The Knockout of the ASIP Gene Altered the Lipid Composition in Bovine Mammary Epithelial Cells via the Expression of Genes in the Lipid Metabolism Pathway

SIMPLE SUMMARY: According to the FDA, a litre of milk contains about 33 g of total lipids, 95% of which are triacylglycerols made up of varying lengths and saturation of fatty acids. The increase in free fatty acids in milk may lead to changes in the flavour of milk and milk products. Bovine mammary epithelial cells (bMECs) function to synthesize and secrete milk and are good in vitro models for cells in milk quality research. In this research, we report for the first time the effect of the ASIP gene knockout in bovine mammary epithelial cells using CRISPR/Cas9 technology. We found that ASIP knockout could down-regulate the expression of PPARγ, FASN, and SCD, thus affecting the saturation of fatty acids in milk and upregulating the expression of FABP4, ELOVL6, and ACSL1, increasing the synthesis of medium-long chain fatty acids. These results may allow the selection of potential targets in future molecular breeding efforts for dairy cows. ABSTRACT: Agouti signalling protein (ASIP) is a coat colour-related protein and also is a protein-related to lipid metabolism, which had first been found in agoutis. According to our previous study, ASIP is a candidate gene that affects the lipid metabolism in bovine adipocytes. However, its effect on milk lipid has not been reported yet. This study focused on the effect of the ASIP gene on the lipid metabolism of mammary epithelial cells in cattle. The ASIP gene was knocked out in bMECs by using CRISPR/Cas9 technology. The result of transcriptome sequencing showed that the differentially expressed genes associated with lipid metabolism were mainly enriched in the fatty acids metabolism pathways. Furthermore, the contents of intracellular triglycerides were significantly increased (p < 0.05), and cholesterol tended to rise (p > 0.05) in bMECs with the knockout of the ASIP gene. Fatty acid assays showed a significant alteration in medium and long-chain fatty acid content. Saturated and polyunsaturated fatty acids were significantly up-regulated (p < 0.05), and monounsaturated fatty acids were significantly decreased in the ASIP knockout bMECs (p < 0.05). The Q-PCR analysis showed that knockout of ASIP resulted in a significant reduction of gene expressions like PPARγ, FASN, SCD, and a significant up-regulation of genes like FABP4, ELOVL6, ACSL1, HACD4 prompted increased mid-to long-chain fatty acid synthesis. Overall, ASIP plays a pivotal role in regulating lipid metabolism in bMECs, which could further influence the component of lipid in milk.