Gestational Diabetes Mellitus Alters DNA Methylation Profiles In Pancreas Of The Offspring Mice And Affect Their Metabolic Phenotype

Objective: Gestational diabetes mellitus(GDM)is one of the most common complications during pregnancy.Although glycometabolism in GDM patients usually return to normal after delivery,the risk of diabetes?obesity and other unfavorable consequence will increase in women with GDM and their offspring later in life.Recent study show that the change of DNA methylation profile play an important role in the future health for both mother and child.However,all these studies examined DNA methylation alterations in samples such as placentas,umbilical cord blood or maternal peripheral blood.Different from the existing research,our work now first provides evidence,that is,GDM alters the DNA methylation patterns in offspring pancreas.Besides,these data depict a comprehensive picture of the adverse DNA methylation profiles and metabolic consequences associated with hyperglycemic intrauterine environment during pregnancy in offspring.Methods Part 1:KS.Cg-Dock7 m +/+ Leprdb/J mice were purchase to reproduce the spontaneous gestational diabetes.While pregnant C57BLKS/J were subjected to a single intraperitoneal injection of freshly prepared streptozotocin to mimic intrauterine hyperglycemia as the condition under gestational diabetes.Detection of fasting blood glucose and random blood glucose were conducted to confirm the establishment of the GDM model.Part 2: We compared the lipid and glucose metabolism among the offspring from different group by detecting fasting blood glucose and random blood glucose.While glucose tolerance test and insulin tolerance test were used to verdict whether the offspring suffered from mild glucose dyregulation or insulin resistance.Part3: In order to confirm that GDM alters DNA methylation profiles in pancreas of the next generation,reduced representation bisulfite sequencing(RRBS)was carried out.To further explore the molecular mechanism by which offspring exposed to hyperglycemic intrauterine environment are at increased risk for developing future obesity and type 2 diabetes,DMRs related genes were analyzed using GO functional analysis,KEGG(Kyoto Encyclopedia of Genes and Genomes enrichment)analysis and PPI(protein-protein interaction,PPI)analysis.Then BSP were commited to confirm the hypermethylation state of the interest gene which have an increasement more than 2 fold in the term of methylation upregulation.In the end,real-time PCR were perform to detect the lower expression of the hypermethylated gene.Results: 1.Although KS.Cg-Dock7 m +/+ Leprdb/J mice have been reported to demonstrate GDM phenotype spontaneous and been recommended as a spontaneous GDM model by the Jax laboratory,the animals in our research never show hyperglycemia during the first and second gestations.While the STZ induced gestational diabetes was successfully committed in the single dose of 150mg/kg,with a remarkable increasement in blood glucose level.2.By contrasted with the normal control group,offspring exposed to uterine hyperglycemia suffered from dyslipidemia and worsened glucose tolerance as aging.3.The results from RRBS demonstrated that GDM triggers wide alterations in DNA methylation in pancreatic genome.Gene ontology showed that the DMR related genes were mostly relating to the process of biological regulation,developmental process,metabolic process,etc.In addition,many DMRs related genes associated with cellular metabolic process were found,such as Kcnq1,Plcb4,Gnas,Stx1 a and Hnf1 b,which are involved in multiple possible signaling pathways susceptible to diabetes and obesity,including insulin secretion,pancreatic secretion,pancreatic development and glucagon signaling pathway,etc.These findings show that DMR related genes do not act as single molecules,but rather play interrelated roles together in the context of networks.In the end,BSP and real-time PCR were combined to confirm the hypermehtylation and lower transcription state of Agap2 gene.Conclusion: In summary,our results demonstrate that hyperglycemic intrauterine environment appears to be involved in DNA methylation alternation in pancreatic genome.Among them several genes that engaged in the process of glucose and lipid metabolism were considered to be responsible for the metabolic dyfunction in the following genetation,especially when it comes to the susceptibility to T2 DM,obesity,and cardiovascular disease in adult offspring.