The Effects Of Ang-(1-7) And Its Receptor Mas On Insulin Resistance Of Rat Ovarian Granulosa Cells

Objective: Cultured primary rat ovarian granulosa cells in vitro,establish insulin resistance model of the rat ovarian granulosa cells and add exogenously angiotensin II（angiotensin II）,angiotensin 1-7（Ang-（1-7））,angiotensin 1-7 receptor Mas agonists and antagonists.By detecting the amount of glucose uptake and lactic acid accumulation in each group of culture medium,to observe the changes of insulin resistance and explore whether Ang-（1-7）can improve insulin resistance in local ovaries granulosa cells by combining its receptor Mas.Methods:1 Construct insulin resistance model of rat ovarian granulosa cell Purchase fresh primary rat ovarian granulosa cells,cultured and passages in vitro.After culturing 4 days with adding dexamethasone and insulin,the amount of residual glucose in the medium was determined by oxidaseperoxidase method,lactic acid accumulation was determined by colorimetric method.Confirm the completion of the insulin resistance model by comparing with the blank control group.2 The rat ovarian granulosa cell were divided into three groups: the first group was blank control model,the second group was added AngⅡ(10^–6mol /L)and the third group was added Ang-（1-7）(10^-5mol / L).After 24 hours of culturing,the amount of residual glucose in the medium was determined by oxidase-peroxidase method,lactic acid accumulation was determined by colorimetric method.With calculating and comparing the contents of glucose and lactic acid in each group（the smaller of the difference indicates that less glucose consumption,less lactic acid production and the more severe insulin resistance）,we investigated whether Ang-（1-7）can antagonize AngⅡ to alleviate local insulin resistance in the ovary.3 The insulin resistance model of rat ovarian granulosa cells was divided into four groups:the first group was blank control model,the second group was added Ang-（1-7）(10^-5mol / L),the third group was added AVE0991（the agonist of angiotensin 1-7 receptor Mas）(10^-5 mol / L),the fourth group was added with Ang-（1-7）+ A799（the antagonist of angiotensin 1-7 receptor Mas）(10^-5 mol / L).After 24 hours of culturing,the amount of residual glucose in the medium was determined by oxidase-peroxidase method,lactic acid accumulation was determined by colorimetric method.With calculating the contents of glucose and lactic acid in each group,comparing with the blank control group and Ang-（1-7）group,we study whether Ang-（1-7）had an effect on local ovarian insulin resistance by binding to its receptor Mas.Results:1 Morphological trait of rat ovarian granulosa cells in vitro: We observed the cell morphological trait under inverted phase contrast microscope,adherence rate was above 90%.Granulosa cells cultured in vitro were slow,observely with inverted microscope,cell was monolayer growth and proliferation after culturing 24 h,the form were spindle or polygonal.The cells were proliferated obviously after 48 h,in the peak of proliferation after2-5d,beginning to degenerate after 7-9d and floating cells increased.2 Insulin resistance model of ovarian granulosa cells induced by dexamethasone + insulin: The fresh primary rat ovarian granulosa cells was divided into two groups.The experimental group was treated with dexamethasone and insulin for 4 days.The remaining glucose contents in the culture medium of blank control model group and the experimental group were:7.4888 ± 0.6624,10.4969 ± 0.7812,the accumulation of lactic acid were:23.7339 ± 0.8934,13.0538 ± 0.8869 respectively.The remaining glucose content in the experimental group was significantly higher than that in the blank control group and the accumulation of lactic acid was lower than that of the blank control group,and the difference was statistically significant（P<0.05）.3 After adding AngⅡ(10^–6mol / L)to granulosa cells of rat ovarian insulinresistance model for 24 h,we measured AngⅡ and blank model group culture difference of glucose containing medium respectively: 1.9856 ± 0.3051,3.4987±0.7036,and the amount of lactic acid accumulation difference respectively: 14.2944±0.7196,20.7581±1.4459.The difference of the content of glucose and lactate in the culture medium was lower than that in the blank control group,and the difference was statistically significant（P <0.05）.After adding Ang-（1-7）(10^-5mol/L)to granulosa cells of rat ovarian insulin resistance model for 24 h,we measured Ang-（1-7）group culture difference of glucose containing medium: 7.4698±1.0264,and the amount of lactic acid accumulation difference: 30.3427±2.6144.The difference of glucose content and lactic acid accumulation in culture medium was higher than that in blank control group and AngⅡ group,and the difference was statistically significant（P <0.05）.4 After adding Ang-（1-7）receptor Mas agonist AVE0991(10^-5mol/L)to granulosa cells of rat ovarian insulin resistance model for 24 h,the difference of glucose content in AVE0991 group,Ang-（1-7）group and blank model control group was measured respectively: 3.5345 ± 0.8142,7.4698 ± 1.0264,3.4987±0.7036,the amount of lactic acid accumulation difference respectively:20.1532±1.8087,30.3427±2.6144,20.7581±1.4459,the difference of glucose content and lactic acid accumulation compared with the blank model group showed no significant difference（P > 0.05）,compared with Ang-（1-7）group,the difference of glucose content and lactate accumulation in culture medium was significantly lower than that in Ang-（1-7）group,the difference was statistically significant（P < 0.05）.After adding Ang-（1-7）+ receptor Mas antagonist A799(10^-5mol/L)to granulosa cells of rat ovarian insulin resistance model for 24 h,the difference of glucose containing in Ang-（1-7）+ A799 group and Ang-（1-7）group was measured respectively: 6.1481 ± 0.6186,7.4698±1.0264,the amount of lactic acid accumulation difference respectively:25.4194 ± 2.1254,30.3427±2.6144,the difference of glucose content and lactic acid accumulation in culture medium was significantly lower than that in Ang-（1-7）group,the difference was statistically（P <0.05）.Conclusions:1 Dexamethasone + insulin can induce the insulin resistance model of rat ovarian granulosa cells,with the glucose uptake and the amount of lactic acid decreased.2 AngⅡ can increase the insulin resistance on local ovarian,and Ang-（1-7）can alleviate insulin resistance.3 Ang-（1-7）+ receptor Mas antagonist A799 can antagonize significantly the effect of improving insulin resistance by Ang-（1-7）,suggesting that Ang-（1-7）may play a role in binding to its receptor Mas.However,after Mas receptor agonist AVE0991 acting on the insulin resistance model of cells,it has no significant effect on local insulin resistance in ovarian rats,the reasons need further exploration.