VPS28 regulates triglyceride synthesis via ubiquitination in bovine mammary epithelial cells

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 critical system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in the regulation of 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 line (MAC-T) and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results showed that VPS28 knockdown significantly upregulated the levels of the fatty acid transporter CD36 molecule (CD36) and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, along with elevated ubiquitin (UB) levels, while reducing proteasome activity. In contrast, VPS28 overexpression increased CD36 levels while not significantly affecting ADFP or TG levels, with a trend toward reduced lipid droplets and increased UB expression and proteasome activity. In addition, inhibition of the ubiquitin-proteasome system and the endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, further increased CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo in a mouse model, where VPS28 knockdown increased mammary CD36, ADFP, UB expression, TG content, and lipid droplets 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 cell model.

Keywords: MAC-T cells; Mouse model; Triglyceride; Ubiquitination; VPS28.

Fig. 1

The cell vitality in each group. Treatments were replicated 4 times. The values are means ± SEM. VPS28^−/−: VPS28 knockdown. VPS28^+/+: VPS28 overexpression. **Denote significant differences (P < 0.01, respectively).

Fig. 2

The effects of VPS28 on the MAC-T cells. (A) The relative protein expression levels. (B) The relative TG content. (C) The Nile Red staining. (D) The three activities of proteasome. CT: control. VPS28^−/−: VPS28 knockdown. VPS28^+/+: VPS28 overexpression. All treatments were independently replicated three times. Data are presented as means ± SEM. Significance levels: * and ** indicate significant differences (P < 0.05, P < 0.01, respectively).

Fig. 3

The effect of proteasome and lysosome inhibition in MAC-T cells. (A) The three activities of proteasome. (B) The relative protein expression levels. (C) The relative TG content. (D) The Nile Red staining. CT: control. EXM: inhibited proteasome activity using epoxomicin. CQ: inhibited lysosomal activity using chloroquine. All treatments were independently replicated three times. Data are presented as means ± SEM. Significance levels: * and ** indicate significant differences (P < 0.05, P < 0.01, respectively).

Fig. 4

Body weight changes of mice in each group (n = 6 per group). Data was collected by weighing each mouse in the treatment groups at regular intervals throughout the study. The error bars represent SD. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey’s test.

Fig. 5

The impact of intraperitoneal injection of VPS28 knockdown on mice. (A) Western blot assay was conducted after VPS28 knockdown to assess CD36, ADFP, and UB protein expression in the mammary gland of mice. (B, C) The levels of TG in the mammary gland and blood of mice were measured (n = 3, error bars represent SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey’s test). (D, E) Representative images illustrating the morphology of the mammary gland using HE staining and Oil Red staining were obtained for each treatment group (n = 3 per group).

Fig. 6

The impact of intraperitoneal injection of EXM on mice was investigated. (A) Western blot assay was conducted after EXM injection to assess changes in CD36, ADFP, and UB protein expression in the mammary gland of mice. (B, C) The levels of TG in the mammary gland and blood of mice were measured (n = 3, error bars represent SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey’s test). (D, E) Representative images illustrating the morphology of the mammary gland using HE staining and Oil Red staining were obtained for each treatment group (n = 3 per group).

Fig. 7

The impact of intraperitoneal injection of CQ on mice was investigated. (A) Western blot assay was conducted after CQ injection to assess changes in CD36, ADFP, and UB protein expression in the mammary gland of mice. (B, C) The levels of TG in the mammary gland and blood of mice were measured (n = 3, error bars represent SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey’s test). (D, E) Representative images illustrating the morphology of the mammary gland using HE staining and Oil Red staining were obtained for each treatment group (n = 3 per group).