Ethanol Metabolism in Embryonic Stem Cells

 描述（由申请方提供）：乙醇（EtOH）消耗导致其代谢为毒性中间体乙醛（AcH）。这种反应性代谢物通过Aldh超家族中的酶进一步代谢为乙酸盐。在大量饮酒期间，例如在狂饮发作期间，AcH不能有效地代谢为乙酸盐，AcH积累，导致细胞损伤和酒精中毒的一些症状。虽然大部分EtOH代谢发生在肝脏中，但其他细胞类型越来越多地显示出代谢EtOH并对其代谢的影响表现出敏感性。干细胞能够代谢EtOH，并且许多干细胞类型表达高水平的EtOH代谢酶。由于乙醇的致癌性和致畸性，干细胞代谢与发育疾病和癌症高度相关。许多代表成体干细胞的遗传和生物能量学回路也存在于胚胎干细胞（ESC）中，使其成为研究干细胞中EtOH代谢的代谢后果的相关模型。然而，EtOH在ESC中代谢的分子机制仍不清楚。与成人肝脏相比，ESCs具有不同的Aldh家族成员表达谱，Aldh1A1，Aldh1A2，Aldh1B1和Aldh2都被假设在干细胞中的EtOH代谢中发挥作用。我们实验室的初步数据表明，在ESCs中，给予1 μM AcH可增加Aldh1A2转录物，50 mM和100 mM EtOH可降低Aldh1A1 mRNA水平。此外，糖酵解底物和前体，和酰基肉毒碱转运蛋白SLC22A5转录增加后处理的ESCs与乙醇，表明能量代谢的变化。因此，我们假设EtOH是由不同的Aldh家族酶在胚胎干细胞代谢相比，成人肝脏，这导致不同的代谢表型作为改变的乙酰胆碱和乙酸形成率的结果。我们将通过使用shRNA技术和非靶向代谢组学的细胞培养方法来解决这一假设。对于目标1，我将确定哪些酶在Aldh家族代谢乙酰胆碱乙酸在胚胎干细胞。我将通过使用针对Aldh1A1、Aldh1A2、Aldh1A3、Aldh1B1和Aldh2的shRNA敲低单个Aldh基因的表达来实现这一点，并确定一旦向细胞中加入EtOH，这些酶将AcH代谢为乙酸的能力。在目标2中，我将使用非靶向代谢组学方法确定添加EtOH后ESC的代谢特征。这将提供一种高通量的方法，用于识别新的代谢物，辅因子和途径，有助于ESCs中的EtOH作用。使用Aldh抑制剂二乙氨基苯甲醛抑制Aldh活性，并通过特异性shRNA敲除显示代谢AcH的个体Aldh家族成员，将揭示ESCs中由EtOH产生AcH改变的途径。该项目的完成将增加我们对干细胞如何代谢EtOH的理解，并可能通过饮食或药物干预缓解EtOH诱导的并发症。

 DESCRIPTION (provided by applicant): Ethanol (EtOH) consumption results in its metabolism to a toxic intermediate, acetaldehyde (AcH). This reactive metabolite is further metabolized to acetate by enzymes in the Aldh superfamily. During times of heavy alcohol consumption, such as during binge drinking episodes, AcH is not efficiently metabolized to acetate and AcH accumulates, leading to cellular damage and some symptoms of alcohol poisoning. While much of EtOH metabolism occurs in the liver, other cell types are increasingly being shown to metabolize EtOH and exhibit sensitivity to the effects of its metabolism. Stem cells are capable of metabolizing EtOH and many stem cell types express high levels of EtOH-metabolizing enzymes. Because of the carcinogenicity and teratogenicity of EtOH, stem cell metabolism is highly relevant to developmental diseases and cancer. Many of the genetic and bioenergetics circuits that typify adult stem cells are also present in embryonic stem cells (ESCs), making them a relevant model for studying the metabolic consequences of EtOH metabolism in stem cells. However, the molecular mechanisms through which EtOH is metabolized in ESCs remain unclear. Compared to the adult liver, ESCs have different expression profiles of Aldh family members, with Aldh1A1, Aldh1A2, Aldh1B1, and Aldh2 all hypothesized to play roles in EtOH metabolism in stem cells. Preliminary data from our laboratory has shown that Aldh1A2 transcripts are increased by administration of 1 µM AcH and Aldh1A1 mRNA levels are decreased by 50 mM and 100 mM EtOH in ESCs. In addition, glycolysis substrates and precursors, and the acylcarnitine transporter SLC22A5 transcripts are increased following treatment of ESCs with EtOH, suggesting changes in energy metabolism. Consequently, we hypothesize that EtOH is metabolized by different Aldh family enzymes in ESCs compared to the adult liver, which leads to different metabolic phenotypes as a result of altered rates of AcH and acetate formation. We will address this hypothesis through a cell culture approach using shRNA technology and untargeted metabolomics. For Aim 1 I will determine which enzymes in the Aldh family metabolize AcH to acetate in ESCs. I will achieve this by knocking down expression of individual Aldh genes using shRNA against Aldh1A1, Aldh1A2, Aldh1A3, Aldh1B1, and Aldh2, and determining the ability of these enzymes to metabolize AcH to acetate once EtOH is added to the cells. In Aim 2 I will determine the metabolic signatures of ESCs following the addition of EtOH using an untargeted metabolomics approach. This will provide a high-throughput approach for identifying novel metabolites, cofactors, and pathways that contribute to EtOH actions in ESCs. Inhibition of Aldh activity using the Aldh inhibitor diethylaminobenzaldehyde and by specific shRNA knockdown against individual Aldh family members shown to metabolize AcH will reveal pathways altered by the production of AcH from EtOH in ESCs. Completion of this project will increase our understanding of how stem cells metabolize EtOH and may provide insight into alleviation of EtOH-induced complications through dietary or pharmacological interventions.