Effect Of Glucose On Energy Metabolism And Casein Synthesis In Bovine Mammary Epithelial Cells

This paper study the effects of different concentration of glucose on glucose uptake and casein synthesis in bovine mammary epithelial cells(BMECs).The relative proliferation rate,glucose intake,the activity of enzymes related to glucose metabolism and casein expression of genes related to casein synthesis were investigated so as to explore the appropriate supply mode of glucose for BMECs,to initially reveal the molecular mechanism of glucose regulating casein synthesis,and to provide a theoretical basis for regulating the synthesis of dairy milk components.The thesis contains three parts of experiment.Trial 1 study the effect of different concentration of glucose(0,7,10.5,14,17.5 and 21 mmol/L)on glucose uptake,ATP content,mRNA expression of glucose transport caarrier and enzyme activities of glucose metabolism in two-dimensional cultured BMECs.After purifying and cultivating the second-generation BMECs were selected,and 0(control),7,10.5,14,17.5 and 21 mmol/L glucose were added to the medium.After 12 hours of treatment,the kits were used to detect the content of glucose and ATP,the activities of hexokinase(HK),pyruvate kinase(PK)and glucose-6-phosphate dehydrogenase(G6PD)in the culture fluid.Real-time quantitative PCR was used to detect,gene expression of glucose transporter 1(GLUT1),glucose transporter 8(GLUT8)and glucose transporter 12(GLUT 12)expression;Trial 2 the effect of different concentration of glucose(0,7,10.5,14,17.5 and 21 mmol/L)on viability and mRNA expression of genes related to casein synthesis in two-dimensional cultured BMECs.Thiazoly blue tetrazolium(MTT)method was used to detect cell viability;real-time quantitative PCR was used to detect genes expression of ?s 1-casein(CSN1S1)and?-casein(CSN3),adenosine 5'-monophosphate-activated protein kinase(AMPK),rapamycin target mammalian target of rapamycin(mTOR),eukaryotic translation initiation factor 4E-binding protein 1(4EBP1),ribosomal p70s6 kinase(S6K1)and Expression of eukaryotic translation initiation factor 4E(eIF4E).Trial 3 study the effect of appropriate glucose concentration(0,14 and 17.5 mmol/L)on casein synthesis in three-dimensional cultured BMECs.Real-time fluorescence quantitative PCR was used to detect the BMECs glucose transporter and the expression of casein synthesis-related genes;Western Blot was used to detect the protein expression levels of CSN1S1 and CSN2 in BMECs.The results of the trial 1 showed that when the glucose concentration was higher than 17.5 mmol/L,the glucose uptake rate of BMECs decreased significantly(P<0.05);the amount of ATP synthesis and the supplementation level of glucose in BMECs showed a dose-dependent effect.Compared with the control group,the 10.5?21 mmol/L glucose treatment groups significantly increased the enzyme activities of HK,PK and G6PD in BMECs(P ?0.05).The mRNA expression of GLUT 1 and GLUT8 in group 14?17.5 mmol/L were significantly higher than the control group(P?0.05),whereas no significant differences were observed in GLUT12 expression level among three groups(P?0.05).The results of trial 2 showed that the addition of glucose significantly promoted the relative proliferation rate of BMCEs.Compared with the control group,the 17.5 mmol/L glucose treatment groups significantly increased the mRNA expression of CSN1S1,CSN3,AMPK,mTOR,S6K1 and eIF4E(P?0.05),7 mmol/L glucose treatment group 4EBP1 mRNA expression was significantly higher than the control group and other glucose treatment group(P?0.05),but when the glucose concentration was 21 mmol/L,the mRNA expression of casein synthesis-related genes decreased trend.The results of trial 3 showed that the gene expression of GLUT1,GLUT8,GLUT12,?-casein(CSN2),CSN3,S6K1 and eIF4E in the 14 mmol/L?17.5 mmol/L glucose group was significantly higher than that of the control group in the three-dimensional culture mode(P?0.05),but the AMPK gene expression was not significantly different from the control group(P?0.05);the mTOR gene expression in the 14mmol/L glucose group was significantly higher than the 17.5mmol/L glucose group and the control group(P?0.05);The gene expression of 4EBP1 in the 17.5 mmol/L glucose group was significantly higher than that in the 14 mmol/L glucose group and the control group(P?0.05).