mTORC2 Regulates Lipogenic Gene Expression through PPAR to Control Lipid Synthesis in Bovine Mammary Epithelial Cells
mTORC2 Regulates Lipogenic Gene Expression through PPAR to Control Lipid Synthesis in Bovine Mammary Epithelial Cells
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mTORC2 通过 PPAR 调节脂肪生成基因表达，从而控制牛乳腺上皮细胞中的脂质合成
DOI：
10.1155/2019/5196028
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发表时间：
2019-01-01
影响因子：
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通讯作者：
Wang, Zhigang
中科院分区：
文献类型：
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作者：
Guo, Zhixin;Zhao, Keyu;Wang, Zhigang
雷帕霉素复合物2（mTORC 2）的机制靶点主要作为胰岛素/PI 3 K信号传导的效应物来调节细胞增殖，并且与细胞代谢相关。然而，mTORC 2在脂质代谢中的功能还不清楚。在本研究中，在原代牛乳腺上皮细胞（pBMEC）中，mTORC 2被ATP竞争性mTOR抑制剂AZD 8055或靶向RICTOR的shRNA灭活。MTT法检测AZD 8055对细胞增殖的影响。采用ELISA法和GC-MS法测定脂质含量。采用RT/real-time PCR和western blot分析方法检测mRNA和蛋白表达水平。我们发现AZD 8055可抑制细胞增殖、mTORC 2激活和脂质分泌。RICTOR被敲低，mTORC 2的激活被shRNA特异性减弱。与对照细胞相比，转录因子基因PPARG和脂肪生成基因LPIN 1、DGAT 1、ACACA和FXR的表达在RICTOR沉默细胞中下调。结果，在处理的细胞中，细胞内三酰甘油（TAG）、棕榈酸（PA）、二十二碳六烯酸（DHA）和其他16种脂肪酸的含量降低; TAG、PA和DHA在细胞培养基中的积累也减少。总体而言，mTORC 2在调节pBMEC中的脂肪生成基因表达、脂质合成和分泌中起关键作用，并且该过程可能是通过PPAR进行的。这一发现提供了一种模型，通过该模型在pBMEC中调节脂肪生成。
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 RICTOR 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. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR 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.