Abstract
Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from − 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.
Highlights
Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase[1] (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism
After 48 h, green fluorescence was observed under fluorescence inverted microscope (Leica, DMI4000B, Germany), which suggested that the pEGFP-C1-LPIN1 was successfully transfected into the buffalo mammary epithelial cells (BMECs) (Fig. 2A)
The overexpression of LPIN1 gene significantly increased the expression of Peroxisome proliferator-activated receptor gamma (PPARG)(~ 4.5 fold) and SREBF1 (~ 13.5 fold) genes related to the regulation of fatty acid metabolism, but had no significant effect on the expression of SREBF2 (P > 0.05; Fig. 2C)
Summary
Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase[1] (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs. Phosphatidic acid phosphohydrolase[1] (LPIN1), as a member of the lipid protein family, was identified during positional cloning of the mutant gene underlying lipodystrophy in the fatty liver dystrophy (fld) m ouse[1]. We hypothesized that LPIN1 plays an important role in the milk fat synthesis of buffalo mammary epithelial cells (BMECs), and that PPARG can regulate its transcription by binding to the potential PPREs in the promoter region of the buffalo LPIN1 gene. This study will provide strong evidence to clarify the role of LPIN1 in the synthesis of buffalo milk fat
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