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Cloning, phylogenetic analysis and distribution of the free fatty acid receptor GPR120 expression in the gastrointestinal tract of housing versus grazing kid goats Tao Ran, Hengzhi Li, Yong Liu, Chuanshe Zhou, Shao-Xun Tang, Xuefeng Han, Min Wang, Zhixiong He, Jinghe Kang, Qiongxian Yan, Zhi-Liang Tan, and Karen A. Beauchemin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06131 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on March 1, 2016

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Running head: Phylogenetic analysis of GPR120 in goats

Cloning, phylogenetic analysis and distribution of free fatty acid receptor GPR120 expression along the gastrointestinal tract of housing versus grazing kid goats Tao Ran*, †, Hengzhi Li*, †, Yong Liu*, ‡, Chuanshe Zhou*, 1, Shaoxun Tang*, Xuefeng Han*, Min Wang*, Zhixiong He*, §, Jinghe Kang*, Qiongxian Yan*, Zhiliang Tan* and Karen. A. Beauchemin§ *

Key Laboratory for Agro-Ecological Processes in Subtropical Region, and

South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, P.R. China; †

Graduate University of Chinese Academy of Sciences, Beijing 100049, P. R. China;

‡

Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de

México, Toluca, Estado de México, C.P. 50090, México §

Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta

T1J 4B1, Canada 1

Corresponding author: Chuanshe Zhou. Address: Institute of Subtropical Agriculture,

the Chinese Academy of Sciences, Changsha, Hunan 410125, P. R. China. E-mail: [email protected]; Tel: +86 731 4615230; Fax: +86 731 4612685.

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ABSTRACT: G-protein-coupled receptor 120 (GPR120) is reported as a long chain

2

fatty acid (LCFA) receptor that elicit free fatty acid (FFA) regulation on metabolism

3

homeostasis. The study aimed to clone the gpr120 gene of goats (g-GPR120), and

4

subsequently investigate phylogenetic analysis and tissue distribution throughout the

5

digestive tracts of kid goats, as well as the effect of housing vs grazing (H vs G)

6

feeding systems on GPR120 expression. Partial coding sequence (CDS) of g-GPR120

7

was cloned and submitted to NCBI (Accession No.: KU161270). Phylogenetic

8

analysis revealed that g-GPR120 shared higher homology in both mRNA and amino

9

acid sequences for ruminants than non-ruminants. Immunochemistry, real-time PCR

10

and Western blot analysis showed that g-GPR120 was expressed throughout the

11

digestive tracts of goats. The expression of g-GPR120 was affected by feeding system

12

and age, with greater expression of g-GPR120 in the G group. We conclude that

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g-GPR120 mediated LCFA chemosensing mechanism is widely present in the tongue

14

and GIT of goats, and its expression can be affected by feeding system and age.

15

KEYWORDS: free fatty acid receptor, gastrointestinal tract, g-GPR120, grazing and

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housing, goats

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17

INTRODUCTION

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Recent findings suggest that the gastrointestinal tract (GIT) can sense nutrients (named

19

‘gut nutrient chemosensing’) via G-protein coupled receptors (GPCRs) expressed

20

throughout the gut epithelium, which presents exciting prospects for human metabolic

21

disease therapy (i.e., obesity and diabetes) and animal production.1 As the GIT

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represents the first site of interaction between ingested nutrients and the host, its

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ability to precisely sense ingested nutrients initiates crucial negative feedback systems

24

that control food intake, absorption and glucose production, important for

25

maintenance of metabolic homeostasis.2 Although endocrine cells scattered

26

throughout the epithelial layer of the GIT represent only a small fraction (< 1 %) of

27

the total number of epithelial cells, they make the GIT a highly specialized

28

chemosensory organ.3 Gut nutrient chemosensing is accomplished by these cells via

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activation of cell membrane GPCRs, which trigger intracellular signaling pathways,

30

including membrane depolarization, elevated calcium levels and second messenger

31

cascades that ultimately result in the release of hormones or changes in gene

32

expression.3

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Free fatty acids (FFAs) are not only an important direct source of energy but they also

34

play key roles in regulating a series of physiological responses because they serve as

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signaling molecules. Recently, a number of GPCRs, including free fatty acid receptors

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FFAR1, FFAR2, FFAR3 and GPR120, have been identified and are thought to be 3


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involved in FFA chemosensing and mediation of the release of gut hormones, such as

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glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK).4,5 It is now apparent

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that FFAR1 (also known as GPR40) and GPR120 respond to medium- and long-chain

40

FFAs, whereas FFAR2 and FFAR3 (also known as GPR43 and GPR41, respectively)

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bind short-chain FFA.6,7 Despite the similarity of GPR120 and GPR40 in ligand

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specificity, they share only 10% amino acid identity with each other, and a distant

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evolutionary relationship has been suggested by phylogenetic analysis.8 Furthermore,

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silencing the expression of GPR120 and not that of GPR40 by small interfering RNA

45

(siRNA) can prevent both fatty acid induced CCK 5 and GLP-1 secretion.4 Hence, it

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can be deduced that GPR120, and not GPR40, is the primary GPCR involved in

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regulating hormone secretion in response to dietary lipids.

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GPR120 was first identified by using GPCR deorphanizing strategy in human genome

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databases. It belongs to the rhodopsin-family GPCR with limited sequence homology

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to other family members.9 GPR120 responds to a variety of long chain FFAs,

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including saturated FFAs with a carbon chain length of 14 to 18 and unsaturated FFAs

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with a chain length of 16 to 22,10 with ω-3 FFAs docosahexaenoic acid (DHA) and

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α-linolenic acid (ALA) being the most potent and common GPR120 agonists.4

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GPR120 mRNA has been proven to be highly expressed in human, mice and porcine

55

intestine.4,11-13 GPR120 is also expressed in a number of other tissues, including

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adipocytes, taste buds, pituitary, lung and pancreas,10,14,15 but to date, the functional

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consequences of GPR120 activation in these tissues remain unclear. It has been 4



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proposed that GPR120 and subsequent signaling pathways are involved in many

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physiological processes, such as stimulating the secretion of a number of

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enteroendocrine hormones (including CCK, GLP-1 and GIP),4,5 regulating adipogenic

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processes (i.e., adipocyte development and differentiation),11 controlling gonadotropin

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secretion,15 and mediating FFAs induced anti-apoptotic effect.16

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As GPR120 plays an important role in regulating energy metabolism, it could be

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critical for preventing the occurrence and development of metabolic disorders such as

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obesity and diabetes.17 However, when compared with rodent and human models,

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much less is known about GPR120 in ruminants. Ruminants have evolved a

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polygastric digestive system, in which digestion of dietary lipids is significantly

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different from the monogastric digestive system of non-ruminants. In the rumen,

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lipids undergo two main processes: hydrolysis of ester bonds into triglycerides and

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hydrogenation of unsaturated bonds, leading to substantially different composition of

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fatty acids (mostly palmitic and stearic acids) leaving the rumen comparted with

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of the diet.18 In contrast, only negligible changes occur anterior to the small intestine

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in non-ruminants.19 Thus, the GPR120 expression pattern and potential role in

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ruminants may differ from that in non-ruminants. Furthermore, ruminants undergo a

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transition from pre-ruminant to a fully functional rumen, as well as a change in nutrient

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supply from high-fat milk diets during the pre-ruminant suckling phase to forage- and

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cereal-based diets containing low levels of long-chain fatty acids (LCFAs) when the

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rumen is fully functional. In current animal production, fats are routinely added to the 5


that

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diet to satisfy the energy requirements of high-yielding animals (mainly growing

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weaned cattle and lactating cows).20 Therefore, study of GPR120 provides

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opportunity to understand ways of improving dietary energetic efficiency in ruminants,

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providing greater flexibility in feeding management. Meanwhile, as lipid metabolism

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of farm animals plays a central role in economically relevant phenotypic traits (such

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as animal health and performance),21 study of GPR120 may greatly extend the

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application of lipid metabolism manipulation strategies in livestock production and

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food supply worldwide. In the present study, we examined cloning and tissue

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distribution of GPR120 (at both mRNA and protein levels) in the GIT of goats, as

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well as its expression pattern under housing and grazing conditions.

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MATERIALS AND METHODS

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Experimental Animal and Management

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All procedures for animal experimentation were carried out in accordance with

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guidelines approved by the Animal Care Committee (Approval Number: 20130108),

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Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha,

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China.

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Because GPR120 expression in goats was not previously studied, the GPR120

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sequences had to be determined through cloning at first, to further study its

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phylogenetic analysis and distribution pattern in the tongue and GIT of goats, and later

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developing changes in goat kids. Four adult Liuyang black goats (a local breed in south

6



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of China) with similar body weight and age (1 year old) were used as experimental

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animals of the first part in this study. These animals were maintained individually in

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metabolic cages under controlled condition with fresh water was available ad libitum.

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A diet consisting of forage and concentrate was offered twice daily in equal amounts at

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0800 and 1800 h to each goat to supply 1.3 × maintenance requirement of

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metabolizable energy according to our previous studies.22 After 15 days of feeding, the

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goats were slaughtered for sample collection.

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Forty-four newly born Liuyang black goat kids (average weight of 1.35 ± 0.12 kg)

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were used to investigate the effect of age and feeding system (housing vs. grazing, H

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vs G) on the expression of free fatty acid receptor GPR120 in the GIT. After birth the

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kids were separated from the nanny and trained to suckle milk from nipple pails.

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Detailed feeding management, ingredients of concentrate starter and forage (mainly

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Miscanthus) has been described in our previous parallel study.23 All goats had free

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access to water.

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Sample Collection

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Epithelium samples of tongue, tissue and mucosa of rumen, abomasum, duodenum,

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jejunum, ileum, colon, cecum and rectum were collected immediately after slaughter.

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Collected samples were wrapped with sterilized tinfoil and snap-frozen in liquid

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nitrogen

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immunohistochemistry analysis, afore-mentioned tissue samples sized about 1 × 1 cm2

119

were collected and washed with phosphate-buffered saline (PBS: 0.85 % NaCl, 1.4 mM

and

stored

at

-80°C

until

RNA

and

7


protein

isolation.

For

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KH2PO4, 8 mM Na2HPO4, pH 7.4),24 fixed in 10% formalin (v/v) for 24 h, embedded in

121

paraffin wax, and stored at 4°C until use.

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RNA Isolation and cDNA Preparation

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Total RNA was extracted from collected mucosal samples using TRIZOL (Invitrogen,

124

California, American) according to the manufacturer’s instructions. After genomic

125

DNA was eliminated by digestion with DNase I (Thermo Scientific, Waltham, USA),

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the RNA quality and quantity was determined. Immediately afterwards, 1 µg of the

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extracted RNA was reverse-transcribed to synthesize tissue specific cDNA using

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PrimeScript™ RT reagent Kit (Takara, Dalian, China). Briefly, a 20 µl reverse

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transcription mixture that contained 1 µg of total RNA, 2 µl 5 × g DNA Eraser Buffer, 4

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µl 5 × PrimeScript Buffer, 1 µl PrimeScript RT Enzyme Mix, 1 µl RT Primer Mix and

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10 µl RNase Free dH2O was prepared. This reaction mixture was incubated for 2 min at

132

42°C, followed by a reverse transcription step for 15 min at 37°C, and a final heating

133

step at 85°C for 5 s to stop the reaction. The prepared cDNA samples were stored at

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-20°C until subsequent cloning of GPR120 and quantitative real-time PCR analysis.

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Primer Design

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Primers for cloning of GPR120 and quantitative real-time PCR analysis were designed

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according to predicted GPR120 gene sequences of Capra hircus (Gene bank:

138

XM_005698245.1) and Ovis aries (Gene bank: XM_004020291.1) reported online.

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β-Actin gene was used as a housekeeping gene in quantitative real-time PCR analysis.

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All primers were synthesized by Sangon Biotech (Sangon Biotech, Shanghai, China), 8



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and the primer sequences are given in Table 1.

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Cloning of GPR120 and Phylogenetic Analysis

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We aimed to clone, characterize, and pave the way for further examination of the

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physiological role of GPR120 in goats. cDNA prepared from goat tongue samples were

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used to clone GPR120 gene. Partial CDS of GPR120 were obtained by PCR

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amplification using primer pair GPR120-C and Phusion Hot Start II High-Fidelity

147

DNA Polymerase (ThermoFisher Scientific, Shanghai, China). Amplified fragments

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were added A tailing using A-Tailing Kit (CWbiotech.com, Beijing, China), cloned

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into pMD™ 18-T Vector Cloning Kit (Takara, Dalian, China) and sequenced (Sangon,

150

Shanghai, China). Briefly, PCR was performed in a 50 µl reaction mixture containing 2

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µl of the tongue first-strand cDNA template, 10 µl 5 × Phusion HF Buffer

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(ThermoFisher Scientific, Shanghai, China), 1 µl 10 mM dNTPs, 0.5 µl (2 U/µl)

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Phusion Hot Start II HF DNA Polymerase (ThermoFisher Scientific, Shanghai, China),

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2 µl forward primer (10 µM), 2 µl reverse primer (10 µM) and 32.5 µl ddH2O. PCR

155

was carried out at 98°C for 30 s, followed by 35 cycles of 10 s of denaturation at 98°C,

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30 s annealing at 60°C, and 1 min extension at 72°C, and finally extension at 72°C for

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10 min.

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Quantitative real-time PCR Analysis

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Quantitative real-time PCR was performed on an ABI-7900HT qPCR system (Applied

160

Biosystems, Foster City, CA, USA) using FG POWER SYBR GREEN PCR MASTER

161

MIX (Applied Biosystems, Foster City, CA, USA). Primer pair GPR120-R was used 9


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for quantitative real-time PCR analysis; quantification of the PCR products of GPR120

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gene were evaluated in comparison with the PCR products of β-actin. The relative

164

changes in mRNA expression levels determined from qPCR were calculated according

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to the 2-△△CT method,25 where -∆∆CT = - (∆CT other tissue samples - ∆CT colon sample at d 0) and

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∆CT = CT samples - CT β-actin.

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Protein Isolation and Western Blot Analysis

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Protein isolation and Western blot analysis were carried out as described

169

previously.26,27 In detail, mucosal samples (0.2 g each) were frozen in liquid nitrogen,

170

crushed into powder, and dissolved in 300 µl RIPA lysate (Applygen Technologies,

171

Beijing, China) with 1% protease inhibitor cocktail (Roche Diagnostics GmbH,

172

Mannheim, Germany ), followed by 30 min of cleavage on ice. Then, samples were

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centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was taken. Protein

174

concentrations were measured utilizing a BCA Protein Assay kit (Hin biotech, Beijing,

175

China). Exact amounts of protein needed for each sample were calculated and mixed

176

with 5 × loading buffer, incubated at 95°C for 5 min and stored at -20°C.

177

Equal amounts of protein of each sample and pre-stained standards (Bio-Rad

178

Laboratories) were separated by electrophoresis in 10% SDS-polyacrylamide gels.

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Separated proteins were transferred onto polyvinylidene difluoride (PVDF)

180

membranes (Bio-Rad, USA) under constant electric current of 200 mA for 70 min.

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The PVDF membranes were then incubated with 5% skim-milk in TBS buffer [10 ml

182

1 mol/L Tris·HCl (pH = 7.5) and 8.8 g NaCl dissolved in 1000 ml distilled water] 10



183

containing 0.2% Tween20 for 1 h to suppress non-specific binding of

184

immunoglobulins. The pre-blocked membranes were incubated with anti-GRP120

185

antibody (Sc-99105, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and

186

diluted 1:200 in 1 × TBST at 4°C overnight, washed in 1 × TBST 3 times (15 min

187

each); incubated with horseradish-peroxidase (HRP)-labeled secondary antibody

188

(1:3,000; Proteintech Group, Inc., Chicago, USA) in 1 × TBST for 1 h at room

189

temperature, and washed in 1 × TBST (3 × 10 min). Bands of GPR120 proteins were

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detected by WesternBright ECL Western Western Blotting HRP Substrate (APGBio,

191

Shanghai, China), and the images were taken and analyzed by AlphaImager 2200

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digital imaging system (Digital Imaging System, Kirchheim, Germany).

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Immunohistochemistry and Immunofluorescence

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Immunohistochemistry was carried out as described by Matsumura et al..14 Briefly,

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pre-embedded samples were cut into sections of 5 µm thickness on a CM3000

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cryostat (Leica Microsystems, Bensheim, Germany) and adhered to positively charged

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adhesion slides (Beyotime, Shanghai, China). Slides were dewaxed, treated with 0.3 %

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Triton X-100 in PBS for 2 h, and then with 0.3% H2O2 for the inhibition of

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endogenous peroxidase activities. After pre-incubation with blocking serum for 1 h,

200

the slides were incubated with anti-GPR120 (Sc-99105, Santa Cruz Biotechnology,

201

Inc., Santa Cruz, CA, USA) at a dilution of 1:400 in 1 × PBS containing 10% normal

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goat serum at 4°C overnight. Control staining was conducted without primary

203

antibody incubation. The subsequent secondary antibody incubation and staining 11


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process were carried out by using an ImmunoCruz™ rabbit ABC Staining System

205

(Sc-2018, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as suggested by the

206

supplier. The antigen-antibody reaction was visualized by 3,3’-di-aminobenzidine

207

(DAB) staining system and the slides were observed using a light microscope.

208

For

209

immunohistochemistry until secondary antibody incubation. After first antibody

210

incubation, the slides were washed thrice for 5 min in 1 × PBS, then incubated with

211

goat anti-rabbit IgG-FITC secondary antibody (Sc-2012, Santa Cruz Biotechnology,

212

Inc., Santa Cruz, CA, USA); 1:500 in 19 PBS/10% NGS/0.3% Triton X-100) for 30

213

min at room temperature. The slides were washed in PBS and cover-slipped using an

214

anti-fading glycerol-based mounting media. Immuno-stained slides were examined

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with a fluorescence upright microscope (Olympus, Tokyo, Japan) with argon and

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He-Ne laser sources.

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Statistical Analysis

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When studying the distribution pattern of GPR120 using four adult goats, one-way

219

ANOVA with Turkey-Kramer multiple comparison test (SPSS Statistical Software)

220

was used for statistical analysis. The effect of feeding system (H vs G) on the

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expression of free fatty acid receptor GPR120 was examined from d 28 to 70, and data

222

were analyzed as a completely randomized design with the MIXED procedures of SAS

223

(SAS Inst. Inc., Cary, NC) with a model that included the fixed effect of feeding system,

immunofluorescence

analysis,

the

process

was

12


the

same

as

for


224

age, and the feeding system × age interaction, with individual animal as the experiment

225

unit as described in our previous study.23 In detail, the SLICE option was used when

226

the feeding system × age interaction was significant to partition and test the effect of

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feeding system within age. To test the effects of age on rumen development of goats

228

from 0 to 70 d, the MIXED procedure of SAS was used, with animal within age as

229

random effect and individual animal as the experimental unit. Orthogonal contrasts

230

were used to test for linear, quadratic and cubic effects of age. Quartic, quintic and

231

sextic effects were not examined, because they could not be interpreted biologically. If

232

there was no feeding system × age interaction from 28 to 70 d, the linear and quadratic

233

effects of age from 0 to 70 d were averaged over the two feeding systems (S and G). If

234

the interaction is significant, the effects of age from 0 to 70 d for S and G groups were

235

presented separately. Statistical significance was defined as P < 0.05.

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RESULTS AND DISCUSSION

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Cloning of GPR120 and Phylogenetic Analysis

238

An 1126 bp transcript that matched the predicted nucleotide sequence of Capra hircus

239

(XM_005698245.1) was amplified and cloned using primer pair g-GPR120-C, and

240

was submitted to NCBI (Accession No.: KU161270). Multiple sequence alignments

241

and phylogenic tree comparison of the CDS and deduced amino acid sequences of

242

GPR120s revealed that g-GPR120 shared greater homology with Ovis aries and Bos

243

taurus than with Sus scrofa, Homo sapiens and Mus musculus in both CDS (Figure 1. 13


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A and Figure 2. A) and amino acid sequences (Figure 1. B and Figure 2. B). Previous

245

studies have shown that the rat GPR120 (rGPR120) shares 85 and 98% sequence

246

identity with human and mouse GPR120 proteins,28 respectively, while the porcine

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GPR120 shares greater homology with human receptor sequences (both CDS and

248

amino acid sequences) than those of mice and rats.12 Therefore, we suggest that there

249

is a certain distance in evolution of the GPR120 between ruminants and

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non-ruminants. However, GPR120 shares high homology within ruminants and within

251

non-ruminants.

252

Tissue Distribution of g-GPR120

253

Immunoreactivity of g-GPR120 was observed in dispersed epithelial cells of the

254

rumen, abomasum, duodenum, jejunum, ileum, cecum and colon (Figures 3 and 4)

255

using immunohistochemistry and immunofluorescence analyses. This was in

256

accordance with previous studies, in which GPR120 immunoreactivity was found

257

dispersed throughout the rat colon.14 Real-time PCR and Western blotting further

258

indicated that g-GPR120 was abundantly expressed in the cecum, colon and rectum at

259

the mRNA level (Figure 5. A), and in the duodenum, ileum, cecum and colon at the

260

protein level (Figure 5. B). Representative bands of Western blot analysis of

261

g-GPR120 throughout the GIT segments are shown in Figure 4. C. These observations

262

are similar to the expression pattern of GPR120 in the GIT of non-ruminants reported

263

previously.4,11-13 It appears that the general mechanisms of intestinal LCFAs

264

absorption are same for ruminants and non-ruminants (the major digestion of lipids 14



265

results from hydrolysis with pancreatic lipase in the small intestine),29,30 even though

266

there is distance in evolution of GPR120 (Figure 2) and difference in fat digestion

267

processes between ruminants and non-ruminants. The relatively low g-GPR120

268

expression in the rumen was likely caused by relatively low concentrations of LCFAs

269

compared with volatile fatty acids (VFAs).

270

The expression of g-GPR120 was also analyzed in the epithelium collected from

271

different parts of tongue by Western blotting. Epithelium collected from the central

272

part of the tongue had abundant expression of g-GPR120 (Figure 6. A).

273

Representative bands of Western blot analysis of g-GPR120 of tongue are shown in

274

Figure 6. B. In non-ruminants, digestion of dietary lipids initiates in the oral cavity by

275

lingual lipase, and the lingual lipase activity of rodents is sufficient to hydrolyze

276

triacylglycerides even during short exposure times.31 It has been revealed that the

277

majority of GPR120-positive taste cells are type II cells in mice.32 In ruminants, the

278

transformation of dietary lipids entering the rumen begins with the hydrolysis of ester

279

linkages by microbial lipolytic enzymes, followed by biohydrogenation of unsaturated

280

fatty acids,29 but it should be noted that the rumination process increases the chance of

281

FFAs being sensed in the oral cavity. Thus, we deduced that g-GPR120 expressed in

282

the tongue of goats would indeed participate in gustatory fatty acid perception as it

283

does in human, rat and mice models.14,31,32 The high expression of g-GPR120 in the

284

middle of the tongue suggests this region, rich in fungiform papilla and foliate papilla,

285

might play a critical role in sensing FFA in the oral cavity of goats. 15


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Effect of Feeding System and Age on g-GPR120 Expression

287

The effect of feeding system (H vs G) on the expression of g-GPR120 was assayed at

288

mRNA and protein levels. As shown in Tables 2 and 3, during d 28 and 70, the

289

expression of g-GPR120 in the duodenum and jejunum were affected (P < 0.01) by

290

the feeding system at both mRNA and protein levels. In the rumen (P > 0.05) and

291

abomasum its expression was affected at the protein level (P < 0.05) but not at the

292

mRNA level (P > 0.05), while in the ileum, cecum and colon, g-GPR120 expression

293

were unaffected (P > 0.05) by the feeding system at both mRNA and protein levels.

294

Meanwhile, the expression of g-GPR120 was affected (P < 0.05) by age in major GIT

295

segments at both mRNA and protein levels, with exception of the rumen, abomasum

296

and rectum at the mRNA level. However, there was no feeding system × age

297

interaction (P > 0.05) on g-GPR120 expression throughout the GIT at both mRNA

298

and protein levels, with exception of the cecum (P = 0.021), colon (P < 0.001) and

299

rectum (P < 0.001) at mRNA level. Our results imply that expression of g-GPR120 is

300

tissue specific, and can be affected by individual and combined effects of feeding

301

system and age, and this effect is post-translationally regulated.

302

It was noteworthy that relatively greater expression of g-GPR120 at mRNA and

303

protein levels occurred in the G group in comparison with the H group in all major

304

segments of the GIT except the rumen. As reported previously, the majority of dietary

305

lipids for grazing ruminants come from chloroplast membranes (mainly in the forms

306

of galactolipids, sulpholipids and phospholipids) ingested; while for animals receiving 16



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concentrates triglycerides are the major dietary lipid.19 In the current study, the G

308

group only consumed fresh pasture, and therefore dietary lipids were likely in the

309

form of galactolipids, sulpholipid and phospholipids; while the H group received

310

relatively greater concentrations of dietary lipids in the form of triglyceride contained

311

in the concentrate supplement. Greater expression of g-GPR120 at the protein level in

312

the rumen was observed in the H group, which might be caused by greater LCFAs

313

concentration in the diet. A previous study reported that the mRNA level of GPR120

314

in the corpus region of stomach tissue was significantly increased in rats fed a high fat

315

diet for either 3 weeks or 6 months.33 This report probably reflected a significant

316

increase in the number of GPR120 positive cells in the corpus region. However, in the

317

remaining segments, relatively greater expression of g-GPR120 was observed in the G

318

group, which we propose can be interpreted in two ways. Firstly, the G group goats

319

may have had adopted a compensatory mechanism in g-GPR120 expression to meet

320

the physiological demands of increased intake of LCFAs under grazing conditions, as

321

a similar compensatory mechanism has also been observed in rats where GPR120

322

mRNA expression in the pituitary gland was increased after 24 h of fasting.15

323

Alternatively, the high fat diet consumed by the H group may have affected negatively

324

feed intake, leading to a reduction in the expression of g-GPR120.

325

possible to measure intake of grazing goats, it is not possible to confirm or refute that

326

speculation.

327

Lipid chemosensing mechanisms in the gut can regulate glucose production and food 17


As it was not

Page 19 of 39


328

intake through a neuronal negative feedback network.34 Fat-rich foods are potent

329

stimulators of cholecystokinin secretion, which in turn restrict feed intake. However,

330

data about effects of dietary fat on cholecystokinin concentrations in the plasma of

331

ruminants is limited. Furthermore, it has been long recognized that rumen

332

fermentation

333

biohydrogenation of fats, and methanogenesis), are affected by lipid concentration of

334

the diet.29 To prevent a depression in feed intake and digestibility, it is generally

335

recommended that total lipid content of ruminant diets not exceed 6% of the dietary

336

dry matter. It has been reported that 10% added fat in a ruminant diet reduces

337

carbohydrate digestion by 50%, causing a reduction in VFA production and a lower

338

acetate/propionate ratio (A/P).35 Under the current conditions, even though no feeding

339

system × age interaction was observed for total VFA (TVFA) and A/P, the feeding

340

system did have an effect on A/P as reported in our previous study.23 Conversely,

341

enhanced expression of GPR120 has recently been observed in the small intestine of

342

diet-induced obese rats fed a high fat diet.36 Similarly, rats fed fish or flaxseed

343

oil-supplemented diets has significantly heightened colonic GPR120 expression.37

344

When the findings of the current study are examined together with those in the

345

published literature, it is clear that the effect of dietary factors on GPR120 expression

346

is inconsistent between ruminants and non-ruminants.

347

Irrespective of the feeding system, from d 0 to 70, the g-GPR120 mRNA expression in

348

the duodenum, jejunum, ileum, cecum and colon was affected by age with a linear

processes

(such

as

fermentation

of

18


carbohydrates,

ruminal


349

increase in the ileum (for both H and G groups, P < 0.001) and colon (for H group, P

350

= 0.003), and a quadratic increase in the jejunum (P = 0.011) and cecum (P < 0.05) for

351

both H and G groups (Table 2); the expression of g-GPR120 at protein level in the

352

rumen, abomasum, duodenum and jejunum increased linearly with age (P < 0.01)

353

while its expression in the cecum increased quadratically with age (P < 0.05) (Table

354

3). Unfortunately, there is a paucity of information about the developmental changes

355

of GPR120 is even for the human and mice models. Our study revealed for the first

356

time that the expression of g-GPR120 in the GIT of kid goats is increased with age

357

postnatally.

358

Most previous studies concerning FFAs chemosensing have been conducted in humans,

359

rats and mice, and FFARs have received considerable attention as potential therapeutic

360

targets for the treatment of metabolic diseases, such as diabetes and obesity.17,38

361

Because GPR120 plays an important role in regulating secretion of GLP-1 4, CCK 5,

362

and ghrelin,39 it is predicted that the stimulation of GPR120 might regulate appetite

363

and systemic energy homeostasis.40,41 Therefore, it is possible that GPR120 may affect

364

energetic efficiency in ruminants, and greater knowledge of the role of GPR120 may

365

help improve efficiency of rearing goats. Indeed, GPR120 has been proven to serve as

366

a LCFA receptor in porcine

367

sequenced partial CDS of g-GPR120 for the first time, and showed that g-GPR120

368

was expressed predominantly in the cecum, colon and rectum at mRNA level, and in

369

the duodenum, ileum, cecum and colon at protein level in goats. Moreover, interaction

12

and chicken.42 In the current study, we cloned and

19


Page 20 of 39

Page 21 of 39


370

between feeding system and age on g-GPR120 mRNA expression was observed in

371

some of GIT segments (the cecum, colon and rectum), indicating that these segments

372

play a critical role in LCFA chemosensing.

373

ABBREVIATIONS

374

GPR120, G-protein-coupled receptor 120; LCFA, long chain fatty acid; FFA, free

375

fatty acid; FFAR, free fatty acid receptor.

376

AUTHOR CONTRIBUTIONS

377

Chuanshe Zhou, Min Wang, Xuefeng Han, Zhixiong He and Zhiliang Tan made the

378

experimental design; Tao Ran, Hengzhi Li and Yong Liu carried out all animal

379

feeding management and the laboratory analysis. Jinghe Kang and Qiongxian Yan did

380

a lot work during sample collection and Western blot analysis. Shaoxun Tang did the

381

analysis of dataset. This manuscript was written by Tao Ran and revised by Karen. A.

382

Beauchemin and Zhiliang Tan, reviewed by all authors.

383

ACKNOWLEDGEMENTS

384

The authors acknowledge Key Laboratory of Subtropical Agro-ecological

385

Engineering, Institute of Subtropical Agriculture, the Chinese Academy of Sciences

386

(CAS) for providing all the experimental materials and apparatus.

387

FUNDING SOURCES

388

This work was jointly supported by the National Natural Science Foundation of China

389

(Grant No. 31320103917), “Strategic Priority Research Program - Climate Change: 20



Page 22 of 39

390

Carbon Budget and Relevant Issues” (Grant No.XDA05020700), “CAS Visiting

391

Professorship

392

2012T1S0009), and Hunan Provincial Creation Development Project (Grant No.

393

2013TF3006).

394

CONFLICT OF INTEREST STATEMENTS

395

The authors declare no competing financial interest.

for

Senior

International

Scientists

(Grant

21


No.

2010T2S13,

Page 23 of 39


396

REFERENCES

397

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398

SYMPOSIUM: Gut chemosensing and the regulation of nutrient absorption and energy

399

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400 401 402 403

(2) Reimann, F.; Tolhurst, G.; Gribble, F. M. G-Protein-Coupled Receptors in Intestinal Chemosensation. Cell Metab. 2012, 15, 421-431. (3) Tolhurst, G.; Reimann, F.; Gribble, F. M. Intestinal sensing of nutrients. Handb.Exp. Pharmacol. 2012, 309-35.

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Sugimoto, Y.; Miyazaki, S.; Tsujimoto, G. Free fatty acids regulate gut incretin

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glucagon-like peptide-1 secretion through GPR120. Nat. Med. 2005, 11, 90-4.

407

(5) Tanaka, T.; Katsuma, S.; Adachi, T.; Koshimizu, T. A.; Hirasawa, A.;

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Tsujimoto, G. Free fatty acids induce cholecystokinin secretion through GPR120. N.-S.

409

Arch. Pharmacol. 2008, 377, 523-527.

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P. G.; Wilson, S.; Ignar, D. M.; Foord, S. M.; Wise, A.; Dowell, S. J. The Orphan G

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Res.-Tokyo 2007, 28, 49-55.

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GPR120 in a murine enteroendocrine cell line STC-1. J. Biol. Chem. 2005, 280,

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(22) Wang, M.; Hu, Y.; Tan, Z. L.; Tang, S. X.; Sun, Z. H.; Han, X. F. In situ ruminal

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phosphorus degradation of selected three classes of feedstuffs in goats. Livest. Sci. 2008,

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117, 233-237.

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(23) Jiao, J.; Li, X.; Beauchemin, K. A.; Tan, Z.; Tang, S.; Zhou, C. Rumen

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development process in goats as affected by supplemental feeding v. grazing:

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age-related anatomic development, functional achievement and microbial colonisation.

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Brit. J. Nutr. 2015, 113, 888-900.

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(24) Sutherland, K.; Young, R. L.; Cooper, N. J.; Horowitz, M.; Blackshaw, L. A.

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Phenotypic characterization of taste cells of the mouse small intestine. Am. J.

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Physiol.-Gast. L. 2007, 292, G1420-8.

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real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25,

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Analysis of Isolated Plasma Membrane Fractions from the Mammary Gland in

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Lactating Cows. J. Agric. Food. Chem. 2015.

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(27) Karaki, S.; Tazoe, H.; Hayashi, H.; Kashiwabara, H.; Tooyama, K.; Suzuki, Y.;

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Kuwahara, A. Expression of the short-chain fatty acid receptor, GPR43, in the human

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colon. J. Mol. Histol. 2008, 39, 135-42. 25


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(28)Tanaka, T.; Yano, T.; Adachi, T.; Koshimizu, T. A.; Hirasawa, A.; Tsujimoto, G.

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Cloning and characterization of the rat free fatty acid receptor GPR120: in vivo effect

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of the natural ligand on GLP-1 secretion and proliferation of pancreatic beta cells. N.-S.

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Arch. Pharmacol. 2008, 377, 515-22.

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(29) Doreau, M.; Chilliard, Y. Digestion and metabolism of dietary fat in farm animals. Brit. J. Nutr. 1997, 78 Suppl 1, S15-35. (30) Mu, H.; Hoy, C. E. The digestion of dietary triacylglycerols. Prog. Lipid Res. 2004, 43, 105-33.

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(31)Galindo, M. M.; Voigt, N.; Stein, J.; van Lengerich, J.; Raguse, J. D.; Hofmann,

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T.; Meyerhof, W.; Behrens, M. G protein-coupled receptors in human fat taste

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perception. Chem. Senses. 2012, 37, 123-39.

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(32) Matsumura, S.; Eguchi, A.; Mizushige, T.; Kitabayashi, N.; Tsuzuki, S.; Inoue,

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K.; Fushiki, T. Colocalization of GPR120 with phospholipase-Cbeta2 and

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alpha-gustducin in the taste bud cells in mice. Neurosci. Lett. 2009, 450, 186-90.

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(33)Widmayer, P.; Goldschmid, H.; Henkel, H.; Kuper, M.; Konigsrainer, A.; Breer,

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H. High fat feeding affects the number of GPR120 cells and enteroendocrine cells in the

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mouse stomach. Front. Physiol. 2015, 6, 53.

496 497 498 499

(34) Rasmussen, B. A.; Breen, D. M.; Lam, T. K. T. Lipid sensing in the gut, brain and liver. Trends Endocrin. Met. 2012, 23, 49-55. (35) Ikwuegbu, O. A.; Sutton, J. D. The effect of varying the amount of linseed oil supplementation on rumen metabolism in sheep. Brit. J. Nutr. 1982, 48, 365-75. 26



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(36) Duca, F. A.; Swartz, T. D.; Sakar, Y.; Covasa, M. Decreased intestinal nutrient

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response in diet-induced obese rats: role of gut peptides and nutrient receptors. Int. J.

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Obesity 2013, 37, 375-381.

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(37) Cheshmehkani, A.; Senatorov, I. S.; Kandi, P.; Singh, M.; Britt, A.; Hayslett,

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R.; Moniri, N. H. Fish oil and flax seed oil supplemented diets increase FFAR4

505

expression in the rat colon. Inflamm. Res. 2015, 64, 809-15.

506

(38) Ichimura, A.; Hasegawa, S.; Kasubuchi, M.; Kimura, I. Free fatty acid

507

receptors as therapeutic targets for the treatment of diabetes. Front. Pharmacol. 2014,

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5.

509

(39) Janssen, S.; Laermans, J.; Iwakura, H.; Tack, J.; Depoortere, I. Sensing of

510

Fatty Acids for Octanoylation of Ghrelin Involves a Gustatory G-Protein. PloS One

511

2012, 7.

512 513

(40) Ichimura, A.; Hara, T.; Hirasawa, A. Regulation of Energy Homeostasis via GPR120. Front. Endocrin. 2014, 5, 111.

514

(41) Hara, T.; Kashihara, D.; Ichimura, A. K., I.; Tsujimoto, G.; Hirasawa, A. Role

515

of free fatty acid receptors in the regulation of energy metabolism. Bba.-Mol. Cell Biol.

516

L. 2014, 1841, 1292-1300.

517

(42) Sawamura, R.; Kawabata, Y.; Kawabata, F.; Nishimura, S.; Tabata, S. The role

518

of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

519

Biochem. Biophys. Res. Commun. 2015, 458, 387-91.

27


Page 28 of 39

Page 29 of 39


520

Figure captions

521

Fig 1. Sequences alignment of G-protein-coupled receptor 120 (GPR120) mRNA (A)

522

and amino acid sequences (B) from Homo sapiens, Mus musculus, Sus scrofa, Bos

523

taurus, Ovis aries and Capra hircus.

524

Fig 2. Phylogenetic analysis of G-protein-coupled receptor 120 (GPR120) mRNA (A)

525

and amino acid sequences (B) from Homo sapiens, Mus musculus, Sus scrofa, Bos

526

taurus, Ovis aries and Capra hircus.

527

Fig 3. Immunohistochemistry analysis of the G-protein-coupled receptor 120 (GPR120)

528

throughout the GIT of goats, and a-h represent the rumen, abomasum, duodenum,

529

jejunum, ileum, cecum and colon, respectively.

530

Fig 4. Immunofluorescence analysis of the G-protein-coupled receptor 120 (GPR120)

531

throughout the GIT of goats, and a-h represent the rumen, abomasum, duodenum,

532

jejunum, ileum, cecum and colon, respectively.

533

Fig 5. Tissue distribution of the G-protein-coupled receptor 120 (GPR120) at the

534

mRNA level (A) and at the protein level (B) of the gastrointestinal tract (GIT) of goats;

535

representative lanes of Western blot analysis of GPR120 could be seen in (C), lane from

536

1 to 7 represent the rumen, abomasum, duodenum, jejunum, ileum, cecum and colon,

537

respectively.

538

bearing a common letter differ (P < 0.05).

539

Fig 6. Distribution of the G-protein-coupled receptor 120 (GPR120) at the protein level

540

of different parts of the tongue of goats (A); representative lanes of Western blot

A-D

Means not bearing a common letter differ (P < 0.05);

28


a-e

Means not


541

analysis of GPR120 could be seen in (B), and lane 1and 2 represents the apical part of

542

tongue, lane 3 and 4 represents the middle part of tongue, lane 5 and 6 represents the

543

rear part of tongue. a-b Means not bearing a common letter differ (P < 0.05).

29


Page 30 of 39

Page 31 of 39


Table 1 Details of primers used in this work Primer name

Primer Sequence (5’-3’)

Product Size (bp)

Annealing Temperature (℃)

1126

60

91

60

111

60

F: AGAGCAAGAAACCAAGTCTCCAA GPR120-C R: CTAGCTGGAAATGACAGACAGGT F: TTCTTCTGGGTGATGGCCTT GPR120-R R: ACCTCCTCCACTCGTTCCTA F: CTTCCAGCCTTCCTTCCTG β-Actin R: ACCGTGTTGGCGTAGAGGT

30



Page 32 of 39

Table 2. Expressions of GPR120 at mRNA level throughout GIT under housing and grazing systems at different developing stages

Item Rumen Abomasum Duodenum Jejunum Ileum Cecum Colon Rectum

System H G H G H G H G H G H G H G H G

Age (d) 0

7

14

0.02

0.04

0.04

0.42

0.14

0.19

0.11

0.15

0.11

0.03

0.06

0.13

0.09

0.18

0.45

1.42

0.62

1.21

1.00

2.42

2.45

1.22

1.67

0.27

28

42

56

70

0.09

0.09

0.13

0.12

0.07

0.08

0.09

0.10

0.47

0.20

0.27

0.27

0.19

0.36

0.14

0.28

0.08

0.09

0.16

0.11

0.12

0.14

0.29

0.15

0.11

0.16

0.15

0.16

0.15

0.28

0.24

0.23

0.14

0.36

0.40

0.55

0.28

0.40

0.41

0.58

0.91

1.21

1.29

1.76

0.92

1.50

0.69

2.06

1.11

1.57

1.91

2.26

2.43

2.02

2.42

2.43

1.29

1.70

1.15

2.54

1.87

2.21

1.70

1.74

SEM1

SEM2

0.024

P value1

P value for Age2

System

Age

System × Age

L

Q

0.021

0.2357

0.3187

0.9171

0.0002

0.4173

0.071

0.083

0.2991

0.4562

0.0654

0.7122

0.5744

0.034

0.038

0.0133

0.0055

0.5370

0.5069

0.6509

0.045

0.033

0.0024

0.0537

0.5567

< .0001

0.0114

0.048

0.049

0.1266

< .0001

0.1441

< .0001

0.1324

0.9938

< .0001

0.0207

0.0014

0.0026

0.0015

0.0005

0.3316

0.0148

0.0002

0.0028

0.9877

0.150 0.262 0.227

0.136 0.191 0.224 0.249 0.191 0.335

< .0001

SEM1=SEM for System × Age (from 28 to 70 d of age); P value1 =P value for both treatment groups from 28 to 70 d of age. SEM2=SEM for age (from 0 to 70 d of age); P value for age2 =P value for age from 0 to 70 d of age. L = Linear effect of age, Q = Quadratic effect of age; H, Housing; G, grazing. 31


0.2389

< .0001

0.071

0.1619

0.0006

0.0035

< .0001

0.0008

Page 33 of 39


Table 3. Expressions of GPR120 at protein level throughout GIT under housing and grazing systems at different developing stages

Item

System

Rumen Abomasum Duodenum Jejunum Ileum Cecum Colon

H G H G H G H G H G H G H G

Age (d) 0 0.16 0.24 0.26 0.34 0.45 0.31 0.56

28

42

70

0.29

0.39

0.36

0.18

0.31

0.25

0.30

0.33

0.33

0.33

0.42

0.48

0.29

0.42

0.34

0.38

0.44

0.46

0.27

0.40

0.46

0.39

0.47

0.51

0.45

0.56

0.46

0.45

0.55

0.58

0.45

0.53

0.54

0.47

0.60

0.59

0.52

0.53

0.62

0.50

0.63

0.65

SEM1

SEM2

0.041

P value1

P value for Age2

System

Age

System × Age

L

Q

0.027

0.0016

0.0054

0.9047

0.0027

0.0598

0.048

0.031

0.0091

0.0475

0.2143

0.0052

0.5748

0.035

0.024

0.0024

0.0065

0.1963

0.0011

0.0635

0.041

0.031

0.0056

0.0006

0.5173

0.0061

0.4381

0.057

0.031

0.3073

0.0829

0.2515

0.1222

0.5927

0.065

0.043

0.2261

0.0665

0.8822

Cloning, Phylogenetic Analysis, and Distribution of ... - ACS Publications

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