ABSTRACT
Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21-encoded protein had PPAR ligand-binding domains (NR_LBD_PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes (ACACA, CD36, ACSL1, GPAT, AGPAT6, DGAT1) was significantly modified (p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21. All kinds of FAs detected in this study were significantly decreased (p < 0.05) after RNAi of PPARG-X17 or PPARG-X21. Overexpression of PPARG-X17 or PPARG-X21 significantly decreased (p < 0.05) the SFA content, while significantly increased (p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.
Subject(s)
Buffaloes , Mammary Glands, Animal , PPAR gamma , Animals , Buffaloes/genetics , Buffaloes/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Mammary Glands, Animal/metabolism , Female , Epithelial Cells/metabolism , Alternative Splicing , Fatty Acids/metabolism , Fatty Acids/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Milk/metabolismABSTRACT
During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPATs affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1, AGPAT3, and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1, AGPAT3, and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1, FASN, GPAM, DGAT2, and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.
