Effects of Combination of Glucose and Lactation Related Hormones on Casein Synthesis in Bovine Mammary Epithelial Cells

MU Xiaojia; LI Dabiao; SUN Mei; CAO Yue; HAO Yihong; YANG Jing

doi:10.3969/j.issn.1006-267x.2022.09.060

Chinese Journal of Animal Nutrition >

2022 , Vol. 34 >Issue 9: 6072 - 6087

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.09.060

RESEARCH PAPER

Effects of Combination of Glucose and Lactation Related Hormones on Casein Synthesis in Bovine Mammary Epithelial Cells

MU Xiaojia ,
LI Dabiao ,
SUN Mei ,
CAO Yue ,
HAO Yihong ,
YANG Jing

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Key Laboratory of Animal Nutrition and Feed Science in Universities of Inner Mongolia Autonomous Region, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China

Received date: 2022-03-06

Online published: 2022-10-17

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Abstract

This experiment was to investigate the effects of combined addition of glucose and lactation-related hormone on casein synthesis in bovine mammary epithelial cells (BMECs) and mechanism of action by using BMECs in vitro as a model. The experiment was divided into nine groups, and different concentrations of glucose, estrogen and prolactin were added to the culture medium, group Ⅰ was the control group without glucose and lactation hormones, group Ⅱ was added with 14.0 mmol/L glucose+200 ng/mL estrogen, group Ⅲ was added with 17.5 mmol/L glucose +100 ng/mL estrogen, group Ⅳ was added with 14.0 mmol/L glucose+100 ng/mL prolactin, group Ⅴ was added with 17.5 mmol/L glucose+200 ng/mL prolactin, group Ⅵ was added with 100 ng/mL estrogen+100 ng/mL prolactin, group Ⅶ was added with 200 ng/mL estrogen+200 ng/mL prolactin, group Ⅷ was added with 14.0 mmol/L glucose+100 ng/mL estrogen+200 ng/mL prolactin, group Ⅸ was added with 17.5 mmol/L glucose+200 ng/mL estrogen+100 ng/mL prolactin, and each group was set with 6 replicates. Cell proliferation rate was detected by MTT method; real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression of glucose transporter carrier, lactation-related hormone receptor, casein synthesis-related genes, tyrosine kinase 2/signal transduction and transcription factor 5 (JAK2/STAT5) signaling pathway and adenosine monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway-related genes; protein blotting (Western blot) was used to detect the expression of α-casein, mTOR, ribosomal p70s6 kinase (S6K) and AMPK proteins and their phosphorylation levels. The results showed as follows:1) compared with the control group, the proliferation rate of BMECs was significantly increased in all the test groups (P<0.05), and the proliferation rate of BMECs in the groups Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅷ and Ⅸ was significantly higher than that in the groups Ⅵ and Ⅶ (P<0.05), and the best results were obtained in the group Ⅷ. 2) The relative expression levels of hormone receptor genes[estrogen α receptor (ER-α), estrogen β receptor (ER-β), G protein-coupled receptor 30 (GPER30) and prolactin receptor (PRLR)], mTOR signaling pathway related genes[mTOR, protein kinase B (AKT), phosphatidylinositol-3-hydroxykinase (PI3K) and eukaryotic translation initiation factor 4E (eIF4E)] and JAK2/STAT5 signaling pathway related genes (JAK2 and STAT5) and к-casein (CSN3) gens in group Ⅷ were the highest, and the phosphorylation levels of mTOR and S6K were the highest, which showed the best promoting effect on BMECs casein synthesis. 3) The relative expression levels of α S1-casein (CSN1S1), β-casein (CSN2) and ribosomal p70s6 (S6K1) genes of BMECs in group Ⅸ were the highest. 4) The phosphorylation level and casein expression of mTOR in groups Ⅴ, Ⅷ and Ⅸ were significantly higher than those in the other test groups (P<0.05). Taken together, the combination addition of glucose 14.0 mmol/L, estrogen 100 ng/mL and prolactin 200 ng/mL has the best effect on the promotion of casein synthesis in BMECs; the combination addition of glucose and prolactin has the greatest effect on casein synthesis.

Key words： cow mammary epithelial cells; glucose; hormone; casein

Cite this article

MU Xiaojia , LI Dabiao , SUN Mei , CAO Yue , HAO Yihong , YANG Jing . Effects of Combination of Glucose and Lactation Related Hormones on Casein Synthesis in Bovine Mammary Epithelial Cells[J]. Chinese Journal of Animal Nutrition, 2022 , 34(9) : 6072 -6087 . DOI: 10.3969/j.issn.1006-267x.2022.09.060

References

[1] 李楠, 高学军.雌激素及其受体对动物乳腺上皮细胞泌乳性能的影响[J].中国畜牧兽医, 2011, 38(4):151-153. LI N, GAO X J.Effects of estrogen and its receptor on lactation function of mammary gland epithelial cell in mammal[J].China Animal Husbandry & Veterinary Medicine, 2011, 38(4):151-153.(in Chinese)
[2] 卢德勋.反刍动物葡萄糖营养调控理论体系及其应用[J].畜牧与饲料科学, 2010, 31(Z1):402-409. LU D X.Theoretical system of glucose nutrition regulation in ruminants and its application[J].Animal Husbandry and Feed Science, 2010, 31(Z1):402-409.(in Chinese)
[3] TOERIEN C A, TROUT D R, CANT J P.Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal s6 kinase 1[J].The Journal of Nutrition, 2010, 140(2):285-292.

[4] 周顺伍.动物生物化学[M].3版.北京:中国农业出版社, 1999:119-134. ZHOU S W.Animal biochemistry[M].3rd ed.Beijing:China Agricultural Press, 1999:119-134.(in Chinese)
[5] BOLSTER D R, CROZIER S J, KIMBALL S R, et al.AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling[J].Journal of Biological Chemistry, 2002, 277(27):23977-23980.

[6] DUBBELHUIS P F, MEIJER A J.Hepatic amino acid-dependent signaling is under the control of AMP-dependent protein kinase[J].FEBS Letters, 2002, 521(1/2/3):39-42.
[7] BURGOS S A, KIM J J M, DAI M, et al.Energy depletion of bovine mammary epithelial cells activates AMPK and suppresses protein synthesis through inhibition of mTORC1 signaling[J].Hormone and Metabolic Research, 2013, 45(3):183-189.
[8] 武雪会, 孙会增, 王迪铭, 等.不同产奶量和乳蛋白率奶牛的血液生化和激素水平分析[J].中国畜牧杂志, 2019, 55(11):99-104. WU X H, SUN H Z, WANG D M, et al.Research on serum biochemical parameters and hormone profiles in cows with extremely different milk yield or milk protein content[J].Chinese Journal of Animal Science, 2019, 55(11):99-104.(in Chinese)
[9] BURGOS S A, DAI M, CANT J P.Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway[J].Journal of Dairy Science, 2010, 93(1):153-161.

[10] KELLY P A, BACHELOT A, KEDZIA C, et al.The role of prolactin and growth hormone in mammary gland development[J].Molecular and Cellular Endocrinology, 2002, 197(1/2):127-131.
[11] COUSO I, EVANS B S, LI J, et al.Synergism between inositol polyphosphates and TOR kinase signaling in nutrient sensing, growth control, and lipid metabolism in chlamydomonas[J].The Plant Cell, 2016, 28(9):2026-2042.

[12] EHMANN U K, PETERSON W D Jr, MISFELDT D S.To grow mouse mammary epithelial cells in culture[J].The Journal of Cell Biology, 1984, 98(3):1026-1032.

[13] 朱河水, 王月影, 王艳玲, 等.大豆黄酮对奶牛泌乳性能及血浆中激素水平的影响[J].中国农学通报, 2006, 22(5):20-22. ZHU H S, WANG Y Y, WANG Y L, et al.Effect of daidzein on the lactational performance and levels of hormones in plasma of dairy cows[J].Chinese Agricultural Science Bulletin, 2006, 22(5):20-22.(in Chinese)
[14] 金亚亚, 李大彪, 孙梅, 等.催乳素对奶牛乳腺上皮细胞酪蛋白合成的影响[J].饲料研究, 2021, 44(14):77-81. JIN Y Y, LI D B, SUN M, et al.Effect of prolactin on casein synthesis in dairy cow mammary epithelial cells[J].Feed Research, 2021, 44(14):77-81.
[15] 金亚亚.雌激素和催乳素对奶牛乳腺上皮细胞酪蛋白合成的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学, 2021:24-38. JIN Y Y.Effects of estrogen and prolactin on casein synthesis in bovine mammary epithelial cells[D].Master's Thesis.Hohhot:Inner Mongolia Agricultural University, 2021:24-38.(in Chinese)
[16] 孙振稳.氨基酸、葡萄糖和胰岛素对奶牛乳腺上皮细胞增殖及乳蛋白合成的影响[D].硕士学位论文.泰安:山东农业大学, 2018:13-23. SUN Z W.Effects of essential amino acids, glucose and insulin on proliferation and protein synthesis in bovine mammary epithelial cells[D].Master's Thesis.Tai'an:Shandong Agricultural University, 2018:13-23.(in Chinese)
[17] 李子南.葡萄糖对奶牛乳腺上皮细胞能量代谢和酪蛋白合成的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学, 2020:15-30. LI Z N.Effect of glucose on energy metabolism and casein synthesis in bovine mammary epithelial cells[D].Master's Thesis.Hohhot:Inner Mongolia Agricultural University, 2020:15-30.(in Chinese)
[18] 邢媛媛.二、三维培养模式下添加激素对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学, 2017:24-34. XING Y Y.Effect of hormone supplementation on milk fat and milk protein synthesis in two and three-dimensional cultured bovine mammary epithelial cells[D].Master's Thesis.Hohhot:Inner Mongolia Agricultural University, 2017:24-34.(in Chinese)
[19] CANT J P, TROUT D R, QIAO F, et al.Milk composition responses to unilateral arterial infusion of complete and histidine-lacking amino acid mixtures to the mammary glands of cows[J].Journal of Dairy Science, 2001, 84(5):1192-1200.

[20] IMBERT-FERNANDEZ Y, CLEM B F, O'NEAL J, et al.Estradiol stimulates glucose metabolism via 6-phosphofructo-2-kinase (PFKFB3)[J].Journal of Biological Chemistry, 2014, 289(13):9440-9448.

[21] 王珊珊.葡萄糖和氨基酸应激对奶牛乳腺上皮细胞β-酪蛋白及信号通路相关蛋白磷酸化表达的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学, 2016:12-34. WANG S S.Effects of glucose and amino acids stress on β-casein and signaling pathways related protein phosphorylation expression of bovine mammary epithelial cells[D].Master's Thesis.Hohhot:Inner Mongolia Agricultural University, 2016:12-34.(in Chinese)
[22] 王苹, 陈怡君, 吴黎雅, 等.性激素水平与ER、PR的表达在乳腺增生病辨证分型中的意义[J].福建医科大学学报, 2005, 39(3):291-294. WANG P, CHEN Y J, WU L Y, et al.The importance of the serum sex-hormone level, expression of estrogen receptor and progesterone receptor in syndrome differentiation of mammary hyperplasia[J].Journal of Fujian Medical University, 2005, 39(3):291-294.(in Chinese)
[23] 陈静, 王根林, 李莲, 等.雌激素对奶牛乳腺上皮细胞增殖及抗氧化性的影响[J].农业生物技术学报, 2013, 21(2):216-222. CHEN J, WANG G L, LI L, et al.Effects of estrogen on proliferation and antioxidation in bovine (Bos taurus) mammary epithelial cells[J].Journal of Agricultural Biotechnology, 2013, 21(2):216-222.(in Chinese)
[24] 田青, 王洪荣.胰岛素、催乳素和氢化可的松对奶牛乳腺上皮细胞增殖和凋亡的影响[J].中国饲料, 2013(2):8-12. TIAN Q, WANG H R.Effects of INS, PRL and HYD on proliferation and apoptosis of mammary epithelial cells of dairy cow in vitro[J].China Feed, 2013(2):8-12.(in Chinese)
[25] 母晓佳, 李大彪.激素及细胞因子调控奶牛乳腺上皮细胞增殖和泌乳的研究进展[J].中国细胞生物学学报, 2021, 43(5):1061-1067. MU X J, LI D B.Research progress of hormones and cytokines regulating the proliferation and lactation of cow mammary epithelial cells[J].Chinese Journal of Cell Biology, 2021, 43(5):1061-1067.(in Chinese)
[26] MULDOON T G.Interplay between estradiol and prolactin in the regulation of steroid hormone receptor levels, nature, and functionality in normal mouse mammary tissue[J].Endocrinology, 1981, 109(5):1339-1346.

[27] HOVEY R C, TROTT J F, GINSBURG E, et al.Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland[J].Developmental Dynamics, 2001, 222(2):192-205.

[28] 田青, 王洪荣.激素协同作用对奶牛乳腺上皮细胞激素受体基因表达的影响[J].中国饲料, 2015(13):11-14. TIAN Q, WANG H R.Effects of hormones synergy on hormone receptor genes expression in cow mammary epithelial cells[J].China Feed, 2015(13):11-14.(in Chinese)
[29] 金亚亚, 李大彪, 孙梅, 等.组合添加雌激素和催乳素对奶牛乳腺上皮细胞酪蛋白合成的影响[J].中国细胞生物学学报, 2021, 43(12):2316-2325. JIN Y Y, LI D B, SUN M, et al.Effects of estrogen and prolactin on casein synthesis of bovine mammary epithelial cells[J].Chinese Journal of Cell Biology, 2021, 43(12):2316-2325.(in Chinese)
[30] ZHAO Z T, LI L, DU P N, et al.Transcriptional downregulation of miR-4306 serves as a new therapeutic target for triple negative breast cancer[J].Theranostics, 2019, 9(5):1401-1416.

[31] GIULIANELLI S, RIGGIO M, GUILLARDOY T, et al.FGF2 induces breast cancer growth through ligand-independent activation and recruitment of ERα and PRBΔ4 isoform to MYC regulatory sequences[J].International Journal of Cancer, 2019, 145(7):1874-1888.
[32] ROSANO C, PONASSI M, SANTOLLA M F, et al.Macromolecular modelling and docking simulations for the discovery of selective GPER ligands[J].AAPS Journal, 2016, 18(1):41-46.

[33] FILARDO E J, GRAEBER C T, QUINN J A, et al.Distribution of GPR30, a seven membrane-spanning estrogen receptor, in primary breast cancer and its association with clinicopathologic determinants of tumor progression[J].Clinical Cancer Research, 2006, 12(21):6359-6366.

[34] 杨振山.雌激素/GPR30信号通路对山羊乳腺上皮细胞增殖与上皮间质转化的影响及其机制研究[D].硕士学位论文.杨凌:西北农林科技大学, 2018:12-18. YANG Z S.Effect and mechanism of estrogen/GPR30 signaling pathway on proliferation and epithelial-mesenhymal transiton in goat mammary epithlial cells[D].Master's Thesis.Yangling:Northwest A&F University, 2018:12-18.(in Chinese)
[35] KOMATSU T, ITOH F, KUSHIBIKI S, et al.Changes in gene expression of glucose transporters in lactating and nonlactating cows[J].Journal of Animal Science, 2005, 83(3):557-564.

[36] ZHAO F Q, KEATING A F.Expression and regulation of glucose transporters in the bovine mammary gland[J].Journal of Dairy Science, 2007, 90(Suppl.1):E76-E86.
[37] FREEMAN M E, KANYICSKA B, LERANT A, et al.Prolactin:structure, function, and regulation of secretion[J].Physiological Reviews, 2000, 80(4):1523-1631.

[38] TAN D Y, JOHNSON D A, WU W, et al.Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo-and hetero-pairs of long and short human PRL receptors in living human cells[J].Molecular Endocrinology, 2005, 19(5):1291-1303.

[39] YONEKURA S, SAKAMOTO K, KOMATSU T, et al.Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells[J].Domestic Animal Endocrinology, 2006, 31(1):88-96.

[40] 杨建英, 张勇法, 王艳玲, 等.大豆黄酮对奶牛产奶量和乳中常规成分的影响[J].饲料研究, 2005(6):30-31. YANG J Y, ZHANG Y F, WANG Y L, et al.Influence of daidzein on milk production and component in dairy cattle[J].Feed Research, 2005(6):30-31.(in Chinese)
[41] 卢志勇, 梁代华, 杨运玲, 等.大豆异黄酮对奶牛乳腺上皮细胞泌乳性能及抗氧化能力的影响[J].饲料与畜牧, 2013(2):25-28. LU Z Y, LIANG D H, YANG Y L, et al.Influence of isoflavones on the lactation and antioxidation of mammary epithelial cells of dairy cow[J].Animal Agriculture, 2013(2):25-28.(in Chinese)
[42] 武开乐, 库西塔别克·买买提依不拉音, 马静, 等.雌激素对奶牛乳腺上皮细胞中酪蛋白和甘油三酯表达的影响[J].饲料工业, 2019, 40(11):36-42. WU K L, HUXTARBEK.MANBETEBRAYIM, MA J, et al.Effects of estrogen on casein and triglyceride expression in bovine mammary epithelial cells[J].Feed Industry, 2019, 40(11):36-42.(in Chinese)
[43] SIGL T, MEYER H H D, WIEDEMANN S.Gene expression analysis of protein synthesis pathways in bovine mammary epithelial cells purified from milk during lactation and short-term restricted feeding[J].Journal of Animal Physiology and Animal Nutrition, 2014, 98(1):84-95.

[44] TIAN Q, WANG H R, WANG M Z, et al.Lactogenic hormones regulate mammary protein synthesis in bovine mammary epithelial cells via the mTOR and JAK-STAT signal pathways[J].Animal Production Science, 2015, 56(11):1803-1809.
[45] FRASOR J, GIBORI G.Prolactin regulation of estrogen receptor expression[J].Trends in Endocrinology and Metabolism, 2003, 14(3):118-123.

[46] 贾祥捷, 李秋玲, 王长法, 等.牛催乳素基因及其单核苷酸多态性与产奶性状关系的研究进展[J].中国畜牧兽医, 2010, 37(3):148-152. JIA X J, LI Q L, WANG C F, et al.Progress of bovine prolactin gene and the relationship of its SNPs and milk production traits[J].China Animal Husbandry & Veterinary Medicine, 2010, 37(3):148-152.(in Chinese)
[47] 綦松智, 吴登俊, 张中显.mTOR对信号通路调控的研究进展[J].中国畜牧杂志, 2010, 46(1):57-60. QI S Z, WU D J, ZHANG Z X.Advance in the mTOR signaling pathway[J].Chinese Journal of Animal Science, 2010, 46(1):57-60.(in Chinese)
[48] VIOLLET B, ANDREELLI F, JØRGENSEN S B, et al.The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity[J].The Journal of Clinical Investigation, 2003, 111(1):91-98.

[49] LIU Y, WANG X M, ZHEN Z, et al.GRP78 regulates milk biosynthesis and the proliferation of bovinemammaryepithelial cells through the mTOR signaling pathway[J].Cellular & Molecular Biology Letters, 2019, 24:57.
[50] BICKERSTAFFE R, ANNISON E F, LINZELL J L.The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows[J].The Journal of Agricultural Science, 1974, 82(1):71-85.

[51] TOERIEN C A, TROUT D R, CANT J P.Nutritional stimulation of milk protein yield of cows is associated with changes in phosphorylation of mammary eukaryotic initiation factor 2 and ribosomal s6 kinase 1[J].Journal of Nutrition, 2010, 140(2):285-292.

[52] APPUHAMY J A D R N, NAYANANJALIE W A, ENGLAND E M, et al.Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells[J].Journal of Dairy Science, 2014, 97(1):419-429.

[53] 秦春霞, 李虹, 张帅, 等.雌激素与葡萄糖能量代谢研究进展[J].现代生物医学进展, 2018, 18(17):3388-3391. QIN C X, LI H, ZHANG S, et al.Research advancement of estrogen and energy metabolism of glucose[J].Progress in Modern Biomedicine, 2018, 18(17):3388-3391.(in Chinese)
[54] ZHOU J Y, JIANG M H, SHI Y, et al.Prolactin regulates LAT1 expression via STAT5(signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows[J].Journal of Dairy Science, 2020, 103(7):6627-6634.

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