DNA Methylation In The Development Of Pancreatic Islets In Rats With Intrauterine Growth Restriction

Objective: Intrauterine growth restriction(IUGR),one of the most common embryo development disorders,is defined as the failure of a fetus to achieve its genetic potential for size.IUGR affects 3-10% of pregnancies and is recognized as a major cause of fetal and neonatal morbidity and mortality.Epidemiological and experimental studies indicate that IUGR newborns are strongly predisposed to the development of metabolic syndrome consisting of insulin resistance,type 2 diabetes mellitus,obesity,hypertension and coronary artery disease in later life.Recent studies have been indicated that metabolic syndrome is associated with IUGR.The mechanism of IUGR resulting in metabolic syndrome continues poorly understood and controversial.Epigenetics has been recognized as a new approach to understand the mechanism of IUGR resulting in metabolic syndrome.DNA methylation,a major part of epigenetics,take place in CpG islands.DNA methylation makes a crucial role in regulating gene expression.DNA chip and various methods to test DNA methylation of single gene loci are used to investigate the DNA methylation.Some studies found that decreased blood glucose and hypo-insulinemia take place in IUGR newborn mouse.Numerous factors are involved in the development of pancreatic islets.DNA methylation may regulate the expression of crucial genes involved in the development of pancreas islets.In order to determine the impact of DNA methylation on IUGR pancreatic islets,first of all,this study developed an IUGR model in rat by maternal protein restriction and selected male offspring at different stages(newborn,3-week-old,12-week-old and 10-month-old)in normal and IUGR groups as research objects.The body weight,pancreatic weight,size and amount of pancreatic islets as well as ? cells were observed and the serum glucose and insulin level were monitored.Secondly,we generated the first DNA methylation map throughout the rat genome in normal pancreatic islet cells,allowing us to identify the changes that occur as a consequence of IUGR.The differentiated loci,as results of DNA methylated chip,were screened by the data library including of genes involved in the development of rat pancreas with the literature mining.Combined with the gene function analysis,candidate DNA methylation sites were validated as objects to test the chip results.Finally,candidate DNA methylation sites were tested by the method for testing the methylation of a single site as well as mRNA expression of genes matched with candidate DNA methylation sites in the pancreatic islets of newborn and old IUGR rats.The aim is to investigate the relationship between DNA methylation and IUGR reduced by intrauterine protein restriction and provide clues to deep understand the mechanism of metabolic syndrome resulted by IUGR.Methods: Maternal protein restriction was used to develope IUGR model in rats.48 Outbred Wistar pregnant rats(230~280g)were provided by Medical Animal Center in Shengjing Hospital of China Medical University.According to the diet(Institute of Zoology,Chinese Academy of Science),the pregnant rats were divided into two groups randomly: 24 rats with normal diet and 24 rats with low protein diet(protein fraction: 8%).The offspring of the rats with normal diet was called as control group.If the newborn weight of rats whose mother with maternal low protein diet was lower than 2x SD of the newborn weight of normal rats,the rats were called as IUGR group.To exclude the influence of estrogen,male offspring at different stages(newborn,3-week-old,12-week-old and 10-month-old)in normal and IUGR groups were selected as research objects.6 rats respectively from 6 pups were used to study at different stages.The body weight,pancreatic weight,size and number of pancreatic islets as well as ? cells were observed,and the serum glucose and insulin level were monitored.Hyperinsulinemic-euglycemic clamp technique was provided to validate whether insulin resistance occur in 10-month-old IUGR rats.MeDIP-chip was first used to investigate genomic DNA methylation in pancreatic islets of newborn IUGR and normal rats(n=3,respectively from 3 pups).Briefly,genomic DNA were extracted from cells using a DNeasy Blood & Tissue Kit(Qiagen,Fremont,CA)and sonicated to random fragments in size about 200-1000 bp with a Bioruptor sonicator(Diagenode).Immunoprecipitation of methylated DNA was performed using BiomagTM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine(Diagenode).The immunoprecipitated DNA was eluted and purified by phenol chloroform extraction and ethanol precipitation.The total input and immunoprecipitated DNA were labeled with Cy3-and Cy5-labeled random 9-mers,respectively,and hybridized to NimbleGen Rat DNA Methylation 3x720 K Promoter Plus CpG Island Arrays(Roche),which is a multiplex slide with 3 identical arrays per slide and each array contains 15,790 CpG Islands annotated by UCSC and 15,287 well-characterized RefSeq promoter regions(from about-3,880 bp to +970bp of the TSSs)totally covered by ~720,000 probes.Scanning was performed with the Axon GenePix 4000 B microarray scanner.NimbleScan v2.5(Roche-NimbleGen)was applied to analyze the data.The differential methylated loci provided by the chip results were analyzed by Gene Ontology analysis and KEGG pathway analysis to confirm the function or biological process of the genes matched with the differential loci.The data library including of genes involved in the development of rat pancreas was established by literature mining.Combined with the gene function analysis,candidate DNA methylation sites were validated as objects to test the chip results.Candidate DNA methylation sites included Pdx-1,Nkx2.2,Nkx6.1,Pax6,Pde3 b and Dnmt3 a.Firstly,MeDIP-real time PCR method was programmed in the pancreatic islets of newborn normal and IUGR rats(n=5,respectively from 5 pups)to to test the chip results and The mRNA expression of Pdx-1,Nkx2.2,Nkx6.1,Pax6,Pde3 b and Dnmt3 a were investigate by real time PCR.Secondly,the methylation of 6 candidate loci and mRNA expression of matched genes were tested in the pancreatic islets of 10-month-old rats.Finally,Nkx2.2 and Nkx6.1 expression in the pancreatic islets of newborn and 10-month-old was detected by the double-labeled immunefluorescent method and Western blot method.Results: Compared with control group,the body weight of newborn IUGR rat was less than the normal group and the body weight of 10-month-old rat was more than the normal group(P<0.05).The pancreas weight of newborn,3-week-old and 12-week-old IUGR rats was less than the normal group and the pancreas weight of 10-month-old IUGR rat was more than the normal group(P<0.05).The pancreas weight/body weight ratio in newborn and 12-week-old IUGR rats decreased but in 10-month-old IUGR rats increased compared with the control group(P<0.05).Both the size and amount of pancreatic islets in newborn and 12-week-old IUGR rats were less than normal rats(P<0.05).Both the fraction and size of ?-cells in IUGR rats at different stages except 3-week-old were less than normal group(P<0.05).The serum glucose level and insulin level in 12-week-old and 10-month-old IUGR rats increased significantly compared with the normal rats(P<0.05).Hyperinsulinemic-euglycemic clamp test showed that the serum glucose levels at all time in 12-week-old IUGR rats were more than normal rats and the serum insulin levels at 60 min and 120 min were obviously more than normal group(P<0.05).SSGIR of IUGR group was less than control group suggesting that insulin resistance of peripheral tissue occurred in the 12-week-old IUGR rats.MeDIP-chip of newborn normal and IUGR pancreatic islets found 254 differentiated DNA methylation sites in promotors and 373 differentiated CpG islands.The differentiated DNA methylation sites in promotors contained 157 hypermethylat-ed sites(61.81%)and 97 hypomethylated sites(38.19%).HCP was the most type in the differentiated DNA methylation sites in promotors.LCP was named as the second on the top one.There were 110 hypomethylated(29.49%)and 263 hypermethylated CpG islands(70.51%)in the differentiated CpG islands including 173 loci in promotor(46.38%)and 165 intergenic loci(44.24%).227 loci matched with genes(60.86%)and 146 loci without matched genes(39.14%)were consisted in the differentiated CpG islands.Gene Ontology analysis was programmed to analyze the function of the genes matched with the differentiated methylated loci in promotor.The results showed that 79 items in biological process,16 items in cellular component and 14 items in molecular function.KEGG pathway analysis revealed 7 pathways,such as protein export,protein processing in endoplasmic reticulum and so on.The data library including of 220 genes involved in the development of rat pancreas was established by literature mining.The 254 differentiated methylated loci in promotor were embedded into the data library to screen the candidate loci.Combined with the function analysis results,6 loci including Pax6,Pde3 b,Pdx1,Nkx2.2,Nkx6.1 and Dnmt3 a were confirmed as the candidate loci.The methylation of candidate loci was tested in pancreatic islet of newborn and 10-month-old rats by MeDIP-real time PCR.The results showed that Pde3 b and Dnmt3 a were hypermethylated,Pax6 was hypomethylated,and Pdx1,Nkx2.2 as well as Nkx6.1 were not changed in IUGR newborn rats.These was consistent with the results of MeDIP-chip.Pax6 mRNA expression increased rather than mRNA expression of the other genes decreased in newborn IUGR rats compared with the normal group.The similar results occurred in the 10-month-old IUGR rats.Nkx2.2 and Nkx6.1 expression in the pancreatic ? cells were declined in newborn IUGR rats but were absence in the 10-month-old rats.Conclusions: Pancreatic islets dysplasia and dysfunction occurred in the IUGR rats and metabolic syndrome occurred in later life.Abnormal DNA methylation in genome and some important loci in IUGR pancreatic islets demonstrated that DNA methylation dysregulation is a strong candidate for propagating the cellular memory of intrauterine events,causing changes in expression of nearby genes and long-term susceptibility to metabolic syndrome.