Effect Of Exenatide On The Renal Of Insulin Resistance Rats Induced By A High-fructose Diet And The Mechanism

Objective: Along with the improved living condition of modern society,food production and diet structure have been changed,resulting in increased intake of fructose.High fructose diet is closely related to metabolic syndrome,which is a risk factor of cardiovascular,cerebrovascular,and chronic kidney disease(CKD).Insulin resistance and hyperinsulinemia are the pathological basis of metabolic syndrome.Glucagon like peptide 1(GLP-1)analogue is a kind of new antidiabetic drugs.It can reduce blood glucose to a constant level through promoting insulin biosynthesis and secretion,protecting islet beta cells,improving insulin sensitivity,inhibiting glucagon secretion,inhibiting appetite and food intake and delaying gastric emptying.However,the effect of GLP-1 analogue on renal diseases in insulin resistant rats is not clear.This project established a high fructose induced insulin resistance rat model and evaluated the protective effect of Exenatide on renal tissue of rats.The corresponding potential mechanism was discussed as well,which would provide theoretical basis for further medication of CKD.Methods: Thirty male Wistar rats(body weight 180g-200g)were randomly assigned to 2 groups: normal control group(Con,n=12)and high fructose diet group(HF,n =18).Normal control group was fed with normal diet and high fructose group was fed with high fructose.Six rats were randomly selected from each group after 8 weeks’ feeding.To evaluate the insulin sensitivity,hyperinsulinaemic-euglycaemic clamp was performed and the glucose infusion rate(GIR)was calculated.Then the rats were killed and the blood and kidney tissue samples were collected.After 8 weeks of high fructose feeding,the insulin resistance rats model was established.The remaining rats in high fructose diet group were randomly assigned to 2 groups:high fructose group(HF)and Exenatide treatment group(HFG).Both groupswere fed high fructose and gave the corresponding drug intervention for 4weeks.After 4 weeks of drug intervention,hyperinsulinaemic-euglycaemic clamp was performed to calculate GIR.Then the rats were killed.Serum were collected for measuring fasting blood glucose(FBG),fasting insulin(FINS),total cholesterol(TC),triglyceride(TG),creatinine(Cr),blood urea nitrogen(BUN),uric acid(UA).Protein product of bone morphogenetic protein 7(BMP-7)in serum was detected by enzyme-linked immuno sorbent assay(ELISA).Kidney tissues were collected for detection of transforming growth factor beta 1(TGF-β1),bone morphogenetic protein 7(BMP-7),Smad4,connective tissue growth factor(CTGF),Toll like receptor 4(TLR-4),nuclear factor kappa B(NF-κB)and p38 mitogen activated protein kinase(p38 MAPK)by Western Blotting.HE staining,electron microscope was used to observe the morphological differences of kidney between groups.Result:1 Changes of indicators after 8 weeks’ HF diet:1.1 Weight No significant difference in the initial body weight between Con and HF group.After 8 weeks of feeding,the average weight of the HF group was slightly higher than that of the Con group.There was no statistical difference between these two groups(P>0.05).1.2 Serological indicators Compared with the Con group,FINS,BUN,Cr,TC,UA and TG of HF group were significantly improved(P<0.05),but there was no statistical difference in FBG between the two groups(P>0.05).1.3 Hyperinsulinaemic-euglycaemic clamp Glucose infusion rate(GIR)of HF group was significantly lower than that in the Con group(P < 0.05).2 Changes of indicators after 4 weeks of drug intervention2.1 Weight After 4 weeks of drug intervention,the weight of HF group was slightly higher than that of the Con group and the HFG group,there was no statisticaldifference among these groups(P>0.05).2.2 Serological indicators Compared with the Con group,FINS,BUN,Cr,TC,UA and TG of HF group were significantly improved(P<0.05).These indicators of the HFG group were significantly higher than that in Con group(P<0.05).These indicators of the HFG group were significantly lower than that in HF group(P<0.05).There was no statistical difference in FBG between the three groups(P>0.05).2.3 Serum BMP-7Compared with the Con group,serum BMP-7 of HF group were significantly decreased(P<0.05).Serum BMP-7 of the HFG group were significantly lower than that in Con group(P<0.05).Serum BMP-7 of the HFG group were significantly higher than that in HF group(P<0.05).2.4 Renal HE staining Renal tissue sections of all rats in each group were observed by light microscope(×400).The glomerular and renal tubules of normal control group were clear,and the structure was normal.In high fructose group,glomerular volume were small,the number of cells increased obviously,renal tubular epithelial cells were swelling,mesangial cells and endothelial cells were hyperplasia,mesangial area was broadening.The above abnormalities were improved in the HFG group.2.5 Transmission electron microscope After 4 weeks of drug intervention,renal tissue of all rats in each group were observed by transmission electron microscopy(×15000).There were no obviously abnormal in the renal structure of Con group.Whereas HF group showed glomerular podocyte foot process structure disorder,widely fused and even disappeared,increased extracellular matrix,thickened basement membrane and uneven thickness.Compared to the high fructose group,the abnormal structure of the glomerular foot cell was improved in the HFG group.2.6 Renal fibrosis Compared with the normal control group,the protein expression ofTGF-β1,Smad4 and CTGF in kidney of high fructose group rats were significantly increased(P<0.05).Protein expression of BMP-7 was significantly decreased(P<0.05).The protein expression of TGF-β1,Smad4 and CTGF was decreased in the HFG group than those in the HF group,while the expression of BMP-7 was increased,and the difference was statistically significant(P<0.05).2.7 The signaling pathway of TLR-4Compared with the normal control group,the protein expression of NF-κB,p38 MAPK and TLR-4 in kidney tissue of high fructose group rats were significantly higher(P<0.05).The protein expression of TLR-4,NF-κB,p38 MAPK in HFG group were significantly decreased than those in the high fructose group(P<0.05).2.8 Hyperinsulinaemic-euglycaemic clamp Glucose infusion rate in high fructose diet group was significantly lower than that in the normal control group(P<0.05).The glucose infusion rate of the HFG group was significantly higher than that of the high fructose group(P<0.05).Conclusion:1 High fructose diet can induce insulin resistance in rats,which is marked by hyperinsulinemia,hyperlipidemia,hyperuricemia and other metabolic disorders.2 Exenatide showed effects of improving insulin sensitivity,reducing blood lipid and protecting renal function in rats fed with high fructose diets.3 Exenatide may reduce renal fibrosis through down-regulating TGF-beta1,Smad4,CTGF and up-regulating BMP-7.4 Exenatide can improving renal function through TLR-4 signal pathway,in which the protein products of TLR-4,NF-κB and p38 MAPK were down-regulated.