Perilipin1 Promotes the Synthesis of Milk Fat by Regulating the Activity of SREBP1 in Bovine Mammary Epithelial Cells


 BackgroundMilk fat content is an important index of milk product quality and one of the main traits of dairy cattle breeding. Perilipin1 is a predominant binding protein that mainly surrounds lipid droplets. Perilipin1 is important in the regulation of lipid metabolism. SREBP1 is a transcription factor that controls the expression of a variety of lipogenic genes and is the main regulator of milk fat synthesis. Here, we investigated the effect and mechanism of Perilipin1 on milk fat synthesis in bovine mammary epithelial cells ( BMECs ). ResultsWe found that the number and volume of lipid droplets increased following periplipin1 overexpression, leading to increased triglyceride accumulation, increased relative expression of lipid synthesis-related genes, decreased expression of lipid lipolysis genes, and increased SREBP1 activity . On the contrary, periplipin1 silencing reduced the number of lipid droplets, inhibited the synthesis of triglycerides, decreased the relative expression of lipid synthesis-related genes, increased the expression of lipid lipolysis genes, and downregulated the activity of SREBPI. ConclusionsPerilipin1 promotes the synthesis of milk fat via up-regulating the activity of SREBP1 in BMECs. These findings laid the foundation for Holstein dairy cows to increase their milk fat content in molecular breeding.


Background
Milk is rich in fat, protein, lactose, multiple vitamins, and mineral elements. It is widely referred to as "white blood" as it provides most of the nutrients required for human life.
Fat is the main energy component in milk, determines many of the organoleptic qualities, manufacturing characteristics, and physical properties of milk and its products [1]. Therefore, milk fat is also considered to be the main target trait in dairy cattle breeding [2]. Milk fat is synthesized in breast epithelial cells (BMECs), and its synthesis process and regulation mechanism are extremely complex [3], it is coordinated and regulated by a variety of factors such as genetics, hormones, physiology, environment, and nutritional levels [3][4][5], while genetic factors are the core factors that determine milk fat synthesis in dairy animals [6]. A large number of genes related to milk fat metabolism have been identified, including LPL, FABP3, ACSL1, ACCα, FASN, SCD, FADS1, AGPAT6, LPIN1, PLIN2, Par, and SREBP1 [5,7], however, our current understanding of the regulation on milk fat synthesis remains limited, it is necessary to further in-depth study and analysis.
As we all know, the PAT protein family is the most abundant protein in lipid droplets (LDs), with highly conserved similar sequences, it has an affinity for the surface of intracellular neutral lipid storage droplets and plays an important role in the biosynthesis and decomposition of LDs [8][9][10]. As a "molecular switch" for regulating lipid metabolism, perilipin1 (PLIN1) is a member of this family, has three subtypes, namely, PLIN1A, B, and C, they have a common N-terminal region but different Cterminal lengths; PLIN1A is the most abundant and is often referred to as PLIN1 [11,12]. The LD coating protein encoded by the PLIN1 is the most abundant protein on the surface of LDs, it binds to lipid droplets in cells and promotes triglyceride (TAG) synthesis in fat cells, increasing the number and volume of LDs, resulting in large LDs [13][14][15]. TAG can be hydrolyzed into diglycerides (also referred to as diacylglycerol, DAG) by fatty triglyceride lipase (ATGL) and further hydrolyzed to glycerol by hormone-sensitive lipase (HSL) [16]. PLIN1 inhibits the basic lipolysis of ATGL and HSL in adipocytes [11,17,18]. However, under starvation and catabolic conditions, PLIN1 supports TAG breakdown by lipase [19,20]. These results show that the PLIN1 plays an important role in LD biosynthesis and lipid metabolism. Therefore, based on the above findings, we used BMECs to explore the regulatory role of PLIN1 in milk fat.
Sterol regulatory element-binding transcription protein 1 (SREBP1, gene name SREBF1) is a transcription factor that plays an important role in cholesterol biosynthesis and fatty acid metabolism, specifically fat biosynthesis. SREBP1 plays an important role in the regulation of milk fat in BMECs [21], and acts as a key positive regulator in the synthesis of milk fat [22]. Many studies have shown that SREBP1 is regulated by many genes [23][24][25][26], and a research has shown that PLIN1 may activate SREBP1 in lipid metabolism [27]. Consequently, we hypothesized that PLIN1 could activate SREBP1 and plays an important role in milk fat synthesis and metabolism in BMECs.

Cell culture and transfection
Mammary epithelial cells were extracted from the mammary gland parenchyma of 4 mid-lactating Holstein cows, as previously described [2,3]. BMECs from the breast tissue were separated by collagenase digestion [3]. Cells were cultured in DMEM/F12 (Gibco, 12500062) complete medium containing 10% fetal bovine serum, 100 μg/mL streptomycin, 100 μg/mL penicillin culture BMEC in the medium. We incubated the cells in a humidified environment of 37°C, 95% air, and 5% CO2 for subsequent experiments. When the cells reach 80% confluence, we used 0.25% trypsin for digestion. Pure breast epithelial cells were isolated after 3-4 generations. Forty-eight hours before cell treatments, we replaced the complete medium with a milk production medium. The cells were seeded in a 6-well plate, and the cells were transfected when the cells reached 50-60% confluency, and the samples were collected after 48 hours for subsequent experiments. All experiments were processed in parallel in triplicate.

Total RNA extraction and real-time PCR
TRIzol reagent (Sigma, Louis) was used to extract total RNA of differently processed

Triacylglycerol assay
We determined the intracellular TAG content in BMECs after 48 h of transfection using the cell/tissue TAG analysis kit (Applygen Technologies). The total protein concentration was measured using the BCA kit (Takara), to calibrate the TAG content.
The TAG values were expressed in micrograms per milligram of protein. All the above operations were performed per the agreement issued by the above manufacturer.

Glycerol assay
The content of intracellular glycerol in BMECs after 48 h of transfection was measured with a tissue cell glycerase assay kit (Applygen Technologies). The total protein concentration measured by the BCA kit (Takara) was used to calibrate the glycerol content. The glycerol values were expressed in micrograms per milligram of protein.
All operations were carried out per the freehand notice issued by the manufacturer.

Western blotting
The total protein in BMECs was collected after 48 h of transfection. The cells were digested from the 6-well plate with 0.25% trypsin (Solarbio), and then the digestion was terminated with DMEM/F12 (Gibco, 12500062) complete medium. We discarded the supernatant after centrifugation. Cell pellets were washed with PBS and supplemented with 200 μL of RIPA buffer containing 1% PMSF (Solarbio) and 10% phosphatase inhibitor cocktail (Roche). They were then placed on ice for lysis for 15 min. We collected 180 μL of supernatant, to be used for western blotting.

Statistical analysis
All experiments were processed in parallel, in triplicate. All the experimental data obtained were sorted by Office Excel and then analyzed by statistical software SPSS17.0. Data were expressed as means ± standard deviation (means ± SD). The independent sample t-test was used for comparative analysis. The P-value (*P < 0.05, **P < 0.01, ***P < 0.001) was used to indicate the significant differences. Figures were generated using the GraphPad Prism 6.01 software.

PLIN1 promotes milk fat synthesis in BMECs
We designed PLIN1 overexpression (op-PLIN1; Figure 1A) and PLIN1 interference (si-PLIN1; Figure 1B) vectors. We found that compared with the control group, op-PLIN1 significantly promoted LD formation ( Figure 1C) and resulted in an increase in TAG synthesis ( Figure 1E). Additionally, si-PLIN1 inhibited LD formation ( Figure 1F) and decreased TAG synthesis ( Figure 1D) compared with the control group. The data above showed that PLIN1 promoted milk fat synthesis in BMECs.

PLIN1 promotes the expression of lipid synthesis-related genes in BMECs
In the op-PLIN1 group, the relative mRNA expression of lipid synthesis-related genes increased (Figure 2A). In the si-PLIN1 group, the relative mRNA expression of lipid synthesis-related genes decreased ( Figure 2B). These data suggest that PLIN1 promoted the expression of lipid synthesis-related genes in BMECs.

PLIN1 inhibits lipid lipolysis in BMECs
Compared with the control group, op-PLIN1 reduced the mRNA expression of HSL and ATGL ( Figure 3A) and caused a significant decrease in the glycerol content of BMECs ( Figure 3C). Additionally, compared with the control group, si-PLIN1 promoted the mRNA expression of HSL and ATGL ( Figure 3B) and led to a decrease in glycerol content in BMECs ( Figure 3D). Based on these results, we concluded that PLIN1 inhibited lipid lipolysis in BMECs.

PLIN1 improves SREBP1 activity in BMECs
To further clarify the regulation mechanism of PLIN1 in milk fat synthesis in BMECs, we tested the relative mRNA and protein expressions of SREBP1. We found that PLIN1 did not affect SREBP1 mRNA levels ( Figure 4A, B). WB results showed that the PLIN1 did not affect the total protein of SREBP1, however, had a significant effect on phosphorylated SREBP1. The expression of phosphorylated SREBP1 in the op-PLIN1 group increased significantly compared with the control group ( Figure 4C). The expression of phosphorylation in the si-PLIN1 group was significantly decreased ( Figure 4D). The above experimental results suggested that PLIN1 increased SREBP1 activity in BMECs.

PLIN1 promotes milk fat synthesis in BMECs by increasing SREBP1 activity
To verify the role of SREBP1 in the regulation of milk fat synthesis by PLIN1, we cotransfected op-PLIN1 and si-SREBP1 (op-PLIN1&si-SREBP1) in BMECs. We found that the relative expression of PLIN1 increased in the op-PLIN1&si-SREBP1 group in BMECs, and SREBP1 decreased ( Figure 5A). Staining with oil red O found that in the op-PLIN1 group, the number of lipid droplets was significantly increased compared with the corresponding control group. The LD number was significantly reduced in the si-PLIN1 group, and in the op-PLIN1&si-SREBP1 group, the number was roughly the same as the number of the control group in the first two groups ( Figure 5C). We further verified this trend by using the TAG enzymatic assay technology ( Figure 5B). The WB results further showed that in the op-PLIN1&si-SREBP1 groups, the expression level of phosphorylated SREBP1 was comparable to that of the control group ( Figure 5D).
These results verified that PLIN1 regulated milk fat synthesis by regulating SREBP1 activity, in BMECs.

Discussion
Milk fat content is an important indicator of milk quality and dairy cattle breeding, with TAG as its main component. It is well known that PLIN1 is highly expressed in white fat, can promote the accumulation of TAG in adipocytes, and plays an important role in lipid biosynthesis and lipid metabolism of adipocytes [3,19]. However, the effect of PLIN1 on milk fat synthesis and its regulatory mechanism remains unclear in BMECs. Therefore, we mainly explored the adipogenic effect and potential mechanism of PLIN1 in BMECs.
PLIN1 is the most abundant protein associated with the LDs of adipocytes and surrounds them [28,29]. PLIN1 prevents basal lipolysis, increases LD formation and TAG accumulation under basal conditions [30]. PLIN1 knockout increases basal lipolysis, reduces the size of LD in adipocytes, and inhibits the accumulation of TAG [31]. We found that PLIN1 promotes the accumulation of TAG in BMECs, consistent with previous results in fat cells of mice and cattle [19,20,28,30].
Additionally, we found that PLIN1 inhibited the expression of HSL and ATGL. As the two most abundant enzymes in adipocytes, HSL and ATGL, have significant hydrolytic activities on different stages of TAG [19,20]. Once PLIN1 is phosphorylated, it actively promotes the action of lipase, predominantly by transporting HSL to the surface of LDs.
As a result, PLIN1 knockouts increase basal lipolysis and reduce LDS size in adipocytes [32], and studies have shown that lack of ATGL leads to lipid accumulation [33,34].
Therefore, we concluded that PLIN1 promoted the synthesis of milk fat by promoting and inhibiting the expression of lipid synthesis-related and lipolysis genes, respectively, in BMECs.
Interestingly, we found that PLIN1 has no affect the expression of SREBP1, however, it can promote its activity. In mice, the fat marker gene SREBP1 remains unchanged; however, its activity was significantly reduced following the knockout of PLIN1 [27,41,42]. In mouse mammary glands, SREBP was shown to be a key molecule in the process of milk fat synthesis [43]. SREBP1 promotes milk fat synthesis and increases glucose transport by upregulating GLUT1 [44]. SREBP1 regulates lipid synthesis by regulating the transcription of SCD1, FABP3, FASN, and other coding genes in BMECs [45]. Collectively, we found that PLIN1 regulated the transcription of lipid genes by promoting SREBP1 activity, thereby affecting the synthesis of milk fat in BMECs.

Conclusions
Our findings showed that PLIN1 regulates the synthesis of milk fat by affecting the activity of SREBP1, which in turn affects the transcription of lipid-related genes in BMECs. These results provide a novel direction or ways to improve milk fat content in dairy cows through molecular breeding.