Maternal High Estradiol During Early Pregnancy Induces Insulin Resistance In Offspring Through Decreased Hypothalamic INSR

Part I Glycometabolism function of the newborns and children resulted from spontaneous conception（SC）,frozen embryo transfer（ET）and fresh ETObjective: To compare the serum estradiol（E₂）of SC,frozen ET and fresh ET mothers during early pregnancy,and analyze the glycometabolism function of their newborns and children.Methods: Chemiluminescent immunoassay was performed to detect serum E₂ levels of SC,frozen ET and fresh ET mothers during early pregnancy.Glucose oxidase method and radioimmunoassay were performed separately to detect glucose and insulin levels of newborns and children resulted from SC,frozen ET and fresh ET,whose clinical characteristics were collected thourgh browsing of medical records and face-to-face interviewing.Results:（1）The blood E₂ level of fresh ET mothers was significantly higher than SC and frozen ET during early pregnancy.（2）No significant difference of the umbilical glucose was shown among SC,frozen ET and fresh ET male newborns,while the umbilical insulin and HOMA-IR scores of fresh ET male newborns were significantly higher than SC and frozen ET male newborns.（3）No significant difference of the umbilical glucose,insulin and HOMA-IR scores was shown among SC,frozen ET and fresh ET female newborns.（4）No significant difference of the fasting glucose was shown among SC,frozen ET and fresh ET male children,while the fasting insulin and HOMA-IR scores of fresh ET male children were significantly higher than SC and frozen ET male children.（5）No significant difference of the fasting glucose,insulin and HOMA-IR scores were shown among SC,frozen ET and fresh ET female children.Conclusion: Fresh ET mothers showed elevated serum E₂ during early pregnancy,male newborns and children resulted from fresh ET showed increased fasting insulin and HOMA-IR scores.Part II Mouse model of maternal high E₂ and observation of weight and glycometabolism in offspringObjective: To explore whether maternal high E₂ during early pregnancy will lead to metabolic disorders in offspring.Methods: Male and female C57BL/6 mice were mated overnight and the next day was declared day 0.5 of pregnancy（E0.5）if a vaginal plug was present in the morning.The pregnant mice were randomly assigned to receive gavage of 100ug/kd/d estradiol valerate dissolved in corn oil or an equal volume of pure corn oil from E5.5 to E11.5,whose offspring were defined as HE and NC group separately.Maternal blood was tested for E₂ level.The offspring were weighted during growth,glucose tolerance test（GTT）and insulin tolerance test（ITT）were performed at 3,8,12 and 24 weeks after birth.Enzyme-linked immuno sorbent assay（ELISA）was performed to detect fasting insulin and leptin of the 24-w male offspring.Results:（1）The serum E₂ of pregnant mice administrated with estradiol valerate increased significantly.（2）The HE male and female mice were lighter than NC group during the first two weeks and showed a catch-up growth thereafter.The HE male mice grew heavier than NC group since 20 w after birth.（3）The HE male mice showed increased area under the curve（AUC）of GTT than NC group at 12 and 24 w after birth,and increased AUC of ITT at 24 w after birth.However,the AUCs of GTT and ITT of HE and NC female mice did not show significant difference.（4）The 24-w HE male mice showed elevated fasting insulin than NC group,while the fasting leptin was similar between two groups.Conclusion: The male offspring with maternal high E₂ gained more weight and developed insulin resistance during growth.Part III The food intake and hypothalamic neuropeptides expression in mouse offspringObjective: To explore the influence on food intake regulation of offspring by maternal high E₂.Motheds: The daily food intake of male offsprinng was monitored from 3 to 24 w.The m RNA expression level of neuropeptide Y（NPY）and proopiomelanocortin（POMC）was detected by quantitative polymerase chain reaction（q PCR）,and immunofluorescence of brain section was performed to detect their protein expression.Results:（1）The HE male mice tended to eat more since 8 w after birth,and their daily food intake exceeded NC group at 20 and 24 w after birth.（2）The hypothalamic Npy m RNA expression was increased in HE male mice,while no significant difference of Pomc m RNA expression was shown between HE and NC male mice.（3）The HE male mice showed larger positive staining area of NPY in hypothalamic arcuate nucleus（ARC）and paraventricular nucleus（PVN）.Conclusion: The male offspring with maternal high E₂ showed increased food intake with enhanced expression of orexigenic neuropeptide NPY.Part IV The hypothalamic INSR expression in mouse offspringObjective: To explore how maternal high E₂ affects hypothalamic INSR expression in offspring.Motheds: q PCR was performed to detect hypothalamic glucose metabolism gene expression in 24-w male offspring.Western Blot and immunofluorescence were performed to detect hypothalamic INSR expression in 24-w and E18.5 male offspring.Neural stem cells（NSCs）were isolated from fetal mouse hypothalami and were induced to differentiate into neurons with different concentrations of E₂.Proteins involved in insulin signaling were detected in the neurons by western blot.Results:（1）The hypothalamic INSR m RNA and protein expression was decreased in 24-w HE male offspring than control group.（2）The hypothalamic INSR m RNA and protein expression was decreased in E18.5 HE male offspring than control group.（3）INSR,p-AKT/AKT and p-ERK/ERK were decreased in neurons with supraphysiologic concentration of E₂.Conclusion: The male offspring with maternal high E₂ showed decreased hypothalamic INSR.Supraphysiologic concentration of E₂ attenuated insulin signaling during hypothalamic NSCs differentiation.Part V The influence of chronic food restriction on metabolic function and hypothalamic gene expression in mouse offspringObjective: To explore whether food restriction would interfere the insulin resistance of HE male mice.Methods: The daily amount of food supply was reduced to 75% of original food intake in 24-w HE male mice,which lasted to 32 w.The 32-w NC,HE and HE with food restriction（HE-FR）male mice were weighted,GTT and ITT were performed to assess their glycometabolic funciton.ELISA was performed to detect the fasting insulin and leptin.q PCR,Western Blot and immunofluorescence were performed to detect hypothalamic INSR expression,q PCR and immunofluorescence were performed to detect hypothalamic NPY expression in three groups.Results:（1）Food restriction significantly reduced the weight of HE male mice to levels similar with NC group.（2）Food restriction significantly decreased the AUC of GTT and ITT in HE male mice to levels similar with NC group.（3）Food restriction significantly decreased fasting insulin and HOMA-IR scores in HE male mice to levels similar with NC group,and the fasting leptin was also decreased.（4）Food restriction significantly decreased hypothalmic INSR m RNA and protein expression in HE male mice to levels similar with NC group,but hypothalamic NPY expression was not affected.Conclusion: Chronic food restriction reversed insulin resistance in offspring with maternal high E₂ and rescued hypothalamic INSR expression.Part VI The promoter DNA methylation status of hypothalamic Insr in mouse offspring with maternal high E₂ before and after food restrictionObjective: To explore how maternal high E₂ affects promoter DNA methylation status of hypothalamic Insr in offsprinng and whether food restriction would exert interference.Methods: Bisulfite genomic sequencing PCR（BSP）was performed to analyze DNA methylation of CpG sites in Insr promoter of E18.5 and 32-w male mice hypothalami.Results:（1）Four CpG sites of the hypothalamic Insr promoter showed elevated methylation in E18.5 HE male mice.（2）Four CpG sites of the hypothalamic Insr promoter showed elevated methylation in 32-w HE male mice,among which 3 sites were consistent with the E18.5 HE male mice.（3）Food restriction reduced the methylation level of 3 CpG sites in hypothalamic Insr of 32-w HE male mice,among which 2 sites were consistent with the hyper-methylated sites of 24-w HE male mice.Conclusion: Maternal high E₂ elevated hypothalamic Insr promoter methylation in male offspring,lasting from fetus to adult,which could be reversed by chronic food restriction.