Effect Of Gestational Diabetes Mellitus On Inflammatory Factors In Placenta And Igf2Imprinting Of Fetal Mouse Pancreas

Part Ⅰ. Effects of gestational diabetes mellitus on expression of inflammatory factors in placentaObjective:To investigate the effects of gestational diabetes mellitus （GDM） on expression of inflammatory factors in placenta, and, examine the effect of high glucose on the secretion of inflammatory factors in human trophoblast cell line （HTR8/SVneo）.Materials and methods:Placentas were obtained from gestational diabetes mellitus （GDM） and control pregnancies. Human trophoblast cell line was cultured with physiological glucose （5mmol/L） or high glucose （25mmol/L） for48hours, followed by treatment with lipopolysaccharide （LPS,1μg/ml） for another period of24hours. Inflammatory factors were measured by ELISA and q-PCR.Results:The expression levels of adiponectin （ADIPOQ）, interleukin5（IL5） and interleukin13（IL13） in GDM placenta were down-regulated （p<0.05）, while interleukin6（IL6） and interleukin8（CXCL8） were up-regulated （p<0.05）. Treatment of human trophoblast cells with high glucose significantly increased the level of IL-6（p<0.05） and decreased the level of adiponectin （p<0.05）.Conclusion:High glucose may induce the alteration of inflammatory factors in placenta and HTR8/SVneo. Part Ⅱ. Effects of gestational diabetes mellitus on expression of Igf2in fetal mouse pancreasObjective:To evaluate the impact of hyperglycemia on expression of inflammatory factors in placenta and the insulin storage in fetal mouse pancreas （FMP）. To clarify the effects of hyperglycemia on expression of Igf2and underlying mechanism.Materials and methods:We established a mouse model of gestational diabetes mellitus. Mouse placenta and FMP were isolated at embryonic day18.5. Expression of insulin, Igf2, Dnmt1, Dnmt3a, Trim28and Aicda were analyzed by q-PCR. Fetal mouse pancreatic insulin storage was evaluated by immunohistochemical analysis and real-time RT-PCR. Igf2DMRs were verified by MassARRAY EpiTYPER.Results:The expression of interleukin6（116） was up-regulated in GDM mouse placenta （p<0.05）. The expression levels of insulin and Igf2were significantly decreased in the FMP of GDM group （p<0.05）, while the expression levels of Dnmtl and Trim28were elevated （p<0.05）. Aicda was also reduced （p<0.05）.There were five hypermethylation CpG sites in Igf2DMR2compared with control （p<0.05）.Conclusion:The hyperglycemia environment in uterus altered the116expression in placenta and impaired fetal mouse pancreatic insulin storage. One potential mechanism underlying impaired fetal pancreatic insulin storage is down-regulation of imprinting gene Igf2. Intrauterine hyperglycemia causes hypermethylation at Igf2DMR via up-regulation of Dnmtl, Trim28and down-regulation of Aicda. Part III. Effect of IL-6and high glucose on fetal mouse pancreasObjective:To investigate possible causes of epigenetic alteration in fetal mouse pancreas and the underlying mechanism.Materials and methods:FMPs were cultured with four different conditions:C （glucose5mmol/L）, C+IL-6（glucose5mmol/L+IL-64000pg/ml）, HG （glucose25mmol/L）, HG+IL-6（glucose25mmol/L+IL-64000pg/ml）. Expression of insulin, Igf2, Dnmt1, Trim28and Aicda were analyzed by q-PCR. Fetal mouse pancreatic insulin storage was evaluated by immunofluorescence analysis and q-PCR. We verified the Igf2DMRs by MassARRAY EpiTYPER as well.Results:The expression of insulin and Igf2were significantly decreased （p<0.05） while Dnmt1、Trim28were increased （p<0.05） in the other three groups compared with group C. The effects of IL-6on the regulation of insulin, Igf2and Dnmt1were more apparent. The expression of Aicda was not significantly changed in each group （p>0.05）. There were four hypermethylation CpG sites （CpG₄, CpG₅, CpG₇and CpG₁1） in Igf2DMR2in the cells treated with IL-6or HG （p<0.05）.Conclusion:Both HG and IL-6have effects on the expression and methylation status of Igf2DMR2. IL-6regulates the expression and methylation status of Igf2by increasing the expression of Dnmt1. However, the effects of HG may be mediated by increased expression of Trim28.