RESEARCH PAPER

Effects of Leucine on Nutrient Transport in Porcine Mammary Epithelial Cells and Regulatory Mechanism

  • ZHU Pengwei ,
  • WU Caichi ,
  • CUI Chang ,
  • ZHENG Xiaoyu ,
  • MA Ziwei ,
  • WANG Jun ,
  • ZHANG Shihai ,
  • GUAN Wutai ,
  • CHEN Fang
Expand
  • College of Animal Sciences, South China Agricultural University, Guangzhou 510642, China

Received date: 2021-07-18

  Online published: 2022-03-14

Abstract

In this study, porcine mammary epithelial cell (pMEC) was used as an in vitro model to investigate the effects of leucine on milk composition synthesis and its molecular mechanism. pMEC cells were treated with 0 (control group), 1, 5 and 10 mmol/L leucine for 24 and 48 h, respectively, the cell viability and the gene expression of milk protein synthesis, amino acid transporters, glucose transporters and fatty acid transporters were analyzed. The expression and phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase 1 (S6K1) in mTOR signaling pathway were further detected to explore the mechanism of leucine regulating milk composition synthesis in vitro. The results showed that leucine treatment 48 h with different concentrations significantly increased the cell viability compared with control group (P<0.05), and the highest cell viability was found when leucine concentration was 1 mmol/L. Compared with control group, the mRNA relative expression levels of milk protein synthesis genes α-s2-casein (CSN1S2)and κ-casein (CSN3)in 1 mmol/L group were significantly increased (P<0.05), and the mRNA relative expression levels of CSN1S2 in 5 mmol/L group was significantly increased (P<0.05). Compared with control group, the mRNA relative expression levels of amino acid transporters solute carrier family 1 member 4 (SLC1A4), solute carrier family 7 member 11 (SLC7A11), solute carrier family 5 member 5 (LAT1), solute carrier family 7 member 7 (SLC7A7) and fatty acid transporter fatty acid transport protein 1 (FATP1) were significantly increased in 1, 5 and 10 mmol/L groups(P<0.05); the mRNA relative expression levels of solute carrier family 38 member 1 (SNAT1), solute carrier family 7 member 2 (SLC7A2), fatty acid transport protein 2 (FATP2), fatty acid binding protein 7 (FABP7) were significantly increased in 1 and 5 mmol/L groups(P<0.05); the mRNA relative expression levels of solute carrier family 1 member 5 (SLC1A5) was significantly increased in 1 and 10 mmol/L groups(P<0.05); the mRNA relative expression levels of solute carrier family 3 member 2 (4F2hc), solute carrier family 3 member 1 (rBAT) and fatty acid translocase (CD36) were significantly increased in 5 and 10 mmol/L groups(P<0.05); in addition, 1 mmol/L leucine treatment could significantly increase the mRNA relative expression levels of FATP1, fatty acid binding protein 3 (FABP3), fatty acid binding protein 5 (FABP5) and acyl-CoA-binding protein (ACBP)(P<0.05); glucose transporter 8 (GLUT8) mRNA relative expression level in 5 and 10 mmol/L groups was significantly decreased (P<0.05). Treatment with 1 mmol/L leucine significantly increased the phosphorylation level of S6K1(P<0.05), however, there was no significant difference in phosphorylation level of mTOR in each leucine group compared with the control group (P>0.05). These results indicate that adding leucine in pMEC medium can significantly increase the cell viability, and promote the expression of milk protein synthesis, amino acid transporter and fatty acid transporter genes.

Cite this article

ZHU Pengwei , WU Caichi , CUI Chang , ZHENG Xiaoyu , MA Ziwei , WANG Jun , ZHANG Shihai , GUAN Wutai , CHEN Fang . Effects of Leucine on Nutrient Transport in Porcine Mammary Epithelial Cells and Regulatory Mechanism[J]. Chinese Journal of Animal Nutrition, 2022 , 34(3) : 1895 -1907 . DOI: 10.3969/j.issn.1006-267x.2022.03.049

References

[1] 宋广鹏.母猪泌乳力的影响因素和提高措施[J].现代畜牧科技,2020(5):13-14. SONG G P.Influencing factors and improving measures of lactation ability of sows[J].Modern Animal Husbandry Science & Technology,2020(5):13-14.(in Chinese)
[2] AKERS R M.Lactation and the mammary gland[M].Oxford:Blackwell Publishing Company,2002.
[3] ARENDT L M,KUPERWASSER C.Form and function:how estrogen and progesterone regulate the mammary epithelial hierarchy[J].Journal of Mammary Gland Biology and Neoplasia,2015,20(1/2):9-25.
[4] KIM S W,WU G Y.Regulatory role for amino acids in mammary gland growth and milk synthesis[J].Amino Acids,2009,37(1):89-95.  
[5] REZAEI R,WU Z L,HOU Y Q,et al.Amino acids and mammary gland development:nutritional implications for milk production and neonatal growth[J].Journal of Animal Science and Biotechnology,2016,7:20.
[6] CAPUCO A V,ELLIS S E.Comparative aspects of mammary gland development and homeostasis[J].Annual Review of Animal Biosciences,2013,1:179-202.
[7] TROTTIER N L,SHIPLEY C F,EASTER R A.Plasma amino acid uptake by the mammary gland of the lactating sow[J].Journal of Animal Science,1997,75(5):1266-1278.  
[8] BOYD D R,KENSINGER R S,HARRELL R J,et al.Nutrient uptake and endocrine regulation of milk synthesis by mammary tissue of lactating sows[J].Journal of Animal Science,1995,73(Suppl.2):36-56.
[9] MURGAS TORRAZZA R,SURYAWAN A,GAZZANEO M C,et al.Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation[J].The Journal of Nutrition,2010,140(12):2145-2152.  
[10] APPUHAMY J A D R N,KNOEBEL N A,NAYANANJALIE W A D,et al.Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices[J].The Journal of Nutrition,2012,142(3):484-491.  
[11] 高海娜.亮氨酸、组氨酸、赖氨酸和蛋氨酸对奶牛乳腺上皮细胞中酪蛋白合成的影响及调控机理研究[D].硕士学位论文.兰州:甘肃农业大学,2016. GAO H N.Effects of Leu,His,Lys and Met on casein synthesis and its manipulating mechanism in bovine mammary epithelial cells[D].Master's Thesis.Lanzhou:Gansu Agricultural University,2016.(in Chinese)
[12] 庞学燕,季昀,田青,等.亮氨酸对κ-酪蛋白合成的影响及相关信号通路的研究[J].中国畜牧杂志,2013,49(9):38-41. PANG X Y,JI Y,TIAN Q,et al.Leucine pair κ-study on the effect of casein synthesis and related signal pathways[J].Chinese Journal of Animal Science,2013,49(9):38-41.(in Chinese)
[13] PRIZANT R L,BARASH I.Negative effects of the amino acids Lys,His,and Thr on S6K1 phosphorylation in mammary epithelial cells[J].Journal of Cellular Biochemistry,2008,105(4):1038-1047.  
[14] QIU Y W,QU B,ZHEN Z,et al.Leucine promotes milk synthesis in bovine mammary epithelial cells via the PI3K-DDX59 signaling[J].Journal of Agricultural and Food Chemistry,2019,67(32):8884-8895.  
[15] MOSHEL Y,RHOADS R E,BARASH I.Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells[J].Journal of Cellular Biochemistry,2006,98(3):685-700.  
[16] MERCIER J C,GAYE P.Early events in secretion of main milk proteins:occurrence of precursors[J].Journal of Dairy Science,1982,65(2):299-316.  
[17] 王超先,王旭贞,管武太,等.母猪妊娠后期日粮添加亮氨酸对断奶仔猪生长性能及乳成分的影响[J].中国畜牧杂志,2016,52(9):48-52. WANG C X,WANG X Z,GUAN W T,et al.Effects of leucine supplementation in late gestation sow diets on growth performance of weanling piglets and milk components of Sows[J].Chinese Journal of Animal Science,2016,52(9):48-52.(in Chinese)
[18] 代文婷.亮氨酸对奶牛乳腺上皮细胞酪蛋白合成影响和机理研究[D].硕士学位论文.长春:吉林大学,2015. DAI W T.Effects of leucine on casein synthesis and its regulating mechanism in bovine mammary epithelial cell[D].Master's Thesis.Changchun:Jilin University,2015.(in Chinese)
[19] 杨金勇.蛋氨酸、赖氨酸及其二肽对奶牛乳腺上皮细胞酪蛋白αs1基因表达的影响[D].硕士学位论文.杭州:浙江大学,2006. YANG J Y.Effect of free amino acids and peptides on the expression of casein αs1 gene in cultured bovine mammary epithelial cells[D].Master's Thesis.Hangzhou:Zhejiang University,2006.(in Chinese)
[20] 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. 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.Taian:Shandong Agricultural University,2018.(in Chinese)
[21] BACKWELL F R,BEQUETTE B J,WILSON D,et al.Evidence for the utilization of peptides for milk protein synthesis in the lactating dairy goat in vivo[J].The American Journal of Physiology,1996,271(4 Pt 2):R955-R960.
[22] KANDASAMY P,GYIMESI G,KANAI Y,et al.Amino acid transporters revisited:new views in health and disease[J].Trends in Biochemical Sciences,2018,43(10):752-789.  
[23] 林叶,侯晓明,田雷,等.必需氨基酸上调乳腺腺泡中L型氨基酸转运载体1的表达[J].中国畜牧兽医,2014,41(3):83-86. LIN Y,HOU X M,TIAN L,et al.Essential amino acid upregulates L-type amino acid transporter 1 expression in mammary acinus[J].China Animal Husbandry & Veterinary Medicine,2014,41(3):83-86.(in Chinese)
[24] ZHOU M M,WU Y M,LIU H Y,et al.Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells[J].Journal of Animal Physiology and Animal Nutrition,2015,99(2):215-220.  
[25] LÓPEZ A,TORRES N,ORTIZ V,et al.Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland[J].American Journal of Physiology.Endocrinology and Metabolism,2006,291(5):E1059-E1066.
[26] LAPIERRE H,GALINDO C E,LEMOSQUET S,et al.Protein supply,glucose kinetics and milk yield in dairy cows[M]//CROVETTO G M.Energy and protein metabolism and nutrition.Wageningen:Wageningen Academic Publishers,2010:275-286.
[27] 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.
[28] 张世海.支链氨基酸调节仔猪肠道和肌肉中氨基酸及葡萄糖转运的研究[D].博士学位论文.北京:中国农业大学,2016. ZHANG S H.Branched-chain amino acids modulate amino acid and glucose transport in intestine and muscle of piglets[D].Ph.D.Thesis.Beijing:China Agricultural University,2016.(in Chinese)
[29] 吕佳栋.低蛋白过瘤胃氨基酸补饲日粮对奶牛乳腺组织氨基酸和葡萄糖转运载体的影响[D].硕士学位论文.泰安:山东农业大学,2017. LV J D.Effects of low protein diets supplemented with rumen-protected amino acid on the expression of amino acid transporters and glucose transporters in the mammary gland of lactating cows[D].Master's Thesis.Taian:Shandong Agricultural University,2017.(in Chinese)
[30] BAUMAN D E,GRIINARI J M.Nutritional regulation of milk fat synthesis[J].Annual Review of Nutrition,2003,23:203-227.
[31] KAZANTZIS M,STAHL A.Fatty acid transport proteins,implications in physiology and disease[J].Biochimica et Biophysica Acta,2012,1821(5):852-857.  
[32] BONEN A,CHABOWSKI A,LUIKEN J J F P,et al.Is membrane transport of FFA mediated by lipid,protein,or both? Mechanisms and regulation of protein-mediated cellular fatty acid uptake:molecular,biochemical,and physiological evidence[J].Physiology,2007,22:15-29.
[33] HUI D Y,LABONTÉ E D,HOWLES P N.Development and physiological regulation of intestinal lipid absorption.Ⅲ.Intestinal transporters and cholesterol absorption[J].American Journal of Physiology:Gastrointestinal and Liver Physiology,2008,294(4):G839-G843.
[34] OSORIO J S,LOHAKARE J,BIONAZ M.Biosynthesis of milk fat,protein,and lactose:roles of transcriptional and posttranscriptional regulation[J].Physiological Genomics,2016,48(4):231-256.  
[35] 李珊珊.必需氨基酸调节奶牛乳腺合成乳蛋白和乳脂肪的作用机制[D].博士学位论文.杭州:浙江大学,2015. LI S S.Regulation role of essential amino acid in synthesis of the protein and fat in mammary cell of dairy cow[D].Ph.D.Thesis.Hangzhou:Zhejiang University,2015.(in Chinese)
[36] SUZUKI T,INOKI K.Spatial regulation of the mTORC1 system in amino acids sensing pathway[J].Acta Biochimica et Biophysica Sinica,2011,43(9):671-679.  
[37] WOLFSON R L,CHANTRANUPONG L,SAXTON R A,et al.Sestrin2 is a leucine sensor for the mTORC1 pathway[J].Science,2016,351(6268):43-48.  
[38] CHE L,XU M M,GAO K G,et al.Valine supplementation during late pregnancy in gilts increases colostral protein synthesis through stimulating mTOR signaling pathway in mammary cells[J].Amino Acids,2019,51(10):1547-1559.
Outlines

/