOVA or Student's t test (SPSS 18.0, Chicago, IL, USA), with P < 0.05 indicating

108

significance.

109

Results

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EGCG reduces slow myosin heavy chain expression in C2C12 cells 7



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After 2 days of treatment with EGCG, the MyHC I and MyHC II protein levels

112

and MyHC I, MyHC IIa, MyHC IIx, and MyHC IIb mRNA expression in C2C12 cells

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were detected by western blot and qPCR, respectively. The protein levels of MyHC I

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but not MyHC II were significantly reduced in cells treated with 25 µM and 50 µM

115

EGCG (Figure 1A). In addition, MyHC I and MyHC IIa mRNA expression was

116

significantly decreased in the 25 µM and 50 µM EGCG treatment groups. Moreover,

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MyHC I mRNA expression was also downregulated in the 5 µM EGCG group, and

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MyHC IIx mRNA expression was significantly reduced with 50 µM EGCG treatment.

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However, none of the tested EGCG doses had a significant effect on MyHC IIb

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mRNA expression (Figure 1B).

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Activity of glucose metabolic enzymes was influenced by EGCG treatment

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Glucose metabolism in skeletal muscle cells normally occurs via two pathways:

123

glycolysis and aerobic oxidation. Hexokinase (HK) is an important enzyme in the first

124

step of glycolysis. Lactate Dehydrogenase (LDH) is a glycolytic enzyme that

125

catalyzes pyruvate to generate lactic acid, and LDH activity can reflect the degree of

126

anaerobic glycolysis. Succinate Dehydrogenase (SDH) is the key enzyme of aerobic

127

respiration in mitochondria. The activities of HK, LDH and SDH were examined in

128

EGCG treated cells in using commercial assay kits. SDH activity was significantly

129

decreased by all doses of EGCG, and 50 µM EGCG treatment decreased HK activity

130

but increased LDH activity (Figure 1C).

131

Mitochondrial biosynthesis in C2C12 cells is decreased by EGCG treatment

132

To

detect

mitochondrial

biosynthesis 8


in

C2C12

cells,

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Peroxisome-proliferators-activated receptor γ coactivator-1α (PGC-1α), Nuclear

134

respiratory factor 1 (NRF-1) and Mitochondrial transcription factor A (mtTFA)

135

protein levels were measured using western blot. EGCG treatment at 25 µM and 50

136

µM significantly decreased PGC-1α and NRF-1 protein levels in C2C12 cells, but

137

there was no significant change in the mtTFA protein levels (Figure 1D). Mito Tracker

138

staining revealed that the 25 µM EGCG-treated cells tended to contain fewer

139

mitochondria than did untreated cells (P=0.083) (Figure 1E).

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EGCG-mediated reduction of slow-twitch muscle fiber formation in C2C12 cells

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may involve ROS-AMPK signaling

142

According to the results showing altered mitochondria biosynthesis and MyHC

143

expression, we selected the 25 µM EGCG treatment to further investigate the

144

intracellular mechanism of EGCG.

145

First, the ROS levels and antioxidant enzyme activities in C2C12 cells were

146

examined after 2 days of EGCG treatment. As shown in Figure 2A, EGCG treatment

147

significantly reduced the intracellular ROS levels. Furthermore, the activities of

148

Catalase (CAT) and Glutathione peroxidase (GSH-Px) were significantly increased by

149

EGCG treatment (Figure 2C and 2D), whereas Superoxide dismutase (SOD) activity

150

was not affected (Figure 2B).

151 152

In addition, AMPK phosphorylation was significantly repressed by 25 µM EGCG (Figure 2E).

153

When cells were treated with 25 µM EGCG, the PGC-1α, MyHC I, NRF-1 and

154

pAMPKα/AMPKα protein levels were significantly reduced. The effect of EGCG on 9



155

the MyHC I and pAMPKα/AMPKα protein levels was reversed by co-treatment with

156

300 µM H2O2 (Figure 3). Similarly, when C2C12 cells were co-treated with EGCG

157

and an AMPK activator, AICAR, the reduction in the NRF-1, MyHC I and

158

pAMPKα/AMPKα protein levels was also reversed (Figure 4). These results suggest

159

that the AMPK signaling pathway at least partly mediates the effects of EGCG on

160

MyHC I expression.

161

4. Discussion

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Epigallocatechin gallate (EGCG) is recognized as a natural antioxidant that has

163

been shown to reduce body weight, alleviate metabolic syndrome, and prevent

164

diabetes and cardiovascular diseases in animal models and humans. 22, 23 In addition to

165

its anticancer24, antimicrobial25 and antihypertensive26 effects, EGCG was recently

166

shown to have anti-Alzheimer’s27 and anti-inflammatory28 activity. In skeletal muscle,

167

EGCG supplementation was reported to relieve insulin resistance, probably through

168

the PI3K-mediated promotion of GLUT4 translocation.29, 30 Moreover, research on

169

skeletal muscle atrophy has shown that EGCG can influence protein synthesis and

170

degradation.31-33 However, whether EGCG has any effect on skeletal muscle fiber type

171

has not been reported.

172

In the present study, we found that EGCG does have an effect on the fiber type of

173

C2C12 cells. With regard to MyHC composition, EGCG decreased the protein and

174

mRNA levels of MyHC I and the expression of MyHC IIa and MyHC IIx mRNA but

175

had no influence on the MyHC II protein levels or MyHC IIb mRNA expression in

176

C2C12 cells. Thus, we believe that EGCG mainly reduces the formation of slow 10


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muscle and has little effect on fast-muscle formation. For the metabolic characteristics

178

of muscle fibers, EGCG mainly decreased the activity of SDH in C2C12 cells,

179

suggesting that EGCG mainly reduces oxidative metabolism and has little effect on

180

glycolysis. These results are consistent with the effect of EGCG on MyHC

181

composition.

182

Research on rats and humans has shown that EGCG can activate AMPK in

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skeletal muscle23, 34 and that AMPK activation increases PGC-1 expression.35 PGC-1α

184

is a major regulator of muscle fiber type, with PGC-1α overexpression being

185

sufficient to drive slow-muscle-fiber formation.19 However, work on C2C12 cells has

186

indicated that EGCG has no significant effect on PGC-1α expression.36 In contrast,

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ROS were reported to promote PGC-1α expression via AMPK activation.15, 16 These

188

studies raise questions regarding the effect of EGCG on AMPK activity and PGC-1α

189

expression and the signaling pathway by which EGCG alters the muscle-fiber type.

190

Our results showed that EGCG decreased both the ROS levels and AMPK

191

phosphorylation in C2C12 cells, with a concurrent reduction in PGC-1α expression.

192

These results suggest that EGCG may downregulate PGC-1α expression in

193

differentiated C2C12 cells by clearing ROS. This outcome is the opposite of what has

194

been observed in vivo. EGCG is widely recognized as a strong antioxidant that

195

efﬁciently scavenges free radicals and prevents ROS formation. In vivo, however,

196

EGCG may also cause the formation of mitochondrial ROS.37 Thus, whether AMPK

197

phosphorylation is activated or repressed by EGCG probably depends on the cellular

198

ROS levels. 11



199

PGC-1α induces nuclear respiratory factor 1 (NRF-1) expression, biosynthesis

200

and oxidative metabolism, and NRF-1 improves mitochondrial transcription factor A

201

(mtTFA) expression, which is necessary for mitochondrial DNA replication and

202

transcription.38 Studies have shown that ROS may increase NRF-1, mtTFA and

203

PGC-1α expression to affect mitochondrial biosynthesis.39-41 According to our study,

204

25 µM EGCG-treated cells showed a tendency (P=0.083) to contain fewer

205

mitochondria, and their PGC-1α and NRF-1 protein levels were down regulated, but

206

there were no changes in mtTFA protein expression. Thus, EGCG probably reduced

207

mitochondrial biosynthesis.

208

To investigate the signaling pathway by which EGCG affects muscle fiber type,

209

differentiated C2C12 cells were treated with EGCG in the presence or absence of 300

210

µM H2O2 or 0.5 mM AICAR. The reduced MyHC I protein levels and AMPK

211

phosphorylation caused by EGCG were reversed by both H2O2 and AICAR. AICAR

212

also reversed the inhibition of NRF-1 by EGCG. The results suggested that the effect

213

of EGCG on C2C12 cell muscle fiber type might be related to ROS, AMPK and

214

PGC-1α.

215

In summary, our data revealed that EGCG affects MyHC I by eliminating ROS.

216

The signaling pathway underlying the effect of EGCG on C2C12 cells appears to

217

involve the reduction of ROS levels, which decreases AMPK activity and PGC-1α

218

expression, ultimately reducing slow-twitch muscle fiber formation and mitochondria

219

biosynthesis.

220 12


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Funding Sources

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This study was supported by the National Basic Research Program of China

223

(2012CB124701) and the National Natural Science Foundation of China (31101780).

224

Conflict of Interest

225

The authors declare no competing financial interests.

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Figure captions Figure 1 Effects of EGCG on MyHC expression, metabolism-related gene expression and mitochondria biosynthesis in C2C12 cells. Cells were cultured in differentiation medium for 6 days and treated with EGCG for the final 2 days. qPCR and western blot were conducted to measure the mRNA and protein levels, respectively, of the indicated genes. β-actin was used as a control. Enzyme assays were performed on cell lysates to measure HK, LDH and SDH activity. (A) MyHC I and MyHC II protein expression in C2C12 cells. (B) MyHC I, MyHC IIa, MyHC IIx and MyHC IIb mRNA expression in C2C12 cells. (C) The effects of EGCG on C2C12 cell metabolism. (D) The effects of EGCG on mitochondrial biosynthesis-related gene expression in C2C12 cells. (E) Mito Tracker staining of C2C12 cell treated with EGCG. All results represent six replicates (n=6), and values are shown as the means ± S.E.M. Columns that do not share a common letter are significantly different (P

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