scholarly journals CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells

2021 ◽  
Vol 104 (1) ◽  
pp. 1123-1135
Author(s):  
A.M. Edick ◽  
J. Audette ◽  
S.A. Burgos
Keyword(s):  
Amino Acid ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Critical Role ◽  
Mammary Epithelial ◽  
Amino Acid Deprivation ◽  
Bovine Mammary Epithelial Cells

GlyRS is a new mediator of amino acid‐induced milk synthesis in bovine mammary epithelial cells

2018 ◽  
Vol 234 (3) ◽  
pp. 2973-2983 ◽  
Author(s):  
Chaochao Luo ◽  
Hao Qi ◽  
Xin Huang ◽  
Meng Li ◽  
Li Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells

scholarly journals Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Yuhao Chen ◽  
Yuze Ma ◽  
Qiang Ji ◽  
Xiaoru Yang ◽  
Xue Feng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Milk Protein ◽  
Mammary Epithelial Cells ◽  
Protein Composition ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Mtorc1 Signaling

Staphylococcus aureus (S. aureus) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus, and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro, and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced β-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs.

scholarly journals Amino Acid Metabolomic Profiles in Bovine Mammary Epithelial Cells under Essential Amino Acid Restriction

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1334
Author(s):  
Laura López-Diez ◽  
Camilo Calle-Velásquez ◽  
Mark D. Hanigan ◽  
Zulma Tatiana Ruiz-Cortés
Keyword(s):  
Amino Acid ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Tca Cycle ◽  
Protein Quantification ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Alternative Pathways ◽  
Multiple Classification Analysis

Mammary epithelial cells (MECs) in culture are a useful model for elucidating mammary gland metabolism and changes that occur under different nutrient disponibility. MECs were exposed to different treatments: 100% EAA for 8 h and 24 h restriction (R); 2% EAA for 8 h and 24 h R; 2% EAA for 8 h and 24 h + 100% EAA for 8 h and 24 h restriction + re-feeding (R + RF). Western blotting and protein quantification was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) software identified the amino acids (AAs) and signaling pathways. The chi-squared test, multiple classification analysis, and analysis of variance were used for the purification and identification of data. Intracellular casein levels were not affected. The KEGG analysis revealed that the important pathways of metabolism of AAs, which were involved in processes related to metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan (fumarate, acetyl-CoA, and tricarboxylic acid (TCA) cycle), were affected by both R and R + RF treatments, mainly through the glutamic-oxaloacetic transaminase-2 enzyme. Additionally, metabolic processes mediated by the mitochondrial malate dehydrogenase, S-adenosylmethionine synthetase, and asparagine synthase proteins positively regulated the carbohydrate pathway, pyruvate, and TCA cycles, as well as the metabolism of alanine, aspartate, and glutamate metabolism (carbohydrate and TCA cycle). We hypothesized that MECs have the capacity to utilize alternative pathways that ensure the availability of substrates for composing milk proteins.

scholarly journals mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Zhixin Guo ◽  
Keyu Zhao ◽  
Xue Feng ◽  
Dandan Yan ◽  
Ruiyuan Yao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Critical Role ◽  
Lipid Synthesis ◽  
Mammary Epithelial ◽  
Lipogenic Gene ◽  
Bovine Mammary Epithelial Cells ◽  
Lipogenic Gene Expression

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

PURB is a positive regulator of amino acid-induced milk synthesis in bovine mammary epithelial cells

2018 ◽  
Vol 234 (5) ◽  
pp. 6992-7003 ◽  
Author(s):  
Nan Huo ◽  
Mengmeng Yu ◽  
Xueying Li ◽  
Chenjian Zhou ◽  
Xin Jin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Positive Regulator

scholarly journals 5-ALA Attenuates the Palmitic Acid-Induced ER Stress and Apoptosis in Bovine Mammary Epithelial Cells

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1183
Author(s):  
Mst Mamuna Sharmin ◽  
Md Aminul Islam ◽  
Itsuki Yamamoto ◽  
Shin Taniguchi ◽  
Shinichi Yonekura
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Palmitic Acid ◽  
Er Stress ◽  
Aminolevulinic Acid ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Protective Effects ◽  
Bovine Mammary Epithelial Cells ◽  
Induced Apoptosis

The conservation of mammary gland physiology by maintaining the maximum number of mammary epithelial cells (MECs) is of the utmost importance for the optimum amount of milk production. In a state of negative energy balance, palmitic acid (PA) reduces the number of bovine MECs. However, there is no effective strategy against PA-induced apoptosis of MECs. In the present study, 5-aminolevulinic acid (5-ALA) was established as a remedial agent against PA-induced apoptosis of MAC-T cells (an established line of bovine MECs). In PA-treated cells, the apoptosis-related genes BCL2 and BAX were down- and upregulated, respectively. The elevated expression of major genes of the unfolded protein response (UPR), such as CHOP, a proapoptotic marker (C/EBP homologous protein), reduced the viability of PA-treated MAC-T cells. In contrast, 5-ALA pretreatment increased and decreased BCL2 and BAX expression, respectively. Moreover, cleaved caspase-3 protein expression was significantly reduced in the 5-ALA-pretreated group in comparison with the PA group. The downregulation of major UPR-related genes, including CHOP, extended the viability of MAC-T cells pretreated with 5-ALA and also reduced the enhanced intensity of the PA-induced expression of phospho-protein kinase R-like ER kinase. Moreover, the enhanced expression of HO-1 (antioxidant gene heme oxygenase) by 5-ALA reduced PA-induced oxidative stress (OxS). HO-1 is not only protective against OxS but also effective against ER stress. Collectively, these findings offer new insights into the protective effects of 5-ALA against PA-induced apoptosis of bovine MECs.

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