Effects Of EGF And Insulin On The Expression Of Main Glucose Transporter In Yak Mammary Epithelial Cells

The aims of this study were to investigate the molecular mechanism of Glucose transporter 1(GLUT1),Glucose transporter 3(GLUT3)and Glucose transporter 8(GLUT8)participated in lactation regulation of yak,understand the effects of epidermal growth factor(EGF)and Insulin(INS)on glucose metabolism.Mammary epithelial cells were cultured in vitro using improved pancreatic enzyme digestion method;GLUT1,GLUT3 and GLUT8 gene were cloned by RT-PCR from purified cells and its biological characteristics were also analyzed.The expression differences of GLUT1,GLUT3 and GLUT8 were detected by the methods of real-time quantitative PCR and Western-blotting at both m RNA and protein levels,and the location of GLUT1,GLUT3 and GLUT8 proteins in yak mammary epithelial cells were detected by the methods of indirect immunofluorescence.The effects of EGF and INS on GLUT1 gene and protein expression was detected by RT-q PCR and Western-blotting.The results as follows:(1)Mammary epithelial cells were cultured successfully in vitro,and determined its best cryopreservation method.(2)We first cloned the yak GLUT1,GLUT3 and GLUT8 gene(Gen Bank accession number: KU902419,KX094456 and KX268646,respectively).They could encode 492,494 and 478 amino acids,respectively.Nucleotide sequence analysis revealed that GLUT1,GLUT3 and GLUT8 genes were conserved.Protein encoded by the three genes have similar physical and chemical properties,are hydrophobic membrane proteins having 12 transmembrane helical region.(3)The expression of GLUT1,GLUT3 and GLUT8 in mammary epithelial cells of yak were extremely remarkably different(P<0.01),of which the expression quantity of GLUT1 was the highest,GLUT8 followed expression,while GLUT3 was the lowest.In addition,GLUT1,GLUT3 and GLUT8 protein are distributed in mammary epithelial cells of yak and mainly in the nucleus.(4)In this study,different concentrations of EGF and insulin were added to the culture medium of yak mammary epithelial cells.When the concentration of EGF was 50 ng / m L,the relative expression level of GLUT1 was the highest,and the difference was significant(P <0.05).The relative expression of GLUT1 decreased with the increase of EGF concentration,and it was lower than the control group(EGF concentration 0 ng / m L).The results suggested that the expression of related genes and proteins was inhibited when the exogenous factor exceeded a certain concentration range,and the optimal concentration of EGF for GLUT1 expression was 50 ng/m L.When the concentration of INS was 500 ng / m L,the relative expression level of GLUT1 was the highest,and the difference was significant(P <0.05),suggesting that INS could promote the expression of GLUT1 in yak mammary epithelial cells with a dosedependent effect.In this study,we found that both EGF and insulin could regulate the expression of GLUT1 gene and protein in yak mammary epithelial cells in vitro.It suggested that EGF and insulin could regulate glucose metabolism by regulating the expression of GLUT1.While it may reveal different biological effects in the process of lactating yak.