| Objective To study the effects of metformin and glucokinase activator(GKAs,HMS5552)with type 2 diabetes(TD2M)mechanism of glucose metabolism in rats.Methods 8 healthy male SD rats were randomly selected from100 rats as the normal control group(NC),fed with normal diet,the rest were fed with high-fat-sugar diet 2 months then injection of streptozotocin 40 mg/kg to developed the model of type 2 diabetes mellitus,twice the fasting blood glucose(FPG)?8.0mmol/L as type 2 diabetes rat model.After modeling,fasting for 12 hours,with sodium pentobarbital anesthetized rats,heart blood detection to measure FPG,triglyceride(TG),total cholesterol(TC),insulin(INS)and glucagon(GC).The rats were randomly divided into seven groups with successful modeling,8 rats in each group.1.Diabete group(DM)2.HMS5552 low dose treatment group(HMS-L,10 mg/kg HMS5552)3.HMS5552 high dose treatment group(HMS-H,30 mg/kg HMS5552)4.Metformin low dose treatment group(MET-L,80 mg/kg metformin)5.Metformin high dose treatment group(MET-H,240 mg/kg metformin)6.Low dose of HMS5552 + low dose metformin treatment group(HMS-L+MET-L,10 mg/kg HMS5552+80 mg/kg metformin)7.HMS5552 low dose + high dose metformin treatment group(HMS-L+MET-H,10 mg/kg HMS5552+240 mg/kg metformin).After grouping,oral glucose tolerance test(OGTT)and oral drug tolerance test(ODTT)were collected from the tail vein.Every day for each drug treatment,NC and DM group with equal volume of saline.During the period of drug treatment,the general situation of rats was observed,and the body weight was measured every week.After 4 weeks of drug treatment and fasting for 12 hours,the tail vein was collected for OGTT and ODTT experiments,rats were killed,heart blood detection to measure FPG,TG,TC,INS and GC.The glucose-6-phosphate dehydrogenase coupling ratio detection method in liver glucokinase(GK)enzymatic activity value;the expression of RT-PCR molecular level detection of GK;HE staining to observe the pathological changes in liver and andpancreas,immunohistochemical staining to observe the glucokinase(GK),insulin(INS)and glucagon(GC)expression.Western-blot to observe the GK,amp activated protein kinase(AMPK),acetyl coenzyme A carboxylase(ACC)and phosphorylation of acetyl coenzyme A carboxylase(P-ACC)expression in protein level.Results(1)Successfully established the model of type 2 diabetes mellitus,FPG>8.0mmol/L.(2)Body weight growth rate of high-fat-sugar rats was higher than that of normal group before STZ injection.For fourteenth weeks,compared with DM group,the difference between HMS-H and MET-H group was significant(P < 0.05),HMS-L+MET-L and HMS-L+MET-H group had significant difference(P < 0.01).(3)The results of OGTT showed that after treatment,compared with DM group,all treatment groups ? AUC0-240 were decreased(P < 0.01).HMS-H,MET-H,HMS-L+MET-Land HMS-L+MET-H group the glucose peak time compared with that before treatment in advance 30 min.(4)ODTT results showed that after treatment,the initial blood glucose of treatment groups decreased to a certain degree compared with that before treatment.The maximum blood glucose level of postprandial 60 min decreased significantly compared with that before treatment.Compared with DM group,the difference between HMS-H and HMS-L+MET-H group was significant(P < 0.05).(5)Serological indicators showed that after treatment,the level of cholesterol(TC)increased in high-fat-sugar rats,but the level of TC in treatment groups were lower than that in the DM group at the same time.After treatment,the level of triglyceride(TG)in groups was higher than that before treatment,but the level of TG in treatment groups were lower than that of the DM group in the same period.Before treatment,the fasting blood glucose(FPG)of high-fat-sugar rats was significantly higher than that in NC group,which showed that the model was successful.After treatment,compared with DM group,the difference between HMS-L and control group was significant(P < 0.05),the rest of the treatment groups significantly different(P < 0.01).After treatment,Compared with DM group,the level of serum insulin(INS)in MET-H group was significantly higher(P<0.01).After treatment,the serum glucagon(GC)level of the rats in each group were lower than that before treatment.Compared with DM group,there was no significant difference in treatment groups(P > 0.05).(6)The activity of GK showed that after treatment,compared with DM group,MET-L and MET-H group had a significant difference(P < 0.05),the other treatment groups significantly(P < 0.01).(7)The results of GKm RNA showed that after treatment,compared with DM group,the expression of treatment groups were significantly increased(P < 0.01).(8)HE staining of liver showed,the liver cells in NC group were intact,and the liver cells were arranged in a radial way around the central vein,and the structure of liver lobules and nuclei was normal.The liver cells in DM group had a fatty change,the nucleus of the liver was nuclear dissolving,and the permutation of the hepatic cord was disordered.The morphology and structure of hepatocytes in the treatment groups were improved in varying degrees.HE staining of pancreas revealed,the structure and shape of pancreatic tissue in NC group were intact,and the cells in islet cells were clearly visible.The cells in the islets were arranged in a cluster like shape,and the nuclei were normal.In DM group,the cells in the islet were reduced and atrophied.In treatment groups the cells in the islet were increased and the degree of atrophy decreased than DM group.(9)The results of immunohistochemical staining showed that Compared with DM group,GK expression was increased in HMS-L group(P < 0.05),HMS-H,HMS-L+MET-L and HMS-L+MET-H group increased significantly(P < 0.01).The results of insulin(INS)staining showed that Compared with DM group,HMS-H,MET-L group the expression increased(P < 0.05),MET-H,HMS-L+MET-L and HMS-L+MET-H group increased significantly(P < 0.01).The results of glucagon(GC)staining showed that Compared with DM group,MET-L group the expression decreased(P < 0.05),the rest treatment groups decreased significantly(P < 0.01).(10)Western blotting showed that except MET-L and MET-H group,the expression of GK in rest treatment groups,was increased significantly(P < 0.01).AMPK blotting results showed that compared with DM group,the expression of AMPK in treatment groups increased significantly(P < 0.01).P-ACC/ACC blotting results showed that compared with DM group,the expression of the difference with HMS-L group was not statistically significant(P > 0.05),expression of HMS-H group differences(P < 0.05),the statistically different between the four groups(P < 0.01).Conclusion(1)To reduce blood glucose,the combined treatment was better than single drug.(2)Advance insulin and reduce glucagon,the combined treatment was better than single drug.(3)HMS5552 can increase the concentration and activity of GK,the concentration of metformin can increase GK,the combined treatment was better than single drug.(4)HMS5552 may reduce blood glucose through AMPK-ACC signaling pathway. |
PDF Full Text Request