The Effects And Possible Mechanisms Of Sphingosine-1-phosphate To Glucose Stimulated Insulin Secretion

Objective:To observe the effects and the underlying molecular mechanisms of glucose-stimulated insulin secretion(GSIS)on rat islets after S1 P treatment,which was used to explore a therapeutic target for diabetes.Method:(1)Opening the chest and abdominal cavity after killing the rat.Collagenase P was injected slowly into the pancreas through the bile duct,and the pancreas was oscillated in a water bath at 37℃ for 11 minutes.The pancreatic islets were obtained by using 10771 centrifugation.The rat islets can be obtained after EDTA combined calcium and magnesium ions and Diapase II digested islets for 5min(β-cells in the majority).(2)RT-PCR was used to detect the expression of S1 P receptors(S1PR1-5)on rat pancreatic islet cells.(3)To observe the effect of different S1 P concentrations on insulin secretion of pancreatic β-cells under different glucose concentrations.Observe the effect of S1 P on insulin secretion after S1PR1-4 bolckers treatments.(4)The changes of voltage-dependent potassium current(Kv current)were observed after S1 P,S1PR1-4 blockers and S1P+S1PR1-4 blockers treatments by patch clamp technique.(5)To observe the changes of intracellular Ca2+ concentration after different S1 P concentrations treatments by calcium imaging technique.(6)To observe the changes of intracellular cAMP concentration after S1 P,S1PR1-4 blockers,S1P+S1PR1-4 blockers and Forskolin treatments.Results:(1)Pancreatic islets obtained by digestion with collagenase P were round and uniform in texture.The pancreatic islet cells obtained by Diapase II digestion were uniform in size,and their membrane was intact.(2)S1PR1-5 was expressed on the surface of islet cells by RT-PCR.(3)Compared with the low glucose(LG)control group,high glucose(HG)control group significantly promoted the secretion of insulin.S1 P did not change the insulin secretion(P> 0.05)under LG condition.S1 P increased insulin secretion significantly in a dose-dependent manner under HG condition(P <0.001).S1PR1-4 blockers did not affect the secretion of insulin,but S1PR1-4 blockers eliminated the effect of S1 P on insulin secretion(P <0.01).(4)S1P inhibited Kv currents under HG condition(P <0.01).On the basis of no effect of S1PR1-4 blockers on Kv currents,S1PR1-4 blockers eliminated the inhibitory effect of S1 P on Kv currents(P <0.05).(5)S1P increased intracellular Ca2+ concentration significantly in a dose-dependent manner under HG condition(P <0.001).(6)Forskolin as a positive control,S1 P inhibited the formation of intracellular cAMP(P <0.01).On the basis of S1PR1-4 blockers having no effect on cAMP,S1PR1-4 blocker eliminated the inhibition of S1 P on cAMP.Conclusion:(1)Islets and islet cells were of good activity.(2)Islet cell surface expressed S1PR1-5.(3)S1P increased GSIS in a dose-dependent manner via S1P/S1 PRs.(4)S1P inhibited the Kv currents via S1P/S1 PRs under HG condition.(5)S1P increased intracellular Ca2+ concentration in a dose-dependent manner under HG condition.(6)S1P inhibited the formation of c AMP via S1P/S1 PRs.In conclusion,S1 P promoted glucose-stimulated insulin secretion mianly via S1P/S1PRs/Kv/Ca2+ pathway.