evaluation-of-lipid-droplet-size-and-fusion-in-bovine-hepatic-cells

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

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

Milk fat globule membrane (MFGM) proteins surround the triacylglycerol core comprising milk fat globules (MFG). We previously detected a decrease in the size of fat globules during conjugated linoleic acid (CLA)-induced milk fat depression (MFD), and other studies have reported that some MFGM proteins play a central role in regulating mammary cellular lipid droplet size. However, little is known about the relationship between MFD, MFG size, and MFGM proteins in bovine milk. The aim of this study was to investigate the profile of MFGM proteins during MFD induced by CLA. Sixteen mid-lactating Holstein cows (145 ± 24 d in milk) with similar body condition and parity were divided into control and CLA groups over a 10-d period. Cows were fed a basal diet (control, n = 8) or control plus 15 g/kg of dry matter (DM) CLA (n = 8) to induce MFD. Cow performance, milk composition, and MFG size were measured daily. On d 10, MFGM proteins were extracted and identified by quantitative proteomic analysis, and western blotting was used to verify a subset of the identified MFGM proteins. Compared with controls, supplemental CLA did not affect milk production, DM intake, or milk protein and lactose contents. However, CLA reduced milk fat content (3.73 g/100 mL vs. 2.47 g/100 mL) and the size parameters volume-related diameter D (3.72 μm vs. 3.35 μm) and surface area-related diameter D (3.13 μm vs. 2.80 μm), but increased specific surface area of MFG (1,905 m/kg vs. 2,188 m/kg). In total, 177 differentially expressed proteins were detected in milk from cows with CLA-induced MFD, 60 of which were upregulated and 117 downregulated. Correlation analysis showed that MFG size was negatively correlated with various proteins, including XDH and FABP3, and positively correlated with MFG-E8, RAB19, and APOA1. The results provide evidence for an important role of MFGM proteins in regulating MFG diameter, and they facilitate a mechanistic understanding of diet-induced MFD.

Milk fat globule membrane proteins are involved in controlling the size of milk fat globules during conjugated linoleic acid-induced milk fat depression.

Milk fat globules (MFGs) surround the triacylglycerol core that composes milk fat. The aim of this study is to induce milk fat depression via dietary conjugated linoleic acid (CLA) supplementation to study MFG size parameters, number and glycerophospholipid composition. Eighteen Holstein dairy cows (136 ± 28 days in milk, 571 ± 37.9 kg body weight, 27.6 ± 2.1 kg milk/day) were selected and randomly assigned to a control or CLA group for a 14-day period. Cows were fed a basal diet (control, n = 8) or the control plus 400 g/day CLA (C18:2 cis-9, trans-11 38.1% and C18:2 trans-10, cis-12 36.8%) (n = 10) for 7 days after which the CLA group was switched to the basal diet for another 7 days along with the control group. Cow performance, milk composition, MFG size and numbers were measured daily. On the seventh day after the start of the experiment, milk samples were identified and the quantification of glycerophospholipid compounds, and RNA were isolated from milk fat samples for a real-time polymerase chain reaction. Compared with control, at Day 7 from the start of feeding, supplemental CLA did not affect milk production (28.09 vs. 28.50 kg/day), dry matter intake (14.9 vs. 15.4 kg/day), or milk protein (3.55/100 vs. 3.70 g/100 ml) and lactose contents (5.11/100 vs. 5.17 g/100 ml). However, although the specific surface area of MFG (2138 vs. 1815 m²/kg) was greater, CLA reduced milk fat content (1.95/100 vs 3.64 g/100 ml on Day 7) and particle size parameters of MFG. The number of MFG gradually decreased until Day 7 of feeding, and then increased by Day 14 (2.96 × 10 on Day 1, 1.63 × 10 on Day 7 and 2.28 × 10 on Day 14) in the CLA group. Compared with control, glycerophospholipid analysis revealed that concentrations of phosphatidylcholine (PC) (e.g., PC [16:0/18:1] 20322 vs. 29793 nmol/L), lysophosphatidylethanolamine (LPE) (e.g., LPE [18:1] 956 vs. 4610 nmol/L) and phosphatidylethanolamine (PE) (e.g., PE [16:0/18:1] 7000 vs. 9769 nmol/L) in milk lipids decreased during CLA feeding. In contrast, concentrations of phosphatidylinositol (PI) (e.g., PI [18:0/18:1] 4052 vs. 1799 nmol/L) and phosphatidylserine (PS) (e.g., PS [18:1/18:2] 9500 vs. 6843 nmol/L) increased. The messenger RNA abundance of fatty acid synthase, diacylglycerol O-acyltransferase 1, glycerol-3-phosphate acyltransferase 4 and phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) were downregulated in the CLA group, confirming published data demonstrating a negative effect of CLA on lipogenesis in the mammary gland. Overall, these results provided evidence for the important role of lipogenic gene expression in the regulation of MFG size, number and glycerophospholipid composition.

Size, number and phospholipid composition of milk fat globules are affected by dietary conjugated linoleic acid.

In mammary epithelial cells, milk fat is synthesized as lipid droplets and secreted in the form of globules. Milk fat globules (MFGs) are covered by a lipid-protein membrane known as the milk fat globule membrane (MFGM). We randomly divided 12 Holstein cows into control and conjugated linoleic acid (CLA) groups. The control group was fed a basal diet, while the CLA group was fed the basal diet + CLA (15 g per kg DM) for 10 days. Cow performance, milk composition, and MFG size were measured daily. On day 10, we extracted MFGM proteins ( = 3) and identified them  quantitative proteomic analysis. We investigated the effects of the MFGM proteins from control and CLA-treated milk on the lipid droplet formation in MAC-T cells. Compared with the control group, the CLA group had reduced milk fat content (3.39 g/100 mL  2.45 g/100 mL) and MFG size parameters ( of 3.85 μm  3.37 μm;  of 3.24 μm  2.83 μm). The specific surface area (SSA) increased in the CLA group. A total of 361 differentially expressed proteins were identified in the CLA group by iTRAQ quantitative proteomic analysis. Among these proteins, 100 were upregulated and 251 were downregulated ( < 0.05). In MAC-T cells, CLA-MFGM proteins increased the diameter of the lipid droplets to 1.32 μm. CLA-MFGM proteins decreased the proportion of the small lipid droplets (15.33%  47.78%) and increased the proportion of the large lipid droplets (25.04%  11.65%). CLA-MFGM proteins promoted lipid droplet fusion. Therefore, MFGM proteins play an important role in the regulation of the lipid droplet size.

Proteomic analysis of milk fat globule membranes from small-sized milk fat globules and their function in promoting lipid droplet fusion in bovine mammary epithelial cells.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

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.

ArticleTotal : 2	Year
Publication title	2023
Publication title	2026

Article	Year
Milk fat globule membrane proteins are involved in controlling the size of milk fat globules during conjugated linoleic acid-induced milk fat depression. Journal of dairy science\| PubMed ID: 36175227	2022
Size, number and phospholipid composition of milk fat globules are affected by dietary conjugated linoleic acid. Journal of animal physiology and animal nutrition\| PubMed ID: 36353940	2022
Proteomic analysis of milk fat globule membranes from small-sized milk fat globules and their function in promoting lipid droplet fusion in bovine mammary epithelial cells. Food & function\| PubMed ID: 36752527	2023
Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro. Journal of dairy science\| PubMed ID: 37678795	2023
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

Jingna Yang

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