Abstract
Simple SummaryThe levels of milk fats and proteins are important indexes used to evaluate milk quality. Generally, feed additives are used to improve milk quality. This study aimed to investigate the effect of β-sitosterol on milk fat and protein gene expression in bovine mammary epithelial cells. β-sitosterol increased the β-casein levels in bovine mammary epithelial cells and promoted the expression of milk fat and protein synthesis-related genes, suggesting the use of β-sitosterol as a potential feed additive to improve milk quality in dairy cows.β-sitosterol, a phytosterol with multiple biological activities, has been used in the pharmaceutical industry. However, there are only a few reports on the use of β-sitosterol in improving milk synthesis in dairy cows. This study aimed to investigate the effects of β-sitosterol on milk fat and protein syntheses in bovine mammary epithelial cells (MAC-T) and its regulatory mechanism. MAC-T cells were treated with different concentrations (0.01, 0.1, 1, 5, 10, 20, 30, or 40 μM) of β-sitosterol, and the expression levels of milk protein and fat synthesis-related genes and proteins were analyzed. β-sitosterol at 0.1, 1, and 10 μM concentrations promoted the mRNA and protein expression of β-casein. β-sitosterol (0.1, 1, 10 μM) increased the mRNA and protein expression levels of signal transducer activator of transcription 5 (STAT5), mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (S6K1) of the JAK2/STAT5 and mTOR signaling pathways. It also stimulated the milk fat synthesis-related factors, including sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor-gamma (PPARγ), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL), and stearyl CoA desaturase (SCD). β-sitosterol (0.1, 1, 10 μM) also significantly increased the expression of growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and hypoxia-inducible factor-1α (HIF-1α)-related genes. Notably, the compound inhibited the expression of the negative regulator, the suppressor of cytokine signaling 2 (SOCS2) at the two lower concentrations (0.1, 1 μM), but significantly promoted the expression at the highest concentration (30 μM). These results highlight the role of β-sitosterol at concentrations ranging from 0.1 to 10 μM in improving milk protein and fat syntheses, regulating milk quality. Therefore, β-sitosterol can be used as a potential feed additive to improve milk quality in dairy cows.
Highlights
The effect of β-sitosterol on the mRNA expression of β-casein in MAC-T cells was determined by Quantitative Real-Time PCR (qPCR) (Figure 1)
The mRNA expression of β-casein in MAC-T cells treated with β-sitosterol at low concentrations (0.1, 1, 5, 10 μM) was significantly upregulated compared to the control (p < 0.05, Figure 1a)
1 μM β-sitosterol significantly increased the p-signal transducer activator of transcription 5 (STAT5)-β protein levels (p < 0.05; Figure 2c,d), consistent with the mRNA levels, while 30 μM β-sitosterol did not affect the p-STAT5-β protein levels in MAC-T cells (Figure 2c,d). These results suggested that β-sitosterol enhanced JAK2/STAT5 signaling pathway expression
Summary
Mammary epithelial cells synthesize caseins and whey proteins in milk [1]. Hormones, and nutrients regulate milk protein synthesis [2,3]. Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) and phosphoinositide 3-kinases/RAC-alpha serine/threonine-protein kinase/mammalian target of rapamycin (PI3K/AKT1/mTOR) pathways are the major pathways regulating milk protein synthesis in mammary epithelial cells [4]. Regulatory elements and enzymes such as sterol regulatory element-binding protein family (SREBPs), stearoylcoenzyme A desaturase (SCD), and fatty acid synthase (FASN) regulate fatty acid synthesis by influencing de novo fat synthesis and absorption and fatty acid intake [5,6]
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