Trans10, cis12 conjugated linoleic acid regulates stearoyl-coenzyme

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Biotechnology and Biological Transformations

Trans10, cis12 conjugated linoleic acid regulates stearoyl-coenzyme A desaturase 1 through SREBP1 in primary goat mammary epithelial cells Tianying Zhang, Cong Li, Lian Huang, Ning Song, Yanhong Cao, Juan J. Loor, Jun Luo, and Huaiping Shi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06358 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Journal of Agricultural and Food Chemistry

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Trans10, cis12 conjugated linoleic acid regulates stearoyl-coenzyme A desaturase

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1 through SREBP1 in primary goat mammary epithelial cells

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Tianying ZHANG†‡ 1, Cong LI† 1, Lian HUANG†, Ning SONG†, Yanhong CAO#,

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Juan J LOOR§, Jun LUO†*, and Huaiping SHI†*

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Short title: T10c12-CLA regulates SCD1 in GMECs

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† Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal

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Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, P.

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R. China

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‡ Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Institute of Basic and

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Translational Medicine, Xi’an Medical University, Xi’an, Shannxi, 710000, P. R.

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China

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# Guangxi Institute of Animal Science, Nanning, Guangxi, 535001, P. R. China

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§ Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of

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Nutritional Sciences, University of Illinois, Urbana, Illinois 61801, United States

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*Corresponding authors:

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Jun Luo, email: [email protected], phone: +86-29-8708-2891, fax: +86-29-

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8708-2892;

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Huaiping Shi, email: [email protected], phone: +86-29-8709-2102, fax:

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+86-29-8709-2164. 1

ACS Paragon Plus Environment


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1 These

authors contributed equally.

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Abstract

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Trans10, cis12 conjugated linoleic acid (t10c12-CLA) is a biohydrogenation

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intermediate in the rumen, which inhibits mammary fatty acid de novo synthesis of

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lactating dairy goats. However, the underlying molecular pathways of t10c12-CLA on

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milk lipid metabolism are not completely understood. The present study investigated

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the lipid regulation mechanisms on goat mammary epithelial cells (GMECs) in

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response to t10c12-CLA. Gene expression analysis indicated sterol regulatory element

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binding transcription factor1 (SREBF1) and its putative target gene stearoyl-CoA

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desaturase (SCD1) were down-regulated (fold change 0.33 ± 0.04, P < 0.05; and 0.19

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± 0.01, P < 0.01; respectively). Concentrations of cellular palmitoleic acid (C16:1) and

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oleic acid (C18:1) were decreased (1.12% ± 0.05% vs. 1.69% ± 0.11%, and 15.70% ±

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0.44% vs. 24.97% ± 0.82%, respectively, P < 0.01), while linoleic acid (C18:2) was

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increased (5.00% ± 0.14% vs. 3.81% ± 0.25%, P < 0.05); and the desaturation indices

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of C16 and C18 were decreased in response to t10c12-CLA (6.90% ± 0.05% vs. 8.00%

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± 0.30%, and 61.41% ± 0.65% vs. 67.73% ± 1.33%, respectively, P < 0.05). Both,

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luciferase activity assay indicated that deletions of the sterol response element (SRE)

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site and the nuclear factor (NF-Y) site in SCD1 promoter region (-511/+65 bp)

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suppressed the regulation effect by t10c12-CLA. Overexpression of SREBF1 partly

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counteracted the inhibitory effect of t10c12-CLA on de novo fatty acid synthesis.

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Overall, t10c12-CLA causes an inhibition of fatty acid synthesis and desaturation, and

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regulates SCD1 through affecting the binding of SREBP1 protein to the SRE and NF-

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Y. 3



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Key words: fatty acid, mammary, t10c12-CLA, SREBP1, SCD1

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Introduction

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In dairy production, the manipulation of dietary fatty acids plays a central role in

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determining the content and composition of milk fat.1-3 Trans10, cis12 conjugated

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linoleic acid is a ruminal biohydrogenation intermediate that can cause milk fat

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depression in dairy animals.4-6 The biohydrogenation theory of “milk fat depression”

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was first proposed in dairy cattle fed high-concentrate diets and polyunsaturated fatty

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acids. This kind of diet decreased concentrations of short-to-medium fatty acids, as a

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consequence of de novo fatty acids synthesis inhibition. In lactating dairy goats, t10c12-

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CLA was also demonstrated to inhibit de novo synthesis and increase the content of

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long-chain fatty acids, as well as improve the energy balance of the animal.4, 7

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However, few studies have focused on the detailed molecular mechanisms underlying

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those responses to t10c12-CLA in dairy goat. In bovine mammary epithelial cells, the

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inhibition of de novo synthesis when exogenous supplementation with t10c12-CLA

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increases occurs through down-regulation of lipogenic genes8 and reducing proteolytic

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activation of sterol regulatory element binding protein 1 (SREBP1; encoded by

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SREBF1).1 SREBP1 acts as one critical transcription factor in mammary gland,

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participating in fatty acid and triacylglycerol synthesis through its control of lipogenic

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genes.9 In a recent study using GMECs, RNA-sequencing revealed transcriptome-wide

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alterations caused by t10c12-CLA,10 indicating a potent effect on transcription as well

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as regulation of the AMPK (protein kinase AMP-activated catalytic subunit alpha 1)

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signaling pathway. Despite the significant effect of t10c12-CLA on the transcriptome,

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and the underlying mechanisms for such responses in GMECs remain to be thoroughly 5



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investigated. Thus, the present study further revealed the regulation mechanism of

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t10c12-CLA on fatty acid synthesis and desaturation in GMECs, providing more

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evidences of the role of SREBP1 on the t10c12-CLA-induced anti-lipogenic effect in

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GMECs.

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Material and Methods

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Chemicals

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The HPLC grade t10c12-CLA (purity ≥ 96.0%) was obtained from a commercial

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reagent supplier (Sigma-Aldrich, St, Louis, MO, USA).

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Ethics statement

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The animal experiments were carried out under the agreement of Institutional Animal

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Use and Care Committee (Northwest A&F University, Yangling, China; permit number:

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15-516; date: September 13th, 2015). All animals in the present study received humane

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care according to the Guide for the Care and Use of Experimental Animals of the

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National Institutes of Health. All surgeries were performed in a way to ensure a

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minimized suffering.

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Preparation of stock solutions

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A stock solution of 30 mM t10c12-CLA and the control were prepared as previously

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described.8 The working concentration of t10c12-CLA was 100 μM; also, the control

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solution was diluted at the same way. 6


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Cell culture and t10c12-CLA incubation

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Primary GMECs separated from Xinong Saanen goats on mid-lactation were cultured

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for about five passages, then were authenticated as described previously.11-12 The

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GMECs were cultured in a growth medium containing 90% DMEM/F12 (Hyclone,

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South Logan, UT, USA), 10% fetal bovine serum (Gibico, Carlsbad, CA, USA), 5 mM

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sodium acetate (Sigma-Aldrich, USA), 5 μg/mL insulin (Sigma-Aldrich, USA), 1

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µg/mL hydrocortisone (Sigma-Aldrich, USA), 10 ng/mL epidermal growth factor 1

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(Invitrogen Corp., Frederick, MD, USA), 100 U/mL penicillin (Harbin Pharmaceutical

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Group, Harbin, China) and 100 mg/mL streptomycin (Harbin Pharmaceutical Group,

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China) in a humidiﬁed atmosphere with 5% CO2 at 37°C. Twelve h prior to the

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incubation with t10c12-CLA, the growth medium was changed to serum-free

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DMEM/F12 medium containing 5 mM sodium acetate, 5 μg/mL insulin, 1 µg/mL

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hydrocortisone, 10 ng/mL epidermal growth factor 1, 100 U/mL penicillin, 100 mg/mL

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streptomycin, 2 μg/mL prolactin (Sigma-Aldrich, USA) and 1 mg/mL bovine serum

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albumin (Sigma-Aldrich, USA). Then the serum-free medium with or without t10c12-

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CLA was incubated with GMECs.

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RNA extraction and gene expression analysis

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The GMECs were treated with or without t10c12-CLA for 24 h. Cell RNA extraction,

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first-strand cDNA synthesis and real-time quantitative PCR (RT-qPCR) were

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performed as methods previously described.13 Ribosomal protein S9 (RPS9) and

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ubiquitously expressed transcript (UXT) were used as internal control genes. Reactions 7



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were performed in a Bio-Rad CFX96 sequence detector (Bio-Rad Laboratories Inc.

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Hercules, CA, USA). The amplification conditions were as follows: 1 cycle at 95°C for

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30 s, followed by 39 cycles at 95°C for 5 s, and 60°C for 30 s; a dissociation curve of

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each sample was generated at 95°C for 10 s and then from 65°C to 95°C with an

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increment of 0.5°C. Data were analyzed using a method of relative quantiﬁcation (2-

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ΔΔCt),

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primer sequences were listed in Supplemental Table 1.

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Cellular fatty acid extraction

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The GMECs were treated with or without t10c12-CLA for 24 h. Fatty acids were

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extracted according to previously described.14 In brief, cellular fatty acids were

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extracted using 2 mL of vitriol/methanol (2.5:1, v/v). Methylated lipid samples were

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analyzed using a gas chromatograph according to the methods of Shi et.al and Wang

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et. al.4, 15 In brief, each sample injection was 2 μL with 1:1 split ratio. The injector and

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detector temperature were 250 °C and 280 °C, respectively. Fatty acids were identified

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based on retention times of standard fatty acid methyl-esters, and concentrations were

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calculated as percentages of summed peak areas.

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Western blot

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The GMECs were treated with or without t10c12-CLA for 24-72 h. Cellular total

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protein was lysed with RIPA (active component including 1% Triton X-100, 1%

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deoxycholate and 0.1% SDS, Solarbio, Beijing, China) containing protease and

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phosphatase inhibitors (Cat. No. 04693132001 and 04906845001, Roche, Mannheim,

and normalized to the geometric mean of internal control genes. The RT-qPCR

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Germany). Sample preparation and western blot were performed as previously

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described.13 Monoclonal mouse anti- SREBP1 (1 μg/mL, ab3259; Abcam, Inc.,

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Cambridge, UK) and β-Actin (0.2 μg/mL, CW0096S, CoWin Biotech Co., Ltd., Beijing,

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China) were used as primary antibodies. Polyclonal goat anti-mouse IgG coupled to

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HRP (0.2 μg/mL, CW0102S, CoWin, China) was used as secondary antibody. Signals

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were collected by chemiluminescent ECL Western blot detection system (Bio-Rad,

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USA). The intensity of the bands was calculated through ImageJ software, and relative

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protein expression was normalized by β-actin as the internal control. Data were

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expressed as the fold change of target proteins in the t10c12-CLA group vs. control

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group.

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Transfection and luciferase activity assay

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The pcDNA3.1-SREBF1 eukaryotic expression vector was constructed basing on the

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coding sequence of goat SREBF1 (GenBank accession ID: NM_001285755). For

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SREBF1 overexpression, GMECs were transfected with pcDNA3.1-(+) empty vehicle

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or pcDNA3.1-SREBF1 plasmid in Lipofectamine 2000 Reagent (Invitrogen Corp.,

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USA) for 5 h on the basis of manufacturers’ instructions. Cells were then incubated

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with serum-free medium for 12 h, followed by incubation with or without t10c12-CLA

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for 24 h.

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The certain promoter region of genes was cloned into pGL3-basic plasmid for luciferase

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activity assay. The goat SREBP1c promoter was obtained from Xu et al16; the goat

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SCD1 promoter (GenBank accession ID: KT266815), as well as its 5’ deletion and site-

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directed deletions for specificity protein 1 (Sp1), sterol regulatory element (SRE), and 9



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the nuclear factor (NF-Y) plasmids were obtained from Yao et al.17 For luciferase

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activity assay, GMECs were transfected with 200 ng of pGL3-basic, pGL3-SREBF1c

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or pGL3-SCD1 luciferase reporter plasmids in Lipofectamine 2000 for 5 h, then treated

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with serum-free medium for 12 h, followed by incubation with or without t10c12-CLA

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for 24 h. Similarly, GMECs were co-transfected with the SCD1 reporter plasmid and

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either the pcDNA3.1-SREBF1 expression plasmid or pcDNA3.1-(+) vehicle, then

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treatment with or without t10c12-CLA before performing luciferase assay. Renilla

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luciferase vector (pRL-TK) was used as an internal control to normalize transfection

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efficiency at a 25:1 ratio of pGL3- luciferase reporter plasmid and pRL-TK vector. The

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relative luciferase activity was a ratio of firefly to renilla luciferase activities assayed

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in a microplate reader through Promega Dual-Luciferase Reporter Assay System

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(Promega Beijing Biotech Co., Ltd. Beijing, China).

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Statistical analysis

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Each experiment included at least three biological replicates. The statistical analysis

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was performed through SPSS 19.0 software (SPSS Inc. Chicago, IL, USA). Multiple

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comparison analysis was performed through ANOVA using the general linear model

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procedure with mean separation via the Duncan test, and different letters represent

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statistical significance at P < 0.05. For only two comparison groups, data were analyzed

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via paired and two-tailed Student’s t test, and statistical significance was declared at P

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< 0.05 (*, P < 0.05；**, P < 0.01).

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Results 10


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Gene expression of lipid metabolism

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Statistical data of 25 pivotal genes expression related to mammary lipid metabolism

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were listed in Supplemental Table 2 and presented as volcano plot in Figure 1.

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Compared with the control, the data indicated that the mRNA abundance of SCD1 was

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down-regulated by CLA along with the key de novo fatty acid synthesis gene FASN

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(Figure 1, P < 0.01). In addition, mRNA expression of SREBF1 was down-regulated in

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response to CLA (Figure 1, P < 0.05). In contrast, mRNA expression of lipid catabolism

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genes such as ACOX1 and CPT1A was up-regulated (Figure 1, P < 0.05; P < 0.01).

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Meanwhile, expression of the fatty acid uptake gene CD36, the lipid droplet coat

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proteins PLIN2 and PLIN3 were up-regulated (Figure 1, P < 0.05).

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Cellular fatty acid composition in response to t10c12-CLA

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Cellular fatty acid composition results indicated that the percentages of palmitic acid

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(C16:0), palmitoleic acid (C16:1), stearic acid (C18:0) and oleic acid (C18:1) were

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down-regulated (Figure 2A-D, P < 0.05) in response to t10c12-CLA. In addition, the

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percentage of linoleic acid (C18:2) was up-regulated (Figure 2E, P < 0.05). Furthermore,

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the desaturation indices of C16 and C18, calculated according to the ratio between the

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desaturation product and the sum of the product and the substrate,18 were decreased

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(Figure 2F, P < 0.05).

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Regulation of SREBP1 expression in response to t10c12-CLA

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Similarly with the mRNA results (Figure 1, P < 0.05), the luciferase activity of

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SREBF1c promoter in t10c12-CLA group was down-regulated (Figure 3A, P < 0.05)

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indicating that transcriptional activity of SREBF1c was inhibited by t10c12-CLA. 11



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However, the protein abundance of precursor SREBP1 (pre-SREBP1), and nuclear

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form of SREBP1 (n-SREBP1) were not affected after incubation of t10c12-CLA for 24

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h (Figure 3B, P > 0.05). Nevertheless, after 72 h incubation, both pre-SREBP1 and n-

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SREBP1 were down-regulated (Figure 3B, P < 0.05).

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Transcriptional regulation of SCD1 expression in response to t10c12-CLA

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To investigate which elements response the transcriptional regulation of SCD1 gene

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when exogenous t10c12-CLA was used, progressive 5’ flanking deletion constructs

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were transfected to narrow the location within the SCD1 promoter. Data revealed that

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promoter activities were high, and the inhibitory effect of t10c12-CLA was maintained

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until the promoter was shortened to -511 bp upstream of the transcription start site (TSS,

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Figure 4A, P < 0.05). When the promoter was truncated to -355 and -109 bp upstream

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of the TSS, the promoter activities were very low and the response to t10c12-CLA was

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not significant (Figure 4A, P > 0.05).

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Following t10c12-CLA incubation, relative luciferase results from cells containing the

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pGL-(-511/+65) luciferase reporter vectors with wild-type or with site-deleted

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constructs of Sp1, SRE, and NF-Y transfected into the GMECs, indicated that deletion

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of Sp1 alone could not eliminate the t10c12-CLA induced inhibition of SCD1 promoter

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activity (Figure 4B, P < 0.01). However, deletion constructs of SRE, NF-Y, SRE

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together with NF-Y, or a combination of Sp1, SRE and NF-Y, completely abolished

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the inhibitory effect of t10c12-CLA (Figure 4B, P > 0.05). Moreover, as for the

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inhibitory degree, deletion constructs of Sp1 changed little of promoter activity on -

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511/+65 bp; however, deletion constructs of SRE or NF-Y seems to result in a marked 12


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decreasing, and the function of NF-Y seems to be stronger (Figure 4B). Thus, deletions

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of SRE and NF-Y on goat SCD1 gene promoter strongly suppressed the transcriptional

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activity in response to t10c12-CLA.

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Overexpression of SREBF1 ablated the inhibitory effect of t10c12-CLA on SCD1

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expression

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To clarify the effect of SREBP1 on the regulation of SCD1 in response to t10c12-CLA,

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an overexpression SREBF1 vector was transfected into GMECs. Compared with

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pcDNA3.1-(+) groups, SREBF1 mRNA expression in the pcDNA3.1-SREBF1 groups

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was up-regulated. Furthermore, SCD1 mRNA expression was up-regulated with up-

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regulation of SREBF1; however, in the presence of t10c12-CLA, up-regulation of SCD1

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is greatly reduced in SREBF1 overexpression groups (Figure 5A, P < 0.05).

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As for SREBP1 protein expression, both pre-SREBP1 and n-SREBP1 were increased

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in the pcDNA3.1-SREBF1 groups; and t10c12-CLA inhibited the effect of up-

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regulation of pre-SREBP1 caused by overexpression of SREBF1 (Figure 5B, P < 0.05).

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Overexpression of SREBF1 up-regulated SCD1 promoter activity of the wild-type pGL-

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(-511/+65), and compensated the inhibitory effect of t10c12-CLA on transcriptional

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regulation of SCD1 to some extent (Figure 5C, P < 0.05).

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Discussions

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It is well-established in dairy cows that increasing the post-ruminal availability of

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t10c12-CLA via abomasal or duodenal infusions results in milk fat depression.6 In a

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previous study with lactating goats, dietary supplementation of t10c12-CLA resulted in

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a significant decrease of milk fat percentage, possibly caused by the down-regulation 13



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of genes involved in mammary lipogenesis.4 In order to explore the detailed

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mechanisms whereby t10c12-CLA alters mammary cell lipid metabolism, we

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conducted an in vitro study using GMECs.

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Regulation of lipogenesis by t10c12-CLA in GMECs

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The down-regulation of FASN and ACACA along with the reduction in C16:0 by

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exogenous CLA agrees with our previous data in which t10c12-CLA decreased the

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protein expression of FASN and increased the phosphorylation of ACACA.10 Thus,

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because of the well-known role of de novo fatty acid synthesis and exogenous uptake

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of C16:0 from plasma19 in the overall process of milk fat synthesis, the present data

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underscore that mammary lipogenesis is controlled at the transcriptional, translational,

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and post-translational level, with exogenous t10c12-CLA eliciting negative effects at

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each of them.

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Goat milk has higher content of monounsaturated fatty acids (MUFA) compared with

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cow milk.20 In lactating goat mammary gland, SCD1 expression increased dramatically

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postpartum and reached a peak value on mid-lactation (~170-fold compared with the

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dry-period).21 SCD is the rate-limiting enzyme of MUFA biosynthesis, catalyzing

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C16:0 or C18:0 to form Δ9 unsaturated carbon-carbon double bonds, namely C16:1 or

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C18:1.2, 22 In this study, the down-regulation of SCD1 level and the decrease in cellular

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MUFA (C16:1 and C18:1) with exogenous CLA confirmed that it inhibits the

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desaturation process in GMECs.

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Our previous study demonstrated that t10c12-CLA activates the AMPK signaling

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pathway in GMECs.10 AMPK plays critical roles in regulating energy balance in 14


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eukaryotic cells. AMPK can directly phosphorylate key substrates that affect cell

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growth and metabolism, as well as long-term metabolic reprogramming.23 Activation

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of AMPK can result in CPT1 activation, and this process plays a pivotal part in

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regulation of LCFA oxidation in mitochondria.24 In bovine Mac-T cells, activated

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AMPK can inhibit the activity of ACACA by phosphorylation.25 ACOX1 is the first

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rate-limiting enzyme on fatty acid β-oxidation, catalyzing acyl-CoA to 2-trans-enoyl-

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CoA and hydrogen peroxide.26 Thus, the up-regulation of CPT1A and ACOX1 caused

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by t10c12-CLA agrees with the role of AMPK in mammary cells. The majority of milk

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fat is secreted through the process of membrane envelopment via cytosolic lipid

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droplets (CLD) synthesized by mammary epithelial cells.27 Adipocyte differentiation-

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related protein (ADRP; gene name: PLIN2) is a CLD-binding protein which plays a key

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role in CLD formation and stability27-28. Considering the up-regulation of CD36 and

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PLIN2 expression with exogenous t10c12-CLA, the increase of C18:2 n6-t can be partly

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explained as a consequence of the inhibition of de novo synthesis inducing exogenous

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fatty acid uptake for triacylglycerol formation and accumulation in GMECs.

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Mechanism of transcriptional regulation of SCD1 by t10c12-CLA

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The previous study provided a strong evidence for a central role of SREBP1 in

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regulating lipid synthesis on goat mammary cells.29 The activated fragment nuclear

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SREBP (nSREBP) can enter the nucleus, activating the sterol responsive element-

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containing genes. In response to t10c12-CLA, down-regulation of SREBF1 expression

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agreed with previous studies in bovine.8 Furthermore, the promoter activities of

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SREBF1 were inhibited by t10c12-CLA, in accordance with the mRNA expression data. 15



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Studies in GMECs revealed a direct regulation of lipogenic genes such as SCD121 and

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FASN30 by SREBP-1, and a direct role of auto-loop regulation in maintaining its basal

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transcription activity.31 Considering that mRNA expression of SCD1, FASN and

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SREBF1 was down-regulated after 24 h of CLA treatment in spite of unchanged n-

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SREBP1, we speculate that t10c12-CLA not only alters its protein expression and

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processing but also may affect the combination of n-SREBP1 to the promoter region of

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its target genes. This is partly supported by the decrease in both pre-SREBP1 and n-

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SREBP1 were decreased at 72 h of CLA treatment.

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In bovine mammary cells, t10c12-CLA has an inhibitory role on the activation of

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SRE.32 Progressive 5’ flanking deletion constructs within the SCD1 promoter indicated

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the inhibitory effect of t10c12-CLA was maintained until the promoter was truncated

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to -511 bp upstream of the TSS. In dairy goats, promoter region within −415 to −109

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bp of SCD1 gene plays critical roles for maintenance of basal transcriptional activity.17,

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21

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to clarify whether t10c12-CLA affects SCD1 transcription through an SRE element. In

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non-ruminants, the neighboring sequence of SREBP target gene promoters contain an

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NF-Y or Sp1-binding site, which contribute to promote the recruitment of a basic

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transcription machinery through NF-Y and Sp1 directly binding to SREBP.33 The

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present study indicated that deletion of SRE or NF-Y alone, or both SRE and NF-Y in

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SCD1 promoter of GMECs strongly suppressed the transcriptional activity in response

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to t10c12-CLA. It is probably due to SRE and NF-Y in SCD1 promoter showed a

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stronger response to t10c12-CLA in regulating the SREBP1 complex on SCD1

Site deletions to -511 to +65 bp of the SCD1 promoter provided an effective method

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promoter. According to our present study, up-regulation of SCD1 mRNA expression

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level was found after transfection of an overexpression vector for SREBF1; furthermore,

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the inhibitory effect of t10c12-CLA on SCD1 transcriptional activity was relieved to

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some extent. This fact underscores that one of the mechanisms whereby t10c12-CLA

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regulates SCD1 expression is probably through affecting the binding of SREBP1

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protein to the promoter region.

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In conclusion, this study greatly expanded our understanding and provided additional

311

evidences to unveil the role of t10c12-CLA on goat milk fatty acid metabolism.

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Through gene expression analysis, fatty acid content assay, promoter activity assay and

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gene overexpression, the detailed regulation mechanism of t10c12-CLA on milk fatty

314

acid synthesis and desaturation in GMECs were identified. Overall, t10c12-CLA

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inhibited de novo fatty acid synthesis and desaturation through downregulation of genes

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involving FASN, ACACA and SCD1. Furthermore, t10c12-CLA regulates SCD1

317

through affecting the binding of SREBP1 protein to the promoter region of SCD1

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involved in SRE and NF-Y site, causing an inhibition of fatty acid desaturation.

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Funding

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This research was jointly supported by the “National Natural Science Foundation of

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China (31272409)”, the “National Post-Doctoral Science Foundation of China

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(2017M613230)”, and the “Science Foundation of Shaanxi province of China

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(2013KTZB02-02-03 and 2016KTZDNY02-05)” .

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Notes 17



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All authors declare no conflict of interest in this study.

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References

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17. Yao, D. W.; Luo, J.; He, Q. Y.; Xu, H. F.; Li, J.; Shi, H. B.; Wang, H.; Chen, Z.; Loor, J. J.,

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epithelial cells via the control of stearoyl-coenzyme A desaturase 1 in an SREBP-1-dependent

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by SREBP-1 and PPARgamma 1 in Dairy Goat Mammary Cells. Journal of cellular physiology

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22. Kelsey, J. A.; Corl, B. A.; Collier, R. J.; Bauman, D. E., The effect of breed, parity, and stage

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fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells. Journal

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30. Li, J.; Luo, J.; Xu, H.; Wang, M.; Zhu, J.; Shi, H.; Haile, A. B.; Wang, H.; Sun, Y., Fatty acid

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32. Ma, L.; Lengi, A. J.; McGilliard, M. L.; Bauman, D. E.; Corl, B. A., Short communication:

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33. Shimano, H., Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Progress in lipid research 2001, 40 (6), 439-52.

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434

Figure Captions

435

Figure 1. Volcano plot of genes involved in lipid metabolism in response to

436

supplementation with t10c12-CLA in GMECs. Cells were treated with t10c12-CLA for

437

24 h, and a total of 25 genes involved in lipid metabolism were detected by RT-qPCR.

438

RPS9 and UXT were used as internal control genes. Data were normalized against the

439

control group and the final result of each gene is presented as log2 |fold change|. The P

440

value was calculated using a student t test and presented as –log10 |P-value|, with dotted

441

lines representing the means of P = 0.05 or 0.01. N = 3.

442

Abbreviations: ACACA = acetyl-CoA carboxylase alpha; ACOX1 = acyl-Coenzyme A

443

oxidase 1; ACSL1 = acyl-CoA synthetase long-chain family member 1; ACSL4 = acyl-

444

CoA synthetase long-chain family member 4; ACLY = ATP citrate lyase; ACSS2 = acyl-

445

CoA synthetase short-chain family member 2; AGPAT6 = 1-acylglycerol-3-phosphate

446

O-acyltransferase 6; ATGL = patatin like phospholipase domain containing 2 (PNPLA2);

447

CD36 = CD36 molecule (scavenger receptor); CPT1A = carnitine palmitoyltransferase

448

1A; DGAT1 = diacylglycerol acyltransferase 1; DGAT2 = diacylglycerol

449

acyltransferase 2; FABP3 = fatty acid binding protein 3; FADS1 = fatty acid desaturase

450

1; FASN = fatty acid synthase; GPAM = glycerin-3-phosphat-acyltransferase 1; HSL =

451

hormone-sensitive lipase; LPL = lipoprotein lipase; PLIN2 = perilipin 2; PLIN3 =

452

perilipin 3; PPARα = peroxisome proliferator activated receptor alpha; PPARγ =

453

peroxisome proliferator activated receptor gamma; SCD1= stearoyl-CoA desaturase 1;

454

SREBF1 = sterol regulatory element binding transcription factor 1; XDH = xanthine

455

dehydrogenase. 24


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456

Figure 2. Effect of t10c12-CLA on the regulation of intracellular fatty acid content in

457

GMECs. Cells were treated with t10c12-CLA for 24 h, lysates were then collected and

458

analyzed using GC. (A-E) Relative changes of intracellular C16:0, C16:1, C18:0, C18:1

459

and C18:2 in response to t10c12-CLA. Data were calculated as ratios of total FA. (F)

460

The desaturation indices of C16 and C18 in response to t10c12-CLA. Data were

461

calculated using the ratio of C16:1/ (C16:0 + C16:1) and C18:1/ (C18:0 + C18:1).

462

Results are presented as means ± standard error of the means, N = 4. *, P < 0.05; and

463

**, P < 0.01. CTR: the control group; CLA: t10c12-CLA group.

464

Figure 3. Measurement of SREBP1c luciferase activity and SREBP1 protein

465

expression in response to t10c12-CLA supplementation in GMECs. (A) Cells were

466

transfected with pGL3-basic or pGL3-SREBP-1c luciferase reporter plasmid followed

467

by t10c12-CLA treatment for 24 h. Cell lysates were assayed for luciferase expression.

468

(B) Protein expression level of SREBP1 with t10c12-CLA treatment for 24 h and 72 h.

469

Expression levels of pre-SREBP1 and n-SREBP1 were determined with specific

470

antibodies using β-Actin as internal control. Results are presented as means ± standard

471

errors of the means, N = 3. *, P < 0.05; and **, P < 0.01. CTR: the control group; CLA:

472

t10c12-CLA group.

473

Figure 4. Effect of t10c12-CLA on promoter activity of the SCD1 gene in GMECs. (A)

474

t10c12-CLA repressed different lengths of SCD1 promoter activity. Cells were

475

transfected with different 5’ flanking deletion constructs of the SCD1 promoter

476

followed by t10c12-CLA treatment for 24 h. (B) Deletions of SRE and NF-Y in the

477

goat SCD1 promoter suppressed transcriptional activity in response to t10c12-CLA 25



478

treatment. Cells were transfected with wild-type pGL-(-511/+65) or with site-deleted

479

constructs of the SCD1 promoter followed by t10c12-CLA treatment for 24 h. Cell

480

lysates were assayed for luciferase expression. Results are presented as means ±

481

standard error of the mean, N = 3. *, P < 0.05; and **, P < 0.01. CTR: the control group;

482

CLA: t10c12-CLA group.

483

Figure 5. SREBP1 overexpression relieved the inhibitory effect of t10c12-CLA on

484

SCD1 expression. Cells were transfected with pcDNA3.1-(+) or pcDNA3.1-SREBF1

485

plasmid followed by t10c12-CLA treatment for 24 h; (A) The mRNA expression levels

486

of SREBF1 and SCD1; (B) The protein expression levels of pre-SREBP1 and n-

487

SREBP1, using β-Actin as internal control. (C) Cells were co-transfected with pGL3-

488

basic or wild-type pGL-(-511/+65) SCD1 reporter plasmid and pcDNA3.1-(+) or

489

pcDNA3.1-SREBF1 plasmid followed by t10c12-CLA treatment for 24 h. Cell lysates

490

were assayed for luciferase expression. Results are presented as means ± standard error

491

of the means, N = 3. The different letters represent significant differences at P < 0.05

492

between the comparison groups. CTR: the control group; CLA: t10c12-CLA group.

26


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493

TOC graphic Legends: This study is aiming at investigating the inhibitory effect of

494

t10c12-CLA on goat mammary de novo fatty acid synthesis. Firstly gene expression of

495

lipid metabolism was determined, and results indicated that expressions of SREBF1 as

496

well as its putative target genes SCD1 were significantly down-regulated; fatty acid

497

composition indicated concentrations of C16:1 and C18:1 were decreased, while C18:2

498

was increased in response to t10c12-CLA. Then constructs with site-deleted luciferase

499

reporter vectors to narrow the location within the SCD1 promoter responsible for the

500

transcriptional regulation of SCD1 were transfected in GMECs, indicating that

501

deletions of the sterol response element (SRE) site in SCD1 promoter suppressed the

502

regulation effect by t10c12-CLA. Finally, overexpression of SREBP1 partly

503

counteracted the inhibitory effect of t10c12-CLA on de novo fatty acid synthesis. We

504

concluded that t10c12-CLA regulates SCD1 through affecting the binding of SREBP1

505

to the SRE and NF-Y.

27



506 507

Figure 1.

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508 509

Figure 2.

29



510 511

Figure 3.

30


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512 513

Figure 4.

31



514

515 516

Figure 5.

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TOC graphic

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Trans10, cis12 conjugated linoleic acid regulates stearoyl-coenzyme

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