State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot 010021, China.
School of Life Sciences, Jining Normal University, Jining 012000, China.
Biomed Res Int. 2019 May 16;2019:5196028. doi: 10.1155/2019/5196028. eCollection 2019.
The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene and the lipogenic genes , , , and was downregulated in silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPAR. This finding provides a model by which lipogenesis is regulated in pBMECs.
雷帕霉素靶蛋白复合物 2（mTORC2）的机械靶点主要作为胰岛素/PI3K 信号的效应物发挥作用，调节细胞增殖，并与细胞代谢有关。然而，mTORC2 在脂质代谢中的功能尚不清楚。在本研究中，通过 ATP 竞争性 mTOR 抑制剂 AZD8055 或针对 的 shRNA 使原代牛乳腺上皮细胞（pBMEC）中的 mTORC2 失活。通过 MTT 测定法检测 AZD8055 对细胞增殖的影响。通过 ELISA 测定法和 GC-MS 分析测定脂质含量。通过 RT/实时 PCR 和 Western blot 分析分别研究 mRNA 和蛋白表达水平。我们发现 AZD8055 抑制细胞增殖、mTORC2 激活和脂质分泌。敲低 ，并通过 shRNA 特异性减弱 mTORC2 激活。与对照细胞相比，沉默细胞中转录因子基因 和脂生成基因 、 、 和 的表达下调。结果，细胞内三酰基甘油（TAG）、棕榈酸（PA）、二十二碳六烯酸（DHA）和其他 16 种脂肪酸的含量在处理细胞中降低；细胞培养物中 TAG、PA 和 DHA 的积累也减少。总之，mTORC2 在调节 pBMECs 中的脂生成基因表达、脂质合成和分泌中起关键作用，该过程可能通过 PPAR 进行。这一发现为 pBMECs 中脂生成的调节提供了模型。
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