从PI3K/AKT通路基因甲基化修饰探讨宫内生长受限致胰岛素抵抗的分子机制

The progressive decrease in insulin sensitivity is the pathological basis of adult metabolic syndrom induced by intrauterine growth retardation (IUGR), but its mechanism is not clear so far. Our research group found out in the early phase study that intrauterine environment, early nutrition and genetic genes played an important role in causing decrease in insulin sensitivity by IUGR in human studies; Our research group also found out the occurrence of abnormal signal transmission with insulin postreceptor was earlier than decrease in insulin sensitivity, and abnormal promoter DNA methylation existed in the gene of the regulatory molecular (PPARγ) in the insulin signalling pathway in the IUGR rat model established through low protein diet during pregnancy.Based on the early phase discovery, we will make use of the established IUGR rat model in this project to take fetal nutritional deprivation - insulin signalling pathway relevant gene promoter DNA methylation - gene expression - insulin sensitivity as studing the major line to explore the molecular mechanism of insulin resistance induced by intrauterine growth retardation. We will analyze the DNA methylation in PI3K/AKT signalling pathway, relevant gene expression and insulin sensitivity changes in IUGR rats and their rat offspring, the changes in the aforementioned indicators in adult IUGR rats with different feeding conditions given at the early stage, and the differences in insulin signal transmission and glucose consumption studies in hepatic cell after in vitro interference with DNA methylation. It aims to demonstrate whether or not the abnormal signal transmission originates from change in DNA methylation with respect to the impact of the change on insulin sensitivity, whether or not the acquired gene marker can be transmitted to the offspring, and whether or not DNA methylation change after birth is reversible to a certain degree in the early life by changing enviroment.

；胰岛素敏感性进行性降低是宫内生长受限（IUGR）成年后易患代谢综合症的病理基础，但其机制尚未阐明。课题组前期研究发现,宫内环境、早期营养及遗传基因在IUGR致胰岛素抵抗中起重要作用；IUGR大鼠生后出现胰岛素受体后信号转导异常先于胰岛素敏感性降低，且信号通路中调节分子启动区存在DNA甲基化异常。基于前期发现，本项目中我们将利用已经建立的IUGR大鼠模型，将胎儿营养剥夺-胰岛素信号通路相关基因启动区DNA甲基化-基因表达-胰岛素敏感性作为研究主线，分析IUGR大鼠及所产子代大鼠DNA甲基化、基因表达和胰岛素敏感性变化；以及早期给予不同喂养条件的IUGR大鼠成年后上述指标的变化；体外干扰DNA甲基化，对肝细胞胰岛素信号转导及葡萄糖消耗的影响。旨在论证信号转导异常是否源自DNA甲基化改变，该改变对胰岛素敏感性影响；这种获得性基因标记是否可传递给子代；生后早期DNA甲基化改变是否具有一定可逆性。

目的：利用孕期低蛋白饮食建立起来的IUGR大鼠模型，探讨IUGR致机体胰岛素敏感性减低的发病机制。方法：采用孕中晚期低蛋白饮食法建立IUGR大鼠模型。哺乳期将IUGR大鼠分为叶酸VB12补充组，和标准饲料组两组。21天断乳后所有仔鼠均给予标准饲料喂养。分别于生后21天、2月、4月对每组仔鼠内眦静脉取血，测定空腹血糖、空腹胰岛素、胆固醇和甘油三酯，并计算胰岛素敏感指数和胰岛素抵抗指数，留取骨骼肌、肝脏组织，提取总RNA和DNA。检测PTEN、PPARγ基因转录量和蛋白表达及基因启动区甲基化情况。并于生后2月、3月提取肝细胞进行体外培养，检测IUGR组和对照组肝细胞葡萄糖代谢情况。IUGR仔鼠2.5月受孕，观察子代胰岛素敏感性变化。结果：⑴妊娠中晚期低蛋白组仔鼠平均出生体重4.37±0.69g（对照组7.03±0.56g）， IUGR发生率为78.67%。⑵生后21天，三组仔鼠的空腹血糖、餐后2小时血糖、空腹胰岛素、胰岛素抵抗指数和胰岛素敏感指数，以及胆固醇和甘油三酯并无统计学差异。生后2月龄、4月龄，IUGR组和IUGR+叶酸VB12补充组餐后2小时血糖、空腹胰岛素、胰岛素抵抗指数，胆固醇和甘油三酯均高于对照组，胰岛素敏感性相对降低，差异有统计学意义。且IUGR组较IUGR+叶酸VB12补充组胰岛素抵抗及胆固醇和甘油三酯升高更明显。⑶ 2月龄开始，IUGR组和IUGR+叶酸VB12补充组肌肉组织中PPARγ mRNA 较对照组表达减少，且IUGR组表达最少，有统计学差异；肝脏组织中PTEN mRNA表达增多，差异同样有统计学意义。生后21天三组肌肉组织中PPARγ mRNA和肝脏组织PTEN mRNA 表达无统计学差异。⑷相关分析发现HOMA-IR随PPARγ mRNA表达降低而升高，随PTEN mRNA表达升高而升高。⑸生后2月和3月，IUGR组大鼠肝细胞活力与对照组相比差异无统计学意义，但肝细胞的葡萄糖摄取率显著低于对照组（P<0.01）。（5）IUGR大鼠3月龄时PPARγ基因启动区甲基化高于对照组。（6）IUGR大鼠所产子代在4月龄时胰岛素敏感性与对照组无统计学差异。结论：孕中晚期低蛋白饮食建立的IUGR模型，青年期胰岛素敏感性已明显降低。生后早期补充叶酸VB12对胰岛素敏感性降低有一定改善作用。PPARγ和PTEN可能是IUGR致胰岛素抵抗的重要调节分子。