Effect of preimplantation embryo culture on adult glucose homeostasis

描述(由申请人提供)：宫内或产后早期的压力对塑造未来的健康起着至关重要的作用。一种最初的应激源，当胚胎对环境变化极其敏感时，对发育中的基因网络、组织和器官进行重新编程，从而使产生的个体容易患病。我们认为，次优植入前条件使成年个体容易受到葡萄糖稳态改变的影响，导致胰岛素抵抗的发生率增加。我们的初步数据显示，在体外培养过程中增加压力会导致小鼠胚胎中基因表达的比例恶化。最重要的是，我们已经确定硫氧还蛋白相互作用蛋白(TXNIP)是植入前应激的标志。体外培养后，胚泡中TXNIP的mRNA和蛋白水平均上调，次优培养条件(Whitten‘s Medium-WM)比优化的培养液(含氨基酸的KSOM，KAA)中的TXNIP上调幅度更大。在成年动物和成年小鼠中，TXNIP的表达仍处于高水平，而且成年小鼠的血糖稳态受到损害。值得注意的是，在WM中培养的成年动物比在KAA中培养的动物有更严重的表型，这表明成年动物保持着对植入前应激的记忆。TXNIP基因的异常表达与控制DNA甲基化的几个基因的表达变化有关，表明这是一种表观遗传调控。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水平受细胞内含有葡萄糖、谷氨酰胺和腺苷化合物的含量的调节；因此，TXNIP似乎将细胞的营养状态与氧化还原状态和随后的代谢调节联系在一起。此外，TXNIP通过下调AKT2/PKB2途径参与外周糖代谢的调节，并与胰岛β细胞质量减少有关。我们认为(1)TXNIP是胚胎代谢感知机制中的一个关键因素，(2)TXNIP在胚胎中的上调改变了体内平衡机制，从而使TXNIP在成人组织中保持升高。(3)成人组织中TXNIP的升高导致了葡萄糖稳态的改变。为了验证这一假设，我们将研究：(1)TXNIP-TXN系统如何响应逐渐恶化的环境(2)负责维持成人组织中TXNIP高表达的机制(3)体外产生的成年小鼠的表型。这项研究很重要，因为这将为植入前胚胎对不同环境条件做出反应的基本机制提供洞察力。此外，这项研究得出的数据可用于监测通过辅助生殖技术(ART)受孕的儿童。因此，本申请是对计划公告(PA-08-104)：辅助生殖技术的不良后果的响应。事实上，已有300多万名儿童在抗逆转录病毒治疗的帮助下受孕，多项研究报告称，这一人群中母婴并发症的发生率有所增加。 公共卫生相关性：宫内或产后早期的压力在塑造未来健康方面发挥着关键作用。当胚胎对环境变化非常敏感时，一种初始应激源会对发育中的细胞、组织和器官进行重新编程，从而使产生的个体更容易患病。这项研究旨在确定在早期胚胎发育期间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用来感知和适应不同环境的机制的新理解。

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.