Effects Of Glucose,Estrogen And Prolactin On Glucose Utilization Capacity And Casein Synthesis Of Bovine Mammary Epithelial Cells

In this study,bovine mammary epithelial cells（BMECs）cultured in vitro were used as a model to investigate the effects of glucose,estrogen and prolactin synergism on glucose utilization capacity and casein synthesis in BMECs and the mechanism of action.Based on the appropriate concentrations of glucose（14 and 17.5 mmol/L）,estrogen（100and 200 ng/m L）and prolactin（100 and 200 ng/m L）,which can promote casein synthesis as derived from the group’s previous study,the L₉（3⁴）orthogonal test was used,and nine groups were divided.Control group（I）was supplemented without glucose and hormones,and 14.0 mmol/L glucose+200 ng/m L estrogen to group II,17.5 mmol/L glucose+100ng/m L estrogen to group III,14.0 mmol/L glucos+100 ng/m L prolactin to group IV,17.5mmol/L glucose+200 ng/m L prolactin to group V,and 100 ng/m L estrogen+100 ng/m L prolactin to group VII.ng/m L prolactin in group V,100 ng/m L estrogen+100 ng/m L prolactin in group VI,200 ng/m L estrogen+200 ng/m L prolactin in group VII,14.0mmol/L glucose+100 ng/m L estrogen+200 ng/m L prolactin in group VIII,17.5 mmol/L glucose+200 ng/m L prolactin in group IX/m L estrogen+100 ng/m L prolactin.MTT method was used to detect cell proliferation rate;q RT-PCR method was used to detect the expression of glucose transporter,lactation-related hormone receptor,casein synthesis-related genes,JAK2/STAT5 and AMPK/m TOR signaling pathway-related genes;Western blotting method was used to detectαs1-casein expression,m TOR,S6K and AMPK phosphorylation levels.The results showed that:1)The combined addition of glucose 14 mmol/L+estrogen 100 ng/m L+prolactin 200 ng/m L had the highest ATP content of BMECs,and the highest gene expression levels of GLUT1,GLUT8 and GLUT12;the combined addition of glucose 17.5 mmol/L+estrogen 200 ng/m L+prolactin 100 ng/m L,the proliferation rate,glucose uptake rate,and HK,PK and G6PDH enzyme activities of BMECs were the highest;2)Combined addition of glucose 14 mmol/L+estrogen 100ng/m L+prolactin 200 ng/m L Hormone receptor genes（ER-α,ER-β,GPER30 and PRLR）,m TOR signaling pathway related genes（m TOR,AKT,PI3K and e IF4E）,JAK/STAT signaling pathway related genes（JAK2 and STAT5）and CSN3 of BMECs The expression level of the gene was the highest,and the phosphorylation level of m TOR and S6K protein was the highest,and the promotion effect on the synthesis of casein of BMECs was the best.3)The expression of CSN1S1,CSN2 and S6K1 genes of BMECs was the highest when the combination of glucose 17.5 mmol/L+estrogen 200 ng/m L+prolactin 100 ng/m L was added.Based on the above results,the combined addition of glucose,estrogen and prolactin increased the uptake and utilization of glucose by BMECs by increasing the proliferation rate,ATP synthesis,and the expression of glucose transporter genes in BMECs;the combined addition of glucose 14 mmol/L+estrogen Hormone 100 ng/m L+prolactin 200 ng/m L is the best combination to promote the expression of casein synthesis-related genes and proteins in BMECs;the synergistic effect of glucose,estrogen and prolactin can increase the hormone receptors of BMECs.Gene expression,up-regulates the gene expression of m TOR signaling pathway and JAK/STAT signaling pathway-related factors,increases m TOR and S6K protein phosphorylation levels,thereby increasing the expression of casein at the transcriptional and translational levels.