The Roles Of Local Cortisol Regeneration And IL-1β In Insulin Resistance Of Granulosa Cells In Polycystic Ovarian Syndrome

AimsInsulin resistance is one of the major characteristic of the PCOS.The increased amount of cortisol and IL-1β contributed to insulin resistance in insulin-traget organs.But till now,no study has investigated the roles of cortisol and IL-1β in the development of insulin resistance of granulosa cells in PCOS.so in our study we aim to explore the effects and relationship of cortisol and IL-1β on the development of insulin resistance in granulosa cells of PCOS.Methods1.Collection of follicular fluid and granulosa cells from PCOS patients（Rotterdam criteria,PCOS with IR: Fasting insulin ≥ 15 μIU/ml or HOMA-IR ≥ 2.0,PCOS without IR: Fasting insulin < 15 μIU/ml and HOMA-IR < 2.0）and non-PCOS patients（only tubal infertility,normal BMI without IR）;2.The cortisol,cortisone,IL-1β,E₂,P₄ and lactate levels in the follicular fluid or cell culture medium were detected with immunoassay kits;3.Western blotting,PCR,immunofluorescence staining and kinetic analysis were performed to examine the expression of 11β-HSD1 and 2 in cultured granulosa cells from non-PCOS patients;4.Real-time PCR was performed to examine 11β-HSDs,GR-α and PTEN mRNA in human granulosa cells from the three groups;5.11β-HSDs reduactase and oxidase activities were detected by the conversion rates of cortisone to cortisol and cortisol to cortisone respectively in cultured granulosa cells from the three groups;6.After three days culture,the granulosa cells retrieved from non-PCOS or PCOS patients were treated with cortisol（0.1 and 1 μM）,IL-1β（0,0.1,1,10 ng/ml）and cortisol（1μM）combined with IL-1β（0.1 ng/ml）for 24 h seperatively,and real time PCR、Western Blot and ELISA were performed to detected 11β-HSDs expressions and activities,genes pertinent to IR （PTEN,Akt phosphorylation,GLUT4 translocation）and P₄ synthesis and key enzymes related to P₄ synthesis（St AR,CYP11A1,3β-HSD）.Results1.The BMI,fasting insulin level,HOMA-IR and hs-CRP were significantly higher in PCOS patients with IR compared with PCOS without IR and non-PCOS patients.E₂ level per oocytes retrieved in PCOS patients with IR was significantly lower than the other two groups.Moreover,P₄ and lactate levels in the follicular fluid were significantly decreased in PCOS patients with IR.The rate of high-quality embryos was only 45% in PCOS patients with IR,which was significantly lower than the non-PCOS patients,suggesting the dysfunction of granulosa cells affecting the quality of embryos.2.Cortisol concentration was about 10-fold higher than that of cortisone in the follicular fluid.Cortisol concentration in the follicular fluid was significantly higher in PCOS patients with IR when compared with either non-PCOS or PCOS patients without IR.There was no significant difference in the cortisone concentration among the three groups.3.The concentration of IL-1β in the follicular fluid was significantly higher in PCOS patients with IR when compared with non-PCOS patients.The lactate concentration in the follicular fluid was significantly lower than that in non-PCOS patients.Meanwhile we found that the IL-1β concentration in the follicular fluid was positively correlated with serum hs-CRP and fasting insulin levels and HOMA-IR,but having a negatively correlation with lactate level in the follicular fluid,indicating that the IL-1β concentration in the follicular fluid was closely correlated with whole-body inflammation status and insulin resistance of whole-body and ovary.4.Immunofluorescence staining,PCR and western blotting showed that both 11β-HSD1 and 11β-HSD2 were detected in the granulosa-lutein cells.Kinetic analysis revealed that the Km values of reductase and oxidase activities of 11β-HSD in granulosa cells were 242.3 ± 69.6 n M（character of 11β-HSD1）and 540 ± 42.4n M respectively（character of 11β-HSD2）,indicating the 11β-HSD1 and 11β-HSD2 being detected in granulosa cells we collected.5.11β-HSD1 but neither 11β-HSD2 nor GR-αmRNA level in the granulosa cells was significantly increased in PCOS patients with IR in comparison with either PCOS patients without IR or non-PCOS patients.11β-HSD1 but not 11β-HSD2 mRNA level in the granulosa cells positively correlated with the cortisol level in the follicular fluid.The reductase and oxidase activities of 11β-HSD were significantly increased and decreased respectively in PCOS patients with IR in comparison with either PCOS patients without IR or non-PCOS patients.All these indicated that the increased 11β-HSD1 expression and reductase activity were the major cause of increased cortisol level in the follicular fluid in PCOS patients with IR.6.Cortisol（0.1 and 1 μM）treatment for 24 h was able to stimulate 11β-HSD1 mRNA and protein expression in cultured granulosa cells from non-PCOS patients.Prior treatment of the granulosa cells with cortisol also increased the cell capacity to convert cortisone into cortisol in a dose-dependent manner.The same concentrations of cortisol had no effect on 11β-HSD2 mRNA abundance.The induction of 11β-HSD1 by cortisol was also observed in the granulosa cells from PCOS patients.7.PTEN mRNA level in the granulosa cells derived from PCOS patients with IR was almost two-fold higher than that in either PCOS patients without IR or non-PCOS patients.The level of PTEN mRNA in granulosa cells positively correlated with cortisol and IL-1β level in the follicular fluid as well as 11β-HSD1 mRNA abundance in the granulosa cells.Treating cultured granulosa cells with cortisol and IL-1β for 24 h separately stimulated PTEN mRNA and protein expression.Prior treatment of the granulosa cells with cortisol and IL-1β separately for 24 hours significantly attenuated the insulin induction of Akt phosphorylation and GLUT4 translocation.8.Cortisol treatment for 24 h could down-regulate St AR expression and P₄ synthesis in cultured granulosa cells,but IL-1β treatment for 24 h was able to stimulate St AR expression and P₄ synthesis.9.Cortisol combined with IL-1β get the similar results in stimulating PTEN expression,Akt phosphorylation and GLUT4 translocation in comparison with either cortisol or IL-1β treatment alone.But when referring to the St AR expression and P₄ synthesis,the cortisol completely suppress the stimulation effect of IL-1β on St AR expression and P₄ synthesis.ConclusionsWe have found that increased 11β-HSD1 expression and reductase activity in the granulosa cells are the major cause of increased cortisol concentrations in the follicular fluid of PCOS patients with IR.Excessive cortisol and IL-1β might impair the insulin signaling pathway and contribute to IR in the granulosa cells in PCOS patients by attenuating insulin-induced Akt phosphorylation and GLUT4 translocation via induction of PTEN expression.Cortisol combined with IL-1β has not aggravated the insulin resistance of granulosa cells.The lower P₄ level in the follicular fluid in the PCOS patients with IR may related with the higher cortisol in the follicular fluid having an effect on inhibiting the St AR expression in granulosa cells.