Effects of L-Histidine and Sodium Acetate on β-Casein Expression in Nutrient-Restricted Bovine Mammary Epithelial Cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Kim J
PROVIDER: S-EPMC8157603 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Non-esterified fatty acids (NEFAs) are pivotal in energy metabolism, yet high concentrations can lead to ketosis, a common metabolic disorder in cattle. Our laboratory observed lower levels of L-histidine in cattle suffering from ketosis, indicating a potential interaction between L-histidine and NEFA metabolism. This relationship prompted us to investigate the metabolomic alterations in bovine mammary epithelial cells (BMECs) induced by elevated NEFA levels and to explore L-histidine's potential mitigating effects. Our untargeted metabolomic analysis revealed 893 and 160 metabolite changes in positive and negative models, respectively, with VIP scores greater than 1 and p-values below 0.05. Notable metabolites like 9,10-epoxy-12-octadecenoic acid were upregulated, while 9-Ethylguanine was downregulated. A pathway analysis suggested disruptions in fatty acid and steroid biosynthesis pathways. Furthermore, L-histidine treatment altered 61 metabolites in the positive model and 34 in the negative model, with implications for similar pathways affected by NEFA. Overlaying differential metabolites from both conditions uncovered a potential key mediator, 1-Linoleoylglycerophosphocholine, which was regulated in opposite directions by NEFA and L-histidine. Our study uncovered that both NEFA L- and histidine metabolomics analyses pinpoint similar lipid biosynthesis pathways, with 1-Linoleoylglycerophosphocholine emerging as a potential key metabolite mediating their interaction, a discovery that may offer insights for therapeutic strategies in metabolic diseases.
| S-EPMC11010852 | biostudies-literature
Project description:Despite the well-characterised mechanisms of amino acids (AA) regulation of milk protein synthesis in mammary glands (MG), the underlying specific AA regulatory machinery in bovine MG remains further elucidated. As methionine (Met) is one of the most important essential and limiting AA for dairy cows, it is crucial to expand how Met exerts its regulatory effects on dairy milk protein synthesis. Our previous work detected the potential regulatory role of seryl-tRNA synthetase (SARS) in essential AA (EAA)-stimulated bovine casein synthesis. Here, we investigated whether and how SARS participates in Met stimulation of casein production in bovine mammary epithelial cells (BMEC). With or without RNA interference against SARS, BMEC were treated with the medium in the absence (containing all other EAA and devoid of Met alone)/presence (containing 0·6 mm of Met in the medium devoid of Met alone) of Met. The protein abundance of ?-casein and members of the mammalian target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways was determined by immunoblot assay after 6 h treatment, the cell viability and cell cycle progression were determined by cell counting and propidium iodide-staining assay after 24 h treatment, and protein turnover was determined by l-[ring-3H5]phenylalanine isotope tracing assay after 48 h treatment. In the absence of Met, there was a general reduction in cell viability, total protein synthesis and ?-casein production; in contrast, total protein degradation was enhanced. SARS knockdown strengthened these changes. Finally, SARS may work to promote Met-stimulated ?-casein synthesis via affecting mTOR and GCN2 routes in BMEC.
| S-EPMC7015878 | biostudies-literature
Project description:This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose-galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.
| S-EPMC9184702 | biostudies-literature
Project description:Precise contact between epithelial cells and their underlying basement membrane is crucial to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG gene (Dag1) expression in cultured mammary epithelial cells. Strikingly, DG loss disrupted laminin-111-induced polarity and beta-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. Dystroglycan re-expression restored these deficiencies. Investigations of the mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in mammary epithelial cells that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity and beta-casein induction. The observed loss of laminin-111 assembly and signaling in Dag1(-/-) mammary epithelial cells provides insights into the signaling changes occurring in breast carcinomas and other cancers, where the binding function of DG to laminin is frequently defective.
| S-EPMC2996718 | biostudies-literature
Project description:MicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows.Here, we found that miR-139 was down-regulated in mid-lactation dairy cow mammary tissues compared with mid-pregnancy tissues. Then, we prioritized two of potential target genes of miR-139 in cow, growth hormone receptor (GHR) and type I insulin-like growth factor receptor (IGF1R) for further functional studies based on their roles in lactation processes. Dual luciferase reporter assays validated direct binding of miR-139 to the 3'- untranslated region (UTR) of GHR and IGF1R. Moreover, over-expression or silencing of miR-139 affected mRNA levels of GHR and IGF1R in cultured bovine mammary epithelial cells (BMECs). Furthermore, over-expression of miR-139 decreased protein levels of ?-casein, proliferation in mammary epithelial cell, and the protein levels of IGF1R and key members of the GHR or IGF1R pathways as well, whereas silencing miR-139 produced the opposite result. Among these signal molecules, signal transducer and activator of transcription-5 (STAT5), protein kinase B (also known as AKT1), mammalian target of rapamycin (mTOR), and p70-S6 Kinase (p70S6K) are involed in ?-casein synthesis, and Cyclin D1 is involved in cell proliferation. In addition, silencing GHR decreased protein levels of ?-casein, IGF1R, and key members of the IGF1R pathway, whereas co-silencing miR-139 and GHR rescued the expression of GHR and reversed GHR silencing effects.Our results demonstrate that GHR and IGF1R are target genes of miR-139 in dairy cow. MiR-139 suppresses ?-casein synthesis and proliferation in BMECs by targeting the GHR and IGF1R signaling pathways.
| S-EPMC5702184 | biostudies-literature
Project description:β-sitosterol, a natural plant steroid, has been shown to promote anti-inflammatory and antioxidant activities in the body. In this study, β-sitosterol was used to protect against lipopolysaccharide (LPS)-induced cell damage in bovine mammary epithelial cells, which are commonly studied as a cell model of mammary inflammatory response and lipogenesis. Results showed that treatment with a combination of LPS and β-sitosterol significantly reduced oxidative stress and inflammation, while increasing the expression of anti-apoptotic proteins and activating the hypoxia-inducible factor-1(HIF-1α)/mammalian target of rapamycin(mTOR) signaling pathway to inhibit apoptosis and improve lipid synthesis-related gene expression. Our finding suggests that β-sitosterol has the potential to alleviate inflammation in the mammary gland.
| S-EPMC10572156 | biostudies-literature
Project description:Acetate is a precursor substance for fatty acid synthesis in bovine mammary epithelial cells (BMECs), and the mTOR signaling pathway plays an important role in milk fat synthesis. However, the mechanism of the regulatory effects of acetate on lipogenic genes via the mTOR signaling pathway in BMEC remains unknown. We hypothesized that acetate can enhance the expression of lipogenic genes and triglyceride (TG) production by activating the mTOR signaling pathway in BMECs. Therefore, the aim of this study was to investigate the network of acetate-regulated lipid metabolism by the mTOR signaling pathway in BMECs. These results showed that TG synthesis was elevated (p < 0.01) in BMECs with acetate treatment. The lipid droplets were increased in the acetate-treated groups compared with those in the control group through the Bodipy staining of the lipids. In addition, the fatty acid profile in BMECs treated with acetate was affected, with an elevation in the proportions of C14:0, C16:0, and C18:0. The mRNA levels of the sterol-response-element-binding protein 1 (SREBP1), stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FAS) genes involved in the lipogenesis and transcriptional factors were upregulated (p < 0.05) in BMECs with acetate treatment. Remarkably, the expression of acetyl-CoA carboxylase α (ACCα) and FAS rate-limiting enzymes involved in lipogenesis was upregulated in BMECs with acetate treatment. Moreover, the addition of acetate enhanced the key protein expression of S6K1, which is related to the mTOR signaling pathway. Taken together, our data suggest that TG accumulation and expression of lipogenic genes induced by acetate are associated with the activation of the mTOR signaling pathway, which provides new insights into the understanding of the molecular mechanism in the expression of mTOR-signaling-pathway-regulated lipogenic genes.
| S-EPMC9558539 | biostudies-literature
Project description: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.
| S-EPMC8614283 | biostudies-literature
Project description:In the present study, the analysis of epithelial cells derived from various sources was undertaken, beginning from the mammary gland tissue through the primary cultures and their subsequent passages. The objective of the study was the comparative analysis of the stage in which the epithelial cells obtained from individuals in different lactation cycles and disparate phases of cell culture growth are the most suitable for morphological research and analysis of gene expression activity. The cultures of primary bovine mammary epithelial cells and passages were identified morphologically using immunocytochemical methods. After positive identification, real-time PCRs were performed for the analysis of the expression level of casein genes, whey protein genes, and butyrophilin gene. The most stable reference genes in real-time PCRs for the mammary gland tissue and cell cultures were also determined. Of the reference genes, the UXT and GAPDH genes appeared to be the most stable ones for the mammary gland tissue samples and epithelial cell cultures. The results obtained allowed concluding that the mammary gland samples collected from heifers constituted the most effective material for the initiation of primary cultures. The primary cultures formed characteristic for the mammary gland tissue dome structures, which images were obtained using confocal microscopy. The highest levels of expression of the CSN1S1, CSN1S2, CSN2, and CSN3 genes were detected in primary cultures. The levels of expression of whey protein genes (LALBA and BGL) were highest in the second passage. The most abundant expression of the BTN1A1 gene was observed in primary cultures and the third passage. On the basis of the whole experiment, it can be concluded that primary cultures and cells of the second passage derived from heifer individuals appeared to be the best materials for the analysis of mammary gland function and gene expression activity.
| S-EPMC4047483 | biostudies-literature
Project description:MicroRNAs play an essential role in mammary gland development, and involution is a factor that limits lactation. Chi-miR-8516 is one of the validated microRNAs that regulates the expression of STC1 and MMP1, which surge during the involution of the mammary gland. This study aims to explore the direct or indirect regulation of STC1 and MMP1 by chi-miR-8516 and the regulation of chi-miR-8516 by circ-140. In goat mammary epithelial cells, we found that chi-miR-8516 takes circ-140 as a sponge and regulates MMP1 expression by targeting STC1 and promoting the phosphorylation of MAPK. The examination of αs1-/β-casein and lipid showed the modulation of the circ-140/chi-miR-8516/STC1-MMP1 axis in casein secretion and lipid formation, which was regulated by the phosphorylation of mTOR and STAT5. This study illustrates an axis that regulates the synthesis of milk components, and explores the pathways in which the axis participates.
| S-EPMC8146108 | biostudies-literature