Supplementing conjugated and non-conjugated L-methionine and acetate alters expression patterns of<i>CSN2</i>, proteins and metabolites related to protein synthesis in bovine mammary cells

Jeon, Seungwoo; Conejos, Jay Ronel V.; Kim, Jung-Eun; Kim, Minjeong; Lee, Jeong Eun; Lee, Baek-Seok; Park, Jin-Seung; Moon, Jungyoon; Lee, Jae-Sung; Lee, Hong-Gu

doi:10.1017/s0022029919000979

Cited by 9 publications

(7 citation statements)

References 36 publications

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“…MAC-T cells were incubated as previously reposted with minor modifications [10,11]. Brief-ly, cells were cultured in 10-cm plates at 37℃ with 5% CO 2 and seeded with growth medium in DMEM/F12 containing 10% fetal bovine serum (Thermo Scientific), 100 units/mL penicillin/ streptomycin, 50 μg/mL gentamycin, 5 μg/mL insulin, and 1 μg/mL hydrocortisone.…”

Section: Cell Culturementioning

confidence: 99%

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells

Kim

Lee

et al. 2020

J Anim Sci Technol

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Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 (ASCT2) and β-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in β-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

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Section: Cell Culturementioning

confidence: 99%

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells

Kim

Lee

et al. 2020

J Anim Sci Technol

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“…This result indicates that 72 h should be used as the optimal incubation time for further tests of Met isomer and precursor efficacy since MAC-T cells were completely differentiated into beta-casein-secreting cells. Previous studies have reported that relative gene expression for beta-casein production is generally increased by supplementation with Met [ 28 , 29 ].…”

Section: Resultsmentioning

confidence: 99%

“…The addition of HMBi upregulated the elongation protein eukaryotic translation initiation factor 3 subunit (EIF3A) but downregulated the ribosomal proteins RPS12 and RPS21. Supplementation with D-Met stimulated phosphoinositide 3 (PI3) kinase, an upstream mTOR activator [ 19 , 28 , 33 , 34 ]. The list of all upregulated and downregulated proteins are listed in Tables 8 and 9 .…”

Section: Resultsmentioning

confidence: 99%

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

Jeon¹,

Conejos²,

Lee³

et al. 2022

J Anim Sci Technol

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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.

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“…It has been reported that Ace has a synergistic effect on secretory proteins when it is supplemented with AA. When Ace and methionine were used together to treat MAC-T cells with an AA-adequate medium, the quantity of the secreted proteins increased compared to that in the group treated with methionine alone [ 25 ]. In our results, a synergistic effect on the relative protein concentration in the cultured medium was observed when both 0.15 mM of His and Ace were used for the treatment under normal conditions.…”

Section: Discussionmentioning

confidence: 99%

“…The results suggest that a supply of Ace with His has stimulatory effects on protein synthesis. Some synergistic effects on β-casein expression were reported when Ace was treated with leucine [ 13 ] or methionine [ 25 ] in nutrient-abundant conditions. Although the protein concentration in the cultured medium was increased after the treatment with a combination of Ace and His, such a combination did not show a synergistic effect on β-casein mRNA expression under the 100%DIF condition.…”

Section: Discussionmentioning

confidence: 99%

Effects of L-Histidine and Sodium Acetate on β-Casein Expression in Nutrient-Restricted Bovine Mammary Epithelial Cells

Kim

Lee

2021

Animals

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Nutrient restriction is a challenging condition for the mammary glands of dairy cows. In this condition, supplementing amino acids and energy sources might be a good strategy to improve the concentration of one of the most important caseins in bovine milk. Therefore, the objective of this study was to investigate the effects of L-histidine (His) and sodium acetate (Ace) in a nutrient-restricted (NR) immortalized bovine mammary epithelial cell line (MAC-T cells). The treatments for the MAC-T cells are as follows: experiment (1) 0–5% diluted basal medium; experiment (2) supplementation of 0–9.6 mM of His or Ace in NR or normal conditions; experiment (3) supplementation of 0–9.6 mM of Ace plus 0.15 mM of His in NR or normal conditions. The 1% diluted medium showed no significant effect on the cell viability with the basal medium; thus, it was selected as the NR condition. The relative expression of β-casein was significantly increased in the NR condition with the inclusion of 0.15 mM His alone or with Ace compared to that in control. The supplementation of Ace increased the β-casein level under normal conditions. However, it did not change the expression of β-casein under the NR condition. The results suggest that His has the potential to increase the β-casein expression under the NR condition.

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Supplementing conjugated and non-conjugated L-methionine and acetate alters expression patterns ofCSN2, proteins and metabolites related to protein synthesis in bovine mammary cells

Cited by 9 publications

References 36 publications

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells

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

Effects of L-Histidine and Sodium Acetate on β-Casein Expression in Nutrient-Restricted Bovine Mammary Epithelial Cells

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