| Gestational diabetes mellitus(GDM)is one of the most common pregnancy complications,which can increase the risk of various maternal complications and adverse pregnancy outcomes,and seriously affect the health of mothers and babies.GDM can continuously affect the long-term health of offspring,which increases the risk of long-term diseases such as abnormal glucose metabolism and obesity in their offspring.And this transgenerational effect is particularly pronounced in female offspring,those female offspring are at higher risk of developing glucose metabolism disorders in childhood and adulthood.However,there is a lack of in-depth systematic research on the dynamic changes and mechanisms of glucose homeostasis in GDM female offspring from birth to adulthood and during gestation and postpartum.Due to special physiological and metabolic changes during pregnancy,pregnant women have an increased demand for various nutrients,with common deficiencies of trace elements such as zinc,selenium,and chromium.Serum levels of trace elements such as zinc,selenium,and chromium in GDM are significantly lower than those in normal pregnant women.But the results of intervention studies have been inconsistent.Studies have found that zinc,selenium,and chromium may regulate the body’s insulin sensitivity through the endoplasmic reticulum(ER)stress mechanism.It might play an important role in maintaining the body’s glucose homeostasis,but the mechanism has not been elucidated.DNA methylation is an important epigenetic modification.Alterations in DNA methylation patterns early in life may lead to long-term dysregulation of gene expression.Studies have shown that the DNA methylation signature of ER stress-related genes is associated with insulin resistance and may be an important mechanism for the transgenerational transmission of GDM,but no in-depth research has been reported.Therefore,based on a diet-induced GDM animal model,the present study aimed to explore the effects of organic yeast zinc,selenium,and chromium supplementation on the growth and development,dynamic changes of glucose homeostasis and insulin sensitivity in GDM rats and their female offspring.The related mechanisms of ER stress and DNA methylation were studied as the following three parts:Part 1 Effects of zinc,selenium and chromium on glucose homeostasis and endoplasmic reticulum stress in gestational diabetes ratsObjection:To investigate the effects of zinc,selenium and chromium supplementation on glucose homeostasis,insulin resistance and ER stress-related mechanisms in dietinduced gestational diabetes rats.Methods:Ninety SPF adult female SD rats(marked as F0)were randomly divided into 9 groups,with 10 rats in each group,namely control group(CTR group),gestational diabetes model group(GDM),model+zinc supplementation group(Zn),model+selenium supplementation group(Se),model+chromium supplementation group(Cr),model+zinc+selenium supplementation group(Zn+Se),model+zinc+chromium supplementation group(Zn+Cr),model+selenium+chromium supplementation group(Se+Cr),and model+zinc+Selenium+chromium supplementation group(Zn+Se+Cr).The CTR group was fed with a normal low-fat diet,and the other 8 groups were fed with high-fat and sucrose diet.Rats in the supplementation group were given the corresponding aqueous solutions of yeast zinc(10 mg/kg.bw),selenium(20 μg/kg.bw)and chromium(20 μg/kg.bw)by gavage every day,while the CTR group and GDM group were treated with equal doses of the yeast aqueous solution without zinc,selenium and chromium the same way.After 6 weeks of feeding,the rats mated with normal male rats until they gave birth naturally,and the supplementation lasted for the entire experimental period.Oral glucose tolerance test(OGTT)was performed pre-pregnancy,gestation 15.5 d and 3 weeks postpartum,respectively.And the area under the curve(AUC)was calculated.Serum oxidative stress indicators were detected by colorimetry.Serum and liver zinc,selenium,and chromium levels were detected by inductively coupled plasma mass spectrometry(ICP-MS).Serum insulin was detected by ELISA.And expressions of hepatic ER stress and insulin signaling pathway-related proteins were detected by Western blot.Results:After 4 weeks of HFS feeding,the body weight of rats in the GDM group was significantly higher than that in the CTR group(P<0.05),and the difference persisted until pre-mating.There was no significant difference in food intake among the groups,but the energy intake of the rats fed with HFS was significantly higher than that of the CTR group(P<0.05).Before mating,the fasting blood glucose among the rats in each group was no significant difference.On the 15.5th day of gestation,compared with the CTR rats,fasting blood glucose and serum insulin levels of the GDM rats were significantly increased,with markedly impaired glucose tolerance and insulin resistance observed in the GDM group.Postpartum,fasting blood glucose,AUC,oxidative stress level and expression level of hepatic ER stress-related proteins including glucose-regulated protein 78(GRP78),protein kinase RNA-like endoplasmic reticulum kinase(PERK),phosphorylated eukaryotic transcription initiation factor 2α(p-eIF2α),transcription factor 4(ATF4),C/EBP homologous protein(CHOP),tribbles 3(TRB3),inositol-requiring enzyme 1α(IRE1α),and phosphorylated c-Jun N-terminal kinase(p-c-Jun)were significantly up-regulated in the GDM group compared with the CTR group.While the expression of phosphorylated protein kinase B,a key molecule in the insulin signaling pathway,was significantly down-regulated(P<0.05).In addition,the serum and liver zinc,selenium and chromium levels of the GDM group significantly decreased compared with the CTR group(P<0.05).Zinc,selenium and chromium supplementation could improve the nutritional status of zinc,selenium and chromium,as well as the impaired glucose tolerance,insulin resistance,oxidative stress and ER stress in GDM rats to varying degrees,while zinc,selenium and chromium cosupplementation did best.Conclusion:Zinc,selenium and chromium supplementation might improve glucose homeostasis,insulin resistance and hepatic ER stress in diet-induced GDM rats.Part 2 Transgenerational effects of zinc,selenium and chromium on glucose homeostasis in female offspring of gestational diabetes rats and its endoplasmic reticulum stress mechanismObjection:To investigate the transgenerational effects of zinc,selenium and chromium supplementation on glucose homeostasis in GDM female offspring and its ER stress mechanism.Methods:The female F0 generation gave birth to the F1 generation naturally.After weaning,the female F1 generation of the CTR group,GDM group and Zn+Se+Cr group were randomly divided into 7 groups and were given different diets and supplementation respectively,named as the F1 generation of the CTR group fed with a low-fat diet(CTRLF)or high-fat and sucrose diet(CTR-HFS);the F1 generation of the GDM group with lowfat diet(GDM-LF)or high-fat and sucrose diet(GDM-HFS)or high-fat and sucrose diet plus zinc,selenium and chromium supplementation(GDM-HFS+S);the F1 generation of the Zn+Se+Cr group with high-fat and sucrose diet without supplementation(GDM+S-HWS)or with continued zinc,selenium and chromium supplementation(GDM+S-HFS+S).The supplementation and treatment of F1 generation female rats were the same as F0 generation.Oral glucose tolerance test was performed pre-pregnancy,gestation 15.5 d and postpartum,respectively.And the area under the curve(AUC)was calculated.Glucose homeostasis and blood lipid levels of F1 and F2 female neonatal rats were detected by colorimetry.Serum oxidative stress indexes of F1 generation adult rats were detected by colorimetry.ICP-MS was used to detect serum and liver zinc,selenium,and chromium levels of F1 generation adult rats.ELISA was used to determine serum insulin content.And expressions of hepatic ER stress and insulin signaling pathway-related proteins were detected by Western blot.Results:The birth weight,length,serum glucose and insulin levels of the GDM-F1 generation were significantly increased compared with the CTR-F1 group(P<0.05),Dyslipidemia was observed in the GDM-F1 rats.Supplementation GDM rats with zinc,selenium and chromium could markedly improve the adverse effects of GDM on their offspring.As for female GDM F1 generation(GDM-HFS group and GDM-HFS+S group)fed with a high-fat and sucrose diet,they developed GDM,and a significantly increased fasting blood glucose level was observed in contrast with the CTR-LF group(P<0.05),as well as impaired glucose homeostasis and insulin resistance.Meanwhile,the F2 generation(GDM-HFS/F2)had significantly higher birth weight,body length,serum glucose and insulin levels than the CTR-LF/F2 group(P<0.05).Compared with the CTR-LF/F2 group,there was no difference in the glucose homeostasis and insulin sensitivity of the GDM+SHFS+S group during pregnancy by which both the F0 and F1 generations were supplemented with zinc,selenium and chromium(P>0.05).And birth length,body weight,serum glucose and insulin levels of the F2 generation(GDM+S-HFS+S/F2)were significantly lower than those of the GDM-HFS/F2 group(P<0.05).Moreover,fasting blood glucose during pregnancy of the GDM+S-HFS group was significantly lower than that of the GDM-HFS group(P<0.05),and the birth blood glucose and insulin of F2 generation were significantly lower than those of the GDM-HFS/F2 group.Moreover,compared with the CTR-LF group,the expression levels of hepatic ER stress-related proteins(GRP78,p-PERK,p-IRE1α,p-eIF2α、ATF4、CHOP、TRB3,p-IRE1αand pc-Jun)of the GDM-HFS group were significantly increased,while the activation of the insulin signaling pathway was significantly inhibited(P<0.05).However,reversed changes were observed in the GDM+S-HFS+S group.Conclusion:Transgenerational effects of zinc,selenium,and chromium supplementation on the improvement of glucose homeostasis in female offspring with GDM did exist.The simultaneous administration of zinc,selenium and chromium to GDM mothers and offspring was more effective in improving glucose homeostasis and hepatic ER stress than the parent or offspring alone.Part 3 Zinc,selenium and chromium improve the hepatic endoplasmic reticulum stress in the female offspring of gestational diabetes rats via the methylation mechanismObjection:The methylation levels and expressions of ER stress-related genes in the liver tissue of GDM female F1 and F2 generations were detected to explore the effect of zinc,selenide and chromium supplementation and the mechanism of its transgenerational effect.Methods:The whole-genome DNA methylation levels in the liver tissue of female F1 and F2 generations newborn female rats were detected by 5-mC DNA ELISA kit.And the methylation levels of CpG island in the promoter region of ER stress-related genes such as endoplasmic reticulum disulfide oxidase 1α(ERO1α)in the liver tissue of female F1 and F2 generation newborn female rats were evaluated by pyrosequencing.The transcription levels of candidate genes and DNA methyltransferases(DNMTs)were detected by fluorescence quantitative PCR technology.Results:The global DNA methylation levels of the whole genome in the liver tissue of female GDM-F1 and GDM+HFS-F2 rats were significantly higher than the control group.The promoter region of the ERO1α gene showed a hypermethylated state with its gene expression was significantly down-regulated in comparison with the control group.It was further found that in the liver tissue of GDM-F1 and GDM+HFS-F2 female rats,only the expression of DNMT1 was significantly up-regulated,but the expression of DNMT3a and DNMT3b did not change.The expression of ER stress-related proteins(GRP78,p-PERK,p-IRE1α,p-eIF2α、ATF4、CHOP、TRB3,p-IRE1α和p-c-Jun)was significantly increased,and the expression of insulin pathway was significantly inhibited.Zinc,selenium and chromium supplementation significantly improved the hypermethylation status of the ERO1α gene promoter region in GDM female offspring,thus alleviating their ER stress and insulin resistance.Conclusion:GDM resulted in altered liver gene methylation status in female offspring,in which the methylation level of the ERO1α promoter region was significantly increased,with a decrease in its gene expression.Zinc,selenium,and chromium supplementation could correct these abnormal methylation modifications.This might be one of the mechanisms of improving hepatic ER stress and insulin resistance. |
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