title: Amino acids regulate mTOR pathway and milk protein synthesis in a mouse mammary epithelial cell line is partly mediated by T1R1/T1R3.
abstract: OBJECTIVE
  The mechanism of dietary amino acids in regulating milk protein synthesis at the translational level is not well understood. Numerous studies have shown that the amino acid signal is transferred through the mammalian target of rapamycin (mTOR) pathway; however, the extracellular amino acid-sensing mechanism that activates mTOR complex 1 is unknown. We tested the hypotheses that the T1R1/T1R3 heterodimer functions as a direct sensor of the fed state and amino acid availability preceding the mTOR pathway and affects milk protein synthesis in mammary epithelial cells.
  
  
  METHODS
  The expression of T1R1 was repressed by T1R1 siRNA in mouse mammary epithelial cells model (HC11). Western blot was used to analyze activity of the mTOR pathway and β-casein expression, and quantitative real-time RT-PCR was used to analyze the change in mRNA abundance of amino acid transporters.
  
  
  RESULTS
  The transcripts and proteins of T1R1 and T1R3 were detected in HC11 cells and mouse mammary gland tissue. siRNA silencing of T1R1 repressed β-casein synthesis in HC11 cells both with and without essential amino acids present in the culture medium. The phosphorylation of mTOR, S6K, and 4EBP1 in T1R1 knockdown HC11 cells declined to 25, 50, and 30 %, indicating T1R1 knockdown repressed the activity of the mTOR pathway. T1R1 knockdown increased the mRNAs coding three important amino acid transporters (SLC1A5 and SLC3A2/SLC7A5). Activation of the mTOR pathway was partially repressed by T1R1 siRNA or SLC7A5/SLC3A2 inhibitor (BCH, 10 mM), and the combination of these two treatments further repressed the activity of this pathway.
  
  
  CONCLUSIONS
  T1R1/T1R3 serves as sensor of extracellular amino acids in mouse mammary epithelial cells and involved in milk protein synthesis regulation.
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China.
authors.forename: YanHong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China.
authors.forename: JunQiang
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China.
authors.forename: XingTang
authors.initials: X
authors.lastname: Fang
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China. chenhong1212@126.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Institute of Cellular and Molecular Biology, School of Life Science, Jiangsu Normal University, Xuzhou, 221116, Jiangsu, China. clzhang@jsnu.edu.cn.
authors.forename: ChunLei
authors.initials: C
authors.lastname: Zhang
mesh_terms: D026962:Amino Acid Transport System ASC / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000494:pharmacology*; D000598:Amino Acids, Cyclic / Q000494:pharmacology; D000818:Animals; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins / Q000096:biosynthesis*; D015778:Minor Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D026962:Amino Acid Transport System ASC; D000596:Amino Acids; D000598:Amino Acids, Cyclic; D002364:Caseins; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; D008894:Milk Proteins; D015778:Minor Histocompatibility Antigens; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; C498850:Slc1a5 protein, mouse; C523212:Slc3A2 protein, mouse; C431926:taste receptors, type 1; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases
keywords: Amino acid sensing; T1R1; T1R3; mTOR