Lauric Acid Accelerates Glycolytic Muscle Fiber ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Bioactive Constituents, Metabolites, and Functions

Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through TLR4 Signaling Leshan Wang, Lv Luo, Zhao Weijie, Kelin Yang, Gang Shu, Songbo Wang, Ping Gao, Xiaotong Zhu, Qian-yun Xi, Yongliang Zhang, Qingyan Jiang, and Lina Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01753 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Journal of Agricultural and Food Chemistry

1

Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through TLR4

2

Signaling

3

Leshan Wang 1, Lv Luo 1, Weijie Zhao，Kelin Yang 1, Gang Shu 1, Songbo Wang 1,

4

Ping Gao 1, Xiaotong Zhu 1, Qianyun Xi 1, Yongliang Zhang 1,2, Qingyan Jiang1,2,*,

5

Lina Wang1,*

6

1

7

Animal Science, South China Agricultural University, Guangzhou 510640,

8

Guangdong, People’s Republic of China

9

2

Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of

National Engineering Research Center for the Breeding Swine Industry, South China

10

Agricultural University, Guangzhou 510640, Guangdong, People’s Republic of China

11

* Corresponding author:

12

Lina Wang

13

E-mail address: [email protected]

14

Qingyan Jiang

15

E-mail address: [email protected]

16

Tel.: (86)-20-8528-4937;

17

Fax: (86)-20-8528-4901.

ACS Paragon Plus Environment


18

ABSTRACT: Lauric acid (LA), which is the primary fatty acid in coconut oil, was

19

reported to have many metabolic benefits. TLR4 is a common receptor of

20

lipopolysaccharides and involved mainly in inflammation responses. Here, we

21

focused on the effects of LA on skeletal muscle fiber types and metabolism. We found

22

that 200µM LA treatment in C2C12 or dietary supplementation of 1% LA increased

23

MHCIIb protein expression and the proportion of type IIb muscle fibers from 0.452 ±

24

0.0165 to 0.572 ± 0.0153, increasing the mRNA expression of genes involved in

25

glycolysis, such as HK2 and LDH2 (from 1.00 ± 0.110 to 1.35 ± 0.0843 and 1.00 ±

26

0.123 to 1.71 ± 0.302 in vivo, respectively), decreasing the catalytic activity of lactate

27

dehydrogenase (LDH), transforming lactic acid to pyruvic acid. Furthermore, LA

28

activated TLR4 signaling and TLR4 knockdown reversed the effect of LA on muscle

29

fiber type and glycolysis. Thus, we inferred that LA promoted glycolytic fiber

30

formation through TLR4 signaling.

31

KEYWORDS: lauric acid, skeletal muscle fiber type, glycolysis, TLR4

32


Page 2 of 31

Page 3 of 31


33

INTRODUCTION

34

Fatty acids are traditionally regarded as fuel that supplies energy to organisms through

35

oxidation. Recent studies have revealed distinct regulatory roles of fatty acids

36

according to the saturation of their hydrocarbon chains. Research suggests that

37

over-ingestion of fatty acids, especially saturated fatty acids (SFAs), is associated with

38

the incidence of various metabolic diseases. Diets rich in SFAs can promote intestinal

39

lipopolysaccharide (LPS) uptake, causing systemic inflammation, insulin resistance

40

and obesity 1. In contrast, unsaturated fatty acids, especially polyunsaturated fatty

41

acids (PUFAs), have multiple positive effects on human health. For instance, omega-3

42

PUFAs can inhibit some key inflammatory mediators 2, decrease free radical

43

formation and down-regulate the expression of various angiogenic factors 3.

44

Recent studies have also shown that medium-chain fatty acids (MCFAs) play

45

completely different physiological roles than long-chain SFAs and PUFAs in animals.

46

MCFAs are a group of fatty acids with 6 to 12 carbons. The uptake of MCFAs is

47

independent of fatty acid-binding proteins in liver and muscle cells, and their

48

metabolism

49

membrane-embedded fatty acid translocase 4. One study revealed that the oxidation of

50

caprilic acid is approximately 5 times faster than that of oleic acid in hepatocytes 5.

51

Furthermore, it has been reported that diets rich in MCFAs (caprilic acid and capric

52

acid) can decrease body weight, naso-anal length and pancreatic islet mass 6. However,

53

few studies have evaluated the regulatory effects of MCFAs on skeletal muscle

54

metabolism and fiber types.

requires

neither

fatty

acid

transport


proteins

nor

plasma


Page 4 of 31

55

Skeletal muscle is one of the most energetic tissues in vertebrates and exhibits highly

56

adaptive and plastic properties. The available reports referring to the regulatory

57

effects of fatty acids on skeletal muscle are mostly focused on long-chain fatty acids

58

(LCFAs). Evidence has shown that oleic acid can reverse palmitic acid-induced

59

structural and metabolic changes in muscle 7. Elevated docosahexaenoic acid (DHA)

60

contents in gastrocnemius muscle can attenuate LPS-induced muscle atrophy 8. In

61

vitro studies revealed that both linoleic acid and oleic acid can stimulate L6 myoblast

62

differentiation, without activation of peroxisome proliferator-activated receptors

63

(PPARs) 9. In C2C12 myoblasts, both of these fatty acids increase cell proliferation

64

and differentiation, although through different signaling pathways

65

suggest that PUFAs also play important roles in muscle physiology. Arachidonic acid

66

(ARA) alters the lipid composition of the membrane and promotes protein synthesis

67

in L6 myoblasts, accompanied by activation of mammalian target of rapamycin

68

complex 1 (mTORC1)

69

growth of myoblasts in a dose-dependent manner and to accelerate the hypertrophy of

70

myotubes

71

catabolized more quickly than LCFAs, exert on skeletal muscle metabolism and fiber

72

type.

73

Lauric acid (LA) is a typical MCFA with 12 carbons and is the primary fatty acid (45–

74

53%) found in coconut oil. Most ingested LA is transported directly to the liver via

75

the portal vein; LA does not require carnitine to cross the mitochondrial membrane

76

and can instead cross via passive diffusion 13. Among all fatty acids, LA contributes

10

. Recent studies

11

. In a C2C12 cell model, ARA was found to promote the

12

. Nevertheless, it is still unclear what effects MCFAs, which are


Page 5 of 31


77

the least to fat accumulation 14. Furthermore, LA shows strong antimicrobial activity,

78

making it a promising agent to replace antibiotics. TLR4 is regarded as receptor for

79

LA, which modulates TLR4-induced inflammatory responses through regulation of

80

the dimerization of TLR4 and its recruitment to lipid rafts in cell membranes 15.

81

To investigate the effects of LA on skeletal muscle fiber type and metabolism, the

82

immortalized mouse skeletal muscle cell line C2C12 was used in the present study.

83

We found that LA stimulated glycogenolysis and glycolysis and activated TLR4

84

signaling to accelerate glycolytic muscle fiber formation.

85

MATERIALS AND METHODS

86

Chemicals

87

LA used for cell treatment was purchased from Sigma (Shanghai, China). LA used for

88

feed additive was purchased from Aike Chemical Company (Jinan, Shandong, China).

89

Opti-MEM medium were purchased from Gibco Co., Ltd. (Gaithersburg, MD, USA).

90

Bovine serum albumin was purchased from Millipore Corp., (Billerica, MA, USA).

91

Kits used for detect enzyme activities and gluocose, glycogen, lactic acid, triglyceride

92

and non-esterified fatty acid were purchased from Nanjing Jiancheng bioengineering

93

Co., Ltd. (Nanjing, Jiangsu, China). TLR4 siRNA were purchased from Suzhou

94

Genepharma Inc. (Suzhou, Jiangsu, China). Lipofectamin RNAiMAX transfection

95

reagent was purchased from Introvigen (Shanghai, China).

96

Cell Culture and Treatment

97

The C2C12 myoblast cell line was cultured in high-glucose Dulbecco’s modified

98

Eagle’s medium (DMEM, Gibco, California, U.S.A.) supplemented with 10% fetal



99

bovine

serum

(FBS,

Gibco, (Gibco,

California, California,

U.S.A.)

U.S.A.)

and a

100

IU/mL

100

ampicillin-streptomycin

101

atmosphere (5% CO2, 37°C). The cells were plated in 12-well plates at a density of

102

8×104 cells/well. When the cells reached 80% confluence, they were induced to

103

differentiate by exchanging the 10% FBS for 2% horse serum (HyClone, Utah, U.S.A.)

104

for 4 days, to form myotubes. Then, the cells were treated with 200 µM LA (Sigma,

105

Missouri, U.S.A.) for 2 days. Before treatment, LA was added to the differentiation

106

medium supplemented with 1% bovine serum albumin, followed by incubation at

107

55°C for 1 h. For siRNA transfection, cells were transfected with TLR4 siRNA at 10 h

108

after seeding on the plate and 24 h before LA treatment, respectively. The transfection

109

procedure was carried out according to the manufacture’s instruction. After treatment,

110

the medium was collected for the measurement of glucose, lactate acid and dissolved

111

oxygen according to the manufacturer’s instructions, while the cells were harvested

112

for protein and RNA extraction according to protocols.

113

Animals and Feeding

114

Male C57BL/6J mice (3-4 weeks old) were purchased from the Guangdong

115

Experimental Animal Center. Animal maintenance and experiments were carried out

116

with the guidance of the Instructive Guidelines for Laboratory Animals of the

117

People’s Republic of China. Mice were housed in separated cages under a 12-h light-

118

dark cycle in an environment with a constant temperature (23 ± 3°C) and humidity

119

(70 ± 5%). After a 3-day acclamation period, the mice were divided into two groups,

120

which were fed with the control diet or a diet supplemented with 1% LA. The


in

Page 6 of 31

constant-humidity

Page 7 of 31


121

formulas of these two diets are shown in Table 1 (both diets were isoenergetic). Feed

122

and water were supplied ad libitum for 31 days before sacrifice. The weight of the

123

feed and mouse body weight were measured every 3-4 days.

124

Sample Collection and Sectioning

125

At the end of the feeding experiment, blood samples were collected via retro-orbital

126

bleeding, and the mice were subsequently killed via cervical dislocation. The blood

127

samples were centrifuged, and serum was collected for glucose, non-esterified fatty

128

acid (NEFA) and triglycerides tests according to the manufacturer’s instructions. The

129

serum fatty acid composition was determined by Qingdao Yixin Testing Technology

130

Service Co., Ltd. Muscle and adipose tissue samples were quickly separated and

131

weighed, then frozen in liquid nitrogen and preserved at -80°C for further analysis.

132

Gastrocnemius muscle sections (15 µm) were obtained using a cryostat microtome

133

(Leica, Germany) at -20°C and were then attached to microslides for histochemical

134

staining.

135

Enzyme activities and glycogen content were measured via colorimetric methods.

136

Combined Staining of ATPase and Succinate Dehydrogenase

137

For combined staining of ATPase and succinate dehydrogenase, muscle sections were

138

first incubated in acid preincubation liquid (5 mM CaCl2, 0.3% acetic acid, pH 4.1)

139

for 10 min. Then, they were washed with Tris-CaCl2 buffer (0.1 M Tris, 18 mM CaCl2)

140

twice for 1 min each, followed by incubation in succinate dehydrogenase incubation

141

liquid

142

N,N-dimethylformamide, 0.23 M Tris, pH 7.0) for 45 min in a 37°C water bath.

(0.1

M sodium

succinate,

0.18 mM nitroblue


tetrazolium,

0.8%


143

Thereafter, the sections were rinsed with distilled water twice for 30 s each, followed

144

by incubation in ATPase incubation liquid (3 mM adenosine 5’-triphosphate disodium,

145

0.2 M Tris, 18 mM CaCl2, 50 mM KCl, pH 9.4) for 30 min in a 37°C water bath. Next,

146

the sections were rinsed with distilled water twice for 1 min each and dried with filter

147

paper. After being incubated in 2% CoCl2 for 5 min, the sections were carefully

148

washed with running distilled water for approximately 5 min, followed by incubation

149

in 2% ammonium sulfide for 30 s and then washing for 10 min in distilled water. The

150

sections were next stained with Ehrlich’s hematoxylin for 5 min in the dark and

151

washed with running tap water for 5 min, after which they underwent a continuous

152

dehydration procedure in 50, 75 and 100% ethanol. Prior to sealing the sections with

153

neutral balsam, they were subjected to xylene treatment for 2 min to obtain optimal

154

images. Images were finally captured with a microscope at 40×, and the cells stained

155

with different colors were counted using ImageJ software (NIH, Maryland, U.S.A.).

156

Western Blotting

157

Muscle samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer

158

containing 1 mM phenylmethysulfonyl fluoride (PMSF) and were then centrifuged to

159

acquire the supernatant. Cells were also lysed in RIPA containing 1 mM PMSF,

160

followed by centrifugation to acquire the supernatant. The obtained protein

161

concentrations were determined with a BCA kit (Thermo, Illinois, U.S.A.). Then, 30

162

µg of protein from every sample was loaded onto a gel for sodium dodecyl

163

sulfate-polyacrylamide gel electrophoresis, which was run for approximately 1 h.

164

Thereafter, the proteins were transferred to polyvinylidene fluoride membranes,


Page 8 of 31

Page 9 of 31


165

followed by washing for 30 min with Tris-buffered saline containing 0.05% Tween 20

166

(TBST) and blocking with 8% non-fat dry milk dissolved in TBST for 2 h at room

167

temperature. Subsequently, the membranes were incubated with antibodies against

168

MHCI, CD36 and PPARβ (Abcam, Massachusetts, U.S.A.), MHCIIb, TLR4, MyD88

169

and MuRF1 (Santa Cruz, California, U.S.A.), β-actin (Bioss, Beijing, China), and

170

p-IKK, IKK, p-mTOR, mTOR, p-S6 and S6 (CST, Massachusetts, U.S.A.). Primary

171

antibody incubation was performed at 4°C overnight, followed by washing with

172

TBST and incubation with corresponding secondary antibodies (1：50,000, Bioworld,

173

Minnesota, U.S.A.) for 1.5 h at room temperature. The resultant bands were finally

174

washed with TBST for 30 min, then dipped in ECL Western Blotting Substrate

175

(Solarbio, Beijing, China), and photographed with a FluorChem M Fluorescent

176

Imaging System (ProteinSimple, California, U.S.A.). Densitometry measurements

177

were performed using ImageJ software, and values were normalized to β-actin.

178

Quantitative Polymerase Chain Reaction

179

RNA was extracted from muscle and cell samples using a general RNA Extraction Kit

180

(Magen, Guangzhou, China) according to the manufacturer’s instructions, and cDNA

181

was synthesized from 1 µg of RNA using M-MLV Reverse Transcriptase (Promega,

182

Wisconsin, U.S.A.) and oligo-dT18 (Takara, Dalian, China), according to standard

183

protocols. Gene quantification was performed by mixing cDNA, the SYBR reagent

184

(Toyobo, Osaka, Japan) and the primers listed in Table 2 (Sangon, Guangzhou, China),

185

running the specific procedures using QuantStudio 3 (Applied Biosystems, California,

186

U.S.A).



187

Statistical Analysis

188

All data are presented as the mean ± standard error of the mean (SEM). In the feeding

189

experiments, every mouse was treated as an independent individual. In the cell

190

experiments, every well was considered an experimental unit. All of the tests were

191

carried out with at least 4 samples and in duplicate. Data analysis was performed, and

192

figures were drawn using GraphPad Prism 6 software (GraphPad Software, Inc.

193

California, U.S.A.). Differences between groups were determined using Student’s

194

t-test, and P

Lauric Acid Accelerates Glycolytic Muscle Fiber ... - ACS Publications

Recommend Documents