Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells원문보기
Kim, Jungeun
(Department of Animal Science and Technology, Sanghuh College of Life Sciences, Konkuk University)
,
Lee, Jeong-Eun
(Institute of Integrated Technology, CJ CheilJedang)
,
Lee, Jae-Sung
(Department of Animal Science and Technology, Sanghuh College of Life Sciences, Konkuk University)
,
Park, Jin-Seung
(Institute of Integrated Technology, CJ CheilJedang)
,
Moon, Jun-Ok
(Institute of Integrated Technology, CJ CheilJedang)
,
Lee, Hong-Gu
(Department of Animal Science and Technology, Sanghuh College of Life Sciences, Konkuk University)
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 es...
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.
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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문제 정의
The objective of this work was to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using MAC-T cells. We hypothesized different effects of PHE and VAL on cellular signaling.
가설 설정
1)Positive values indicate protein abundance fold change compared to control group.
However, it is still uncertain whether PHE or VAL regulate protein synthesis and energy-mediated cellular signaling in mammary epithelial level. Therefore, we hypothesized that PHE and VAL might have different effects on protein synthesis-related and energy-mediated cellular signaling. To test this hypothesis, gene transcription and proteomic analyses were used to investigate these effects of PHE and VAL in immortalized bovine mammary epithelial (MAC-T) cells.
제안 방법
Cell viability was analyzed to investigate whether the addition of PHE or VAL had a negative effect on MAC-T cells.
LC-MS/MS analysis was performed using a nanoAcquity system, equipped with a Symmetry C18 (5 μm, 5 mm × 300 μm) precolumn and a CSH C18 (1.7 μm, 25 cm × 75 μm) analytical column (Waters).
Real-time PCR assays were performed using an AccuPower® 2X GreenStar™ qPCR MasterMix (Bioneer, Seoul, Korea), with cycling conditions consisting of an initial incubation at 95℃ for 3 min, followed by 40 cycles of 95℃ for 10 s, 55 to 65℃ for 30 s, and 72℃ for 30 s.
10). Thus, further analyses were performed to investigate the effects of PHE or VAL on protein synthesis-related responses and energy-mediated cellular signaling in MAC-T cells.
Therefore, we hypothesized that PHE and VAL might have different effects on protein synthesis-related and energy-mediated cellular signaling. To test this hypothesis, gene transcription and proteomic analyses were used to investigate these effects of PHE and VAL in immortalized bovine mammary epithelial (MAC-T) cells.
대상 데이터
Cell viability, concentrations of cell lysates and medium protein and gene expression were analyzed by Duncan’s multiple range test. Each experiment had three replicates. Differences between treatments were considered significant at p < 0.
The immortalized bovine mammary epithelial cell line, MAC-T cells (University of Vermont, Burlington, VT, USA), was used in this study. MAC-T cells are functional cells that secrete milk components once differentiated.
이론/모형
Cell viability, concentrations of cell lysates and medium protein and gene expression were analyzed by Duncan’s multiple range test.
5-fold. Gene-ontology analysis was performed using PANTHER (gene list analysis, http://www.pantherdb.org).
성능/효과
Proteomic analysis explained the relationship between energy metabolism and mammary cell translation elongation due to PHE and VAL treatment. In conclusion, PHE and VAL affected protein synthesis-related and energy-mediated cellular signaling differentially in immortalized bovine mammary epithelial cells. The current study may help increase our understanding of the milk protein synthesis-related roles of AAs in mammary epithelial levels in vitro.
Because mammary glands lack glucose-6-phosphate, mammary epithelial cells need to transport glucose to meet their energy requirements. Our results showed the stimulation of GLUT1 gene expression after the addition of 0.9 mM PHE. These results suggested that PHE can contribute to energy metabolism by increasing glucose uptake.
Results showed that the addition of both PHE and VAL did not affect MAC-T cell viability (p > 0.10).
후속연구
Our results suggested that the addition of PHE may not regulate the transport of other AAs into cells. Further research is required to understand the regulation of AAs by PHE. VAL is transported by both ASCT2 and LAT1 in the basolateral membrane.
참고문헌 (27)
1. Kim SG Buel GR Blenis J Nutrient regulation of the mTOR complex 1 signaling pathway Mol Cells 2013 35 463 73 10.1007/s10059-013-0138-2 23694989
4. Doelman J Curtis RV Carson M Kim JJM Metcalf JA Cant JP Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2B ε but milk protein yield is not increased during an imbalance J Dairy Sci. 2015 98 4499 508 10.3168/jds.2014-9051 25912861
5. Doepel L Hewage II Lapierre H Milk protein yield and mammary metabolism are affected by phenylalanine deficiency but not by threonine or tryptophan deficiency J Dairy Sci. 2016 99 3144 56 10.3168/jds.2015-10320 26851853
7. Li SS Loor JJ Liu HY Liu L Hosseini A Zhao WS et al Optimal ratios of essential amino acids stimulate β -casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants J Dairy Sci. 2017 100 6676 88 10.3168/jds.2017-12681 28571990
10. Jeon SW Conejos JR Kim J Kim MJ Lee JE Lee BK et al Supplementing conjugated and non-conjugated L-methionine and acetate alters expression patterns of CSN2, proteins and metabolites related to protein synthesis in bovine mammary cells J Dairy Res. 2020 87 70 7 10.1017/S0022029919000979 32114997
11. Wang T Jeon SW Jung US Kim MJ Lee HG L-lactate dehydrogenase B chain associated with milk protein content in dairy cows Animals 2019 9 442 10.3390/ani9070442 31311116
12. Peng DQ Lee JS Kim WS Kim YS Bae MH Jo YH et al Effect of vitamin a restriction on carcass traits and blood metabolites in Korean native steers Anim Prod Sci. 2018 59 2138 46 10.1071/AN17733
13. Ishihama Y Oda Y Tabata T Sato T Nagasu T Rappsilber J et al Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein Mol Cell Proteomics 2005 4 1265 72 10.1074/mcp.M500061-MCP200 15958392
14. Swanepoel N Robinson PH Erasmus LJ Effects of ruminally protected methionine and/or phenylalanine on performance of high producing Holstein cows fed rations with very high levels of canola meal Anim Feed Sci Tech. 2015 205 10 22 10.1016/j.anifeedsci.2015.04.002
15. Swanepoel N Robinson PH Erasmus LJ Impacts of adding ruminally protected phenylalanine to rations containing high levels of canola meal on performance of high producing Holstein cows Anim Feed Sci Tech. 2016 216 108 20 10.1016/j.anifeedsci.2016.03.017
16. Swanepoel N Robinson PH Erasmus LJ Production responses of high producing Holstein cows to ruminally protected phenylalanine and tyrosine supplemented to diets containing high levels of canola meal Anim Feed Sci Tech. 2018 243 90 101 10.1016/j.anifeedsci.2018.07.006
17. Zhang S Zeng X Ren M Mao X Qiao S Novel metabolic and physiological functions of branched chain amino acids: a review J Anim Sci Biotechnol. 2017 8 10 10.1186/s40104-016-0139-z 28127425
18. Jackson SC Bryson JM Wang H Hurley WL Cellular uptake of valine by lactating porcine mammary tissue J Anim Sci. 2000 78 2927 32 10.2527/2000.78112927x 11063318
20. Dong X Zhou Z Wang L Saremi B Helmbrecht A Wang Z et al Increasing the availability of threonine, isoleucine, valine, and leucine relative to lysine while maintaining an ideal ratio of lysine: methionine alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription J Dairy Sci. 2018 101 5502 14 10.3168/jds.2017-13707 29550128
23. Sans MD Tashiro M Vogel NL Kimball SR D’Alecy LG Williams JA Leucine activates pancreatic translational machinery in rats and mice through mTOR independently of CCK and insulin J Nutr. 2006 136 1792 9 10.1093/jn/136.7.1792 16772439
24. Burgos SA Kim JJ Dai M Cant JP Energy depletion of bovine mammary epithelial cells activates AMPK and suppresses protein synthesis through inhibition of mTORC1 signaling Horm Metab Res. 2013 45 183 9 10.1055/s-0032-1323742 22972179
25. Dai WT Wang QJ Zou YX White RR Liu JX Liu HY Short communication: Comparative proteomic analysis of the lactating and nonlactating bovine mammary gland J Dairy Sci. 2017 100 5928 35 10.3168/jds.2016-12366 28457551
26. Valvona CJ Fillmore HL Nunn PB Pilkington GJ The regulation and function of lactate dehydrogenase a: therapeutic potential in brain tumor Brain Pathol. 2016 26 3 17 10.1111/bpa.12299 26269128
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