Study On The Role Of DBP Protein In Circadian Rhythm And Glucose Metabolism

Objective: To explore the role of DBP(D site albumin promoter binding protein,DBP)in circadian rhythm and glucose metabolism,preliminarily reveal the mechanism of liver DBP regulating AKT signal pathway on glucose metabolism and T2 DM during circadian rhythm disorder,and provide theoretical basis for the prevention and treatment of T2 DM in occupational shift workers.Methods: Male C57BL/6J mice were induced by high-fat diet+streptozotocin(STZ)to establish T2 DM mice model.The normal light cycle was used in the normal group,and the circadian rhythm disorder group was continuously exposed to repeated light phase shift for 4 weeks.The experimental mice were divided into four groups: normal control group(control group),type 2 diabetes+normal circadian rhythm group(T2DM group),normal+disordered circadian rhythm group(shift group),type 2 diabetes+disordered circadian rhythm group(T2DM+shift group),with 12 mice in each group.The weight of mice was recorded and the weight gain of mice was calculated every other week.The blood glucose value was measured every other week.The oral glucose tolerance test(OGTT)was performed on the next day after fasting for 12 hours in the circadian rhythm disorder group after four weeks of rhythm disorder.The insulin tolerance test(ITT)was performed on the next day after fasting for 8hours in the four groups of mice and the area under the blood glucose curve(AUC)was calculated;After anesthesia,blood was taken from the abdominal aorta of mice and liver tissue was taken.The expression of DBP protein in the blood samples of mice was detected by ELISA,and the expression of Dbp,Per2,Cry1 in the liver of mice was detected by q RT-PCR,as well as the expression of Irs-1,Ras,Akt in the AKT signal pathway in the liver of mice;Western blot was used to analyze the expression level of AKT signal pathway IRS-1,RAS,AKT protein and p-IRS-1,p-RAS,p-AKT protein in mouse liver tissue.Results:(1)The effect of shift on the weight change of four groups of mice:compared with control group,the weight increase of T2 DM group,shift group and T2DM+shift group decreased(P<0.05);Compared with T2 DM group,the weight gain of T2DM+shift group mice increased(P<0.05).(2)Effect of shift on glucose metabolism:By monitoring the blood glucose values of four groups of mice,compared with the control group,the blood glucose values of T2 DM group,shift group and T2DM+shift group increased(P<0.05);Compared with T2 DM group,the blood glucose value of T2DM+shift group decreased(P<0.05).Results of OGTT test: OGTT test was carried out on mice in each group at the 4th week.Compared with control,the AUC of OGTT blood glucose in T2 DM group increased significantly(P<0.05);Compared with T2 DM group,the AUC of OGTT in T2DM+shift group increased significantly(P<0.05).ITT test results: the mice in each group were subjected to ITT test at the 4th week.Compared with control,the AUC of ITT blood glucose in T2 DM group increased significantly(P<0.05);Compared with T2 DM group,the AUC of ITT blood glucose in T2DM+shift group increased significantly(P<0.05).(3)Effect of shift on DBP expression: The results of q RT-PCR showed that compared with control,the expression of Dbp gene in shift group,T2 DM group and T2DM+shift group decreased,and the expression of Dbp gene in T2DM+shift group decreased significantly(P<0.01);Compared with T2 DM group,the expression of Dbp gene in T2DM+shift group was significantly lower(P<0.05).Western blot showed that the expression level of DBP protein in shift group was lower than that in control group,and that in T2DM+shift group was lower than that in T2 DM group.ELISA results showed that compared with control group,the DBP concentration in T2 DM group,shift group and T2DM+shift group decreased significantly(P<0.05).(4)Shift and genes related to liver biorhythm in mice: The results of q RT-PCR showed that the expression of Cry1 gene in shift group and T2 DM group was significantly lower than that in control group(P<0.05);Compared with T2 DM group,Cry1 gene expression in T2DM+shift group was significantly lower(P<0.05).Compared with control,the expression of Per2 gene in shift group,T2 DM group and T2DM+shift group increased significantly(P<0.01);Compared with T2 DM group,the expression of Per2 gene in T2DM+shift group was significantly higher.(5)Shift and liver IRS-1-RAS-AKT signal pathway: q RT-PCR detection results: Compared with control,the expression of Irs-1gene in shift group,T2 DM group and T2DM+shift group was significantly lower(P<0.05).Compared with control,the expression of Ras gene in shift group and T2DM+shift group decreased significantly(P<0.05).Compared with control,Akt gene expression in shift group,T2 DM group and T2DM+shift group was significantly lower(P<0.05);Compared with T2 DM group,Akt gene expression in T2DM+shift group decreased significantly(P<0.01).Western blot results showed that compared with control group,the expression levels of IRS-1,RAS and AKT protein in shift group decreased,and compared with T2 DM group,the expression levels of IRS-1,RAS and AKT protein in T2DM+shift group decreased.(6)Phosphorylation level of IRS-1-RAS-AKT signal pathway protein in liver: Western blot results showed that the expression level of p-IRS-1,p-RAS and p-AKT protein in shift group was lower than that in control group,and the expression level of p-IRS-1,p-RAS and p-AKT protein in T2DM+shift group was lower than that in T2 DM group.Conclusion: The circadian disturbance can affect the body weight,blood glucose,glucose tolerance and insulin secretion of normal and T2 DM mice.It may cause the expression of the biological clock gene Cry1 gene to decrease and the expression of Per2 gene to increase by down-regulating the expression of DBP in normal and T2 DM mice,Thus,the phosphorylation level of IRS1-RAS-AKT signal pathway protein in normal and T2 DM mice was down-regulated,which led to the disorder of blood glucose regulation and affected the occurrence and development of T2 DM.