Objective Acyl-CoA cholesterol acyltransferase 2 (ACAT2) is a key enzyme involved in cholesteryl ester synthesis. This study aimed to elucidate its regulatory mechanism in milk fat synthesis in bovine mammary epithelial cells (bMECs).

Method A lactation model of bMECs was established to characterize the temporal expression pattern of ACAT2. Subsequently, ACAT2 overexpression and interference experiments were performed in bMECs. The mRNA expression levels of genes related to milk fat synthesis, cell proliferation, and apoptosis were analyzed by quantitative real-time PCR (qRT-PCR). In addition, cell proliferation rate, apoptosis rate, and intracellular contents of triacylglycerol (TAG) and cholesteryl esters (CE) were measured.

Result Overexpression of ACAT2 significantly upregulated the mRNA expression of key milk fat synthesis-related genes, including ACACA, FASN, SREBP1, and SCD1 (P < 0.05). Meanwhile, the expression of the proliferation-related gene PCNA and the anti-apoptotic gene BCL2 was markedly increased (P < 0.01), whereas the expression of the pro-apoptotic gene BAX was significantly decreased (P < 0.05). At the phenotypic level, ACAT2 overexpression resulted in a significant increase in intracellular TAG and CE contents (P < 0.01), a markedly enhanced cell proliferation rate (P < 0.01), and a reduced apoptosis rate (P < 0.05). In contrast, interference with ACAT2 expression produced opposite effects.

Conclusion ACAT2 acts as a positive regulator of milk fat synthesis in bMECs. By promoting cell proliferation, suppressing apoptosis, and enhancing the expression of key lipogenic genes, ACAT2 facilitates intracellular lipid accumulation. These findings provide a basis for elucidating the molecular mechanisms underlying milk fat synthesis and for molecular breeding in dairy cattle.