| Objective: In current society,with the development of economics and the transformation of life style,psychological stress,such as anxiety,tension and depression is ever-growing and seriously affects the health of human.Both epidemiologic and experimental animal studies demonstrate that chronic psychological stress exerts adverse effects on the initiation and/or progression of many diseases,including neuropsychiatric disorders,cardiovascular diseases,tumorigenesis,and type 2 diabetes.Interestingly,it has been reported that maternal stress,such as stress experienced during pregnancy,could alter body weight and glucose metabolism in offspring.However,it is unclear whether paternal stress could affect glucose metabolism in their offspring.In the present study,using a mouse model of restraint stress,we address how paternal stress exerts a transgenerational effect in mammals,and further explore its underlying mechanisms.Methods: 1.Construction of animal model for paternal psychological stress: Male C57BL/6 mice aged 8 weeks were randomly assigned to control or stress group.Stressed mice(Stress-F0)were individually subjected to 2 hr/day of immobilization stress in 50 ml conical centrifuge tube for 2 weeks,while control mice(Control-F0)were left undistributed and allowed to contact with each other.After 2-week restraint,mice were placed with females(1:1)for another 2 days.Then,males were removed,and pregnant females were left alone.2.Analysis of offspring’s glucose metabolism: We investigated the blood glucose levels and performed pyruvate tolerance tests,insulin tolerance tests and glucose tolerance tests in two groups of mice(Control-F1 and Stress-F1).Besides,body weight,food intake,oxygen consumption and other metabolic data were determined.3.Gene expression analysis: message RNA and protein levels of phosphoenolpyruvate carboxykinase(PEPCK)and glucose-6-phosphatase(G6Pase),two key enzymes in hepatic gluconeogenesis,were analyzed by quantitative real-time PCR and Western blot.4.Molecular mechanisms: a: MicroRNA expression array was performed using livers from two groups of mice to screen potential MicroRNA that regulates PEPCK expression.b: DNA methylation status at the promoter region of target MicroRNA was investigated using livers from offspring and sperm from fathers.c: In vitro fertilization-embryos transfer: to exclude the maternal oocyte and gestational effects,IVF-ET was employed.Sperm from Control-F0 or stress-F0 males were collected 14 days after the last restraint stress and co-incubated with oocytes from a common donor.Then the embryos were evenly transferred into the bilateral uterine horns of the same recipient.The methylation changes at the promoter region of target MicroRNA were detected in fetal livers of IVF-ET mice.5.Potential role of glucocorticoids in the transgeneration effects of paternal stress: a: Stressed fathers were treated with glucocorticoid receptor antagonist – RU486 or vehicle control.Then DNA methylation changes and offspring’s glucose metabolism were investigated.b: Male C57BL/6 mice were treated with glucocorticoids analog-dexamethasone or vehicle control,and then then DNA methylation changes and offspring’s glucose metabolism were investigated.Results: 1.Compared with Control-F1 mice,Stress-F1 mice presented with elevated blood glucose(both during fed and fasting conditions).Stress-F1 mice also showed a higher glucose excursion during pyruvate tolerance tests,pointing to hyperactive hepatic gluconeogenesis.However,insulin sensitivity was not changed in Stress-F1 mice,as evidenced by glucose and insulin tolerance tests.Besides,other metabolic changes were not observed in the Stress-F1 mice.2.While mRNA levels of these two enzymes were unaffected,protein levels of PEPCK were significantly increased in the Stress-F1 mice,pointing to a posttranscriptional regulatory mechanism.Besides,mRNA and protein levels of G6 Pase were not affected.3.By MicroRNA expression array and quantitative real-time PCR analysis,we found that miR-466b-3p,which potentially targets 3’-untranslated region of PEPCK gene,was significantly down-regulated in the livers of Stress-F1 mice.4.Through dual luciferase reporter assays,it showed that miR-466b-3p could bind to the 3’-untranslated region of PEPCK gene and inhibit its protein expression.5.The elevated DNA methylation status at the promoter region of miR-466b-3p in livers of Stress-F1 mice and sperm from Stress-F0 mice contributed to its down-regulation.6.The methylation changes in the miR-466b-3p promoter were also detected in fetal livers of IVF-ET mice,suggesting that psychological stress could reprogram the epigenome of germline cells and be passed onto the offspring.7.DNA methylation at the miR-466b-3p promoter suppresses the binding of certain transcriptional factors and is correlated with repressive histone modifications,leading to the down-regulation of miR-466b-3p expression.8.DNA methylation in the sperm from Stress-F0 mice and abnormal metabolic phenotypes of Stress-F1 mice were largely reversed by paternal treatment of RU486.9.Blood glucose levels and hepatic gluconeogenesis were increased in offspring fathered from dexamethasone-treated mice.Therefore,treatment of dexamethasone in fathers phenocopied the role of psychological stress.Conclusion: Taken together,we described a mouse model in which paternal exposure to psychological stress leads to hyperglycemia in the offspring,due to enhanced hepatic gluconeogenesis and overproduction of PEPCK.Mechanistically,we identified an epigenetic alteration at the promoter region of miR-466b-3p,leading to its down-regulation,which post-transcriptionally inhibited PEPCK protein expression.Therefore,our study further demonstrate the roles of transgeneration in the regulation of glucose metabolism,which might be helpful to understand the pathogenesis of type 2 diabetes and related metabolic disorders.In addition,our data point to the need for evaluating the role of this non-Mendelian form of inheritance in non-communicable human diseases. |
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