Effect Of SHBG On The Expression Of Insulin Transduction Pathway Related Factors In Gestational Diabetes Mellitus

Objective:Gestational diabetes mellitus(GDM)is defined as “any degree of glucose intolerance developing or first detected during pregnancy.” The American diabetes association(ADA)has redefined gestational diabetes as "the second and third trimester of diabetes".Its morbidity is ever mounting year by year worldwide together with obesity and type II diabetes.In our country,IADPSG diagnosis standard is adopted,and the incidence of GDM is nearly 20%.Gestational diabetes has become one of the most common complications during pregnancy.The study of Waters,PM et al.[1] found according to IADPSG diagnostic criteria,neonatal birth weight,body fat rate and umbilical C peptide(C-peptide cord)is higher,the cesarean delivery rate of the pregnant women and the incidence of preeclampsia is higher.Gestational diabetes mellitus has a fatal or non-fatal adverse outcome of pregnancy.Insulin resistance refers to the biological effects after a certain amount of insulin to its specific receptor is lower than normal,characterized by peripheral tissue,especially the liver,fat,muscle tissue barriers of glucose intake and a increase in hepatic glycogen output.As the progress through the pregnancy,the placenta lactation element(HPL),prolactin(PRL),sugar cortical hormone,progesterone,auxin,etc all can adersely affect insulin,placenta can secrete insulin enzyme,accelerate insulin degradation,the resulting state of insulin resistance is the pathophysiologic basis of gestational diabetes.Sex hormone binding globulin(SHBG)plays an important regulatory role in insulin resistance in gestational diabetes mellitus and is an independent risk factor for gestational diabetes mellitus.The level of SHBG in pregnant women was negatively correlated with the severity of GDM.SHBG provides a new basis for further understanding the pathogenesis of GDM insulin resistance and individualized therapy for gestational diabetes mellitus.In this study,the PI3K/AKT pathway associated with insulin resistance was selected to analyze the correlation between SHBG and the representative factors of this pathway in the placenta,and explore its possible target.A cell-level insulin resistance model was constructed,and the expression of SHBG and PI3K/AKT pathway related factors was verified by Real-time PCR and Western Blot.Finally,transient transfection of insulin resistance model cells was carried out to observe the effects of SHBG expression on the proliferation,apoptosis,insulin resistance and the expression of PI3K/AKT pathway related factors in insulin resistance model.In order to further understand the biological function of SHBG and explore the possible mechanism of SHBG in insulin resistance signal transduction pathway in gestational diabetes mellitus(GDM),this paper provides a basis for the treatment of gestational diabetes mellitus with SHBG in the future.Methods: 1.Using Real time-PCR and Western blot to analyze the expression of SHBG and PI3K/Akt in the GDM placenta and the normal placenta.Fifty pregnant women with GDM grade A1 who were diagnosed by obstetrics in Shengjing Hospital affiliated to China Medical University,who had regular antenatal examination,single baby and delivered female baby and were treated with GDM diet to control blood glucose were included in this study(The screening criteria were oral 75 g glucose tolerance test(OGTT)at 24 ~ 28 weeks.The diagnostic threshold was fasting.,the blood glucose levels at 1 h and 2 h after taking glucose were 5.1 mmol / L 10.0 mmol / L and 8.5 mmol / L,respectively.Those who exceeded the threshold of one item or more were diagnosed as GDM.)50 healthy pregnant women who were hospitalized at the same time as normal term,OGTT negative at the same period of pregnancy,single baby and female baby with regular antenatal examination were taken as the healthy control group.Any pregnant woman with complications such as hypertension,asthma,arthritis,preeclampsia,pregestational diabetes,and macrovascular complications is excluded.2.RPMI 1640 medium with different insulin concentrations was used to the placental trophoblastic cells,the medium glucose content was detected at different time points by the method of glucose-hexokinase and the placental trophoblastic cell viability of different insulin concentrations was detected by the method of CCK8,and to determine the optimal concentration and time of insulin resistance;then the changes of cell morphology was observed by the oil red O and HE staining;on the basis of the established model the method of glucose-hexokinase was used to detect the consumption of glucose at different time to determine the duration of the insulin resistance model.3.SHBG was transfected to the constructed insulin model cells,Real-Time PCR and Western blot were used to detect the expression and correlation of GLUT1,GLUT3,GLUT4 PI3Kp85α IRS1 and IRS2 in normal cells,model cells and transfected cells.Glucose consumption was measured by glucose-hexokinase assay,and the effect of overexpression of SHBG on insulin resistance of insulin resistance model cells was observed.The protein expression of SHBG,GLUT1,GLUT3,GLUT4 PI3Kp85α RS1 and IRS2 was observed by immunofluorescence staining in each group.The effect of overexpression of SHBG plasmid on the proliferation of HTR8-SVneo cells was observed by CCK8.After transfection of SHBG plasmid,the apoptosis index of each group was analyzed and the difference was statistically analyzed.Results:1.The expression of m RNA and protein in SHBG in gestational diabetes mellitus group was significantly lower than that in normal group.The related factors of PI3K/Akt insulin resistance pathway except for the significant increase of GLUT1,GLUT3,GLUT4,IRS1,IRS2 PI3Kp85α were significantly lower than those in normal controls.There was a significant positive correlation between the expression of SHBG m RNA and the expression of IRS1,IRS2,PI3Kp85α.2.HTR8/SVneo cells were exposed to 10 ^-7 mol / L insulin for 48 h,and the glucose consumption was the least,so the insulin resistance model cells were established.Compared with normal cells,the proliferation of model cells decreased and lipid droplets increased significantly.Insulin resistance still existed within 60 hours after the establishment of the model.3.The expression of SHBG in insulin resistance model group was significantly lower than that in control group.After transfection of SHBG,with the increase of SHBG expression,the expression of GLUT3,GLUT4,IRS1,IRS2 and PI3Kp85α increased significantly except for the significant decrease of GLUT1 m RNA and protein levels(p < 0.05).The glucose content in the supernatant of the transfected SHBG group was 4.61 ±0.63 mol ·L-1 and that in the negative control group was 6.15 ±0.08 mol ·L-1.Glucose content in SHBG transfected group was significantly lower than that in negative control group(p < 0.05).The cell proliferation rate was 0.713 ±0.050 in SHBG group and 0.457 ±0.028(p < 0.05)in negative control group,but there was no significant difference in apoptosis rate between the two groups.In control group,SHBG was distributed in cell membrane and cytoplasm.The SHBG in the SHBG cell is concentrated in the cell membrane.In the control group,IRS-1 was mainly distributed in the nucleus.The distribution of IRS-1 in the insulin resistance model group was transferred to the cytoplasm.In transfected cells of SHBG,the IRS-1 refocused toward the cell nucleus.Conclusion:1.SHBG was successfully transfected into insulin resistance model cells by cationic liposome.2.The low expression of SHBG and high expression of GLUT-1 were found in insulin resistance model cells,while the expression of other PI3K/AKT insulin pathway related factors was decreased.3.Overexpression of SHBG inhibited the expression of GLUT-1 m RNA and protein,and promoted the expression of GLUT-3,GLUT-4,IRS-1 and IRS-2 PI3Kp85 m RNA and protein.4.SHBG IRS-1 and IRS-2PI3K-p85 α were positively correlated in insulin resistance cell model.5.SHBG in HTR8-SVneo is distributed in cell membrane and cytoplasm,the higher the expression of SHBG,the more the aggregation of the cell membrane.6.Insulin can stimulate irs-1 to release from the nucleus to the cytoplasm,and transfected with the expression of SHBG can enable irs-1 to reassemble the nucleus.7.Overexpression of SHBG in insulin resistance model cells can effectively promote cell proliferation.8.Overexpression of SHBG in insulin resistance model cells inhibited early apoptosis,but did not affect the total apoptosis rate.9.Overexpression of SHBG in insulin resistance model cells significantly increased glucose metabolism.