Abstract
VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs), and is involved in ubiquitination. Ubiquitination is a crucial system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in regulating lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell (MAC-T) line and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results indicated that VPS28 knockdown significantly upregulated the fatty acid transporter CD36 (CD36 molecule) and the adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, alongside elevated expression of ubiquitin (UB) protein and reduced proteasome activity. In contrast, VPS28 overexpression increased CD36 levels without significantly affecting ADFP and TG levels, showing a trend toward reduced lipid droplets and increased UB expression and proteasome activity. Furthermore, the inhibition of the ubiquitin-proteasome system and endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, resulted in a further elevation of CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo by a mouse model, where VPS28 knockdown enhanced CD36, ADFP, UB expression, TG content, and lipid droplets in mammary glands, without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine in vitro cell model.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- VPS28
- Vacuolar Protein Sorting 28
- MAC-T
- Bovine Mammary Epithelial Cell Line
- CD36
- Fatty Acid Transporter CD36
- ADFP
- Adipose Differentiation-Related Protein
- TG
- Triglyceride
- UB
- Ubiquitin
- UPS
- Ubiquitin-Proteasome System
- ELP
- Endosomal-Lysosomal Pathway
- ESCRT
- Endosomal Sorting Complexes Required for Transport
- EXM
- Epoxomicin
- CQ
- Chloroquine
- HE
- Hematoxylin-eosin
- BW
- body weight
- FBS
- Fetal Bovine Serum
- NC
- negative control
- WB
- Western Blot
- OCT
- Optimal Cutting Temperature.