The Effect Of Insulin Secretion In Min6 Cells By Glucagon Through DAG-PKC Signaling Pathway

Objective:Further to define the role and mechanism of insulin secretion by glucagon, through exploring the insulin secretion and the expressinon of DAG and protein kinase C(PKC) of pancreatic β cell-MIN6 cell stimulated by different concentration of Glucagon and Glucose.Methods: 1.The mouse islet cell tumor MIN6 in vitro was divided when cultured to certain cell density the intracellular supernatant of DAG was collected through the stimulated Glucagon and Glucose, used enzymelinked immunsorbent assy to detect the concentration of DAG.2.Transfected the sensitive fluorescent biosensor CKAR(expressed PKC) and PCDNA3.1, using the Fluorescence resonance energy transfer(FRET) technique to observed the fluorescence of cells and detect in real time within living cells under different processing intracellular the PKC concentration. 3. using ELISA to detect the insulin secretion.Results: 1. The result of DAG concentration of MIN6 cell supernatant using enzyme-linked immunosorbent assay:(1) In the condition of 2.8mmol/L and 16.7mmol/L glucose group, along with the glucagon increased, the DAG concentration is increased too, and the tendency is clear. Compared with the 2.8mmol/L glucose group, the tendency of DAG co ncentration of 16.7mmol/L glucose group is more obviously. There is none the tendency of 0mmol/L glucose group.(2) compared with the different group of glucagon: without glucose, the DAG concentration of the middle glucagon group was higher than none glucagon group(P<0.05); under the low glucose group, the DAG concentration of the high middle and low glucagon group was increased than none glucagon group(P<0.05); compared with low and middle glucagon group, the DAG concentration of high glucagon was higher. Under the high glucose: compared with the group of 0ng/L glucagon, the DAG concentration of high and middle glucagon group was higher.(P<0.001), the DAG concentration of high glucagon group was higher than low and middle glucagon group(P<0.001,P<0.05). 2 Based FRET technique under different processing MIN6 living cells in real time quantitative detection of intracellular PKC :(1) transfected empty plasmid, with the stimulated of 16.7 Glucose,1000ng/L Glucagon the ratio of CFP/YFP was no significant changed, P>0.05. Transfected CKAR:(1) without glucose, stimulate with 100ng/L and 500ng/L glucagon, the ratio of CFP/YFP was rised, the tendency is gently, there is significant changed between them( P<0.05); the stimulate with 1000ng/L glucagon, the curve of CFP/YFP was rised, the ratio of CFP/YFP is no significant changed, but it is decreased.(2) with the 2.8mmol/L glucose, stimulate with 100ng/L and 500ng/L glucagon, the ratio of CFP/YFP was rised, the tendency is gently, there is significant changed between them( P<0.05); the stimulate with 1000ng/L glucagon, the curve of CFP/YFP is rised, the tendency of CFP/YFP ratio is obviously, the ratio of CFP/YFP is significant changed compared with 100ng/L and 500ng/L glucagon group( P<0.05).(3) with the16.7mmol/L glucose, stimulate with 100ng/L and 500ng/L glucagon, the ratio of CFP/YFP was rised, the tendency is gently, there is no significant changed between them( P>0.05); the stimulate with 1000ng/L glucagon, the curve of CFP/YFP is rised, the tendency of CFP/YF P ratio is obviously, the ratio of CFP/YFP is significant changed compared with 100ng/L and 500ng/L glucagon group( P<0.05). 3. The result of insulin secretion of MIN6 cell supernatant using enzyme- linked immunosorbent assay: compared with the different group of Glucagon(1) In the condition of 0mmol/L, 2.8mmol/L and 16.7mmol/L glucose group, along with the increased glucagon, the insulin secretion is increased, and the tendency of insulin secretion is more and more obviously along with the glucose concentra tion from low to high. The tendency of insulin secretion of 16.7mmol/L glucose group is maximum.(2)without glucose, the insulin concentration of the high glucagon group, middle glucagon group and low glucagon group was higher than none glucagon group(P<0.01); the insulin concentration of the high middle was increased than middle glucagon group and low glucagon group(P<0.05); under the low glucose group, the insulin concentration of the high middle glucagon group was increased than none glucagon group(P<0.01); compared with low glucagon group, the insulin concentration of high glucagon was higher; Under the high glucose: compared with the group of 0ng/L glucagon, the insulin concentration of high and middle glucagon group was higher.(P<0.05), the insulin co ncentration of high and middle glucagon group was higher than low glucagon group(P<0.01), compared with the group of middle glucagon, the insulin concentration of high glucagon group was higher.(P<0.01).Conclusion:1. Glucagon can increase the insulin sec retion through increasing the DAG and PKC concentration in MIN6 cells. The action way was glucose concentration-denpendent.