Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (−EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase β-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase β-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower β-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.

氨基酸部分通过激活哺乳动物雷帕霉素靶标（mTOR）信号通路而在调节乳蛋白合成中起关键作用。然而，细胞外AA感应受体在此过程中的参与尚不清楚。在非反刍动物中，已经公认AA味道1受体成员1/3（TAS1R1 / TAS1R3）异二聚体有助于大多数1- AA的感测。该受体在牛乳腺细胞中是否具有功能尚不清楚。这项研究的目的是确定通过TAS1R1 / TAS1R3必需的AA信号传导及其在调节牛原发性乳腺上皮细胞和Mac-T中mTOR信号传导途径和酪蛋白mRNA丰度中的作用细胞系。分别用完全Dulbecco改良的Eagle's培养基（+ EAA），不含EAA的培养基（-EAA）或仅添加10种必需AA的培养基刺激牛乳腺上皮细胞。在所有治疗中，非必需氨基酸水平均相同。设计并转染靶向TAS1R1的小分子干扰RNA进入牛原代乳腺上皮细胞（bPMEC）。必需氨基酸或精氨酸，Val，Leu，His，Phe，Met和Ile的完全混合物的补充分别导致更大的mTOR磷酸化。在Val，Leu，Trp，Met和Ile的存在下，核糖体蛋白S6激酶β-1的磷酸化作用更大。缬氨酸，Leu，Met和Ile导致更大的eIF4E结合蛋白1磷酸化。尽管+ EAA和一些单独测试的AA诱导了细胞内钙的增加，但Met和Val是最有效的。敲除TAS1R1会降低同时用Val和Met培养的bPMEC中细胞内钙的含量。当TAS1R1时，mTOR，核糖体蛋白S6激酶β-1和eIF4E结合蛋白1的磷酸化降低在补充了Val和Met的bPMEC中被淘汰。此外，TAS1R1的小干扰RNA沉默导致较低的β-酪蛋白（CSN2）丰度。TAS1R1 / TAS1R3受体可能通过提高细胞内钙浓度来感知细胞外AA并激活牛乳腺细胞中的mTOR信号传导。此机制似乎在Met和Val诱导的CSN2 mRNA丰度变化中起作用。必须进行进一步的体内研究以评估该机制在乳腺中的相关性。