The Role Of The Crosstalk Between Netrin-1 And Autophagy In Diabetes

Background:The incidence of diabetes showed a trend of increase in the world,it serious threat to human health.The insulin resistance and relative shortage of insulin is important factors in the development of type 2 diabetes,the main cause of these two factors including apoptosis,oxidative stress,inflammation,etc.,but there is no clear its specific reasons.Autophagy is a degradation pathways relying on lysosome,damaged organelles within the cell and misfolded proteins were degraded through the system.Autophagy can be activated by hunger,lack of oxygen,inflammation,and other factor in islet beta cells,skeletal muscle cells,fat cells,liver cells and nerve cells,and plays a role in a variety of cells.Autophagy can effect the islet beta cell activity and function by activating the endoplasmic reticulum stress.Survival factor Netrin-1 expressed in multiple tissues in the body,such as the pancreas,kidney,lung,and widely involved in inflammation and tissue damage,a variety of pathophysiological process of angiogenesis and so on,in coronary heart disease,pneumonia,acute pancreatitis,it played an important role in diseases such as tumor.Studies have shown that it can promote the secretion of insulin,Netrin-1 plays a protective role in the occurrence and development of diabetes.There are more and more evidence suggests that autophagy played an important role in the occurrence and development of diabetes.Objective: To test the hypothesis that Netrin-1 and autophagy can effect each other,and these interactions may have good influences on the development of diabetes,and clarify the specific mechanism of its function.Methods and Results:In vivo: A total of 12 SPF 8-week healthy male C57BL/6J mice were selected and randomly divided into a normal control group and a diabetes model group(n = 6,respectively)after 1 week's adaptive feed.The normal control group was fed with the standard diet;and the diabetes model group was fed with fat diet for 4 weeks and injected with streptozotocin(STZ)(75mg/kg)into the abdominal cavity after 12hours' fasting.All mice received abdominal glucose tolerance test 5 weeks later.The results showed that the fasting blood-glucose(FBG)in C57BL/6J mice fed with fat diet and injected with STZ increased slightly and the 30 min,60 min and 120 min postprandial blood glucose increased significantly.The weight of the mice was measured and the changes in FBG levels in mice were detected every week.The results showed that the mice in the diabetes model group fed with fat diet in the first 4weeks were much heavier than those in the normal control group.The difference in weight between the two groups gradually increased after the injection of STZ.The difference was statistically significant.Four weeks later,abdominal glucose tolerance test was conducted again.The results showed that the FBG in C57BL/6J mice fed with fat diet and injected with STZ increased slightly and the 30 min,60 min and 120 min postprandial blood glucose increased significantly.Western Blot and Real time-PCR were applied to detect the expression of Netrin-1 in the mice pancreas in the two groups.The results showed that the expression of Netrin-1 in the mice pancreas was normal in the diabetes model group and higher in the normal control group.The expression of Netrin-1 mRNA in the mice pancreas was normal in thediabetes model group and much higher in the normal control group.In vitro:Netrin-1 gene was transfected to ins-1 cells with viruses.The expressions of LC3-II and P62 before and after the transfection of Netrin-1 gene were detected by Western Blot and Real time-PCR.The results showed that Netrin-1 can obviously promote autophagy.Ins-1 cells were treated with rapamycin(100nm)for 0h,0.5h,2h and 4h respectively after being transfected with Netrin-1 gene.The expressions of LC3-II and P62 at different time points were detected by Western Blot.The results showed that the cells reached the peak of autophagy.The ins-1 cells transfected with Netrin-1 gene and the ins-1 cells in the control group was treated with no glucose(0mmol/L),normal glucose(11.11 mmol/L)and high glucose(25 mmol/L)for 4h and treated with or without rapamycin for 2h.Western Blot was applied to detect the expressions of LC3-II and P62 after the treatment with 3 different concentrations of glucose for different times.The results showed that no glucose and high glucose can promote autophagy.Netrin-1 in high concentration can promote the secretion of insulin,while autophagy can inhibit such promotion.When autophagy is strong,Netrin-1 can inhibit the enhancement of autophagy.The ins-1 cells transfected with Netrin-1 gene and the ins-1 cells in the control group was treated with no glucose(0mmol/L),normal glucose(11.11 mmol/L)and high glucose(25 mmol/L)for 4h respectively and then treated with rapamycin for 0h,0.5h,2h and 4h.The secretion of insulin and Netrin-1 after treatment with 3 different concentrations of glucose for different times was detected by ELISA.The results showed that no glucose and high glucose can promote the secretion of Netrin-1.When Netrin-1 is in low concentration,autophagy can promote the secretion of Netrin-1.When Netrin-1 is in high concentration,autophagy can inhibit the secretion of Netrin-1.Meanwhile,the results also showed that the interaction between Netrin-1 and autophagy can promote thesecretion of insulin.In addition,Netrin-1 also showed a regulatory effect on insulin in abnormal glucose concentration.Conclusion: Netrin-1 and autophagy may affect each other and keep themselves in balance;this balance is important because netrin-1 significantly influences B-cell protection under different glucose concentrations.Consequently,netrin-1 may be a useful therapeutic molecule for treating diabetes because of its modulation of autophagy and insulin secretion.