The Regulation Mechanism Of DOK1/AKT/SREBPIc Signaling Pathway In Progress Of Pre-diabetes To Diabetes

Objective: To observe the role and mechanism of DOK1 gene in progress of pre-diabetes to diabetes.Methods:(1)Establishment of C57/BL mouse with prediabetes by high-fat diet,establishment of C57/BL mouse with type 2 diabetes model by high-fat di et combined with STZ injection,and were divided into control group,pre-diabe tes group and diabetes group.The expressions of DOK1 gene in the tissue of h eart,liver,spleen,lung,kidney,skeletal muscle,pancreas,fat and colon of eac h group detected by qRT-PCR.(2)At the cellular level,BRL-3A cells,3T3-L1 cells,C2C12 cells with insulin resistance model were established by palmitic a cid,and DOK1,IRSI,IRSII,PI3 K,p-PI3 K,AKT,p-AKT,and SFEBPIC protei ns were detected by Western blotting.(3)The Ad-DOK1 was transfected into B RL-3A cells,3T3-L1 cells and C2C12 cells to up-regulate the expressions of D OK1.The cell proliferation was detected by CCK-8,and the multiplicity of inf ection of adenovirus was detected by flow cytometry.Western blotting was use d to detect the protein expression of IRS1,IRS1,PI3 K,p-PI3 K,AKT,p-AKT and SREBPIc.Results:(1)The results of qRT-PCR showed that the expression of DOK1 in the pre-diabetes group and diabetic group was lower than control group(P<0.05).(2)In the cell insulin resistance model,the expression of DOK1 was also lower than control group(P<0.05).There is no change in the expression of insulin signaling pathway proteins IRSI and IRSII(P>0.05),the ratios PI3 K and p-PI3 K also has no change(P>0.05),while the ratios of AKT and p-AK T were down-regulated(P<0.05),and the protein expression of SREBPIc was si gnificantly up-regulated(P<0.05).(3)The results of CCK-8 showed that adenovi rus transfection of BRL-3A cells,3T3-L1 cells,and C2C12 cells to up-regulate d DOK1 will affect the cell's proliferation ability as the adenovirus concentratio n increases and the transfection time prolongs.Analysis of optimal MOI values and adenovirus transfection time of three cells by ROC curve combined cell survival rate with multiplicity of infection of adenovirus.The optima conditions for BRL-3A cells,3T3-L1 cells and C2C12 cells is MOI=60,MOI=100,MOI=150.the transfection time of all three cells is 48 hours.After up-regulation DOK1,there is no change in the expression of insulin signaling pathway proteins IR SI and IRSII(P>0.05),the ratios PI3 K and p-PI3 K also has no change(P>0.05),while the ratios of AKT and p-AKT were up-regulated(P<0.05),and the pr otein expression of SREBPIc was significantly down-regulated(P<0.05).Conclusion: The DOK1 gene may be involved in the pathogenesis process of pre-diabetes to diabetes.The DOK1 gene may influence the pathogenesis of diabetes through the DOK1/AKT/SREBPIc signaling pathway.