Taurine promotes milk synthesis via the GPR87-PI3K-SETD1A

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Bioactive Constituents, Metabolites, and Functions

Taurine promotes milk synthesis via the GPR87-PI3K-SETD1A signaling in BMECs Mengmeng Yu, Yang Wang, Zhe Wang, Yanxu Liu, Yang Yu, and Xuejun Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06532 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Journal of Agricultural and Food Chemistry

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Taurine promotes milk synthesis via the GPR87-PI3K-SETD1A signaling in BMECs

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Mengmeng Yu#,†, Yang Wang#,‡, Zhe Wang‡, Yanxu Liu‡, Yang Yu‡, Xuejun Gao*,†

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†Agricultural

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‡

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College of Guangdong Ocean University, Zhanjiang, 524088, China

The Key Laboratory of Dairy Science of Education Ministry, Northeast Agricultural

University, Harbin, 150030, China

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# Mengmeng Yu and Yang Wang are joint first authors.

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* To whom correspondence should be addressed. Phone: +86-451-55190542; Fax: +86-451-

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55190244; E-mail: [email protected]

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ORCID: Xuejun Gao, 0000-0002-8241-6346

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ABSTRACT Taurine, a β-aminosulfonic acid, exerts many cellular physiological functions. It is still

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unknown whether taurine can regulate milk synthesis in mammary gland. Therefore, in this study

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we investigated the effects and mechanism of taurine on milk synthesis in mammary epithelial

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cells (MECs). Bovine MECs (BMECs) cultured in FBS-free OPTI-MEMⅠmedium were treated

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with taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4 mM). Taurine treatment led to increased milk

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protein and fat synthesis, mTOR phosphorylation, and SREBP-1c protein expression, in a dose-

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dependent manner, with an apparent maximum at 0.24 mM. Gene function study approaches

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revealed that the GPR87-PI3K-SETD1A signaling was required for taurine to increase the mTOR

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and SREBP-1c mRNA levels. Taurine stimulated GPR87 expression and cell membrane

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localization in a dose dependent manner, suggesting a sensing mechanism of GPR87 to

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extracellular taurine. Collectively, these data demonstrate that taurine promotes milk synthesis via

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the GPR87-PI3K-SETD1A signaling.

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Keywords

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mammary epithelial cells, GPR87, milk synthesis, PI3K, SETD1A, taurine

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ACS Paragon Plus Environment


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INTRODUCTION

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Taurine, 2-aminoethane sulfonic acid, is an abundant ß-aminosulfonic acid in many mammalian

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tissues. It presents as a free form, accounting for approximately 0.1% of the total body mass. 1-

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3

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intracellular calcium concentration, ion channel function, glucose and lipid homeostasis, cellular

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redox homeostasis, and anti-antioxidant and anti-inflammatory responses.4-6 The nutritional

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requirements for taurine are met by absorption from food intake and partly by synthesis from

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methionine/cysteine in liver and other tissues. Taurine is considered as a conditionally essential

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amino acid, since newborn mammals are unable to synthesize enough taurine from dietary

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precursors, and the requirements have to rely on dietary food. 1 Taurine has dramatical impacts

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on human health,1,7 and can be used as a novel agent for treatment of diseases of muscle, diabetes,

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obesity, inflammatory diseases, cardiovascular diseases, and neurological disorders.3,4,8 Many of

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the energy drinks also include high doses of taurine.9 Taurine is approved as a growth-promoting

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or antioxidant additive in animal husbandry, mainly used in aquatic, cat, and dog diets. 10-12

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However, there were only a few studies that reported the regulatory roles of taurine on the growth

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of poultry, pig, and ruminants. Increasing evidences have shown that taurine is beneficial to the

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performance of broiler chickens. 13,14 Taurine inhibits the disfunction in mouse mammary

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epithelial cells (MECs) treated with lipopolysaccharide, 15 suggesting that taurine might also be

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beneficial to milk synthesis in mammary gland.

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Previous studies indicate that taurine exerts important physiological functions in modulation of

The mechanism of action of taurine has not been fully understood. Cells accumulate taurine via

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the Na+-dependent taurine transporter (TauT). 16 Taurine can also enter cells via the Na+-

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independent β-amino acid transporter PAT1.16,17 Previous study reveal that taurine can regulate the

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PI3K/AKT, AKT/FOXO1, JAK2/STAT3, and mTOR/AMPK signaling pathways.8,18,19 Taurine

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can inhibit reactive oxygen species (ROS) generation within the respiratory chain and affects

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mitochondrial bioenergetics.4 Two novel modified uridines containing taurine have been found in

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mammal mitochondrial tRNAs, and these modification have crucial roles for translation. 20,21

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Extracellular taurine can affect neuronal progenitor cells through binding to the glycine and γ-

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aminobutyric acid (GABA) receptors.22 Despite these reports, it is still largely unknown how cells

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are enabled to sense taurine to regulate cellular function.


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Milk production in mammary gland is mainly dependent on the milk synthesis and proliferation

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abilities of MECs.23,24 Amino acids such as methionine, leucine and lysine have been established

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to be stimulators to activate members (mTOR, SREBP-1c, etc.) of the signaling pathways leading

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to milk synthesis in and proliferation of MECs. 25-27 Amino acids stimulate these signaling

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pathways via biomolecular sensors, amino acid transporters, and G-protein coupled receptors

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(GPCR).28,29 Some GPCRs can sense amino acids, such as GPCR class C, group 6, member A

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(GPRC6A) and taste receptor type 1 member 1 (T1R1)/taste receptor type 1 member 3 (T1R3).

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They sense extracellular amino acids, leading to activated downstream signaling pathways such as

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the PI3K/mTOR signaling. 30,31 Recent reports point that GPR87 is overexpressed in multiple

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cancers, but it is still unknown whether GPR87 is associated with the amino acid sensing

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pathways. 32,33 Little is known whether taurine can regulate milk synthesis in MECs and whether

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taurine can be sensed by a certain GPCR to activate downstream signaling for milk synthesis.

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SET domain containing 1A (SETD1A) is a histone methyltransferase that predominantly

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trimethylates histone H3 lysine 4 (H3K4) at active promoters near the transcription start sites. 34

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The trimethylation H3K4 (H3K4Me3) is an epigenetic marker widely used for promoter

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activation detection. 35 Previous reports showed that SETD1A-mediated H3K4 trimethylation is

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involved in cell proliferation, differentiation, and metabolism, indicating that SETD1A plays a key

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role in cell physiology by regulating gene expression. 36,37 However, it has not been reported

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whether SETD1A is associated with milk synthesis.

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In our previous studies, it has been demonstrated that amino acids (methionine, leucine, and

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lysine) are key regulators of milk synthesis in bovine MECs (BMECs). 27,38,39 Since taurine is a

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beta-amino acid, though it is not incorporated into proteins, it is possible that taurine might also

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promote milk synthesis. In this study, we show that taurine promotes milk synthesis in BMECs,

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and further uncover the molecular mechanism that taurine activates the mTOR and SREBP-1c

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signaling pathways via the GPR87-PI3K-SETD1A signaling. Our findings may aid in the

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application of taurine in milk production.

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MATERIALS AND METHODS

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Chemicals

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Taurine (purity˃99%) was purchased from Sigma (T0625, Sigma-Aldrich, St. Louis, US).



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Cell culture conditions Three mid-lactation healthy Holstein dairy cows (three years old and in the second parity) were

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chosen for culture of the primary BMECs as the previous report. 40 Procedures involving animals

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were reviewed and approved by the Animal Care and Use Committee of Northeast Agricultural

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University. Briefly, cows were slaughtered and tissue pieces from mammary glands were cultured

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in DMEM/F12 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum

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(FBS) (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. For

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3–4 weeks, cells were grown from the periphery of these tissue pieces, including BMECs,

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fibroblasts, and myoepithelial cells. BMECs were purified from other cells after three to four

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passages due to the different sensitivities of these cells to trypsin digestion. Cell purities were

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identified by means of morphology and immunofluorescence observation. Cells expressing

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cytokeratin 18 and β-casein were identified as BMECs. BMECs cultured in 5-15 passages were

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stored in liquid nitrogen for future use. To observe the effects of taurine, cells were plated at

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~1×105 cells·cm-2, and the culture medium was replaced with FBS-free OPTI-MEMⅠmedium

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(Gibco, Carlsbad, CA, USA) at 24h before treatment. Then cells were treated with taurine (0, 0.08,

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0.16, 0.24, 0.32, and 0.4 mM) for 24 h. To inhibit PI3K activity, LY294002 (S1737, Beyotime,

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Beijing, China) (15 μM), a specific PI3K inhibitor, was added to the culture medium.

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Cell number counting

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The numbers of BMECs were counted by using a CASY TT Analyser System (Scharfe System

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GmbH, Reutlingen, Germany), following the manufacturer’s instructions. Briefly, cells were

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diluted 1:100 with the electrolyte solution. An aliquot (100 μl) of the cell suspension was aspirated

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into a capillary. Cell numbers were automatically calculated according to the sizes of cells when

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cells were passing through a precision measuring pore. The dimensions of cell debris, dead cells

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and living cells were less than 7.63 μm, between 7.63 and 11.75 μm, and larger than 11.75 μm,

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respectively. Living cells could be considered as an electrical isolator due to an intact cell

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membrane. The resistance were measured, which reflected the true sizes of cells, and cursor

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positions (between 11.75 to 50.00 μm) were set to calibrate the living cells.

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Western blotting


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Western blotting analysis was conducted essentially as described previously. 41 Briefly, proteins

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were isolated from cells via ice-cold RIPA lysis solution (Beyotime). Protein concentrations were

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quantified using a BCA assay kit (Beyotime). Equal amounts of protein samples (10-30 μg/lane)

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were electrophoresed in 8-12% SDS-polyacrylamide gels. The proteins were then transferred to

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nitrocellulose membranes. Next the membranes were reacted with various primary antibodies.

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Primary antibodies: α-casein (bs-8245R), β-casein (bs-0813R), p-PI3K (bs-5570R), PI3K (bs-

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2067R), all were rabbit polyclonal antibodies, Bioss, Beijing, China; p-mTOR (Ser2448) (#5536),

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mTOR (#2983), both were rabbit monoclonal antibodies, Cell Signaling Technology, Danvers,

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MA, USA; p-AKT1 (Thr308) (sc-135650), AKT1 (sc-1618), SREBP-1c (sc-365513), β-actin (sc-

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47778), all were mouse monoclonal antibodies, Santa Cruz, Santa Cruz, CA, USA; GPR87

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(ab77517), GPRC6A (ab138994), SETD1A (ab70378), H3K4Me3 (ab8580), H3(ab1791), all were

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rabbit polyclonal antibodies, Abcam, Cambridge, MA, USA. Membranes were further probed with

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appropriate secondary antibodies linked to horseradish peroxidase (Bioss). The signals were

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visualized using an enhanced chemiluminescence kit (Applygen, Beijing, China). The

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relative intensities of the bands were quantified using Image J software. All the values were

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normalized to β-actin or histone H3.

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Detection of triglyceride in the culture medium

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Triglyceride (TG) can be secreted by BMECs into the culture medium. TG amounts in the

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culture medium were measured using the TG GPO-POD Assay kit (Applygen), according to the

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manufacturer’s protocol. Briefly, a series of glycerol standards at known concentrations and

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aliquots (10 μl/one sample) of the cell medium were placed in a 96-well plate and mixed with

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working solutions (190 μl/one sample) and incubated at 37 ℃ for 10 min. In the reaction, TGs

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were broken down into glycerol by lipase, then glycerol was changed into 3-phosphate glycerol

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by glycerol kinase, next 3-phosphate glycerol was oxidized by glycerol oxidase to produce

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hydrogen peroxide, which reacted with a substrate and converted it to phenylquinone imine under

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catalase catalysis. The light densities of the products were monitored at 550 nm in a microplate

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reader, and thereby TG amounts were calculated.

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Detection of lipid droplet formation



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Lipid droplets in cells were detected by a fluorescence optical imaging method. 42,43 Briefly,

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cells were grown on 12 mm glass coverslips to 30-50% confluence. Cells were then fixed in 3.7%

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(w/v) paraformaldehyde in phosphate-buffered saline for 10 min. Next Bodipy 493/503 (4,4-

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difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) (D3922, Invitrogen, Carlsbad, CA,

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USA) (1 μg/mL) was used to incubate the cells for 15 min to label lipid droplets. Finally, cell

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nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (C02-04002, Bioss). Lipid

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droplets formed in the cytoplasm were observed using a laser scanning confocal microscope

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(TCS-SP2 AOBS, Leica, Heidelberg, Germany).

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siRNA transfection

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Small interfering RNAs (siRNAs) targeting GPR87, GPRC6A and SETD1A were purchased

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from a company (GenePharma, Suzhou, China). Transfection was conducted using Lipofectamine

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3000 (Invitrogen). One of the three siRNAs targeting different regions of GPR87, GPRC6A or

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SETD1A mRNA was selected for further use by transfection experiments and western blotting

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analysis. siRNA sequences used for further transfection experiments: GPR87-siRNA, 5’-

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GCCUGGACCCAAUCAUUUATT-3’; GPRC6A-siRNA, 5’-

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GCUCUGAGGUGUGUUUCUATT-3’; SETD1A-siRNA, 5’-

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GCGAUCUGCUGAAGUUAAATT-3’. RNA sequence of the negative control (NC), 5’-

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UUCUCCGAACGUGUCACGUTT-3’. Twenty-four hours post-transfection, cells were harvested.

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qRT-PCR

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Total RNA was isolated from cells using Trizol Reagent (Invitrogen). RNA concentrations

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were measured on a NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific, Waltham,

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MA, USA). Total RNA sample (1μg) was used for reverse transcription with a thermoscript

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reverse transcriptase (TaKaRa, Dalian, China). Real time quantitative PCR (qRT-PCR) reactions

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were performed with a qRT-PCR Kit (TaKaRa) on a qRT-PCR system (ABI PRISM 7300,

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Applied Biosystems, Foster City, CA, USA). qRT-PCR primers: mTOR, F: 5’-

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TGCCTTCACAGATACCCAG-3’, R: 5’-GTAGCGATCAATGCTTAT-3’; SREBP-1c, F: 5’-

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CAGTAGCAGCGGTGGAAGTG-3’, R: 5’-GAGAGACAGAGGAAGACGAGTG-3’; GPR87, F:

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5’-TTGGACCTTGGTACTTCA-3’, R: 5’-GTAGCGATCAATGCTTAT-3’; and β-actin, F: 5’-

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AAGGACCTCTACGCCAACACG-3’, R: 5’-TTTGCGGTGGACGATGGAG-3’. The relative


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mRNA expression of these target genes were normalized using the endogenous β-actin as control

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and quantified using the 2-ΔΔCt method.

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Immunofluorescence assay

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Immunofluorescence assay of GPR87 subcellular localization was carried out according to a

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previous report. 38 Briefly, cells were treated with different concentrations of taurine for 24 h, then

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cells on coverslips were fixed in 3.7% paraformaldehyde in PBS for 15 min. Cells were further

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blocked in blocking buffer (5% BSA plus 0.1% Triton X-100 in TBS) for 1.5 h. Next cells were

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incubated sequentially with primary antibody against GPR87 (ab77517, Abcam) and then

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fluorescein isothiocyanate (FITC)-labeled secondary antibody (A0562, Beyotime). Finally, cells

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were counterstained with DAPI. Images were acquired with a confocal microscope (TCS-SP2

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AOBS, Leica).

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Chromatin immunoprecipitation assays

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H3K4Me3 binding of mTOR and SREBP-1c were analysed using ChIP. Briefly, cells were

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maintained in 10 mL DMEM/F12 medium containing 10% FBS, until the cell confluency reached

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40-50 %. The culture medium was then replaced with FBS-free OPTI-MEMⅠmedium (Gibco,

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Carlsbad, CA, USA) at 24 h before treatment. Cells were next treated with taurine (0.24mM) for

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24h, and the cell confluency reached 80-90 %. Then formaldehyde (270 μL, 37 %) was added to

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the culture medium (the final concentration was 1 %). Cells were then incubated at 37 °C for 10

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min to cross-link the genomic DNA with proteins. Then cells were precipitated and treated with

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SDS Lysis Buffer (500 μL). Cell lysates were further treated with ultrasound (ultrasonic power

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was 20 W, and the ultrasonic probe was 2mm), 10 seconds each time, 3-4 times in total. Cell

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lysates were centrifuged and the supernatants were used for further ChIP analysis using a ChIP

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assay kit (P-2078, Beyotime) with antibody against H3K4Me3 (ab8580, Abcam). An antibody

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specific to RNA polymerase II (to enrich the GAPDH promoter DNA) served as a positive control,

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and IgG served as a negative control. Primers against mTOR in ChIP-PCR and ChIP-qPCR: F: 5’-

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GTATTAGCATTGAACCCAC-3’, R: 5’-GAAACTTAGCTCCAGCAT-3’; Primers against

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SREBP-1c, F: 5’-TACTCAGGATGGCTGGATT-3’, R: 5’-GAAGCAGTGAGCAGAGGC-3’.

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For ChIP-qPCR, Ct values of enrichment were normalized to total input, and the percentage of

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input immunoprecipitated with H3K4Me3 was calculated.



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Statistical Analysis All studies were repeated in three to five independent experiments. Results are shown

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as the means with standard error of representative data. Statistical significance of differences

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between groups was assessed using Student’s t-test, one-way analysis of variance, or chi-

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square test. A p value < 0.05 or < 0.01 was considered significant.

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RESULTS

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Taurine promotes cell proliferation in a dose-dependent manner.

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BMECs were treated with different concentrations of Taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4

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mM) for 24h. Cell number was counted using a cell counter. As taurine concentration was

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increased from 0 to 0.24 mM, cell number was increased gradually and peaked at 0.24 mM. When

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taurine concentration was greater than 0.24 mM, cell number gradually declined (Figure 1B).

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These data revealed that taurine promotes proliferation of BMECs in a dose-dependent manner.

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Taurine promotes the synthesis of milk protein and fat.

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The synthesis of milk protein and fat in cells treated as above were also determined. Western

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blotting analysis detected that, with the increasing of taurine concentration, both the protein levels

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of α-casein (Figures 2A and 2B) and β-casein (Figures 2A and 2C) in cells were significantly

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increased, and peaked at 0.24 mM, followed by a gradual decline. Taurine treatments also

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significantly increased the amounts of TG in the culture medium (Figure 2D) and promoted lipid

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droplet accumulation in cells (Figure 2E), with the maximal effects both at 0.24 mM, followed by

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a gradual decline. These data revealed that taurine stimulates milk synthesis in BMECs in a dose-

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dependent manner.

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Taurine stimulates the major signaling pathways leading to milk synthesis.

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The effects of taurine on the major signaling pathways leading to milk synthesis were further

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determined by western blotting and qRT-PCR analysis. Taurine treatments as above significantly

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increased the phosphorylation of mTOR (Figures 3A and 3B), and upregulated the protein levels

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of SREBP-1c (Figures 3A and 3C). The mRNA levels of mTOR (Figure 3D) and SREBP-1c

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(Figure 3E) were also significantly increased , with all the maximal increases presented in the 0.24

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mM taurine treatment.

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Taurine stimulates the mTOR and SREBP-1c pathways in a PI3K-dependent manner


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We further investigated how taurine regulates the mTOR and SREBP-1c pathways. Our

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previously reports point that amino acids stimulate these pathways through the activation of

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PI3K.38,39,44 We speculated that PI3K activation might be needed for taurine to regulate these

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signaling pathways. Since taurine promotes milk synthesis with an apparent maximum at 0.24 mM,

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the taurine concentration of 0.24 mM was chosen for subsequent studies. Western blotting

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analysis detected that AKT phosphorylation was totally abolished in cells treated with the PI3K

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inhibitor LY294002 (15 μM) (Figures 4A and 4B). mTOR phosphorylation (Figures 4A and 4C)

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and SREBP-1c protein expression (Figures 4A and 4D) stimulated by taurine were both abolished

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by LY294002 treatment. These data reveal that taurine stimulates mTOR phosphorylation and

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SREBP-1c expression in a PI3K-dependent manner.

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Taurine stimulates the PI3K-SETD1A-H3K4Me3 signaling pathway

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The mechanism via which taurine stimulates the mRNA expression of mTOR and SREBP-1c

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was further explored. We first detected the effects of taurine on the protein levels of SETD1A and

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its target H3K4Me3, which might control these two mRNA expression. Taurine significantly

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increased SETD1A (Figures 5A and 5C) and H3K4Me3 (Figures 5A and 5D) protein levels, in a

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PI3K-dependent manner (Figures 5A and 5B). We then knocked down SETD1A in cells by

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siRNA transfection (Figures 6A and 6B). SETD1A knockdown canceled the stimulation of taurine

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on H3K4Me3 (Figures 6A and 6C), p-mTOR (Figures 6A and 6D), and SREBP-1c (Figures 6A

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and 6E) protein levels. To show that taurine modulates H3K4Me3 for the gene transcription of

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mTOR and SREBP-1c, we further observed the binding of H3K4Me3 to these gene promoters by

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ChIP-qPCR. ChIP-PCR detected that H3K4Me3 could bind to these gene promoters (Figures S1A

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and S1B), and ChIP-qPCR further detected that taurine significantly increased these binding

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(Figure 6F). Together, these data reveal that taurine stimulates the gene expression of mTOR and

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SREBP-1c via the PI3K-SETD1A-H3K4Me3 signaling.

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GPR87 is a key mediator of taurine-stimulated PI3K activation.

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Our previous mass spectrometric data showed that the expression of GPRC6A and GPR87 were

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both upregulated in BMECs treated with an amino acid (methionine) (unpublished data), therefore

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we hypothesized that GPRC6A and/or GPR87 might be required for the activation of taurine on

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activation of PI3K and its downstream signaling. GPRC6A knockdown did not suppress PI3K



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activation stimulated by taurine, indicating that GPRC6A is not required for taurine to activate

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PI3K (Figure 7A). GPR87 knockdown largely abolished the stimulation of taurine on p-PI3K

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(Figures 7B-7D), p-mTOR (Figures 7B and 7E), and SREBP-1c protein levels (Figures 7B and

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7F). These data demonstrate that GPR87 is required for taurine to activate PI3K and downstream

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signaling pathways.

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Taurine stimulates GPR87 expression and cell membrane localization

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We next investigated whether taurine affects GPR87 expression and subcellular localization in

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BMECs by western blotting, qRT-PCR, and immunofluorescence assays. Taurine dose-

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dependently increased GPR87 protein (Figures 8A and 8B) and mRNA levels (Figure 8C), with

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the peak increase both present at 0.24 mM. Taurine markedly increased the cell membrane

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localization of GPR87, also with an apparent maximum at 0.24 mM. These data demonstrate that

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taurine is an effective stimulator of GPR87 expression and cell membrane localization, suggesting

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a sensing mechanism of GPR87 to extracellular taurine.

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DISCUSSION

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Taurine can be used as a feed additive in livestock production, however, the role of taurine and

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molecular mechanism of its action is largely unclear, and it is unknown whether taurine can

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promote milk synthesis in mammary gland.45,46,31 Here we show that taurine dose-dependently

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promotes milk synthesis in BMECs. Taurine stimulates the gene expression of mTOR and

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SREBP-1c, which are members of the main signaling pathways leading to milk synthesis. We next

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demonstrate that the PI3K-SETD1A-H3K4Me3 signaling pathway is required for taurine to

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stimulate the gene expression of mTOR and SREBP-1c. We further uncover that GPR87 is

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required for taurine to activate this PI3K signaling pathway. Taurine dose-dependently stimulates

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GPR87 expression and cell membrane localization, suggesting a sensing mechanism of GPR87 to

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extracellular taurine. Our experimental data reveal that taurine promotes milk synthesis via the

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GPR87-PI3K-SETD1A signaling.

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When the concentrations of taurine were below 0.24 mM, taurine dose-dependently increased

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cell numbers, milk protein synthesis, TG secretion, and lipid droplet formation in BMECs.

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Taurine also increased the mRNA levels of mTOR and SREBP-1c and protein levels of p-mTOR

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and SREBP-1c, which two are well established key members of the signaling pathways associated


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with milk protein and fat synthesis. 47 The mRNA levels of mTOR and protein levels of p-mTOR

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were both stimulated by taurine whereas the protein levels of mTOR were similar in all groups, in

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consistent with our previous observation. 27,38,39 Since mTOR is an intracellular signaling hub that

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regulates cell homeostasis, we speculate that the protein level of mTOR (which might be detected

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as the unphosphorylated form of mTOR) needs to maintain a relatively stable level to rapidly

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control mTOR phosphorylation. Under favorable conditions, more mTOR mRNA leads to

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increased mTOR phosphorylation, while under unfavorable conditions less mTOR mRNA leads to

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decreased mTOR phosphorylation.

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With the increase of taurine concentration (> 0.24 mM), the induction of taurine on milk

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synthesis gradually declined, indicating that an excess of taurine may have negative effects.

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Previous reports showed that taurine (1-16 mg/mL) treatment reduced both activities of catalase

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and superoxide dismutase in human neuronal cells and led to cell apoptosis. 48 The metabolism of

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taurine has not been fully studied. Previous reports showed that taurine could be excreted in urine,

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about 70% as taurine and 25% as sulfate. Excessive taurine could be oxidized to isethionic acid

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and sulfoacetaldehyde, which might be cytotoxic. 49,50 Based on these reports and our

302

experimental data, we consider that taurine promotion on milk synthesis is dose-dependent, and

303

excessive taurine may produce secondary metabolites which might exhibit harmful effects.

304

PI3K inhibition and SETD1A knockdown experiments demonstrate that PI3K and its

305

downstream signaling molecule SETD1A are required for taurine to stimulate the gene

306

transcription and subsequent activation of mTOR and SREBP-1c. H3K4Me3 produced by

307

SETD1A bound to mTOR and SREBP-1c genes at active promoters and triggered their mRNA

308

expression leading to enhanced corresponding signaling pathways. These data demonstrated that

309

taurine, like other amino acids (Met, Leu, etc.), activates the gene transcription and subsequent

310

activation of mTOR and SREBP-1c in a PI3K-dependent manner. 27,51,52 Our data further reveal

311

that taurine modulates the mTOR and SREBP-1c pathways via the SETD1A-H3K4Me3 signaling

312

which is an epigenetic determinant in gene expression, 53-55 suggesting that taurine epigenetically

313

modulates signaling pathways associated with milk synthesis. It is still unknown the molecular

314

mechanism through which PI3K activation increases the protein level of SETD1A or leads to

315

SETD1A protein modification, which remains as an open question.



316

GPR87 but not GPRC6A (which is one amino acid sensor) knockdown abolished the

317

stimulatory effects of taurine on the activation of PI3K and its downstream signaling. GPR87 was

318

previously deorphanized to be a lysophosphatidic acid receptor, but new endogenous ligands of

319

GPR87 still need to be described, and the function and mechanism of GPR87 are still not clearly

320

defined. 32,56,57 We further showed that taurine dose-dependently stimulated GPR87 expression

321

and cellular membrane localization, suggesting a sensing mechanism of GPR87 to extracellular

322

taurine. These data demonstrate that GPR87 is required for taurine to activate PI3K, furthermore

323

taurine also positively regulates the GPR87-PI3K signaling for milk synthesis. We hypothesize

324

that taurine might be a ligand of GPR87, which needs to be explored in future study. To our best

325

known, it is the first report that GPR87 as a GPCR mediates amino acid (taurine) signaling to

326

mTOR and SREBP-1c.

327

Taken together, current study provides preliminary evidence that taurine dose-dependently

328

promotes milk synthesis in BMECs, and uncovers the molecular mechanism that taurine

329

stimulates the GPR87-PI3K-SETD1A signaling to produce H3K4Me3 and epigenetically

330

regulates mTOR and SREBP-1c gene expression. Our findings will facilitate the application of

331

taurine in milk production.

332

AUTHOR INFORMATION

333

Corresponding Author

334

*(X.G.) Phone: +86-451-55190542; Fax: +86-451-55190244; E-mail: [email protected]

335

ORCID

336

Xuejun Gao: 0000-0002-8241-6346

337

FUNDING

338

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

339

China (No. 31671473 and 31472162).

340

NOTES

341

The authors declare no competing financial interest.

342

REFERENCES


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deorphanized and shown to be a lysophosphatidic acid receptor. Biochem Biophys Res Commun.

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2007, 363, 861-866.

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FIGURE LEGENDS

514

Figure 1 Effects of taurine on cell numbers

515

BMECs were treated with different concentrations of taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4

516

mM). (A) Molecular structure of taurine. (B) Cell numbers were counted with a cell counter. Data

517

were the mean ± SE from five independent experiments. Values with different superscripted

518

lowercase letter indicate significant difference (p < 0.05).

519

Figure 2 Effects of taurine on milk protein and fat synthesis in BMECs

520

(A) BMECs were treated as in Figure 1. Western blotting analysis were performed to measure the

521

indicated protein levels. (B and C) Relative folds of α-casein (B) and β-casein (C) (protein/β-actin)

522

protein levels from the western blots in (A) were quantified by gray scale scan. (D) TG amounts in

523

the culture medium were monitored using a detection kit. (E) Lipid droplets in cells were observed

524

by fluorescence staining. DAPI (blue), Lipid droplet (green), scale bar represents 15 μm. Data

525

were the mean ± SE from five independent experiments. Values with different superscripted

526


527

Figure 3 Effects of taurine on the mTOR and SREBP-1c signaling pathways

528

(A) Cells were treated as in Figure 1. Western blotting analysis were performed to measure the

529

indicated protein levels. (B) The ratios of p-mTOR to mTOR in (A) were quantified by gray scale

530

scan. (C) Relative folds of SREBP-1c levels (protein/β-actin) in (A) were quantified by gray scale

531

scan. (D and E) qRT-PCR analysis of mTOR (D) and SREBP-1c (E) mRNA levels in cells. Data

532

were the mean ± SE from three independent experiments. Values with different superscripted

533


534

Figure 4 PI3K is required for taurine to activate the mTOR and SREBP-1c signaling

535

pathways

536

(A) BMECs were treated with LY294002 (15 μM) and taurine (0.24mM) for 24 h. Western

537

blotting analysis were performed to measure the indicated protein levels. (B) The ratios of p-AKT

538

to AKT were quantified by gray scale scan. (C) The ratios of p-mTOR/mTOR were quantified by

539

gray scale scan. (D) Relative folds of SREBP-1c levels from the western blots were quantified by

540

gray scale scan. Data were the mean ± SE from three independent experiments. Values with

541

different superscripted lowercase letter indicate significant difference (p < 0.05).



542

Figure 5 PI3K is required for taurine to activate the SETD1A-H3K4Me3 signaling pathway

543

Cells were treated as in Figure 4. (A) Western blotting analysis were performed to measure the

544

indicated protein levels. (B) p-AKT/AKT ratios were quantified by gray scale scan. (C and D)

545

Relative folds of SETD1A (C) and H3K4Me3 (D) protein levels (protein/H3) from the western

546

blots were quantified by gray scale scan. Data were the mean ± SE from three independent

547

experiments. Values with different superscripted lowercase letter indicate significant difference (p

548

< 0.05).

549

Figure 6 Effects of SETD1A knockdown on H3K4Me3 and the mTOR and SREBP-1c

550

signaling pathways

551

(A) BMECs were transfected with a SETD1A siRNA and treated with taurine (0.24mM) for 24 h.

552

Western blotting analysis were performed to measure the indicated protein levels. (B and C)

553

Relative folds of SETD1A (B) and H3K4Me3 (C) Protein levels (protein/H3) from the western

554

blots were quantified by gray scale scan. (D) p-mTOR/mTOR ratios were quantified by gray scale

555

scan. (E) Relative folds of SREBP-1c protein levels (protein/β-actin) were quantified by gray scale

556

scan. (F) ChIP-qPCR analysis of the binding of H3K4Me3 to the gene promoters of mTOR and

557

SREBP-1c in cells stimulated by taurine (0.24mM). Data were the mean ± SE from three

558

independent experiments. Values with different superscripted lowercase letter indicate significant

559

difference (p < 0.05).

560

Figure 7 Effects of GPR87 knockdown on the activation of PI3K by taurine

561

(A) BMECs were transfected with a GPRC6A siRNA and treated with taurine (0.24mM) for 24 h.

562

Western blotting analysis of the indicated protein levels. (B) Cells were transfected with a GPR87

563

siRNA and treated with taurine (0.24mM) for 24 h. Western blotting analysis were performed to

564

measure the indicated protein levels. (C) Relative folds of GPR87 protein levels (protein/β-actin)

565

from the western blots in (B) were quantified by gray scale scan. (D) p-PI3K/PI3K ratios from the

566

western blots in (B) were quantified by gray scale scan. (E) p-mTOR/mTOR ratios from the

567

western blots in (B) were quantified by gray scale scan. (F) Relative folds of SREBP-1c levels

568

from the western blots in (B) were quantified by gray scale scan. Data were the mean ± SE from

569

three independent experiments. Values with different superscripted lowercase letter indicate

570

significant difference (p < 0.05).


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Figure 8 Effects of taurine on GPR87 expression

572

(A) BMECs were treated as in Figure 1. GPR87 protein levels were measured by western blotting

573

analysis. (B) Relative folds of GPR87 protein levels from the western blots were quantified by

574

gray scale scan. (C) qRT-PCR analysis of GPR87 mRNA levels in cells. (D) Expression of

575

GPR87 in cells was observed by immunofluorescence assay. GPR87 (green), DAPI (blue). Scale

576

bar = 25μm. Data were the mean ± SE from three independent experiments. Values with different

577

superscripted lowercase letter indicate significant difference (p < 0.05).

578

Figure S1 ChIP-PCR analysis of the binding of H3K4Me3 to the gene promoters of mTOR and

579

SREBP-1c

580

(A and B) ChIP-PCR analysis of the binding of H3K4Me3 to the gene promoters of mTOR (A)

581

and SREBP-1c (B). M: DL2000 DNA Marker; PC: Positive control; NC: Negative control; 1-2:

582

the target sequences of interest (mTOR, -1485~-1285bp, -1170~-950bp; SREBP-1c, -913~-733bp,

583

-1510~-1325bp) were amplified by PCR with different primers, and the effective primers (lane 1)

584

were further used for ChIP-qPCR.

585 586 587 588 589 590 591 592 593 594 595



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Figure 8

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Figure S1

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Taurine promotes milk synthesis via the GPR87-PI3K-SETD1A

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