Study On The Role Of Insulin In Proliferation Of Goose Primary Hepatocytes By MTOR Signling Pathway

Abstract:The mammalian target of rapamycin (mTOR) is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from growth factors, nutrients, environmental stress and energy to regulate cell proliferation via different cellular processes. The mTOR signaling pathway also affects the development of the early embryo and embryonic stem cells (ESCs), and metabolism disease and involved in cancers, insulin has a positive regulatory role in the process of cell proliferation. Thus this study use goose liver cells as samples, constructs a model of mTOR signaling pathways are suppressed, and detection indicators related to cell proliferation, explore the effect of mTOR signaling pathways in the process of goose hepatocellular stesatosis induced by insulin(cultured with 0 nmol/L,50nmol/L, 100nmol/L and 150nmol/L insulin).The results are listed as follows:1 Arfter insulin treatment, the DNA synthesis rate and the cell viability showed an upward trend with an increasing insulin concentration compared with the control group. In addition, the ration of the number of Brdu-positive cells after the treatment of 150 nmol/L insulin increased up to 60% compared with the control group. Meanwhile, insulin at all three concentrations increased the mRNA level of Cyclin D1, Cyclin D2, Cyclin D3 in a dose-dependent manner,but it increased the mRNA level of p21 and p27 significantly. Compared to the control group, insulin at all three concentrations increased the mRNA level of, mTOR, Rptro,4EBP1, and S6K in a dose-dependent manner.The result of western blot showed insulin stimulated of the protein expression level of S6K and p-S6K.2 After rapamycin treatment, rapamycin at all three concentrations decreased the activity of Cyclin D1 in a dose-dependent manner compared with the control group (P< 0.05). Meanwhile, rapamycin increased the activity of p21 significantly. Compared with the control group, the mRNA expression level of Cyclin D decreased obviously. On the contrary, p21 mRNA expression level increased obviously.Treatment with rapamycin significantly decreased the protein activity of mTOR,4EBP1, and S6K (P< 0.05).Compared with the control group, the mRNA expression level of mTOR, Rptor, 4EBP1 decreased obviously (P< 0.05) and the protein expression levels of S6K and P-S6K were reduced.3 The protein activity of Cyclin D1 after treatment of insulin with 30 nmol/L rapamycin is lower significantly than that of insulin treatment. Meanwhile, the result of p21 after treatment of insulin with rapamycin is greater significantly than that of insulin treatment. Compared to the insulin treatment, the treatment of insulin with rapamycin, decreased the mRNA level of Cyclin D1, Cyclin D2, Cyclin D3, significantly. The mRNA level of p21 and p27 after the treatment of insulin with inhibitors together are all greater than that of single insulin treatment significantly. Compared to the result of insulin treatment, the mRNA level of mTOR, Rptor,4EBP1, and S6K after the collective treatment of insulin at 50 nmol/1 or150 nmol/L with 30 nmol/L rapamycin,all decreased, significantly. After collective treatment with insulin and 30 nmol/L rapamycin, the protein activity of mTOR,4EBP1, and S6K is lower than that after single insulin treatment and and the protein expression levels of S6K and P-S6K were reduced.Above results show that insulin can promote cell proliferation, rapamycin also can inhibit the liver cell proliferation, and insulin with rapamycin can eliminate the effect of insulin on liver cell proliferation.