The Effect Of Metformin On The Expression Of INSR And IRS-1 In Hippocampus Of T2DM Rats

Objective: To observe the effect of hippocampal neurons ultrastructure in hippocampus,the related protein about insulin signaling pathway,the leves of expression of insulin receptor(INSR)protein and insulin receptor substrate-1(IRS-1)in type 2 diabetes(T2DM)rats accepted the treatment with metformin,to investigate the effect of metformin on hippocampus neuronal cells in T2DM rats.Methods: Selected 24 healthy male SPF Goto-Kakizaki(GK)rats and 10 healthy male SPF Wistar rats,adaptively feeded for 2 weeks.After 2 weeks,all the GK random blood glucose was higher than 11.1mmol/l,which were conform to T2DM model standard,according to the random number table,the rats will be randomly divided into model group and metformin group,each group was 12 rats,Wistar rats were selected as normal control group.Metformin group was given metformin(85mg/kg/d,intragastric administration),the normal control group and model group were given the same volume of normal saline(once/d,intragastric administration),and the mode of administration continued for 8 weeks.The weight and fasting blood glucose were measured before administration and after 8 weeks of administration.After 8 weeks of administration,oral glucose tolerance test(OGTT)was administered to rats by intragastric administration of glucose 2g/kg,and calculate the Area under the concentration time curve(AUC).Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate(0.3ml/100g),and the hippocampus was removed after decapitation.The ultrastructure of hippocampal neurons was observed by transmission electron microscope(TEM).Western blot method was used to detect and analyze the expression levels of INSR and IRS-1 protein in hippocampus of each group.Results:1 Comparison of Weight: There was no significant difference in weight between the three groups before administration(P>0.05).Compared the weight among the normal control group,model group and metformin group rats weight was lower obviously,the difference were statistically significant(P<0.01,P<0.01),but the weight difference between model group and metformin group were not statistically significant(P>0.05)(Shown in Table 1).2 Comparison of FBG: Before administration,compared with the normal control group,model group and metformin group rats FBG was significantly higher,the difference were statistically significant(P<0.01,P<0.01).The level of FBG in model group and metformin group was similar,the difference were no statistically significant(P>0.05).After 8 weeks of treatment,the level of FBG in model group was higher than that of normal control group,the difference was statistically significant(P<0.01);The level of FBG in metformin group was lower than that in the model group,the difference was statistically significant(P<0.01).Compared with the normal control group,there were no significant differences in FBG levels of rats in metformin group(P>0.05)(Shown in Table 2).3 OGTT experiments showed: After 8 weeks of treatment,in the experiment of OGTT,the blood glucose of rats in the normal control group and metformin group returned to normal after 120 min and 0min blood glucose levels are similar,the difference was not statistically significant(P>0.05,P>0.05).The 120 min blood glucose of rats in model group was higher than 0min glucose,the difference was statistically significant(P<0.01).The 120 min blood glucose of rats in model group were significantly higher than that of normal control group and metformin group,the difference was statistically significant(P<0.01,P<0.01).The AUC value of OGTT in the model group was significantly higher than that in the normal control group,the difference was statistically significant(P<0.01).Compared with model group,metformin group OGTT test AUC value was significantly lower,the difference was statistically significant(P<0.01)(Shown in Table 3).4 TEM observation results showed:1)In the normal control group: The hippocampal neurons were morphologically normal,nucleus was oval or round,nucleolus was clear and located in the center of the nucleus,chromatin was normal and distributed evenly,nuclear membrane integrity and smooth,clear structure.There were abundant organelles in the cytoplasm.Mitochondrial structure clear and oval,mitochondrial cristae were numerous and distinct.The normal rough endoplasmic reticulum has not swelling and degranulation phenomenon.A large amount of glycogen was clearly visible.2)In the model group: Irregular morphology of neuronal cells,nuclear pyknosis,nucleolar migration and disappearance or atrophy,chromatin margination or disappearance,heterochromatin and electron density increased.The nuclear deformation of jagged or wavy folds,double membrane structure was not clear,nuclear membrane space disappeared.The organelles in the cytoplasm were reduced and vacuolated.The structure of the mitochondria was unclear,and the inner cristae were reduced,broken or disappeared,and showed vacuolar degeneration.Rough endoplasmic reticulum swelling,blurred,disordered and appeared degranulation,glycogen disappeared significantly.3)In the metformin group: The damage of neuron ultrastructure was lighter,the morphology of neurons was normal,nucleolus was obvious,most of the cells were evenly distributed,a few cells have condensed chromatin,nuclear membrane integrity but we can find slightly serrated change.The size and morphology of mitochondria were normal,the membrane structure and cristae were clear,and there were few vacuolar degeneration.The rough endoplasmic reticulum showed slight swelling,individual cell degranulation,glycogen was clearly visible(Shown in Fig.1).5 Western blot results showed: compared with normal control group,the expression of INSR and IRS-1 protein in hippocampus of model group was significantly lower,the difference was statistically significant(P<0.01,P<0.01).Compared with the model group,the expression level of INSR and IRS-1 protein in hippocampus of metformin group was higher,the difference was statistically significant(P<0.01,P<0.01);Compared with the normal control group,the expression of INSR protein in the hippocampus of metformin group decreased,the difference was statistically significant(P<0.01),the difference of IRS-1 protein expression was not statistically significant(P>0.05)(Shown in Fig.2,3,4).Conclusions:1 The ultrastructural damage and the expressions of INSR and IRS-1 decreased in the hippocampus of T2DM rats,suggesting that the occurrence and development of diabetic cognitive impairment may be closely related to INSR and IRS-1.2 Metformin could obviously improve the ultrastructure damage of hippocampal neurons of T2DM rats,and increase the expression of INSR and IRS-1 in hippocampus of T2DM rats.