父代高血糖改变子代肝脏核受体PPARα启动子的甲基化──异常代谢环境经父系跨代传递的一种新机制？

Either paternal or maternal diabetes is one of the important risk factors for development of metabolic abnormalities in the offspring. Long term consequences of diabetic pregnancy in women on next generation have been thoroughly studied. Recently, our group discovered that paternal hyperglycemia can accelerate metabolic disorders and hepatic steatosis in their offspring, accompanied by changes in transcriptional and methylation levels of nuclear receptor PPARalpha even at the condition of normal diet feeding. The mechanism underlying how paternal diabetes aggravate lipid metabolic abnormalities is rarely reported. Based on our previous study, this project will use male normal and DNA methyltransferase 1 (Dnmt1) hypomorphic defect mice to investigate how paternal hyperglycemia aggravates metabolic abnormalities and hepatic lipid deposition in offspring; By utilization of methylated DNA immunoprecipitation with sequencing (MeDip-Seq), and bisulfite sequencing, we explore whether PPAR alpha DNA methylation is involved in transgenerational transmission of paternal hyperglycemia; Bioinformatic database, luciferase report system, plasmid transfection and site-specific methylation modification are used to explore changes in PPAR alpha promoter CpG island methylation states and the the role in transcriptional activation and metabolism regulation. The completion of current proposal will clarify the the potential mechanism underlying paternal hyperglycemia increase hepatic lipid deposition and other metabolic disorders in the offspring, and provide a scientific basis for preventive and therapeutic intervention for adverse outcome caused by this abnormal metabolic programming.

父/母亲任意一方患糖尿病是子代发生代谢异常的重要风险因素。女性妊娠期糖尿病对子代的影响已有系统研究；课题组新近发现，父代高血糖能加速子代代谢紊乱的发生，子代正常饮食即出现异常肝脂质沉积和核受体PPARα转录及甲基化水平的改变。父代糖尿病如何诱发子代肝脂质代谢异常，DNA甲基化是否参与其中？目前鲜有报道。本研究在新近发现的基础上利用正常和DNA甲基转移酶1亚等位基因缺陷的雄性小鼠研究父代高血糖对子代肝脏形态、功能、表观遗传特征和基因表达的影响；甲基化DNA免疫共沉淀结合芯片分析、亚硫酸盐测序等探讨父代高血糖调节子代PPARα的机制；利用荧光素酶报告系统、质粒转染及位点特异甲基化修饰等技术探讨PPARα启动子CpG岛特定位点甲基化改变在转录激活和代谢调控中的作用，以期阐明父代高血糖加重子代肝脂质沉积和代谢紊乱的具体途径和可能机制，为父系介导的异常代谢编程所致不良结局的干预提供科学依据。

课题组新近发现，父代高血糖能加速子代代谢紊乱的发生，出现异常肝脂质沉积和核受体PPARα转录及甲基化水平的改变。本项目在此新发现的基础上利用小鼠模型进一步明确了父代高血糖对子代肝脏形态、功能、表观遗传特征和基因表达的影响；利用甲基化DNA免疫共沉淀结合芯片分析、亚硫酸盐测序等探讨父代高血糖调节子代PPARα的机制；利用荧光素酶报告系统、质粒转染及位点特异甲基化修饰等技术探讨PPARα启动子CpG岛特定位点甲基化改变在转录激活和代谢调控中的作用。研究发现：父代高血糖诱发子代肝脏异常脂质沉积和脂代谢紊乱；父代高血糖诱发子代代谢异常不依赖于体重的增加；上述改变与PPARα信号通路受损有关；父代高血糖子代肝脂质沉积和脂代谢紊乱与PPARα启动子甲基化改变有关；PPARα启动子区域SP1结合位点在PPARα转录激活过程中起着关键的调控作用；父代高血糖致子代肝脏PPARα启动子区域CpG岛A中第13号CpG位点（CpG13）甲基化水平显著升高，阻碍了SP1的转录激活作用，致PPARα的表达下调。以上发现初步阐明了父代高血糖加重子代肝脂质沉积和代谢紊乱的具体途径和可能机制，为父系介导的异常代谢编程所致不良结局的干预提供了科学依据。项目基本按原计划执行和完成，针对执行过程中部分与原设想不一致的研究结果，调整并增加了以下3部分内容：①肥胖导致认知障碍分子机制的研究：发现小胶质细胞激活及极化表型改变、神经元突触损伤是糖尿病/肥胖动物模型的重要变化之一；②石杉碱甲（Hup A）对肥胖小鼠学习记忆和认知功能改善的药理学效应及相关机制：Hup A可能通过增强大脑皮层和海马胰岛素信号和增加脑胰岛素水平来改善肥胖小鼠的空间记忆缺陷; ③ 母代高脂饮食复合BPA暴露对雌性子代生殖功能的影响及其机制：发现母代高脂饮食复合BPA暴露对雌性F1和F2子代下丘脑垂体性腺轴（HPG）呈现明显抑制作用且与下丘脑 Kiss1启动子CpG位点甲基化水平改变有关。从科研贡献，成果转换，国际交流，人才培养等方面均已达到国家自然科学基金结题的标准。