| 1-Deoxynojirimycin(DNJ)is an alkaloid isolated from mulberry leaves.DNJ has long been regarded as an alpha glycosidase inhibitor.In the previous study,we found DNJ may have the effect of increasing insulin sensitivity,therefore,the present study systematically and insightfully investigated whether DNJ could improve insulin resistance.The main content of the present study are as follows:The present study developed a method to separate DNJ from mulberry leaves:mulberry leaves were extracted in boiled water,the extract water was passed over a cation exchange resin,an anion exchange resin column respectively,then the product was purified by adsorbent,concentrated,and finally crystallized in mixed solvent,the purity of DNJ was over 95%.This method is simple and economic,and can prepare hundred grams of DNJ,it also lay foundation for its industrialization application.db/db mice,a typical insulin resistance animal model,were used in the study to investigated the effect of DNJ on improving insulin resistance.db/db mice were received intravenously DNJ(20,40,80 mg-kg-1·day-1)for four weeks,monitor blood glucose,body weight for weeks,did the glucose tolerance test and insulin tolerance test in the last week of experiment,measured the serum insulin levels after animal experiment,calculated the insulin resistance index.After 4-week treatment of DNJ,the blood glucose and body weight of db/db mice were markedly reduced,glucose tolerance and insulin tolerance of db/db mice were significantly improved,the serum insulin levels of db/db mice also declined significantly,insulin resistance index were decreased obviously.These results demonstrated that DNJ can significantly improve insulin resistance of db/db mice.In order to explore the mechanism of DNJ improving insulin resistance,the present study investigated the effect of DNJ on glucose transporting in skeletal muscle and epididymis adipose.The expression and translocation of Glucose transporter 4(GLUT4)in skeletal muscle and epididymis adipose of db/db mice were determined by Western blot,and the key protein expression and phosphorylation in insulin PI3K/AKT signaling pathway which regulated GLUT4 translocation were also analyzed.The results showed that DNJ didn't affect the GLUT4 expression in skeletal muscle and epididymal adipose of db/db mice,but DNJ could significantly promote GLUT4 translocation in the skeletal muscle and epididymal of db/db mice,although DNJ could not increase the total IR-?,IRS 1,PI3K,and AKT expression,DNJ could promote the phosphorylation of IR-? on Tyr1361,IRS1 on Tyr612,PI3K on p85,and AKT on Ser473.These results demonstrated that DNJ can promote GLUT4 translocation via activating insulin PI3K/AKT signaling pathway in skeletal muscle and epididymal adipose tissue,increase glucose uptake,thus improve insulin resistance.Glycogen synthesis plays an important role in the hepatic glucose metabolism,the present study investigated the effect of DNJ on hepatic glycogen synthesis,and explored the related mechanism.The present study measured the hepatic glycogen of all animals,and determined the key enzymes of hepatic glucose metabolism:pyruvate kinase(PK),hexokinase(HK),glycogen phosphorylase(GP),glucose-6-phosphatase,(G6Pase)and phosphoenolpyruvate carboxykinase(PEPCK),the expression and phosphorylation of the key protein in insulin PKB/GSK-3 were analyzed by Western blot.The results showed that DNJ treatment significantly increased the content of hepatic glycogen of db/db mice and the activities of glycolytic enzyme PK and HK,markedly suppressed the activities of gluconeogenesis enzyme GP,G6Pase and PEPCK,the results also showed that DNJ didn't affect the expression of total protein of PI3K,AKT,glycogen synthase kinase 3 beta(GSK-3p)and glycogen synthase(GS),but DNJ significantly up-regulated the phosphorylation of p85-PI3K,Ser473-AKT,Ser9-GSK-3p and down-regulated the phosphorylation of Ser645-GS.These results demonstrated that DNJ can modulate key enzymes of hepatic glucose metabolism and activate insulin PKB/GSK-3? signaling pathway to improve insulin resistance,thus promote hepatic glycogen storage,improve hepatic glucose metabolism.db/db mice suffer from lipid metabolism disorders and nonalcoholic fatty liver disease(NAFLD),which not only endanger the health of liver,but also increase the insulin resistance,the present study evaluated the effect of DNJ on lipid metabolism disorders and NAFLD.The presentd study measured serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),High-density lipoprotein cholesterol(HDL-C),alanine transaminase(ALT),aspartate transaminase(AST)and hepatic TG,H&E stain was carried out to observe the hepatic pathology changes,the expression of tumor necrosis factor alpha(TNF-a),interleukin-1(IL-1)and interleukin-6(IL-6)in the liver of db/db mice were analyzed by enzyme linked immunosorbent assay(ELISA).The results showed that the DNJ significantly reduced the serum TC,TG,LDL-C,ALT,AST and hepatic TG in db/db mice,but didn't affect the serum HDL-C,DNJ significantly improved the hepatic steatosis in db/db mice,and reduced the expression of TNF-?,IL-6,IL-1 in the liver tissue of db/db mice.These results demonstrate that DNJ can improve lipid metabolism and NAFLD in db/db mice,and these effects may be related to the suppressing TNF-?,IL-1 and IL-6 expression by DNJ.Diabetic nephropathy(DN)is a common complication of diabetes,the present study investigated the effect of DNJ on DN of db/db mice,and explored the related mechanism.The present study measured the serum creatinine(Cr),blood urea nitrogen(BUN),advanced glycation end products(AGEs),superoxyde dismutase(SOD)and malondialdehyde(MDA);H&E stain and periodic acid-schiff stain(PAS)stain were carried out to evaluated the effective of DNJ on kidney pathological structure changes in db/db mice;The expression of transforming growth factor-?1,(TGF-?1)and vascular endothelial growth factor(VEGF)in glomeruli were analyzed by immunohistochemical method.The results showed that DNJ significantly reduced serum Cr,BUN,AGEs,MDA,and increased activities of serum SOD;H&E stain and PAS stain results showed DNJ improved the kidney pathological structure changes in db/db mice,reduced the accumulation of extracellular matrix(ECM)and expansion of mesangial in the glomerular of db/db mice;Immunohistochemical results showed DNJ significantly reduced TGF-?1 and VEGF expression in the glomeruli of db/db mice.These results demonstrate that DNJ can improve diabetic nephropathy,and this effect is related to DNJ improving imbalanced redox state and suppressing TGF-?1 and VEGF expression in the glomeruli.The last chapter of the present study nvestigated the synergetic effect of DNJ and rosiglitazone(RSG)on improving insulin resistance.The present study measured the blood glucose,body weight,serum insulin,insulin resistance index and glucose tolerance test.These results showed that DNJ,RSG and DNJ&RSG significantly decreased the fasted blood glucose,body weight,serum insulin and insulin resistance index of db/db mice and improved glucose tolerance test of db/db mice;Compared with DNJ and RSG,fasted blood glucose,area under curve of insulin resistance index and of DNJ&RSG decreased significantly.These results demonstrate that DNJ and RSG can improve insulin resistance synergistically.In conclusion,the present study successfully developed a simple and economic method to prepare DNJ,systematically and insightfully investigated the effect and mechanism of DNJ on improving insulin resistance,the present study demonstrated DNJ can improve insulin resisitance of db/db mice and ecplored the mechanism,meanwhile,the present study found DNJ can improve NAFLD and alleviate DN,furthermore,the present study also found DNJ and RSG can improve insulin resistance synergistically.These research findings of the present study lay a solid foundation for the further development of novel insulin sensitization agent. |
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