The Multigenerational Effects Of Insulin Resistance And Glucose Metabolic Disorder Induced By Developmental BPA Exposure And The Relevant Epigenetic Mechanisms

Bisphenol A (BPA) exposure during early development can induce epigenetic change, and lead to glucose homeostasis disruption and insulin resistance in adult offspring. Since the primordial germline of F1generation is directly exposed to BPA and epigenetic information can be inherited through the male germline, the possibility that BPA has multigenerational effects of glucose intolerance and insulin resistance on F2generation through permanent altered epigenetic information is an unavoidable question. In the present study, FO maternal rats was given BPA orally below the reference dose as40μg/kg/day during gestation and lactation, then the subsequent F1and F2generations were obtained in the absence of BPA exposure. We carried out the ipGTT and ipITT test to reflect the glucose homeostasis sensitively, as well as the relative hormone level and the relative gene expression to further demonstrate the findings. The BPA-treated F2generation was found to exhibit significant glucose intolerance and insulin resistance in ipGTT and ipITT, as well as the down-regulation of glucokinase (Gck) gene in liver compared with the control group. DNA methylation pattern of Gck promoter in the hepatic tissue of the F2generation and in the sperm of the F1generation of were then performed. The Gck promoter in F2hepatic tissue became completely methylated in the all CpG sites compared with five unmethylated sites in the controls. However, there is only CpG site-314was differently methylated between BPA and controls in sperm. The global DNA methylation level was significant decreased both in the BPA-treated F2hepatic tissue and Fl sperm. In conclusion, FO maternal BPA exposure during gestation and lactation can induce impaired glucose homeostasis and insulin resistance in the F2offspring by the epigenetic change through the transmission of sperm. Meanwhile, there are no significant correlation between insulin resistance in F2generation and the metabolic disorder/abnormal behavioral development in F1generation. The F2liver global DNA methylation level and F1sperm global DNA methylation level are also not correlated with the metabolic disorder/abnormal behavioral development in F1generation. Theser resulst indicate that the mulitgeneration of BPA are directly induced by FO maternal BPA exposure. However, the underlying epigenetic modifications in the sperm of F1generation remain to be further elucidated.Partl:Effects of BPA exposure of FO maternal rats during gestation and lactation on the glucose and insulin tolerance level in F2generation.Objective:To investigate the effects of BPA exposure of FO maternal rats during gestation and lactation on the glucose and insulin tolerance level in F2generation.Methods:Two females and one male SD rats were placed together in one cage for breeding. Pregnancy was confirmed when the sperm positive was observed or the vaginal plug was found in the female rats in the next morning. FO pregnant rats were randomly divided into control and BPA-exposed group. FO maternal rats were were administered corn oil or BPA (40μg/kg body weight/day) dissolved in corn oil respectively by oral gavage respectively during the whole gestation and lactation periods. F1generation of male rats and the subsequent F2generation were not given BPA treatment. Litter size were recorded when weaned, birth weight and food intake were measured continuely to21week old of F2rats. Intraperitoneal insulin tolerance test (ipITT) and intraperitoneal glucose tolerance test (ipGTT) were performed at9weeks and20weeks after birth to evaluate the level of insulin resistance and impaired glucose tolerance in F2rats. Fasting blood glucose levels were measured by blood glucose monitoring system. Fasting insulin level, blood leptin and adiponectin levels were determined by ELISA kit. And insulin resistance index (HOMA-IR) was calculated according to the glucose and insulin level. Meanwhile, the correlation between glucose tolerance level in F2generation and the metabolic disorder/abnormal behavioral development in F1generation was examined.Results:The mortality of F2generation litter was not affected by the BPA exposure in F0dam. Litter size, sex ratio and birth weight in F2generation offspring did not differ across the groups, the difference were not statistically significant (p>0.05). Compared with the control group, BPA exposed F2generation also did not show significant difference in body weight and food intake at each time point of the entire life time (p>0.05).There were also no significant differences of the fasting glucose, leptin and adiponectin levels between BPA exposure group and the control group in F2generation (p>0.05). However, compared with the control group, BPA exposed F2generation manifested significantly higher fasting insulin levels at20weeks old (p <0.05). By calculating the insulin resistance index, the HOMA-IR level in BPA treated F2group was also significantly increased (p<0.05). Meanwhile, the results of ipITT and ipGTT unveiled the the glucose intolerance and insulin tolerance in F2generation of BPA group with the enlarged AUC (p<0.05). Meanwhile, there is no correlation between glucose tolerance level in F2generation and the metabolic disorder/abnormal behavioral development in F1generation. Conclusion:BPA exposure of FO maternal rats during gestation and lactation can lead to the glucose intolerance and insulin resistance in the F2generation. Part2:Effects of BPA exposure of F0maternal rats during gestation and lactation on the expression of insulin resistance-related genes and the methylation pattern of Gck in F2hepatic tissue.Objective:To investigate the effects of BPA exposure of FO maternal rats during gestation and lactation on the expression of insulin resistance-related genes and the methylation pattern of Gck in F2hepatic tissue.Methods:Two females and one male SD rats were placed together in one cage for breeding. Pregnancy was confirmed when the sperm positive was observed or the vaginal plug was found in the female rats in the next morning. FO pregnant rats were randomly divided into control and BPA-exposed group. FO maternal rats were were administered corn oil or BPA (40μg/kg body weight/day) dissolved in corn oil respectively by oral gavage during the whole gestation and lactation periods. F1generation of male rats and the subsequent F2generation were not given BPA treatment. The relative expression levels of Gck, NR4A3, NR4A1, IRS-2and SREBP-lc were determined by the real-time quantitative PCR assay. Western blotting was used to determine the Gck protein levels for the further verification of Gck mRNA alternation. Global DNA methylation level in the F2hepatic tissue was measured by using the total DNA methylation detection kit. The methylation changes in the promoter region of Gck in F2liver was performed by bisulfite sequencing PCR. Meanwhile, the correlation between global DNA methylation level in F2liver and the metabolic disorder/abnormal behavioral development in F1generation was examined.Results:Compared with the control group, BPA exposed F2generation also did not show significant difference in the relative expression levels of NR4A3, NR4A1, IRS-2and SREBP-lc genes (p>0.05). However, compared with the control group, BPA exposed F2generation manifested significantly decreased expression level of Gck gene (p<0.05). The Gck protein level of BPA treated F2group was also significantly decreased companied with the mRNA change (p<0.05). The Global DNA methylation level in the BPA-treated F2hepatic tissue was significantly decreased compared with the controls. When assessing the methylation changes in the promoter region of Gck, the control F2rats exhibited unmethylated status in the CpG sites-314,-280,-93,-4,+5of promoter region, however, the Gck promoter in BPA-treated F2offspring rats showed a fully methylated status. Meanwhile, there is no correlation between global DNA methylation level in F2liver and the metabolic disorder/abnormal behavioral development in F1generation.Conclusion:BPA exposure of F0maternal rats during gestation and lactation can lead to the decrease expression of Gck and the alernaton of methylation pattern of Gck promoter in F2hepatic tissue. Part3:Effects of BPA exposure of F0maternal rats during gestation and lactation on the the methylation pattern of Gck in the sperm of Fl generation.Objective:To investigate the effects of BPA exposure of FO maternal rats during gestation and lactation on the the methylation pattern of Gck in F1sperm. Methods:Two females and one male SD rats were placed together in one cage for breeding. Pregnancy was confirmed when the sperm positive was observed or the vaginal plug was found in the female rats in the next morning. FO pregnant rats were randomly divided into control and BPA-exposed group. FO maternal rats were were administered corn oil or BPA (40μg/kg body weight/day) dissolved in corn oil respectively by oral gavage during the whole gestation and lactation periods. F1generation of male rats was not given BPA treatment. The global DNA methylation level in the F1sperm was measured by using the global DNA methylation detection kit. The methylation changes in the promoter region of Gck in F1spermr was performed by bisulfite sequencing PCR. Then the Gck expression level in F1sperm was analysized by the real-time quantitative PCR. Meanwhile, the correlation between the global DNA methylation level in F1sperm and the metabolic disorder/abnormal behavioral development in F1generation was examined.Results:The Global DNA methylation level in the BPA-treated F1sperm was significantly decreased compared with the controls. When assessing the methylation changes in the promoter region of Gck of sperm, the control group exhibited unmethylated status in the CpG sites-314of promoter region, while other CpG sites are methylated, however, the Gck promoter in BPA-treated F2offspring rats showed a fully methylated status at all the CpG sites in the promoter region. Meanwhile, there is no correlation between the global DNA methylation level in F1sperm and the metabolic disorder/abnormal behavioral development in F1generation.Conclusion:BPA exposure of FO maternal rats during gestation and lactation can lead to alernation of methylation pattern of Gck promoter in F1sperm.