Effect Of Insulin Therapy For Gestational Diabetes Mellitus On Offspring Long-term Metabolic Health And Mechanism Involyed

Part I Insulin therapy for GDM mouse and the metabolic phenotype of offspringObjective:To investigate the growth trajectory and metabolic phenotype of the GDM offspring with insulin therapy,and determine the effect of insulin therapy on GDM offspring disease susceptibility in adult life.Materials and methods:we established a mouse model of diabetes in the latter half of pregnancy to mimic human GDM with high incidence during the third trimester of gestation,and treated maternal hyperglycemia with insulin(INS group).Pups from GDM and INS group were fostered by normal females until the age of 3 weeks.We examined the birth weight,body weight trajectory(from 3-week to 20-week).Next,we analyzed the fasting and fed blood glucose levels,glucose tolerance and insulin tolerance in adult offspring by blood glucose meter.Further,we investigated fasting insulin level and HOMA-IR by using ELISA kit,serum lipid level by biochemical analyzer,serum leptin by radioimmunoassay.At 8 weeks of age,offspring were divided into two groups either receiving normal chow diet or high fat diet.The phenotype ofHFD offspring were investigated and analyzed as described above.Results:Our study found that a combination of short acting insulin(0.35 IU/day continuous delivery via pumps)and long acting insulin provided optimal glycemic control in GDM dams.Birth weight was similar among the three groups.Notably,increased body weight and most metabolic abnormalities were seen in GDM-F1 adult offspring.GDM-F1 exhibited significant glucose intolerance at 8 weeks,impaired insulin tolerance at 16 weeks,increased fasting insulin level and HOMA-IR.Insulin therapy was associated with normal weight trajectory,serum glucose and lipid metabolism in adult offspring.Moreover,Insulin therapy of GDM resulted in a distinct rescue of glucose intolerance and insulin tolerance in INS-F1 offspring.However,these associations were observed only in NCD offspring.HFD abolished this protection.When fed with HFD after 8 weeks of age,a significant increase in body weight,fasting insulin levels,glucose intolerance,impaired insulin tolerance and abnormal lipid levels were seen in INS-F1 males offspring.Conclusion:Insulin therapy resulted in a significant improvement of metabolic disorders in offspring fed with NCD.But importantly,in response to HFD,the offspring developed significantly metabolic disorders.Sex difference may influence the effect of insulin therapy,female offspring got more benefits from GDM therapy.The findings indicated that predisposition to metabolic disorders still persisted in offspring even with efficient insulin therapy of GDM,and was significantly enhanced by HFD challenge.Part II Effect of insulin therapy for GDM on offspring pancreatic islets function and DNA methylation statusObjective:To investigate the pancreatic islet morphology,ultrastructure,and function.To analyze the genome DNA methylation status in offspring pancreatic islets and assess the alterations of candidate genes which may contribute to metabolic phenotypes in offspring.Materials and method:Pancreas were isolated from the offspring to evaluate the ratio of pancreas/body weight and islets morphology by HE.Pancreatic islets were isolated from 20-week-old offspring mice.Islet ultrastructure was analyzed by transmission electron microscope.Islet function in vitro was tested by glucose-stimulated insulin secretion test(GSIS).Male offspring receiving NCD at 20 week of age were chosen for genome-wide DNA methylation analysis.Genomic DNA of pancreatic islets was extracted from offspring per groups,and different methylation regions were analyzed by methylated DNA Immunoprecipitaton sequencing(MeDIP-seq).Results:We found that there were no significant differences about the ratio of pancreas/body weight,islet morphology,and ultrastructure among three groups.In vitro,insulin secretory response to 5.6 mM glucose was similar among all groups.However,islets of GDM-F1 males or females showed impaired insulin secretion when exposure to 16.7 mM glucose.Defective insulin response to high glucose(16.7 mM)was also seen in INS-F1 males.In the MeDIP-seq,we found global cytosine methylation status was also altered in INS-F1 offspring pancreatic islets.In INS-F1,1479 element-associated hypermethylated genes,326 were also hypermethylated in GDM-F1 islets,and 1602 element-associated hypomethylated genes,493 were also hypomethylated in GDM-F1.KEGG analysis showed that the differentially methylated genes in GDM-F1 and INS-F1 mainly encoded the ion channels in islet ?-cell,and were involved in insulin secretion.The genes selected for validation were the following:Abcc8(encoding sulfonylurea receptor 1,Surl,belonged to ATP-binding cassette superfamily of transporters),Cacna1c(Cav1.2,encoding one subunit of L-type Ca2+channels in islets ?-cell),Cacna1e(Cav2.3,encoding R-type Ca2+ channel),and Cacnalg(Cav3.1,encoding T-type Ca2+).These genes directly involved in regulating the first and second phase of insulin secretion.Conclusion:GDM offspring from insulin therapy group did not show significant abnormal morphology and ultrastructure.Defect of insulin secretion could contribute to glucose intolerance in INS-F1 offspring.DNA methylation-mediated epigenetic mechanism may involve in the pancreatic islet dysfunction and disease susceptibility in INS-F1 offspring.Part III Effect of insulin therapy for GDM on offspring pancreatic islets ion channel gene expression and DNA methylationObjective:To investigate the ion channel Abcc8?Cav1.2?Cav2.3 and Cav3.1 gene expression,DNA methylation status and ion-channel mediated insulin secretion in GDM offspring with insulin therapy,evaluate the disease risk in INS-F1 offspring from the perspective of epigenetics.Materials and method:Pancreatic islets were isolated from 20-week-old offspring following intraductal collagenase infusion.The gene expression of Abcc8?Cav1.2?Cav2.3 and Cav3.1 was analyzed by real-time quantitative PCR.The protein level of Abcc8?Cav1.2?Cav2.3 was determined by western-blot.Ion-channel mediated insulin secretion was tested by KATP/L-type Ca2+ channel agonists and inhibitors.Pancreatic islets of normal fetal mice at embryonic day 17 were isolated and cultured for 6 days in vitro in media containing different concentrations of glucose,ion channel genes and DNA methylation related genes were determined by real-time quantitative PCR.The methylation status of promotor regions in Abcc8?Cavl.2?Cav2.3 was analyzed by pyrosequencing.Human fetal side of placenta tissue was sampled following delivery.The DNA methylation status of promotor regions in ion channel genes was analyzed via MethylTargetTM.Results:Abcc8?Cav1.2?Cav2.3 gene and protein levels were reduced in GDM-F1 and INS-F1 isolated pancreatic islets,while Cav3.1 expression was normal.Differential ion channel gene expression was more obvious in male offspring than that in female offspring.In INS-F1 females,only Cav2.3 showed decreased expression.Abcc8,Cavl.2 and Cav2.3 showed significant higher DNA methylation status in islets of INS-F1 males.Compared with GDM-F1 males,altered DNA methylation status in Cavl.2 and Cav2.3 were improved to different degree in INS-F1 males.We found that the effect of insulin therapy on DNA methylation in the three targets genes with sex-specific difference,INS-F1 females only showed a moderate hypermethylated level in Cav2.3 promoter regions.Further,we found that responses to KATP/L-type Ca2+channel agonists and inhibitors were significantly reduced in GDM-F1 and INS-F1 male offspring.Treating fetal islets with high glucose(16.7 mM)for 3-day,the increased Abcc8,Cav1.2 and Cav2.3 DNA methylation levels and reduced gene transcription persisted during subsequent culture at 5.6 mM glucose.Moreover,we examined DNA methylation enzyme(Dnmts)and demethylation enzyme(TETs)expression in fetal islet after short high glucose exposure.We found that Dnmtl,Tet2 and Tet3 were persistently altered even return to normal glucose culture.There were also DNA methtylation differences in the fetal side of the placenta between GDM women who received glycemic-control treatment and and women who had normoglycemic and normal glucose tolerance.We found that the DNA methylation levels at specific CpG locations of Cavl.2,Cav1.3,Cav2.3 and Cav3.1 were higher in the GDM group with glycemic control compared with women with normal glycemia.Conclusion:Impaired ion-channel mediated insulin secretion may contribute to glucose tolerance in GDM offspring with insulin therapy.One potential candidate underlying insulin secretary dysfunction was down-regulated ion channel gene Abcc8?Cavl.2?Cav2.3 in pancreatic islets.Transient intrauterine hyperglycemia caused hypermethylation at Abcc8?Cav1.2?Cav2.3 promoter regions in INS-F1 offspring,which may be a epigenetic mechanism for reduced gene expression.In vitro fetal islets culture confirmed the high glucose effect on ion channel gene expression and DNA methylation.Results from small-scale clinical sample analysis indicated that there might be still altered DNA methylation modification in offspring even GDM mother who received treatment.