Mechanism Of Ezetimibe Improve Glucose Metabolism In Db/db Mice

BACKGROUND:The prevalence of type2diabetes is rising at an alarming rate in China, and there is an increasing trend in younger adults. Type2DM patients usually develop abnormal lipid metabolism concomitantly, and blood glucose control alone often fails to diminish the complications in the major blood vessels such as coronary artery in these patients. Thus, control of blood lipid is required in the glucose lowering therapy. Statins are the most widely used in the type2DM combined hyperlipidemia. But with the gradual increase of reported adverse effects of statins on glucose metabolism in patients with type2DM, there are also increased safety concerns in clinical application. Ezetimibe is the first agent of a novel class of selective cholesterol absorption inhibitors and can be applied alone or in combination with statins in the treatment of type2DM with concomitant hyperlipidemia. Studies have shown ezetimibe cannot only reduce the blood lipid but improve the glucose metabolism in type2DM patients. Ezetimibe has a perspective application in the prevention and treatment of cadiovascual disease in patients with type2DM.However,the the specific mechanism of glucose lowing is still unclear.There are disorders in the first phase insulin secretion of P cells in the early stage of type2 DM, and it has been shown to play an important role in development of DM. In recent years, studies have shown that β cells can also develop to insulin resistance, and there is insulin signal transduction pathway in β cells. db/db mice is a congenital obese mouse model of type2DM, is the ideal type2DM model for experimental study. In this study db/db mice is used to evaluate effect of ezetimibe on glucose metabolism.More importantly, evaluation first-phase insulin secretory function of β cells in vitro with islet perifusion system after ezetimibe treatment,which have not been investigated.The effects of ezetimibe on insulin signal pathway of β-cell in db/db mice, which have not been official reported.OBJECTIVES:Through the db/db2DM mouse model,this study aims to:1.To observe the beneficial effect of ezetimibe on glucose metabolism (through evaluating FBG, HbAlc ISI,IPGTT change after ezetimibe intervention).2.To explore glucose lowing mechanisms of ezetimibe:the role of restoring first-phase insulin secretion,the role of increasing p cells mass, the effect of Ezetimibe on insulin signal pathway of β-cell.METHODS:A total of40male db/db mice aged8weeks (specific pathogen free) and weighing35-48g)were randomly assigned into2groups (n=20per group).These animals were treated with ezetimibe at10mg/kg(ezetimibe group) or placebo (5%gum Arabic,db/db control group) for6weeks. In addition,20aged matched db/m mice without diabetes in the same litter served as controls(db/m control group). Before and after6-week treatment, the Physiological and metabolic parameters were determined. After6week treatment,10mice were randomly selected from each group and received intraperitoneal glucose tolerance test(IPGTT). After6week treatment, in vitro perfusion of pancreatic islets was performed with islet perifusion system. After ezetimibe treatment for6weeks, quantitative analysis the content of β-cell with Image-Pro Plus5.0.1after immunohistochemistry for insulin..After6weeks’treatment,IRS-1expression and tyrosine phosphorylation level were determined with Western blotting.Meanwhile,Western blot method was adopted to detect IR expression and tyrosine phosphorylation level、IRS-1expression and tyrosine phosphorylation level、 PTP1B、GLUT-2、GCK expressions of the β-cell in db/db mice. All values were expressed as mean±standard deviation.Means among groups were compared with one way analysis of variance, and those between two groups were compared with LSD test. Data before and after experiment were compared with paired test.RESULTS:l.At baseline, there were no marked differences in the FBG, HbAlc, FSI, TC LDL-C, TG, and FFA between ezetimibe group and control group. The db/dbmice aged8weeks had the FBG of>15mmol/L. After treatment with ezetimibe, the development of hyperglycemia was alleviated,and the blood glucose and HbAlc at the end of treatment in the db/db mice was markedly lower than that in the untreated db/db mice (P<0.05).After ezetimibe treatment for6weeks, the TC, LDL-C, TG,and FFA in the ezetimibe group were markedly reduced as compared to the untreated db/db mice (P<0.05).The ISI at baseline was comparable between ezetimibetreated mice and untreated db/db mice, but the ISI in the ezetimibe-treated mice was significantly higher than that in the untreated db/db mice at the end of treatment.2. At120min after glucose tolerance test, the blood glucose in the ezetimibe group was markedly reduced (P<0.05), and the AUC in the ezetimibe group was significantly lower than that in the untreated db/db mice (P<0.05).At30min after test, the plasma insulin increased and the AUCINS0-30in the ezetimibe-treated mice was remarkably higher than that in the untreated db/db mice. 3. When compared with untreated db/db mice,the insulin secretion was not significantly increased in the ezetimibe-treated mice in the perfusion of pancreatic islets with low glucose solution, but the insulin secretion was markedly elevated at1min after perfusion with16.7mMglucose solution. This suggests that ezetimibe can improve the first phase insulin secretion, which, however, was still lower than that in the db/m group.4. After ezetimibe treatment for6weeks, quantitative analysis showed the content of β cells in the pancreatic islet was at a very low level in the db/db mice, but ezetimibe treatment could significantly reduce thep cell loss. After ezetimibe treatment for6weeks, the P-cell staining intensity in the ezetimibe group was significantly higher than that in db/db group.5. Western blot method test revealed that the expressions of IR and tyrosine phosphorylation level、IRS-1and tyrosine phosphorylation level of db/db group decreased significantly compared to those of db/m group. After ezetimibe treatment for6weeks, the expressions of IR and tyrosine phosphorylation level、IRS-1and tyrosine phosphorylation of ezetimibe group increased1.5-fold.6. Compared to db/m group, the expression level of PTP1B of db/db group increased2-fold. After ezetimibe treatment for6weeks, PTP1B expression levels of ezetimibe group decreased1.3times than that in db/m group.7. Compared to db/m group, the expressions of GLUT-2,GCK of db/db group decreased significantly compared to those of db/m group. After ezetimibe treatment for6weeks, the expressions of GLUT-2,GCK of ezetimibe group increased1.5-fold and1.8-fold respectively.CONCLUSIONS:1. Ezetimibe-treated db/db mice not only reduce the blood lipids, but also alleviate blood high glucose, improve the glucose tolerance and elevate the insulin sensitivity.2. Ezetimibe can alleviate blood high glucose and protect the function of β cells by improving first phase insulin secretion.3. Ezetimibe-treated db/db mice alleviate blood high glucose and protect the function of β cells by reducing the β cell loss and increasing the β cell content.4. Ezetimibe inhibits expression of PTP1B, and promote IR the the expressions level of IR tyrosine phosphorylatiom IRS-1tyrosine phosphorylation,thereby improve insulin resistance.5Ezetimibe-treated db/db mice alleviate blood high glucose and recover insulin secretion induced by glucose,which may be mediated by increasing the expressions of GLUT-2,GCK after ezetimibe treatment.