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.
Highlights
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]
We found that the addition of a 5 mM dose of Methylaminoisobutyric acid (MeAIB) could better inhibit the Sodium-coupled neutral amino acid transporter 2 (SNAT2) that has more than 50% inhibition efficiency, while it has minimized the non-specificity that SNAT analogs and PAT1 show less than 20% inhibition efficiency compared with control group in this porcine cell line
The results suggest that suppression of SNAT2 in Intestinal porcine epithelial cells (IPEC)-J2 cells reduced cell proliferation, which was supported by G1 arrest and the decrease in protein synthesis
Summary
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]. 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]. 2018, 19, x FOR PEER REVIEW substrate, suppresses the expression of SNAT2 in cells and has been used as a means of inhibiting the upstuabksetroaften, asutuprparlessysestsetmheAexspurbessstrioanteosf[S2N]. Cancer cells and skeletal muscle myoblast cells. AT2 in cells and has been used as a means of inhibiting the uptSaNkeAoTf2niastuwrealllsdyostceummAenstuedbsitnrahteusm[2a]n. It has beeSnNdAeTm2 oisnwsterlaltdedoctuomseenntseedinintrhaucmellaunlacranacnearbcoellilcs aanmdinskoealectiadl lmevueslcsleanmdyothbelanstrecgelulsl.aCteutrhreenatmlyino aciitdhsaisgbneaelnindgempaotnhswtraaytesdintoflsueennsceiinngtrpacreoltleuilnartuanrnaboovleicr aamndincoeallcigdrolewvethls[a4n–d8]t.hSenevreegraullastteutdhieesamshinoowed thaactidSNsigAnTa2linhgaspaathlowwaylseivneflluoefnceixnpgrepsrsoitoeninitnurcnoomveprleatnedmceeldl giar,owbutht [i4ts–8a]c. Tchonissesrtuveddy iwnahsucmonandsuc[4te].dTthoisinsvtuedstyigwataesthe rocloenodfutchtedSNtoAinTv2esttriagnasteptohreterrolaeftoefrththeeSNadAdTi2titornanosfpMorteeAr IaBfteinr tcheelladgdroitwiotnho, fpMroetAeiInBtiunrcneollvgerro,wanthd, its replartoetdeimn tTuOrnRovsiegrn, anlidngitsinretlhaeteidntmesTtOinRalspigonracliinnegeipnitheeliinatleIsPtiEnCal-Jp2ocrceilnlse. epithelial IPEC-J2 cells
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