Effect Of Insulin On The Fetal Programming Of Mouse Early Embryonic Development And Its Mechanism Involved

The objectives of the present study were(1) to reveal whether an altered intrauterine endocrine milieu in diabetic mice model in vivo and preimplantation embryos exposed to insulin in vitro would cause long term alterations in growth and development,as well as insulin secretion and action later in adult life;(2) to demonstrate the possible trans-generational effect by examining the alterations in growth and insulin secretion and function in F1 and F2 offspring,and(3) to elucidate whether this action involves epigenetic modification of imprinted genes via changes in DNA methylation.In addtion, DNA microarray was employed to identify the differentially expressed genes in fetuses from control and diabetic mice.1.Effect of insulin on the metabolic programming of mouse embryonic development1.1 Establishment of diabetic mouse model by streptozotocin injection in different dosesTo establish type 1 diabetes mellitus(T1DM),mice were received intraperitoneal injection of streptozotocin(STZ) at the doses of 150mg/kg b.w.and 200mg/kg b.w.. Blood and urine glucose levels were detected by glucose and urine analyzing papers respectively.Pancreatic histological changes of islets were observed under light microscope.Serum insulin concentration was detected by radioimmunoassay.The female diabetic mice were mated to the normal male mice,the mating ability and fetal mortality were observed.The glucose concentration in the control group did not show alteration across the investigation period,while that of the STZ group was increasing with the time, reaching stabilized level 3 weeks after STZ injection.Significantly increased(P＜0.001) serum glucose concentration associated with significantly decreased(P＜0.001) serum insulin levels were observed in the high STZ dose group,with obviously impaired pancreatic isletβcells.In the low STZ dose group,serum insulin concentration was decreased(P＜0.01),while serum glucose level increased or unaltered.In increased glucose group(Glucose＞18mmol/L),the success mating rate,abortion rate and fetal mortality were 46.9%,73.9%and 20%,respectively.The results indicate that STZ can induce diabetes mellitus,and affect the female success mating rate and fetal development.1.2 Effect of maternal low insulin and high glucose environment on programming of fetal development and pancreatic islet functionSTZ-induced diabetic mice were mated to the normal male mice to produce F1 offspring.The adult female F1 mice were bred to give birth to F2 offspring.The alteration of fetal development and pancreatic islet function were observed to reveal the maternal effect.The F1 offspring from diabetic mice exhibited malformation rate of 9%, and 26.5%lower birth weight compared with the control group(P＜0.01).The serum glucose concentration was 24.7%higher(P＜0.05),while the serum insulin concentration was 26%lower than that of the control(P＜0.05).The F2 offspring did not show significant alterations in serum glucose level,but the concentration of serum insulin was 14.3%lower than that of the control(P＜0.05).Adult offspring of both F1 and F2 from diabetic mice displayed insulin resistance and higher apoptotic ratio in pancreas.The result demonstrate that altered maternal endocrine and metabolic status(low insulin and high glucose) could exert trans-generational effect on growth and development,as well as insulin secretion and function in F1 and F2 offspring.1.3 Exposure of Mouse Preimplantation Embryos to Insulin influences fetus development and pancreatic islet programming2-cell embryos were cultured in either 0 or 0.25μg/ml insulin to the blastocyst stage and then transferred into pseudo-pregnant recipient mice.The newboms were raised until sexual maturity and were bred with normal males to produce F1 offspring.The ratio of blastocyst in insulin-exposed group is 16.4%higher than that of the control(P＜0.05), and the birth weight of insulin-exposed group was 17.8%higher than that of the control group(P＜0.05).The differences of serum glucose and insulin between insulin-exposed and control group were not significant,but the treated group demonstrated higher apoptotic ratio in pancreas and insulin resistance in adulthood.The concentration of serum insulin in F1 of treated group was 14.3%lower than that of the control(P＜0.05) with insulin resistance in adulthood.The results indicate that insulin can stimulate early-embryo development and induce alterations in pancreas function,with lasting consequences of insulin tolerance in the offspring in adult life.2.Mechanisms involved in the effect of early insulin intervention2.1 Alterations in the expression and methylation levels of imprinted genes H19 and Igf2 of fetuses from Streptozotocin-Induced Diabetic mice The mRNA expression and methylation levels of the growth-related imprinted genes, H19 and Igf2 in fetuses of Streptozotocin-Induced Diabetic mice were analyzed.Total RNA and DNA of E14 fetuses were extracted.The relative abundance of Igf2 mRNA in STZ treated group was decreased by 15.4%(P＜0.05).Real-time reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts,which is associated with 19.1%higher methylation ratio of all CpGs in the targeted region of genomic DNA.The results indicate that maternal diabetes would affect fetal development via altering the expression of impriting genes.The modified genomic DNA methylation status of imprinting genes may account for the change of gene expression.2.2 Exposure of Mouse Preimplantation Embryos to Insulin Alters the Methylation Status of Imprinted Genes H19 and Igf22-cell embryos were cultured in either 0 or 0.25μg/ml insulin to the morula and blastocyst stage,and then transferred into pseudo-pregnant recipient mice.The DNA and RNA of morula,blastocyst and E14 fetuses were extracted to reveal the alterations of expression and methylation levels of the growth-related imprinted genes,H19 and Igf2. Real-time reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression of Igf2 and H19 in insulin-exposed morula was 4.9 and 5.7 fold of control counterparts,in insulin-exposed blastocyst,that was 1.8 and 2.3-fold respectively. In the insulin-exposed fetuses,both of the mRNA expression of Igf2 and H19 were 1.5 fold of the control group(P＜0.05).The methylation ratio of all CpGs in the targeted region of genomic DNA increased by 86.4%,35.4%and 19.2%in the insulin-exposed morula,blastocyst and E14 fetuses,respectively compared with the control counterparts (P＜0.05).The results indicate that insulin exposure during the preimplantation stage modifies the genomic DNA methylation status of imprinted genes,alters the expression and affects fetal development.3.Identification of differentially expressed genes in fetuses from control and diabetic mice using DNA microarrayTotal RNA of E14 diabetic fetuses were extracted and cDNA were reverse transcribed.Cy3 and Cy5 fluorescent dyes were labeled to diabetic and control cDNA respectively,and used to hybridize mouse oligo-array containing 24,859 genes.Data were analyzed by using Agilent microarray scanner system.A total of 397 genes were found to be differentially expressed between the fetuses of diabetic and control mice.328 genes showed a more than 2-fold increase in the expression compared with the control, while 69 genes showed a more than 2-fold down regulation.For example,Otx2 and Robo3,proteins that are involved in cell differentiation,nervous system and brain development,are down-regulated in the fetuses of the diabetic mice.Ras112 and Rrad, that are associated with diabetes,are up-regulated in the fetuses of the diabetic mice.The present study indicate diabetes mellitus can induce differentially expressed genes,and affect fetal development.