miR-148a and miR-17–5p synergistically regulate milk TAG synthesis via <i>PPARGC1A</i> and <i>PPARA</i> in goat mammary epithelial cells

<p>MicroRNA (miRNA) are a class of ‘18–25’ nt RNA molecules which regulate gene expression and play an important role in several biologic processes including fatty acid metabolism. Here we used S-Poly (T) and high-throughput sequencing to evaluate the expression of miRNA and mRNA during early-lactation and in the non-lactating (“dry”) period in goat mammary gland tissue. Results indicated that miR-148a, miR-17–5p, <i>PPARGC1A</i> and <i>PPARA</i> are highly expressed in the goat mammary gland in early-lactation and non-lactating periods. Utilizing a Luciferase reporter assay and Western Blot, <i>PPARA</i>, an important regulator of fatty acid oxidation, and <i>PGC1a (PPARGC1A)</i>, a major regulator of fat metabolism, were demonstrated to be targets of miR-148a and miR-17–5p in goat mammary epithelial cells (GMECs). It was also revealed that miR-148a expression can regulate <i>PPARA</i>, and miR-17–5p represses <i>PPARGC1A</i> in GMECs. Furthermore, the overexpression of miR-148a and miR-17–5p promoted triacylglycerol (TAG) synthesis while the knockdown of miR-148a and miR-17–5p impaired TAG synthesis in GMEC. These findings underscore the importance of miR-148a and miR-17–5p as key components in the regulation of TAG synthesis. In addition, miR-148a cooperates with miR-17–5p to regulate fatty acid metabolism by repressing <i>PPARGC1A</i> and <i>PPARA</i> in GMECs. Further studies on the functional role of miRNAs in lipid metabolism of ruminant mammary cells seem warranted.</p

Akt Serine/Threonine Kinase 1 Regulates <i>de Novo</i> Fatty Acid Synthesis through the Mammalian Target of Rapamycin/Sterol Regulatory Element Binding Protein 1 Axis in Dairy Goat Mammary Epithelial Cells

Akt serine/threonine kinase acts as a central mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, regulating a series of biological processes. In lipid metabolism, Akt activation regulates a series of gene expressions, including genes related to intracellular fatty acid synthesis. However, the regulatory mechanisms of Akt in dairy goat mammary lipid metabolism have not been elaborated. In this study, the coding sequences of goat <i>Akt1</i> gene were cloned and analyzed. Gene expression of <i>Akt1</i> in different lactation stages was also investigated. For <i>in vitro</i> studies, a eukaryotic expression vector of <i>Akt1</i> was constructed and transfected to goat mammary epithelial cells (GMECs), and specific inhibitors of Akt/mammalian target of rapamycin (mTOR) signaling were applied to GMECs. Results showed that Akt1 protein was highly conserved, and its mRNA was highly expressed in midlactation. <i>In vitro</i> studies indicated that Akt1 phosphorylation activated mTOR and subsequently enhanced sterol regulatory element binding protein 1 (SREBP1), thus increasing intracellular triacylglycerol content. Inhibition of Akt/mTOR signaling down-regulated the gene expression of lipogenic genes. Overall, Akt1 plays an important role in regulating <i>de novo</i> fatty acid synthesis in goat mammary epithelial cells, and this process probably is through the mTOR/SREBP1 axis