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D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter beta-casein, proteins and metabolites linked in milk protein synthesis in bovine mammary epithelial cells
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...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Korean Society of Animal Sciences and Technology
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9184702/ https://www.ncbi.nlm.nih.gov/pubmed/35709129 http://dx.doi.org/10.5187/jast.2022.e37 |
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author | Jeon, Seung-Woo Conejos, Jay Ronel V. Lee, Jae-Sung Keum, Sang-Hoon Lee, Hong-Gu |
author_facet | Jeon, Seung-Woo Conejos, Jay Ronel V. Lee, Jae-Sung Keum, Sang-Hoon Lee, Hong-Gu |
author_sort | Jeon, Seung-Woo |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-9184702 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Korean Society of Animal Sciences and Technology |
record_format | MEDLINE/PubMed |
spelling | pubmed-91847022022-06-14 D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter beta-casein, proteins and metabolites linked in milk protein synthesis in bovine mammary epithelial cells Jeon, Seung-Woo Conejos, Jay Ronel V. Lee, Jae-Sung Keum, Sang-Hoon Lee, Hong-Gu J Anim Sci Technol Research Article 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. Korean Society of Animal Sciences and Technology 2022-05 2022-05-31 /pmc/articles/PMC9184702/ /pubmed/35709129 http://dx.doi.org/10.5187/jast.2022.e37 Text en © Copyright 2022 Korean Society of Animal Science and Technology https://creativecommons.org/licenses/by-nc/4.0/This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Jeon, Seung-Woo Conejos, Jay Ronel V. Lee, Jae-Sung Keum, Sang-Hoon Lee, Hong-Gu D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter beta-casein, proteins and metabolites linked in milk protein synthesis in bovine mammary epithelial cells |
title | D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
title_full | D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
title_fullStr | D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
title_full_unstemmed | D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
title_short | D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
title_sort | d-methionine and 2-hydroxy-4-methylthiobutanoic acid i alter
beta-casein, proteins and metabolites linked in milk protein synthesis in bovine
mammary epithelial cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9184702/ https://www.ncbi.nlm.nih.gov/pubmed/35709129 http://dx.doi.org/10.5187/jast.2022.e37 |
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