Abstract：
The uptake of AA in mammary tissues is affected by prolactin (PRL). To investigate whether PRL-induced AA uptake is involved in L-type AA transporter 1 (LAT1), we analyzed the changes of AA in the medium of dairy cow mammary epithelial cells in the presence of PRL or PRL plus BCH, an inhibitor of LAT1. Then Western blot and luciferase assay were used to detect the regulation mechanism of PRL on LAT1 expression and function. Our results showed that Thr, Val, Met, Ile, Leu, Tyr, Lys, Phe, and His are LAT1 substrates and could be transported into mammary epithelial cells via LAT1. PRL stimulation increased the uptake of most AA into mammary epithelial cells of dairy cows, however, inhibition of LAT1 transport activity reduced PRL-induced AA uptake, suggesting that the effect of PRL on AA transport depends on LAT1 expression and function. PRL stimulation upregulated LAT1 expression and plasma membrane location not only in dairy cow mammary epithelial cells, but also in mouse mammary epithelial cell line HC11. Western blot showed that PI3K-AKT-mTOR signaling could be activated in PRL-stimulated mammary epithelial cells. Treatment of cells with LY294002 decreased PI3K-AKT-mTOR activation, as well LAT1 expression, that in turn decreased milk protein synthesis. Luciferase assay showed PRL treatment increased the promoter activity of LAT1 promoter fragment -419∼-86 bp. Treatment of cells with LY294002, an inhibitor of PI3K, or SC79, an activator of AKT abolished or promoted the transcriptional activity of this promoter fragment in the presence of PRL. These results suggested that the -419∼-86 bp fragment of LAT1 promoter mediates the action of PI3K-AKT-mTOR signaling on LAT1 transcription in mammary epithelial cells of dairy cows, which in turn increased LAT1 expression and AA uptake.
摘要：
乳腺组织中AA的摄取受催乳素（PRL）的影响。为了研究PRL诱导的AA摄取是否与L型AA转运蛋白1（LAT1）有关，我们分析了在PRL或PRL加BCH存在下奶牛乳腺上皮细胞培养基中AA的变化，LAT1的抑制剂。然后用Westernblot和荧光素酶法检测PRL对LAT1表达和功能的调控机制。我们的结果表明，Thr，Val,Met,Ile,Leu,Tyr,Lys,Phe,和His是LAT1底物，可以通过LAT1转运到乳腺上皮细胞中。PRL刺激增加了奶牛乳腺上皮细胞对大多数AA的摄取，然而,抑制LAT1转运活性降低了PRL诱导的AA摄取，提示PRL对AA转运的影响取决于LAT1的表达和功能。PRL刺激不仅上调奶牛乳腺上皮细胞的LAT1表达和质膜定位,而且在小鼠乳腺上皮细胞系HC11中。Westernblot显示PI3K-AKT-mTOR信号可在PRL刺激的乳腺上皮细胞中被激活。用LY294002处理细胞可降低PI3K-AKT-mTOR的活化,以及LAT1表达式，这反过来又减少了牛奶蛋白质的合成。荧光素酶实验表明，PRL处理增加了LAT1启动子片段-419~-86bp的启动子活性。用PI3K抑制剂LY294002处理细胞，或SC79，AKT的激活剂在PRL存在下消除或促进了该启动子片段的转录活性。这些结果表明，LAT1启动子的-419~-86bp片段介导PI3K-AKT-mTOR信号对奶牛乳腺上皮细胞LAT1转录的作用，这又增加了LAT1表达和AA摄取。
