Hao Qi, Wang Zhe, Wang Lulu, Han Meihong, Zhang Minghui, Gao Xuejun
College of Animal Science, Yangtze University, Jingzhou434023, People's Republic of China.
College of Life Science, Northeast Agricultural University, Harbin150030, People's Republic of China.
Br J Nutr. 2022 May 20:1-11. doi: 10.1017/S0007114522001544.
Several amino acids can stimulate milk synthesis in mammary epithelial cells (MEC); however, the regulatory role of isoleucine (Ile) and underlying molecular mechanism remain poorly understood. In this study, we aimed to evaluate the regulatory effects of Ile on milk protein and fat synthesis in MEC and reveal the mediation mechanism of Brahma-related gene 1 (BRG1) on this regulation. Ile dose dependently affected milk protein and fat synthesis, mechanistic target of rapamycin (mTOR) phosphorylation, sterol regulatory element binding protein 1c (SREBP-1c) expression and maturation, and BRG1 protein expression in bovine MEC. Phosphatidylinositol 3 kinase (PI3K) inhibition by LY294002 treatment blocked the stimulation of Ile on BRG1 expression. BRG1 knockdown and gene activation experiments showed that it mediated the stimulation of Ile on milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c expression and maturation in MEC. ChIP-PCR analysis detected that BRG1 bound to the promoters of and , and ChIP-qPCR further detected that these bindings were increased by Ile stimulation. In addition, BRG1 positively regulated the binding of H3K27ac to these two promoters, while it negatively affected the binding of H3K27me3 to these promoters. BRG1 knockdown blocked the stimulation of Ile on these two gene expressions. The expression of BRG1 was higher in mouse mammary gland in the lactating period, compared with that in the puberty or dry period. Taken together, these experimental data reveal that Ile stimulates milk protein and fat synthesis in MEC via the PI3K-BRG1-mTOR/SREBP-1c pathway.
几种氨基酸可刺激乳腺上皮细胞（MEC）中的乳汁合成；然而，异亮氨酸（Ile）的调节作用及其潜在分子机制仍知之甚少。在本研究中，我们旨在评估Ile对MEC中乳蛋白和脂肪合成的调节作用，并揭示与Brahma相关基因1（BRG1）在此调节过程中的介导机制。Ile剂量依赖性地影响牛MEC中乳蛋白和脂肪合成、雷帕霉素机制性靶标（mTOR）磷酸化、固醇调节元件结合蛋白1c（SREBP-1c）表达与成熟以及BRG1蛋白表达。用LY294002处理抑制磷脂酰肌醇3激酶（PI3K）可阻断Ile对BRG1表达的刺激。BRG1敲低和基因激活实验表明，它介导了Ile对MEC中乳蛋白和脂肪合成、mTOR磷酸化以及SREBP-1c表达与成熟的刺激作用。染色质免疫沉淀PCR（ChIP-PCR）分析检测到BRG1与 和 的启动子结合，染色质免疫沉淀定量PCR（ChIP-qPCR）进一步检测到Ile刺激可增加这些结合。此外，BRG1正向调节H3K27ac与这两个启动子的结合，而对H3K27me3与这些启动子的结合起负向作用。BRG1敲低阻断了Ile对这两个基因表达的刺激。与青春期或干奶期相比，哺乳期小鼠乳腺中BRG1的表达更高。综上所述，这些实验数据表明，Ile通过PI3K-BRG1-mTOR/SREBP-1c途径刺激MEC中的乳蛋白和脂肪合成。
