Effects Of The Leptin Gene Methylation On Glucose Metabolism In Gestational Diabetes Mellitus

Background:Gestational diabetes mellitus (Gestational Diabetes Mellitus, GDM) is a type ofdiabetes or abnormal glucose tolerance during pregnancy (except those who have diabetesand then pregnancy) characterized by abnormal elevation of postprandial blood glucose andhyperinsulinemia. Nowadays, GDM is drawing more and more attention for its seriousconsequence to the mother and fetus during the pregnancy. Most traditional research arefocused on hyperinsulinemia and insulin resistance, however, with the recognition of thefunction of leptin, we found relationships between leptin and insulin that there are two-wayinteraction between insulin and leptin in many tissues. There are high expression of leptinand obvious leptin resistance in the mother and in the intrauterine fetus in GDM patients aswell. Therefore, the effect of leptin is drawing so much on GDM.As an epigenetic mechanism, the methylation of DNA is chemical modification thatcauses the cytosine to transform the methylcytosine under the catalysis of DNAmethyltransferase. This modification inhibits gene expression by inhibiting the binding ofsome DNA-binding factors with their cognate DNA recognition sequences, implementingthe silenced state, affecting transcriptional elongation in addition to its characterized role ininhibiting transcriptional activation．The promoter and CpG island of the leptin genelocated in chromosome7is a tissue-specific differentially methylated regiontiallymethylated region. The methylation regulates the leptin gene expression negatively.The goals of this study is, thus, to access a suitable animal model for GDM to simulatethe characteristics of carbohydrate metabolism and abnormal levels of insulin and leptin,expression of leptin gene and the methylation of gene promoter in liver and placenta tissues.And explore the effect of progesterone on the methylation of leptin gene promoter and thegene expression in trophoblast cells, to provide basis for pathogenesis of GDM.Objective:Investigate the relationship of leptin gene promoter methylation and abnormal glucosemetabolism in gestational diabetes mellitus, and explore the pathogenesis of gestational diabetes mellitus from epigenetic perspective.Methods:(1) Compare the level of glucose tolerance and serum insulin and leptin of C57BL/Ksjdb/+mice and normal C57BL/Ksj pre-pregnancy, pregnancy and the postnatal period, andanalyze the feasibility that whether C57BL/Ksj db/+mice can be used as gestationaldiabetes mellitus animal model;(2) Use Western Blot method to measure the leptin expression of pre-pregnancy,different periods of pregnancy, postpartum in hepatic tissues and the expression level inplacenta at different stages in normal mice and GDM mice, and understand the correlationsof leptin gene promoter methylation and expression of leptin in these tissues;(3) Cultivate human trophoblast cells in vitro in different progesterone concentration,to investigate the effect of progesterone on leptin gene promoter methylation, and thecorrelation of leptin gene promoter methylation and glucose metabolism.Results:Experiment1:(1) Compared with normal pregnant rats, the glucose tolerance of C57BL/Ksj db/+inpregnant rats significantly reduced, and insulin levels significantly elevated with pregnancyprogressed (P <0.05), which is significantly higher than that of normal mice during thesame periods，and restored to normal after delivery;(2) the serum leptin of C57BL/Ksj db/+pregnant rats was significantly higher thanthat of pre-pregnancy (P <0.05), but from mid-pregnancy had no significant correlationwith gestational age.Experiment2:(1) Leptin expression of normal pregnant rats in liver tissue was higher than that ofpre-pregnancy (P <0.05), and there is no significant difference between different gestationperiods (P>0.05), but leptin expression in placental tissue increased significantly with theprogress of pregnancy (P <0.05). Leptin expression of C57BL/Ksj db/+pregnancy rat liverand placenta tissue is higher than normal pregnant rats at the same periods(P <0.05), butthere was no significant difference with different gestational periods (P>0.05);(2) There was no difference of methylation levels of liver tissue leptin gene promoterin C57BL/Ksj db/+female rat and normal female rats between pregnancy and non-pregnancy. The methylation level of leptin gene promoter in placenta of C57BL/Ksjdb/+pregnant rat was lower than that in normal pregnant rats.Experiment3:(1) The trophoblast cells early showed round or oval cell growth state. It manifested asa polygon or long fusiform with tiled growth, and the immunohistochemical findingsshowed cytokeratin staining positive, negative vimentin staining cells is close to95%,which can be used for follow-up study;(2) The methylation levels of trophoblast cells in leptin gene promoter regiondecreases with the rise of medium levels of progesterone;3. Leptin expression levels oftrophoblast cells and progesterone concentrations were positively correlated.Conclusions:(1) The characteristics of carbohydrate metabolism of C57BL/Ksj db/+female rat andabnormal expression of insulin, leptin expression is similar to gestational diabetes mellitus,and can be a good animal model.(2) The expression of leptin in liver and placental of C57BL/Ksj db/+and normal miceis significantly elevated in pregnancy, and leptin level have significant relationship withleptin gene methylation in placenta.(3) Progesterone can reduce leptin gene methylation levels, and is involved inregulation of leptin expression of trophoblast cells.