Phytol Promotes the Formation of Slow-Twitch Muscle Fibers through

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Phytol promotes the formation of slow-twitch muscle fibers through PGC-1#miRNA but not mitochondria oxidation Kelin Yang, Lina Wang, Gan Zhou, Xiajing Lin, Jianlong Peng, Leshan Wang, Lv Luo, Jianbing Wang, 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.7b01048 • Publication Date (Web): 27 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Journal of Agricultural and Food Chemistry

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Phytol promotes the formation of slow-twitch muscle fibers through

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PGC-1α/miRNA but not mitochondria oxidation

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YANG kelin#, WANG lina#, ZHOU gan#, LIN xiajing, P ENG jianlong, WANG leshan,

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Luo lv, WANG jianbin, SHU gang, WANG Songbo, GAO Ping, ZHU xiaotong, Xi

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qianyun, Zhang Yongliang, JIANG qingyan*

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College of Animal Science and National Engineering Research Center for Breeding

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Swine Industry, South China Agricultural University, Guangzhou 510640, Guangdong,

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China

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#

These authors contributed equally to this work.

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*Address proofs and correspondence to:

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Pro. Qingyan Jiang

12

College of Animal Science

13

South China Agricultural University

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Wushan Avenue, Tianhe District, Guangzhou, 510642

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P.R. China.

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E-mail address: [email protected]

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Tel./fax: +86 20 85284901.


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Abstract

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Phytol is a side-chain of chlorophyll belonging to the side-chain double

20

terpenoid. When animals consume food rich in chlorophyll, phytol can be

21

broken down to phytanic acid after digestion. It was reported that, feeding

22

animals with different varieties and levels of forage could significant

23

improve pH and marbling score of steer and lamb carcass, but the internal

24

mechanism for this is still not reported. Marbling score and pH of muscle

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was mainly determined by skeletal muscle fiber type, which is due to

26

expression of different myosin heavy chain (MHC) isoforms. Here, we

27

provide evidence that phytol can indeed affect the diversity of muscle

28

fiber types both in vitro and in vivo and demonstrate that phytol can

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increase the expression of MHC I ( p < 0.05), likely by upgrading the

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expression of PPARδ, PGC-1α and related miRNAs. This fiber type

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transformation process may not be caused by activated mitochondrial

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metabolism but by the structural changes in muscle fiber types.

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Keywords: Phytol, skeletal muscle fiber type, PGC-1α, PPARδ



34

Introduction

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The recently study has admitted that, skeletal muscles is the most

36

important tissue of regulating the systemic energy homeostasis, and the

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different fiber types have the different capacity to this regulatory function.

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The slow-oxidative muscle is thought to have more active in removing

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glucose form blood and higher level of athleticism, and the fast-glycolytic

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fiber is often associated with obesity and type II diabetes.1 Meanwhile in

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the agricultural industry, muscle fiber characteristics, especially fiber type

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composition, can be a major factor for meat quality evaluation. Many

43

studies had shown that muscle fiber characteristics could influence the

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meat quality in meat color,2 tenderness of meat,3 the meat pH and

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water-hold capacity4, 5and the intramuscular fat content.6, 7 According to

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the shrinkage rate and metabolic features, skeletal muscle fibers of

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mammals can be classified into three types, Fast Glycolytic (FG), Fast

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oxidative-Glycolytic (FOG), Slow oxidative (SO).8 According to the

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polymorphism of myosin heavy chain, skeletal muscle fibers of mammals

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can be classified to type I (SO), IIa (FOG), IIx (FG) and IIb (Middle type)

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9, 10

, and these four different muscle fiber types can combination and

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transformation in the following way: I ↔ IIa ↔ IIx ↔ IIb.1 It is widely

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believed that the meat quality of oxidative muscle fibers is better than that

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of the glycolysis type, and the slow-twitch fiber type is better than the fast

55

twitch type.11 Recently, studies showed that exercise, cold temperatures,


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energy metabolism level, and nutrition level can influence the skeletal

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muscle fiber type.12 Thus, it is possible to regulate skeletal muscle fiber

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type through certain feed additives.

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Recent studies have shown that capsaicin, apple polyphenols, carnitine

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and ractopamine hydrochloride could influence the skeletal muscle fiber

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type via influence intracellular metabolism.13-16 Phytol, a side-chain of

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chlorophyll, belongs to the side-chain double terpenoid.17,

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animals consume feed rich in chlorophyll, phytol can be released after

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feed digestion.19 In mammals, phytol can be metabolized to phytanic acid.

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Bovine studies demonstrated that green fodder supplement could

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significantly increase the content of phytanic acid and its degradation in

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muscle, fat, and milk.20-22 Phytol and its metabolic products not only

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supply the energy source for oxygenization in animals and supply the raw

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materials for vitamin E and vitamin K but also act as a key regulatory

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molecules

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differentiation.23 Phytanic acid could stimulate glucose uptake in skeletal

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muscles in primary porcine myotubes.24 Mice fed with a diet containing

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phytol exhibited increased metabolic products and activated PPARα.25, 26

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On the other hand, four widely known signal pathways, Ca2+ related

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signaling,27

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signaling,31, 32 and PKC signaling,33 which play important roles in fiber

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type diversity, could all be related by PGC-1α.1 PGC-1α is a

in

modulating

glucolipid

AMPK/PGC-1α/PPARs

metabolism

signaling,28-30


and

18

When

adipocyte

Ras/MAPK


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transcriptional coactivator of several nuclear receptors that regulate key

79

metabolic steps in energy homeostasis.34 It is highly expressed in tissues

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such as heart and skeletal muscle and regulates mitochondrial biogenesis

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via the regulation of genes involved in fatty acid oxidation and oxidative

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phosphorylation.35

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Phytol as a long-branched fat acid that rich in greed fodder, it was

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reported that, feeding animals with different varieties and levels of greed

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fodder could significant improve the pH and marbling score of steer and

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lamb carcass. Marbling score and pH of muscle was mainly determined

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by skeletal muscle fiber type.36-41 Is phytol being the active component in

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greed fodder to show the effects on skeletal muscle? Does it work in pigs？

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There is still having no report. So we hypothesis that phytol could

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influence the skeletal fiber type of pigs through specific signaling

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pathway. In this study, the effects of phytol on skeletal muscle fiber type

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were detected in mice and pigs in vivo and the C2C12 cell line in vitro,

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and its possible intracellular signaling pathways were preliminarily

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explored.


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Materials and methods

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Cell culture and treatment

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The C2C12 cells (immortalized mouse skeletal muscle cell line) were

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cultured in Dulbecco's modified Eagle's medium (DMEM) (Lot: 1776588,

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Gibco BRL, Carlsbad, CA, USA) supplemented with fetal bovine serum

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(FBS) (Lot: 1581730, Gibco BRL, Carlsbad, CA, USA) at 10%

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concentration and 100 IU/mL ampicillin-streptomycin (Lot: 1772658,

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Gibco BRL, Carlsbad, CA, USA) in a humidified atmosphere with 5%

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CO2, humidity of 100% at 37°C. Cells were planted at 3×105 per well in a

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6-well dish. At 80% confluence, cells were differentiated into myotubes

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using DMEM supplemented with pure horse serum (Lot: 1671319, Gibco

106

BRL, Carlsbad, CA, USA) at 2% concentration and 100 IU/mL

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ampicillin-streptomycin. After 6 days of differentiation, cells were treated

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with phytol (Sigma-Aldrich, Louis, Mo, USA) at concentrations of 0 µM,

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5µM, 50 µM and 100 µM for 4 days. The cells were harvested and stored

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at -80℃.

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Animals and feeding protocol

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Thirty male KunMing mice (13 ± 1 g) were obtained from Guangdong

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Experimental Animal Center. Animal maintenance and experiments were

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in accordance with “Instructive Notions with Respect to Caring for

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Laboratory Animals” established by the Ministry of Science and

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Technology of the People’s Republic of China. The KunMing mice were



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placed in individual cages and divided into three groups (n = 10) after

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preliminary feeding with the stander experimental mouse chow

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(XIETONG, Jiangning, Jiangsu, CN); mice were then fed diets containing

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0%, 0.05%, or 0.5% phytol for four weeks before sacrificed. Mice were

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raised with food and water ad libitum and 12 hours cycle light conditions,

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room temperature at 25℃, and 60% humidity.

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Two hundred and forty crossbred (Duroc × Yorkshine × Landrace, day

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156, half male and half female) finishing pigs with an average body

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weight of 85.02 ± 1.15 kg were divided into four groups, with dietary

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supplementation of 0, 0.025%, 0.05% or 0.1% phytol for 35 days after

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preliminary feeding for 3 days and sacrificed at 190 age in days. Each

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group had six repeats, with each repeat containing random 10 pigs form

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total pigs (5 barrow pigs and 5 females). Pigs were bred in a closed, leak

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plate piggery; all repeating groups were bred in an individual sty, with

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free food and water ingestion. The composition of the basal diets is

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shown in Table 1.

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Sacrifice and muscle conservation

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The mice was sacrificed at nearly 8 weeks old 9:00 to 11:00, four weeks

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later feed with phytol by cervical dislocation (n = 10), and the

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gastrocnemius was carefully separated from both hindlimbs. After being

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quickly frozen in liquid nitrogen, samples were stored at -80°C for

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ATPase staining, RNA and protein extraction.


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Ten most close to the average weight pigs in each group were sacrificed

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at 190 age in days 9:00 to 14:00, four weeks later feed with phytol by

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jugular vein exsanguination (n = 10), and samples were taken from the

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longissimus dorsi, triceps brachii, gluteus medius and psoas. Samples

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were frozen in liquid nitrogen and then stored at -80°C. Some of the

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samples were prepared for ATPase staining, the rest were prepared for

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RNA and protein extraction.

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ATPase histochemistry

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Muscles stored in -80℃ were placed in a cryostat (CM1850, Leica, GER)

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to thaw at -20℃. The muscle was incised into 1×1×0.5 cm pieces along

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the vertical direction, oriented in OCT™, serially sectioned (10 µm) in

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the cryostat at -20℃ and selected by microslide. Four sections were cut

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from each sample. Sections were dried at room temperature, incubated in

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acid pre-incubated liquid (5 mM CaCl2, 0.3% glacial acetic acid, pH 4.4)

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for 10 min, eluted by Tris-CaCl2 eluent buffer (0.1 M Tris, 18 mM CaCl2)

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twice for 1 min and then incubated at 37℃ in pre-heating SDH eluent

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buffer

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N,N-dimethyl-formamide, 0.23 M Tris, pH 7.4) for 45 min. Then,

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sections were washed twice with distilled water and then incubated at 37℃

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in pre-heating ATPase eluent buffer (3 mM adenosine 5'-triphosphate

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disodium salt, 0.2 M Tris, 18 mM CaCl2, 50 mM KCl, pH 9.4) for 30 min.

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After two washes in distilled water, sections were incubated in 2% CoCl2

(0.1

M

sodium

succinate,

0.18


mM

NBT,

0.8%


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for 4 min and then washed carefully twice with distilled water. Sections

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were then incubated in 2% ammonium sulfide for 30 sec followed by

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careful washing twice with distilled water. After staining with Ehrlich’s

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hematoxylin for 5 min in the dark and washing, sections underwent

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sequential dehydration with 50%, 75%, and 100% alcohol prior to

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fixation by neutral balsam. The section staining color is related to the

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activity of ATPase, the higher the ATPase activity, the deeper the section

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staining. So the finest and darkest muscle fibers are counted as MHC I,

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the middle dark fibers are counted as MHC IIa and the lightest are

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counted as MHC IIb. Images were captured by a microscope at 10× and

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40×. Each section was captured in 4 different regions. Images were

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analyzed with Matic Image Advanced 3.2, and the numbers of the three

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different fiber types were counted in the 4 regions.

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Transcriptome analysis

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Sus scrofa muscle samples used in the transcriptome analysis were

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randomly selected from 0% phytol groups (n = 3, mixed from 10) and

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0.05% phytol groups (n = 3, mixed from 10). The transcriptome analysis

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process was carried out in accordance with the manufacturer's

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instructions (CapitalBio Corporation, Beijing, China). The Sus scrofa

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transcript

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(ftp://ftp.ensembl.org/pub/release-73/fasta/sus_scrofa/cdna/).

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sequencing reads were mapped onto the reference gene set using Bowtie

set

was

provided


by

ENSEMBL The

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(Bowtie parameter: -v 3 -all -best -strata).42 A Perl script was written to

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process the mapping result and generate the gene expression profile.

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InterPro domains43 were annotated by InterProScan44 Release 36.0, and

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functional assignments were mapped onto Gene Ontology (GO).45 The

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Database for Annotation, Visualization and Integrated Discovery (DAVID;

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http://david.abcc.ncifcrf.gov/) and KO (KEGG Orthology) -Based

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Annotation System (KOBAS; http://kobas.cbi.pku.edu.cn/) was applied to

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obtain the differentially expressed genes (2-fold change) and to cluster

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genes based on functional similarities.46 Genes were compared with

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Kyoto Encyclopedia of Genes and Genomes database (KEGG, release 58)

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by using BLASTX at E values ≤1e-10. Then, a Perl script program was

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used to retrieve KO information from the blast results, and associations

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between genes and pathways were established. The criterion for detection

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of differentially expressed genes was 2-fold change in expression level.

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False discovery rate was controlled at 1%.

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Western blotting

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Muscle samples and cells were lysed in RIPA lysis buffer containing 1

200

mM PMSF. The protein concentration was determined using BCA protein

201

assays (Thermo, IL, USA). After separation on 10% sodium dodecyl

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sulfate (SDS)-polyacrylamide gel electrophoresis gels, the proteins were

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transferred to polyvinylidene fluoride (PVDF) membranes and then

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blocked with 6% (wt/vol) nonfat dry milk in Tris-buffered saline



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containing Tween 20 for 2 h at room temperature. Subsequently, the

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PVDF membranes were incubated with the indicated antibodies,

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including rabbit anti-β-actin (1:2000, Bioss), mouse anti-slow skeletal

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myosin heavy chain (1:3000, ABCAM), mouse anti-slow MyHC (1:3000,

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ABCAM), mouse anti-MyHC II (1:3000, Millipore), rabbit anti-PGC-1α

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(1:3000, CST), mouse anti-CaMKIIα (1:500, Santa Cruz), rabbit

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anti-MEF2a (1:3000, CST), PPARδ (1:3000, ABCAM), CD36 (1:500,

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Santa Cruz). Primary antibody incubation was performed at 4℃ overnight

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followed by incubation with the appropriate secondary antibody (1:50000;

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Bioworld) for 2 h at room temperature. Protein expression was measured

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with FluorChem M Fluorescent Imaging System (ProteinSimple, Santa

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Clara, CA, USA) and normalized to β-actin expression.

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qPCR

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Total RNA was extracted from muscle samples and cells using TRIzol

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reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's

220

instructions. After treatment with DNase I (Takara Bio Inc., Shiga, Japan),

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total RNA (2 µg) was reverse transcribed to cDNA in a final 20 µL by the

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

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random primers oligo-dT18 or looped antisense primer according to the

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manufacturer's instructions. β-actin and GAPDH were used as candidate

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housekeeping genes. SYBR Green Real-time PCR Master Mix reagents

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(Promega, Madison, WI, USA) and both sense and antisense primers (200


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nM for each gene) were used for real-time quantitative polymerase chain

228

reaction (qPCR). qPCR reactions were performed in a Mx3005p

229

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

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Table 2.

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Statistical analysis

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All data are expressed as the means ± standard error of the mean (S.E.M.).

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Significant differences between the control and the treated groups were

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determined by one-way ANOVA test and student's t test. One-way

235

analysis of variance was used to test the interaction between control

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group and the different dose of treatment groups and the student's t test

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was used to test the two different treatments in transcriptome (SPSS 18.0,

238

Chicago, IL, USA). Significant differences were set at p < 0.05.

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Results

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The histochemistry shows that phytol caused the skeletal muscle fiber

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type switch

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The ATPase staining of mice gastrocnemius showed that 0.05% and 0.5%

244

phytol increased the proportion of MHC I (p < 0.05), and there was no

245

significant influence on the proportion of MHC IIa and MHC IIb (p >

246

0.05) (Fig. 1).

247

In pigs, we chose 4 different muscles, the glycolysis muscles longissimus

248

dorsi and gluteus medius, which are mainly composed of IIb muscle fiber

249

types, and the oxidation muscles triceps brachii and psoas, which are

250

mainly composed of MHC I and MHC IIa muscle fiber types. The results

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of ATP staining are shown in Fig. 2. In the two glycolysis muscles,

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longissimus dorsi and gluteus medius, all doses of phytol had no effect on

253

muscle fiber types (p < 0.05), but 0.05% phytol had a trend of enhancing

254

the proportion of MHC I (p = 0.074). In the two oxidation muscles, 0.05%

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and 0.1% phytol significantly enhanced the proportion of MHC I (p
0.05). In the triceps brachii, 0.05% phytol significantly enhanced

259

the proportion of MHC I (p < 0.05); 0.025% phytol significantly

260

enhanced the proportion of MHC IIa (p < 0.05), but all doses of phytol

261

had no influence on MHC IIb (p > 0.05).


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Phytol could influence the expression of MHCs

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In mice gastrocnemius, 0.5% phytol significantly enhanced the protein

264

expression of MHC I (p < 0.05) and decreased the protein expression of

265

MHC II (p < 0.05) (Fig. 3).

266

In the two glycolysis muscles of pigs, 0.1% phytol significantly enhanced

267

the protein expression of MHC I (p < 0.05) and MHC II (p < 0.05) in

268

longissimus dorsi, but all doses of phytol had no significant influence on

269

protein expression in the gluteus medius (p > 0.05). In the two oxidation

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muscles, only 0.1% phytol significantly enhanced the protein expression

271

of MHC II (p < 0.05) in psoas and decreased MHC I and MHC II in

272

triceps brachii (p < 0.05) (Fig. 4.).

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Transcriptome analysis shows that phytol affected the PGC-1α

274

signaling pathway

275

Sus scrofa muscle samples were used for the transcriptome analysis as the

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reference for how phytol functions in muscle fiber type adaptation. As a

277

result, 21,682 expressed genes were identified, comprising 78.81% of the

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Sus scrofa transcripts provided by ENSEMBL (Fig. 5). There were 137

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differentially expressed (fold ≥ 2) genes between 0% phytol and 0.05%

280

phytol, including 33 upregulated and 104 downregulated genes (Table 3).

281

The GO analysis using DAVID revealed that there were 118 genes in the

282

database, and all of these genes were in three categories, including 18

283

functions in biological process (BP), 6 functions in cellular component



284

(CC), and 7 functions in Molecular Function (MF). The KEGG (threshold

285

of gene number ≥ 3, p < 0.05) showed that the differentially expressed

286

genes participated in 4 signaling pathways included Insulin resistance ,

287

AMPK signaling pathway, Epithelial cell signaling in Helicobacter pylori

288

infection and Adherens junction (Table 4). The KOBAS (threshold of

289

gene number ≥ 2, p < 0.05) showed that the differentially expressed

290

genes participated in 6 signaling pathways included Herpes simplex

291

infection, Cytokine-cytokine receptor interaction, AMPK signaling

292

pathway, Jak-STAT signaling pathway, Malaria and PPAR signaling

293

pathway (Table 5). AMPK signaling pathway appeared in these two kinds

294

of analysis results, and the core molecule of AMPK signaling PGC-1α

295

and stearoyl-CoA desaturase (SCD) have a 2.05-fold and 2.07-fold

296

upregulate expression, but the crucial rate limiting enzymes relating to

297

glycolytic pathway, 6-phosphofructo-2-kinase (Pfkfb3), has a 0.21-fold

298

down regulate expression after feed by phytol.

299

PPARs/PGC-1α signaling pathway may be involved in the influence

300

of phytol

301

To explore the intracellular signaling pathway of phytol, Ca2+ signaling

302

pathway molecule CaMKII and MEF2, the PPAR signaling pathway

303

molecule PPARα, PPARδ and PGC-1α were detected. In addition, the

304

mitochondriogenesis core molecule TFAM, the energy sensor Sirt1, the

305

oxidation respiratory related gene COX4, NFAT and NRF1, the fatty acid


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transporters CD36 and FABP3 were detected to determine the alternation

307

in muscle metabolism.

308

In mice, as shown in the Western blot Fig. 6, both 0.05% and 0.5% phytol

309

increased CaMKIIa protein expression (p < 0.05), and 0.05% phytol also

310

increased the protein expression of the slow muscle-related gene PGC-1α

311

(p < 0.05). This result was supported by the qPCR result in Fig. 7, which

312

showed that 0.05% phytol increase the mRNA expression quantity of the

313

slow muscle-related gene PGC-1α, PPARδ, CaMKIIa, and NRF1 (p
0.05).

317

In pigs, we chose the longissimus dorsi to test the influence of phytol. We

318

found that 0.1% phytol significantly enhanced the protein expression of

319

PGC-1α (p < 0.05), and 0.025% and 0.05% phytol significant enhanced

320

the protein expression of MEF2a (p < 0.05) (Fig. 8); however, the mRNA

321

expression of these two genes was not influenced by phytol (Fig. 9 B and

322

D). On the other hand, 0.025% phytol significantly enhanced the mRNA

323

expression of the Ca2+ related signaling molecule NFAT (p < 0.05), and

324

0.1% phytol significantly enhanced the mRNA expression of PPARα (p

327

0.05) (Fig. 9), which were also seen in the results from mice.



328

Phytol may promote the expression of MHC I via PGC-1α and miRs

329

in C2C12 cells

330

The above results show that phytol may affect the PGC-1α signaling

331

pathway but not via activated mitochondrial metabolism, as previously

332

reported. Therefore, further studies were carried out in the C2C12 cell

333

line, and the reported slow fiber type related miRNA miR-499 and

334

miR-208b were detected to support the alternation in skeletal MHCs as

335

added indicators.47, 48

336

As expected, both 5 µM and 50 µM phytol significantly enhanced the

337

protein level of MHC I (p < 0.05), and at the same time 50 µM phytol

338

significantly decreased the protein level of MHC II in C2C12 cells (p

Phytol Promotes the Formation of Slow-Twitch Muscle Fibers through

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