Sphingosine 1-phosphate Glucose-dependently Stimulating Insulin Secretion In Rat And The Underlying Mechanism

Objective:Sphingosine 1-phosphate（S1P）is a metabolite of sphingosine phosphorylation catalyzed by sphingosine kinase（Sph K）,has adjust from cell differentiation and apoptosis of a variety of cellular functions,and able to metabolic disease,organ transplantation,multiple sclerosis,cancer,nerve inflammation and other beneficial effects.Studies have shown that S1P has the effect of promoting insulin secretion,reducingβ-cell apoptosis,and improvingβ-cell function.However,the electrophysiological mechanism and characteristics of S1P-induced insulin secretion remain unclear.In this study,we investigated the effects of S1P on insulin secretion of rat islets and its electrophysiological mechanism,as well as the effects of S1P on blood glucose in mice.In addition,we also explored the cell signaling pathways related to these effects,providing theoretical basis and experimental data support for exploring its use as an intelligent hypoglycemic drug.Methods:（1）Normal male Wistar rats were sacrificed acutely.Collagenase P solution was injected into the pancreas through the common bile duct at a constant speed.The isolated Pancreatic tissue was digested in a 37°water bath for 15 minutes.The rat islets were obtained by density gradient centrifugation and further digested by DispaseⅡto obtain isletβcells.（2）Insulin secretion experiment,the effect of S1P on insulin secretion of isletβcells was observed under different glucose concentrations,and the effect of specific signal pathway blockers on insulin secretion of isletβcells was observed.（3）Patch clamp experiment was conducted to investigate the effects of S1P onβcell action potential duration and voltage-dependent calcium channel current,the effects of S1P and specific signal pathway blockers onβcell voltage-dependent potassium channel,and the effects of S1P on CHO cell voltage-dependent potassium channel.（4）c AMP measurement,using radioimmunoassay to study the effect of S1P on c AMP content in rat pancreatic isletβcells.（5）incubating rat pancreatic isletβwith Ca²⁺-specific fluorescent dye Fura-2 AM.（6）CCK8 assay was used to detect the effects of S1P on the proliferation and survival of INS-1 and Cho-KV2.1 cells induced by high glucose and high lipids,and to study the possible mechanism of S1P on the proliferation and survival ofβcells.（7）Glucose tolerance test（OGTT）was used in C57BL/6 mice to detect blood glucose levels at each time point,and ELISA method was used to detect plasma insulin secretion level at each time point.Results:（1）Under the condition of low glucose concentration of 2.8mmol/L,different concentrations of S1P（5μmol/L,10μmol/L,20μmol/L）had no effect on rat insulin.Under the condition of high glucose concentration of 16.7mmol/L,S1P（5μmol/L、10μmol/L,20μmol/L）significantly promoted insulin secretion in rats in a dose-dependent manner.（2）S1P can prolong the action potential duration ofβcells and inhibit voltage-dependent potassium（Kv）channels.（3）Using Forskolin as a positive control,S1P inhibited the production of intracellular c AMP.（4）S1P increased intracellular Ca²⁺levels but did not significantly activate voltage-dependent calcium channels（VDCC）.（5）S1P elevates Ca²⁺levels mainly through extracellular Ca²⁺influx rather than releasing intracellular Ca²⁺stores.（6）By CHO-Kv2.1 cells,it was found that S1P does not directly inhibit Kv channels.（7）S1P inhibits Kv channel currents and promotes insulin secretion through the PLC/PKC signal transduction pathway.（8）S1P can improve the viability of INS-1 cells induced by high glucose and high fat,and has a protective effect on INS-1 cells.（9）S1P could not improve the viability of CHO-Kv2.1cells induced by high glucose and high fat,and had no obvious protective effect on CHO-Kv2.1 cells.（10）S1P increased plasma insulin levels in C57BL/6 mice and decreased blood glucose levels in C57BL/6 mice.Conclusions:Under the condition of high glucose concentration,S1P could significantly promote insulin secretion in rats in a dose-dependent manner.S1P inhibits Kv channel currents and promotes insulin secretion through the PLC/PKC signal transduction pathway.S1P increased intracellular Ca²⁺levels but did not significantly activate voltage-dependent calcium channels（VDCC）.S1P can improve the viability of INS-1 cells induced by high glucose and high fat,and has a protective effect on INS-1 cells.S1P can promote insulin secretion from isletβcells in C57BL/6 mice and improve glucose tolerance in C57BL/6mice.In addition,this study suggests that the S1P receptor may be a potential target for the treatment of type 2 diabetes,which also provides useful clues for further exploration of S1P receptor agonists as new drugs for the treatment of type 2 diabetes.