Effect of preimplantation embryo culture on adult glucose homeostasis

描述（由申请人提供）：子宫内或产后早期的压力在塑造未来健康方面发挥着重要作用。当胚胎对环境变化极其敏感时，就会出现最初的应激源，它会对发育中的基因网络、组织和器官进行重新编程，从而导致个体容易患病。我们认为，次优的植入前条件容易使成年个体的葡萄糖稳态发生改变，从而导致胰岛素抵抗的发生率增加。我们的初步数据表明，体外培养过程中压力的增加会导致小鼠胚胎中基因表达的相应恶化。最重要的是，我们已经确定硫氧还蛋白相互作用蛋白（txnip）作为植入前应激的标志物。体外培养后囊胚中 Txnip mRNA 和蛋白水平上调；次优培养条件（惠顿培养基 - WM）比优化培养基（含有氨基酸的 KSOM，KAA）中的培养导致更强烈的 txnip 上调。 Txnip 表达在成年动物中仍然升高，并且成年小鼠的葡萄糖稳态受损。值得注意的是，在 WM 中培养的成年动物比在 KAA 中培养的动物具有更严重的表型，这表明成年动物中保留了植入前应激的记忆。异常的 txnip 基因表达与控制 DNA 甲基化的几个基因的表达改变有关，表明存在表观遗传调控。 Txnip 是硫氧还蛋白 (txn) 的主要抑制剂，硫氧还蛋白是一种关键的细胞抗氧化剂。 Txnip 水平受细胞内葡萄糖、谷氨酰胺、含腺苷化合物含量的调节；因此，txnip 似乎将细胞营养状态与氧化还原状态和随后的代谢调节联系起来。此外，txnip 通过下调 AKT2/PKB2 通路参与外周葡萄糖代谢的调节，并与胰腺 β 细胞质量减少相关。我们认为（1）txnip是胚胎代谢传感机制的关键要素，（2）胚胎中txnip的上调改变了体内平衡机制，使得txnip在成人组织中保持升高（3）成人组织中txnip的升高导致葡萄糖稳态的改变。为了检验这一假设，我们将研究：(1) txnip-txn 系统如何响应逐渐恶化的环境 (2) 负责维持成年组织中 txnip 表达升高的机制 (3) 体外产生的成年小鼠的表型。这项研究很重要，因为它将提供对植入前胚胎响应不同环境条件的基本机制的深入了解。此外，从这项研究中获得的数据可用于监测通过辅助生殖技术（ART）受孕的儿童。因此，本申请响应计划公告 (PA-08-104)：辅助生殖技术的不良后果。事实上，已有超过 300 万儿童在 ART 的帮助下受孕，多项研究报告称，该人群中孕产妇和胎儿并发症的发生率有所增加。 公共卫生相关性：子宫内或产后早期的压力在塑造未来健康方面发挥着重要作用。当胚胎对环境变化极其敏感时，就会出现最初的应激源，它会对发育中的细胞、组织和器官进行重新编程，从而使个体容易患病。这项研究旨在确定早期胚胎发育过程中逐渐恶化的压力如何影响未来的健康。从这项研究中获得的知识可能会导致对胚胎感知和适应不同环境的机制有新的理解。

DESCRIPTION (provided by applicant): Stress in utero or during the early post natal period plays a cardinal role in shaping future health. An initial stressor, occurring when the embryo is exquisitely sensitive to environmental changes, reprograms the developing gene networks, tissue, and organs so that the resulting individual is predisposed to disease. We hold that suboptimal preimplantation conditions predispose adult individuals to altered glucose homeostasis, leading to an increased incidence of insulin resistance. Our preliminary data show that increased stress during in vitro culture results in proportionate deterioration of gene expression in mouse embryos. Most importantly, we have identified thioredoxin-interacting protein (txnip) as a marker of preimplantation stress. Txnip mRNA and protein levels are upregulated in blastocyst after in vitro culture; suboptimal culture conditions (Whitten's medium- WM) results in a more intense txnip upregulation than culture in optimized medium (KSOM with amino acid, KAA). Txnip expression remains elevated in adult animals and adult mice have impaired glucose homeostasis. Significantly, adults animals cultured in WM have a more severe phenotype than animals cultured in KAA indicating that a memory of the preimplantation stress is maintained in the adult animal. The aberrant txnip gene expression is associated with altered expression of several genes that control DNA methylation, suggesting an epigenetic regulation. Txnip is the principal inhibitor of thioredoxin (txn), a key cellular antioxidant. Txnip levels are regulated by cellular contents of glucose, glutamine, adenosine containing compounds; txnip appears therefore to link the cellular nutritional state with redox state and subsequent metabolic regulation. In addition, txnip is involved in the regulation of peripheral glucose metabolism via downregulation of AKT2/PKB2 pathway and is associated with decreased pancreatic beta cell mass. We propose that (1) txnip is a key element of the embryonic metabolic sensing mechanism and that (2) txnip upregulation in the embryo modifies homeostatic mechanisms so that txnip will remain elevated in adult tissues (3) elevation of txnip in adult tissues results in alteration of glucose homeostasis. To test this hypothesis, we will investigate: (1) how the txnip-txn system responds to progressively worsening environment (2) the mechanisms responsible to maintain elevated txnip expression in adult tissues (3) the phenotype of adult mice generated in vitro. This study is important because will offer insight into basic mechanisms utilized by the preimplantation embryo to respond to different environmental conditions. In addition, data derived from this study can be used to monitor children conceived by assisted reproductive technology (ART). As such, this application is responsive to the Program Announcement (PA-08-104): Adverse outcome of Assisted Reproductive Technologies. Indeed, more than 3 million children have been conceived with the help of ART and multiple studies have reported an increased incidence of maternal and fetal complications in this population. PUBLIC HEALTH RELEVANCE: Stress in utero or during the early post natal period plays a cardinal role in shaping future health. An initial stressor, occurring when the embryo is exquisitely sensitive to environmental changes, reprograms the developing cells, tissues and organs so that the resulting individual is predisposed to disease. This research is designed to define how progressively worsening stress during early embryo development affect future health. Knowledge gained from this research may lead to novel understanding of the mechanisms used by the embryo to sense and to adapt to different environments.