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Published on: February 10, 2023
mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial
Zhixin Guo1, Keyu Zhao1, Xue Feng1
1State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot 010021, China.
Abstract:
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 PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.
Insights
Mechanistic target of rapamycin complex 2 (mTORC2) regulates lipid metabolism in bovine mammary cells. Inhibiting mTORC2 reduced cell proliferation, lipid synthesis, and secretion, likely via PPARγ, impacting lipogenic gene expression.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Mechanistic target of rapamycin complex 2 (mTORC2) is known to regulate cell proliferation and metabolism.
- Its specific role in lipid metabolism, particularly in bovine mammary epithelial cells, remains largely uncharacterized.
Purpose of the Study:
- To investigate the function of mTORC2 in lipid metabolism within primary bovine mammary epithelial cells (pBMECs).
- To elucidate the molecular mechanisms by which mTORC2 influences lipogenesis and lipid secretion.
Main Methods:
- Inhibition of mTORC2 using AZD8055 and RICTOR shRNA in pBMECs.
- Assessment of cell proliferation via MTT assay.
- Quantification of lipid content using ELISA and GC-MS.
- Analysis of gene and protein expression through RT/real-time PCR and Western blotting.
Main Results:
- AZD8055 treatment inhibited cell proliferation, mTORC2 activation, and lipid secretion.
- RICTOR knockdown specifically attenuated mTORC2 activation.
- Downregulation of PPARγ and key lipogenic genes (LPIN1, DGAT1, ACACA, FASN) was observed in RICTOR-silenced cells.
- Intracellular and secreted lipid content, including triacylglycerol (TAG), palmitic acid (PA), and docosahexaenoic acid (DHA), significantly decreased.
Conclusions:
- mTORC2 plays a crucial role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs.
- This regulation is likely mediated through the PPARγ pathway.
- The study provides a model for understanding lipogenesis regulation in bovine mammary epithelial cells.
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