a-protocol-for-size-based-enrichment-lipid-droplets-from-bovine

A Protocol for Size-Based Enrichment of Lipid Droplets from Bovine Mammary Epithelial Cells and Mammary Tissues

This protocol describes a sequential differential centrifugation approach to enrich lipid droplets (LDs) of different sizes from bovine mammary epithelial ...

Henan Agricultural University

Phosphatidylcholine cytidine transferase α (CCTα) is a key rate-limiting enzyme in the CDP-choline pathway, the primary pathway for phosphatidylcholine (PC) synthesis in mammals. This study investigated the role of CCTα in lipid droplet (LD) formation, phospholipid synthesis, LD fusion, and lipophagy in bovine mammary epithelial cells (BMECs) through CCTα gene knockout (CCT-KO) and overexpression (CCT-OE). CCTα mRNA expression was significantly increased in bovine mammary gland tissue after lactation. In BMECs, CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on LD surfaces in the presence of linoleic acid. Compared with normal BMECs (NC), CCTα knockout (CCT-KO) cells had significantly greater LD diameters (1.53 μm vs. 1.68 μm,  < 0.05), lower proportions of small LDs (<1 µm; 11.39% vs. 5.42%), and higher proportions of large LDs (>3 µm; 0.67% vs. 2.88%). In contrast, CCTα overexpression (CCT-OE) decreased the diameter of LDs to 1.18 μm ( < 0.01), increased the proportion of small LDs to 35.48%, and decreased the proportion of large LDs to 0.24%. CCTα knockout significantly decreased the PC content and the ratio of PC to PE, whereas CCTα overexpression increased the PC content and the ratio of PC to phosphatidyl ethanolamine (PE) ( < 0.05). The lipidomics analysis indicated that PC synthesis was significantly influenced by CCTα gene expression. Live cell observations showed that CCTα knockout promoted the fusion of small LDs into large LDs. In cells with CCT α overexpression, the expression of the microtubule-associated protein 1 light chain 3 (LC3) protein and the number of lysosomes was elevated, and the lysosomal phagocytosis of LDs was observed through transmission electron microscopy, thus indicating that CCTα overexpression enhanced lipophagy. In conclusion, these results suggest that CCTα plays a role in regulating LD formation by influencing PC synthesis, LD fusion, and lipophagy in BMECs.

Phosphatidylcholine Cytidine Transferase α (CCTα) Affects LD Formation Through Fusion and Lipophagy in Bovine Mammary Epithelial Cells.

The size of fat globules in milk, which is influenced mainly by the size of lipid droplets (LDs) in bovine mammary epithelial cells, affects the quality of the milk. The present study aimed to explore how lipophagy affected the catabolism of LDs in bovine mammary epithelial cells.

Lipophagy modifies the catabolism of intracellular triglycerides by preferentially targeting small-sized lipid droplets in bovine mammary epithelial cells.

Milk fat globules (MFGs) are derived from lipid droplets (LDs) in the mammary epithelial cells of ruminants and released into milk. This study aimed to detect the differences in LDs and MFGs to reveal the mechanism by which LDs transform into MFGs in the mammary cells of dairy goats. The mammary gland tissue and milk of lactating dairy goats were collected and analyzed using lipidomics. The results showed that a total of 1167 and 675 lipid molecules were detected in the LDs and MFGs, respectively. The top four lipids in terms of their contents in the LDs were triglycerides (TGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and diglycerol (DG), whereas the top four in the MFGs were TG, PE, PC, and phosphatidylinositol (PI). Furthermore, compared to the LDs, the contents of TG and DG in the MFGs were significantly increased ( < 0.05), while PC content was significantly decreased ( < 0.05). We also found that diglycerol (16:0:18), diglycerol (O-19:0:16), triglycerides (14:0:0_16:0), and phosphatidylethanolamine (P-15:0:20:3) were the key characteristic lipids in the transformation of LDs into MFGs. This study reveals the unique lipid profiles of LDs and MFGs and provides a theoretical basis for understanding the mechanism behind low-fat milk syndrome.

Article	Year
Phosphatidylcholine Cytidine Transferase α (CCTα) Affects LD Formation Through Fusion and Lipophagy in Bovine Mammary Epithelial Cells. International journal of molecular sciences\| PubMed ID: 40076755	2025
Lipophagy modifies the catabolism of intracellular triglycerides by preferentially targeting small-sized lipid droplets in bovine mammary epithelial cells. Journal of the science of food and agriculture\| PubMed ID: 40944479	2026
Lipidomics Reveals Differences in Lipid Composition Between Lipid Droplets and Milk Fat Globules in Dairy Goat Mammary Tissue. Animals : an open access journal from MDPI\| PubMed ID: 41302011	2025

Yuxin Fan

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