Abstract:
Background Diabetes mellitus is a chronic disease with high incidence all over the world and the number of diagnosed diabetes in China has exceeded 100 million. Several studies have shown that p, p'-dichlorodiphenyldichloroethylene (p, p'-DDE) exposure can interfere with glucose metabolism and insulin secretion, and has a correlation with the increased incidence of type 2 diabetes mellitus. In addition, exposure to p, p'-DDE during pregnancy can cause impaired glucose tolerance and induce diabetes in offspring.
Objective This study aims to investigate the effects of prenatal exposure to p, p'-DDE on insulin like growth factor 2 (IGF2)/H19 gene imprinting and islet function in the offspring of SD rats, and to study whether folic acid as a methyl donor has an intervention effect on the process.
Methods The primary islet cells were isolated by retrograde perfusion method, and were tested for purity, survival rate, and insulin secretion function. Sexually mature SD rats at 8-10 weeks old were caged and mated, and pregnant rats were randomly divided into three groups:control group, p, p'-DDE group, and folic acid group, with eight rats in each group. On gestational day 8-15, the rats in the p, p'-DDE group and the folic acid group were given p, p'-DDE (20 mg/mL) at 5 mL/kg (body weight) by gavage; the rats in the control group were given the same volume of corn oil; the rats in the folic acid group were fed a diet supplemented with folic acid (3.5 mg/kg). At birth, pups were culled to four males and four females in each litter and were fed an ordinary diet after weaning. Weights of the pups were weighed in the first, third, and eighth weeks after birth. Eight weeks after birth, DNA and RNA were extracted to detect the methylation level of IGF2/H19 imprinting region by bisulfite genomic sequencing and the expression levels of IGF2 and H19 mRNA by real-time quantitative PCR. The pups at 8 weeks old were subject to glucose tolerance test. The blood glucose levels in the caudal vein were measured before and 15, 30, 60, and 120 min after intragastric administration. At the same time, the serum insulin levels in the blood of orbit were determined by ELISA before and 2 h after intragastric administration.
Results The purity was (90±5)%, the survival rate was more than 90%, and the primary islet cells secreted insulin. The methylation level of IGF2/H19 imprinting region in the p, p'-DDE group offspring(51.5±3.8)% was lower than that in the control group offspring(55.9±3.3)% (P=0.031), but had no significant difference compared with the folic acid group offspring(52.8±1.9)%. The expression level of IGF2 mRNA in the p, p'-DDE group offspring(265.4±70.6)% was higher than those in the control group offspring (100%) (P=0.002) and the folic acid group offspring(101.8±65.9)% (P=0.002). No significant difference was found in H19 mRNA expression level among the three groups. There was also no significant difference in body weight in the first, third, and eighth weeks among the three groups. The blood glucose level at 15 min after intragastric administration in the p, p'-DDE group offspring(10.89±1.17) mmol/L was higher than those in the control group offspring(9.29±1.18) mmol/L (P=0.026) and the folic acid group offspring(9.25±0.95) mmol/L (P=0.022). There was no significant difference in serum insulin level before and 2 h after intragastric administration among the three groups.
Conclusion Prenatal exposure to p, p'-DDE could reduce the methylation level of IGF2/H19 imprinting region and up-regulate the transcription of IGF2 mRNA, and then impair islet function in the offspring of SD rats. Folic acid plays an intervention role in this process.
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