The Study Of Mechanism And Reglatrive Of Insulin Secretion By Glucagon Through The CAMP Signaling Pathway In MIN6 Cells

Objective:By measuring the impact of different concentrations of glucagon(Gc)and glucose(Glu)on intracellular c AMP and insulin secretion,investigate the mechanism of Gc of the c AMP pathway in insulin secretion.Methods: 1.Cultured mouse pancreatic ? cell line MIN6 cells,the cells were grown to a certain density passaging experiment packets;2.Electroporation was transfected into ICUE3(c AMP-expressing plasmid)and negative control plasmid PCDNA3.1 into MIN6 cells,when the condition of none Glu(0 mmol / L),low Glu(2.8 mmol / L)and high Glu(16.7 mmol / L),the MIN6 cells were treated with Gc concentrations of none Gc(0 ng / L),low Gc(500 ng / L),high Gc(1000 ng / L),and then addition of Isoprenaline Hydrochloride(ISO),Fluorescence resonance energy transfer(FRET)technique based on fluorescence biosensor(Biosensor)was used to detect intracellular c AMP levels in different treatment stages;3.The supernatant was collected with or without the addition of ISO at different Glu concentrations and different Gc concentrations.Insulin release and intracellular c AMP levels were measured by enzyme-linked immunosorbent assay(ELISA).4.Statistical analysis of data collation.Results: 1.FRET technology real-time quantitative detection of different Glu concentration and different Gc concentration environment MIN6 live cells c AMP content of the results: None Glu: ISO group CFP / YFP was higher than those none Gc group(P <0.05);Low Glu: ISO group CFP / YFP was higher than those none Gc group,low Gc group and high Gc group(P <0.05),high Gc group was higher than none Gc group(P <0.05);High Glu: ISO group CFP / YFP was higher than those none Gc group,low Gc group and high Gc group(P <0.05),high Gc group was higher than none Gc group(P <0.05),low Gc group was higher than none Gc group(P <0.05).The results of comparison of the effects of different concentrations of Gc on c AMP production at different concentrations of Glu: none Glu: high Gc add ISO group was higher than high Gc group and low Gc group(P <0.05),low Gc add ISO group was higher than high Gc group and low Gc group(P <0.05);Low Glu: high Gc add ISO group was higher than high Gc group and low Gc group(P <0.05),low Gc add ISO group was higher than high Gc group and low Gc group(P <0.05),high Gc group was higher than low Gc group;High Glu: high Gc add ISO group was higher than low Gc add ISO group,high Gc group and low Gc group(P <0.05),low Gc add ISO group was higher than high Gc group and low Gc group(P <0.05).2.Different treatment groups were detected by ELISA the c AMP levels of MIN6 cells: In the same Glu and Gc concentration : ISO group was higher than none ISO group(P <0.05),high Gc group was higher than low Gc group and none Gc group(P <0.05).low Gc group was higher than none Gc group(P <0.05).3.Different treatment groups were detected by ELISA the Insulin secretion of MIN6 cells: ISO group was higher than none ISO group(P <0.05),high Gc group was higher than low Gc group and none Gc group(P <0.05).low Gc group was higher than none Gc group(P <0.05).4.The correlation between c AMP content and Ins secretion in MIN6 cells was analyzed:correlation coefficient r=1.129R~2=(0.813 P <0.05);The was a positive correlation between c AMP content and Ins secretion in MIN6 cells.Conclusion: 1.Gc increase the concentration of cAMP in mouse pancreatic ?-cell line MIN6 cells in a concentration gradient to promote Insulin secretion;2.This effect is more pronounced after the addition of ISO stimulation,indicating Gc promotes Ins secretion through the c AMP signaling pathway;3.Gc promoting insulin secretion by c AMP signaling pathways have Glu– dependent.