mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells

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.

1. Materials and methods 1.1 Isolation of primary bovine mammary epithelial cells Mammary tissues were obtained from 3 Chinese Holstein cows after their slaughter in a commercial cattle slaughter farm per time. The primary bovine mammary epithelial cells (pBMECs) were isolated and cultured by the adherent culture of small-sized cow mammary tissue. After mammary tissues were surgically removed from the slaughtered cow, they were placed in sterile, ice-cold phosphate-buffered saline (PBS) that was supplemented with 300 U/mL penicillin G and 100 mg/mL streptomycin (V900929,Inc. St. Louis,MO,USA) and transported immediately to the laboratory. The mammary tissues were trimmed of visible fat and connective tissue and washed with PBS several times until the solution became pellucid and devoid of milk. Then, the mammary tissues were cut into small pieces (approx. 1×1×1 mm 3 ) and established as a primary culture, from which bovine mammary epithelial cells (BMECs) were isolated. The isolated cells were purified by differential digestion method with trypsin, and purified cells were identified. Cell morphology was examined by light microscopy.

Isolation of primary bovine mammary fibroblasts
The bovine mammary fibroblasts were isolated and cultured by the adherent culture of small-sized cow mammary tissue. The isolated cells were purified by differential digestion method with trypsin, and purified bovine mammary fibroblasts were identified as negative control cells for detecting CSN2 and VIM (vimentin) by RT-PCR with the primers (Table S1), and for examination of CSN2 and VIM (vimentin) by immunofluorescence. Cell morphology was examined by light microscopy.
1.3 Transcription of KRT8, KRT18, CSN2 and VIM by RT-PCR in pBMECs and in bovine mammary fibroblasts Total RNA was isolated using RNAzol (9109, TaKaRa Co. Ltd., Dalian, China) from pBMECs and bovine mammary fibroblasts, respectively, and was reverse-transcribed with an oligo (dT)12-18 primer using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AE311, TransGen Biotech Co. Ltd., Beijing, China). The transcription of KRT8, KRT18 and CSN2 was detected by RT-PCR in pBMECs, and the transcription of VIM, CSN2 was detected by RT-PCR in bovine mammary fibroblasts. An input of 1 μg total RNA was used for each reaction.
cDNA sequences were amplified with the primers in Table S1. The RT-PCR program was as follows: 94°C for 3 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. The 10-µL PCR mixture contained 5 µL 2×TransTaq® HiFi PCR SuperMix II (AS131, TransGen Biotech Co. Ltd. Beijing, China), 0.5 µL 10 mM of each of the forward and reverse primers, 1 µL template cDNA, and 3 µL deionized water. The PCR products were electrophoresed, and photographs were taken on an UV transilluminator (UVItec, London, UK).

Results
Primary bovine mammary epithelial cells (pBMECs) and bovine mammary fibroblasts were isolated by the adherent culture of small-sized cow mammary tissues, respectively. The morphology of pBMECs was of a typical epithelial cell ( Figure S1 (a), (b)), and the morphology of these fibroblasts was of a typical fibroblast with a spindle shape and several protrusions ( Figure S1 (c)). The biomarker genes KRT18, KRT8, and CSN2 were transcribed ( Figure S1 (d)), and the biomarker proteins KRT7, KRT18, and CSN2 were expressed ( Figure S1 (e)) in pBMECs. The biomarker gene VIM and protein VIM (vimentin), which is a biomarker of fibroblasts, were expressed, while CSN2 and CSN2 was not expressed in bovine mammary fibroblasts ( Figure S1 (f, g)). (e) KRT7, KRT18, and CSN2 were expressed in pBMECs.
(f) VIM was transcribed, and CSN2 was not in bovine mammary fibroblasts. M: (g) VIM was expressed, and CSN2 was not in bovine mammary fibroblasts.