The Study On The Role And Mechanism Of The Second Messenger PKA In Insulin Secretion In MIN6 Cells Using FRET-based Fluorescence Biosensor Technology

Objective: To detected the changes of protein kinase A(PKA)and insulin secretion before and after the addition of PKA signaling pathway stimulant isoprenaline(ISO)in MIN6 cells with different concentrations of glucose and glucagon(GC),so as to further explore the role of PKA in GC promoted insulin secretion of MIN6 cells.Methods: 1.Cultured the mouse MIN6 cells in vitro and exchanged every other day.Grew to a certain density and passaged for amplification;2.Divided the cells into three groups and added three glucose of0mmol/L(no glucose),2.8mmol/L(low glucose)and 16.7mmol/L(high glucose).Based on this,three levels of GC of 0ng/L,500ng/L and 1000ng/L were set to intervene.PKA expression-sensitive plasmid AKR and negative control plasmid PCDNA3.1 were transferred to MIN6 cells respectively by electroporation.Then,using biosensor technology based on fluorescence resonance energy transfer(FRET)to quantitatively detect PKA changes in cells before and after the addition of PKA signaling pathway stimulant ISO;3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of insulin in cell supernatants of different treatment groups before and after adding stimulant ISO;4.Collected data and performed statistical analysis.Results: 1.Real-time quantitative detection of PKA content in cells after different concentrations of GC intervention under different concentrations of glucose environment based on FRET technology:(1)No glucose: 500ng/L GC stage increased compared to blank treatment stage and 0ng/L GC stage.1000ng/L GC stage increased compared to blank treatment stage and 0ng/L GC stage.ISO stage increased compared to all stages(P<0.05);(2)Low glucose: 0ng/L GC stage increased compared to blank treatment stage.500ng/L GC stage compared to blank treatment stage and 0ng/L GC stage increased.1000ng/L GC stage increased compared with blank treatment stage and 0ng/L GC stage.ISO stage increased compared with each stage(P<0.05);(3)High glucose: 0ng/L GC stage increased compared to blank treatment stage.500ng/L GC stage was higher than blank stage and 0ng/L GC stage.1000ng/L GC stage was higher than blank stage and0ng/L GC stage.ISO stage was higher than each stage(P<0.05);(4)Comparison of effects of different concentrations of GC on PKA formation under different glucose concentrations: In low and high glucose environment,the increase rates of 1000ng/L GC stage were both higher than that of 500ng/L GC stage(P<0.05);2.Detection of different concentrations of GC on the secretion of insulin in each group of cells by ELISA:(1)Insulin secretion without ISO : In no glucose group 1,1000ng/L GC was higher than 500 and0ng/L after intervention,and 500ng/L GC was higher than 0ng/L(P<0.05).In low glucose group 1,1000ng/L GC was higher than 500 and 0ng/L after intervention,and 500ng/L GC was higher than 0ng/L(P<0.05).In high glucose group 1,1000ng/L GC was higher than 500 and 0ng/L after intervention,and500ng/L GC was higher than 0ng/L(P<0.05);(2)Insulin secretion after adding ISO: In no glucose group 1,1000ng/L GC was higher than 500 and 0ng/L after intervention,and 500ng/L GC was higher than 0ng/L(P<0.05).In low glucose group 1,1000ng/L GC was higher than 500 and 0ng/L after intervention,and500ng/L GC was higher than 0ng/L(P<0.05).In high glucose group 1,1000ng/L GC was higher than 500 and 0ng/L after intervention,and 500ng/L GC was higher than 0ng/L(P<0.05);(3)Comparison of insulin secretion before and after the addition of ISO: No glucose: 1000ng/L GC was higher than 500ng/L after intervention(P<0.05);Low glucose: 1000ng/L GC was higher than 500ng/L after intervention,and500ng/L GC was higher than 0ng/L(P<0.05);High glucose: 1000ng/L GC was higher than 500 and 0ng/L after intervention,and 500ng/L GC was higher than 0ng/L(P<0.05).Conclusions: 1.GC promotes insulin secretion by increasing the concentration of PKA in MIN6 cells in a concentration gradient manner;2.The effect of GC in increasing PKA and insulin secretion was more evident after adding stimulant ISO.It shows the promotion effect of GC is achieved through the second messenger signal system PKA pathway;3.The role of GC in promoting insulin secretion by increasing cAMP concentration has a certain glucose dependency.