Effects Of Glucose And Amino Acids Stress On β-casein And Signaling Pathways Related Protein Phosphorylation Expression Of Bovine Mammary Epithelial Cells

The aim of this study was to investigate the effects of glucose (Glc) and amino acids (AAs) stress and explore which stress is more sensitive on pro-liferation, β-casein and signaling protein expression in vitro cultured bovine mammary epithelial cells. To explore how does this process through the acti-vation of AMP-activated protein kinase (AMPK) and inhibiting mammalian target of rapamycin (mTOR) signaling pathways to regulate protein synthesis, and to provide theory basis and technology for regulating the milk protein synthesis. The CMEC-H cells were cultured in specific medium (without glucose and AAs) and starved overnight, then three levels of glucose (0,2.5, or 17.5 mmol/L) and AAs (0,1.03, or 7.2 mmol/L) in a 3 x 3 factorial design were added to the medium, the treatment groups:Glc 0-AAs 0, Glc 0-AAs 1.03, Glc 0-AAs 7.2, Glc 2.5-AAs 0, Glc 2.5-AAs 1.03, Glc 2.5-AAs 7.2, Glc 17.5-AAs 0, Glc 17.5-AAs 1.03, Glc 17.5-AAs 7.2. CMEC-H cells prolifer-ation was detected by thiazolyl blue(MTT) method, β-casein and signaling protein expression were detected by Western-Blot.This study included four experiments. The purpose of experiment 1 was to examine the effects of glucose and AAs stress on bovine mammary epithe-lial cells relative growth rate. The experiment 2 was to examine the effects of glucose and AAs stress on bovine mammary epithelial cells ATP. The experi-ment 3 was to examine the effects of glucose and AAs stress on the expression of β-casein in bovine mammary epithelial cells. The experiment 4 was de-signed on the basis of the experiment 1,2 and 3 to test the mechanism, we detect the effects of glucose and AAs stress on the signaling proteins phos-phorylation level in bovine mammary epithelial cells.The result of this study were showed as follows:(1) The concentration of glucose or amino acid has a significant effect on cell proliferation (P< 0.01). Cell proliferation was down-regulated with the glucose and/or AAs stress, and when the glucose or AAs was reduce the same proportion of 85.71%, the glucose stress is more sensitive than AAs.(2) The concentration of glucose or amino acid has a significant effect on cellular ATP generation (P< 0.01). The ATP content of CMEC-H was down-regulated with the glucose and/or AAs stress, and when the glucose or AAs was reduce the same proportion of 85.71%, the glucose stress is more sensitive than AAs.(3) The concentration of glucose or amino acid has a significant effect on β-casein expression of CMEC-H (P< 0.01). The β-casein expression was down-regulated with the glucose and/or AAs stress, and when the glucose or AAs was reduce the same proportion of 85.71%, the glucose stress is more sensitive than AAs.(4) The concentration of glucose or amino acid has a significant effect on AMPK phosphorylation expression of CMEC-H (P< 0.01). The AMPK phosphorylation expression was up-regulated with the glucose and/or AAs stress, and when the glucose or AAs was reduce the same proportion of 85.71%, the glucose stress is more sensitive than AAs.(5) The concentration of glucose or amino acid has a significant effect on mTOR, Raptor, S6K1,4EBP1 phosphorylation expression of CMEC-H (P< 0.01). With the stress of glucose and/or AAs, the phosphorylation datas of mammalian target of rapamycin (mTOR, Ser2481), regulatory associated pro-tein of mTOR (Raptor, Ser792), ribosome protein subunit 6 kinase 1(S6K1, Thr389),4E binding protein 1 (4EBP1, Thr37) were down-regulated. The glu-cose stress is more sensitive than AAs.In conclusion, glucose or amino acid stress through the activation of AMPK and inhibiting mammalian target of rapamycin (mTOR) signaling pathways to regulate protein synthesis, the glucose stress is more sensitive than AAs on milk protein synthesis.