Effect Of Insulin On FoxO1 Nucleus-cytoplasmic Shuttling In MIN6 Cell

IntroductionFox O1 is one of the transcription factors of islete beta cells. It participates in variety signal pathways and regulates the differentiation, proliferation and apop tosis. Studying Fox O1 is significant to understanding the mechanism of diabetes.The MIN6 cells or β cells isolated from mice were incubated with 3m M glucose keeps the maintain of Fox O1 in the nucleus by Fox O1-EGFP real-time imaging。 In addition,it can be detectable that the Fox O1 fluorescence in cytoplasm increased but decreased in the nucleus after incubated with 16.7mmol/L and 30mmol/L glucose respectively,or 116.16ng/m L and 1161.6ng/m L insulin for 10-20 min.It has demonstrated that stimulates MIN6 cells with 25mmol/L glucose for 30 min, Fox O1 is rapidly phosphorylated,and transfered to the cytoplasm from nucleus. The effect is time(0.5-2h) dependence and dose(2.5-20mmol/L) dependence. The MIN6 cell were incubated with 0.58ng/m L 、 5.80ng/m L 、 580ng/m L and 1161.6ng/m L insulin, the similar results were obtained, the Fox O1 nucleus-cytoplasmic shuttling was not observed.To explore the effect of insulin on Fox O1 nucleus- cytoplasm transferring, this experiment stimulated the min6 cells with different concentrations of insulin at different times and observed the Fox O1 expression sites by confocal laser scanning microscopy. ObjectiveThe expression of the position changes of Fox O1 stimulation at different time in different concentrations were observed by immunocytochemical staining with confocal laser scanning microscopy. Methods1. mouse insulinoma MIN6 cells cultured with DMEM(high glucose) medium(containing 15%FBS,100U/m L penicillin,100μg/m L streptomycin) in 25 ml culture bottle placed in a cell incubator 37℃, 5% CO2.2. Incubating the MIN6 cells with 25mmol/L and 5.5mmol/L glucose respectively for 12 h and detecting the secretion of insulin.3. Stimulates the cells with 50 μIU/m L, 100 μIU/m L and 150 μIU/m L insulin for 12, 24 and 48 hours.4. immunocytochemical staining combined with confocal laser scanning microscopy to obscure the cellular localization of Fox O1. Results1. The Fox O1 is mainly expressed in the cytoplasm cultured with 25mmol/L glucose, and the concentration of endogenous insulin is 74.37ng/m L; Fox O1 translocates from cytoplasm to nucleus after the 5.5mmo/L cultured, and the concentration of endogenous insulin is 37.46ng/m L.2. compaired with the control group, the ratio of nuclear and cytoplasmic Fox O1 fluorescence of 50μIU/m L insulin group for 12, 24 and 48 h has been increased, except the 12 h group, the difference is statistically significant(P < 0.05); The ratio of nuclear and cytoplasmic Fox O1 fluorescence of 100μIU/m L insulin group for 12, 24 and 48 h has been increased, and the difference is statistically significant(P < 0.05); The ratio of nuclear and cytoplasmic Fox O1 fluorescence of 100μIU/m L insulin group for 12, 24 and 48 h has been increased, and the difference is statistically significant(P < 0.05). The difference within 50μIU/m L, 100μIU/m L and 150μIU/m L groups for different times is statistically significant(P < 0.05) respectively. Conclusions1. Fox O1 was mainly expressed in the cytoplasmat at the level of 25mmol/L glucose,and translocated to the nucleus at the level of 5.5mmol/L glucose.2. Exogenous insulin stimulates Fox O1 nucleus translocation to the cytoplasm, and dependent on the insulin concentration or the time.