Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through

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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

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Journal of Agricultural and Food Chemistry

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Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through TLR4

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Signaling

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

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

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Lina Wang1,*

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1

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Animal Science, South China Agricultural University, Guangzhou 510640,

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Guangdong, People’s Republic of China

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2

Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of

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

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

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* Corresponding author:

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Lina Wang

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E-mail address: [email protected]

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Qingyan Jiang

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E-mail address: [email protected]

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

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

ACS Paragon Plus Environment


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

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

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lipopolysaccharides and involved mainly in inflammation responses. Here, we

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

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

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

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

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

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

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

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

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

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formation through TLR4 signaling.

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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metabolism

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

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

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

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

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

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metabolism and fiber types.

requires

neither

fatty

acid

transport


proteins

nor

plasma


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

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

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

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

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

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

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

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differentiation, without activation of peroxisome proliferator-activated receptors

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

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and differentiation, although through different signaling pathways

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

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

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

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complex 1 (mTORC1)

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

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myotubes

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

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type.

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

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

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

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

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. Recent studies

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

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


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

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

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

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

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

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

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We found that LA stimulated glycogenolysis and glycolysis and activated TLR4

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signaling to accelerate glycolytic muscle fiber formation.

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MATERIALS AND METHODS

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Chemicals

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

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

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

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

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

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

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

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

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reagent was purchased from Introvigen (Shanghai, China).

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Cell Culture and Treatment

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

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



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bovine

serum

(FBS,

Gibco, (Gibco,

California, California,

U.S.A.)

U.S.A.)

and a

100

IU/mL

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ampicillin-streptomycin

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

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

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

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

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

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

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

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

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

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

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

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for protein and RNA extraction according to protocols.

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Animals and Feeding

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

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Experimental Animal Center. Animal maintenance and experiments were carried out

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

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

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

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

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


in

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constant-humidity

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

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

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

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Sample Collection and Sectioning

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

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

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

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

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serum fatty acid composition was determined by Qingdao Yixin Testing Technology

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

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

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

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

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staining.

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Enzyme activities and glycogen content were measured via colorimetric methods.

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Combined Staining of ATPase and Succinate Dehydrogenase

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

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

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

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

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liquid

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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%


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

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

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

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

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

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

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

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

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

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

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

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

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

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Western Blotting

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Muscle samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer

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

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

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followed by centrifugation to acquire the supernatant. The obtained protein

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

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

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

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Thereafter, the proteins were transferred to polyvinylidene fluoride membranes,


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

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

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temperature. Subsequently, the membranes were incubated with antibodies against

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

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

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

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

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

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

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

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

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

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

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Quantitative Polymerase Chain Reaction

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

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

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

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

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

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

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

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U.S.A).



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Statistical Analysis

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

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

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

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

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

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

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t-test, and P

Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through

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