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The Regulatory Role of Meaib in Protein Metabolism and the Mtor Signaling Pathway in Porcine Enterocytes

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The Regulatory Role of Meaib in Protein Metabolism and the Mtor Signaling Pathway in Porcine Enterocytes UC Davis UC Davis Previously Published Works Title The Regulatory Role of MeAIB in Protein Metabolism and the mTOR Signaling Pathway in Porcine Enterocytes. Permalink https://escholarship.org/uc/item/8nn544jf Journal International journal of molecular sciences, 19(3) ISSN 1422-0067 Authors Tang, Yulong Tan, Bie Li, Guangran et al. Publication Date 2018-03-02 DOI 10.3390/ijms19030714 Peer reviewed eScholarship.org Powered by the California Digital Library University of California International Journal of Molecular Sciences Article The Regulatory Role of MeAIB in Protein Metabolism and the mTOR Signaling Pathway in Porcine Enterocytes Yulong Tang 1,2, Bie Tan 1,3,*, Guangran Li 1, Jianjun Li 1, Peng Ji 3 ID and Yulong Yin 1,* 1 Laboratory of Animal Nutritional Physiology and Metabolic Process, Key Laboratory of Agro-ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China; [email protected] (Y.T.); [email protected] (G.L.); [email protected] (J.L.) 2 Hunan Co-Innovation Center of Animal Production Safety (CICAPS), Changsha 410128, China 3 Department of Nutrition, University of California, Davis, CA 95616, USA; [email protected] * Correspondence: [email protected] (B.T.); [email protected] (Y.Y.); Tel.: +86-731-8461-9706 (Y.Y.) Received: 3 January 2018; Accepted: 28 February 2018; Published: 2 March 2018 Abstract: Amino acid transporters play an important role in cell growth and metabolism. MeAIB, a transporter-selective substrate, often represses the adaptive regulation of sodium-coupled neutral amino acid transporter 2 (SNAT2), which may act as a receptor and regulate cellular amino acid contents, therefore modulating cellular downstream signaling. The aim of this study was to investigate the effects of MeAIB to SNAT2 on cell proliferation, protein turnover, and the mammalian target of rapamycin (mTOR) signaling pathway in porcine enterocytes. Intestinal porcine epithelial cells (IPEC)-J2 cells were cultured in a high-glucose Dulbecco’s modified Eagle’s (DMEM-H) medium with 0 or 5 mmoL/L System A amino acid analogue (MeAIB) for 48 h. Cells were collected for analysis of proliferation, cell cycle, protein synthesis and degradation, intracellular free amino acids, and the expression of key genes involved in the mTOR signaling pathway. The results showed that SNAT2 inhibition by MeAIB depleted intracellular concentrations of not only SNAT2 amino acid substrates but also of indispensable amino acids (methionine and leucine), and suppressed cell proliferation and impaired protein synthesis. MeAIB inhibited mTOR phosphorylation, which might be involved in three translation regulators, EIF4EBP1, IGFBP3, and DDIT4 from PCR array analysis of the 84 genes related to the mTOR signaling pathway. These results suggest that SNAT2 inhibition treated with MeAIB plays an important role in regulating protein synthesis and mTOR signaling, and provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine. Keywords: sodium-coupled neutral amino acid transporter 2; mTOR; protein turnover; porcine enterocytes 1. Introduction Amino acid transporters are membrane transport proteins; their major role is transporting amino acids and modulating gene expression and the signal transduction pathway by sensing amino acid levels [1]. The SLC38 family of transporters represents a main branch of solute carrier families in mammals, and the transporters can be subdivided into two groups, namely system A and system N[2,3]. Sodium-coupled neutral amino acid transporter 2 (SNAT2) has a very broad tissue distribution profile and is characterized as a system A transporter, which plays various roles in different tissues and has dual transport/receptor functions [4,5]. α-Methylaminoisobutyric acid (MeAIB), a system A Int. J. Mol. Sci. 2018, 19, 714; doi:10.3390/ijms19030714 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 714 2 of 9 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 10 substrate, suppresses the expression of SNAT2 in cells and has been used as a means of inhibiting the uptakesubstrate, of natural suppresses system the Aexpression substrates of [SNAT22]. in cells and has been used as a means of inhibiting the uptakeSNAT2 of natural is well system documented A substrates in human [2]. cancer cells and skeletal muscle myoblast cells. Currently it has beenSNAT2 demonstrated is well documented to sense intracellularin human cancer anabolic cells aminoand skeletal acid levelsmuscle and myoblast then regulate cells. Currently the amino acidit has signaling been demonstrated pathways influencing to sense intracellular protein turnover anabolic amino and cell acid growth levels [and4–8 ].then Several regulate studies the amino showed thatacid SNAT2 signaling has pathways a low level influencing of expression protein inturnover complete and media,cell growth but [4–8]. its activity Several could studies be showed enhanced that SNAT2 has a low level of expression in complete media, but its activity could be enhanced by by withdrawing amino acids, which provide evidence of SNAT2’s ability to regulate amino acid withdrawing amino acids, which provide evidence of SNAT2’s ability to regulate amino acid homeostasis as a transporter [9,10]. Potential regulatory mechanisms of SNAT2 in amino acid nutrition homeostasis as a transporter [9,10]. Potential regulatory mechanisms of SNAT2 in amino acid has demonstrated it is involved in eukaryotic initiation factor 2 phosphorylation, increased gene nutrition has demonstrated it is involved in eukaryotic initiation factor 2 phosphorylation, increased transcription, and internal ribosome entry site-mediated translation [9,10]. The intestinal porcine gene transcription, and internal ribosome entry site-mediated translation [9,10]. The intestinal epitheliumporcine epithelium comprises comprises a large surface a larg areae surface lined byarea a singlelined by layer a single of columnar layer of intestinal columnar epithelial intestinal cells withepithelial the expression cells with of the a varietyexpression of transporters of a variety inof apicaltransporters and basolateral in apical and membranes; basolateral these membranes; transporter proteinsthese transporter play complex proteins and interactiveplay complex roles and in controllinginteractive roles the absorption in controlling and the metabolism absorption of and amino acidsmetabolism [11,12]. Weof amino have previouslyacids [11,12]. cloned We have and previously described thecloned SNAT2 and genedescribed in the the small SNAT2 intestine gene ofin piglets,the whichsmall is intestine highly evolutionarilyof piglets, which conserved is highly in evolutionarily humans [4]. Thisconserved study in was humans conducted [4]. This to investigate study was the roleconducted of the SNAT2 to investigate transporter the role after of the the SNAT2 addition transp of MeAIBorter after in the cell addition growth, of protein MeAIB turnover,in cell growth, and its relatedprotein mTOR turnover, signaling and its in related the intestinal mTOR signaling porcine epithelialin the intestinal IPEC-J2 porcine cells. epithelial IPEC-J2 cells. 2.2. Results Results 2.1.2.1. SNAT2 SNAT2 Inhibition Inhibition Decreased Decreased CellCell Growth and Intracellular Intracellular cAMP cAMP Concentration Concentration in inIPEC-J2 IPEC-J2 Cells Cells ImmunoblottingImmunoblotting of of IPEC-J2 IPEC-J2 treated treated with with MeAIB MeAI causedB caused a significant a significant decrease decrease in SNAT2 in expressionSNAT2 (pexpression< 0.05). mTOR (p < 0.05). phosphorylation mTOR phosphorylation was more was strongly more strongly inhibited inhibited compared compared with with the controlthe control group (pgroup< 0.05) (p (Figure < 0.05) 1(Figure). There 1). was There no was change no change in total in mTOR total mTOR abundance. abundance. FigureFigure 1. 1.MeAIB MeAIB induces induces thethe inhibition of of SNAT2 SNAT2 and and mT mTOROR phosphorylation phosphorylation in inIPEC-J2 IPEC-J2 cells. cells. Data Data areare expressed expressed as as mean mean± ± StandardStandard Deviation (SD), (SD), nn == 4 4independent independent experiments. experiments. * p *<p 0.05< 0.05 versus versus control treatment. control treatment. MeAIB significantly reduced IPEC-J2 cell growth, reaching statistical significance at two days post-MeAIBMeAIB significantly addition (p < reduced 0.05) (Figure IPEC-J2 2A). cellIPEC-J2 growth, cells reachingwere subjected statistical to cell significance cycle analysis at two using days post-MeAIBflow cytometry addition after treating (p < 0.05) with (Figure MeAIB2A). for IPEC-J2 48 h, and cells Figure were 2B subjected showed that to cell the cyclenumber analysis of cells using in flowthe cytometryG1 phases increased after treating and the with proportion MeAIB for of 48cell h,s in and the Figure G2 and2B S showedphases substantially that the number decreased of cells (p in the< 0.05). G1 phases increased and the proportion of cells in the G2 and S phases substantially decreased (p < 0.05). Int. J. Mol. Sci. 2018, 19, 714 3 of 9 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 10 The intracellular intracellular cGMP cGMP level level of of IPEC-J2 IPEC-J2 treated treated with with MeAIB MeAIB did did not not show show a significant a significant effect effect (p >(p 0.05);> 0.05) by; bycontrast,
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