Mechanisms of AHR Metabolic Toxicity

摘要 这项研究将解决芳烃受体(AHR)生物学中的一个主要知识缺口：如何 环境毒素2，3，7，8-四氯二苯并对二恶英(二恶英， TCDD)导致代谢毒性，包括葡萄糖生成抑制、能量衰竭和肝脏 脂肪变性。我们将研究NAD耗竭在AHR代谢毒性中的作用。NAD是一种小分子 在细胞氧化还原反应中很重要，是PARPS和sirtuins催化活性所必需的底物， 调节能量代谢、基因组稳定性和衰老的酶。这项研究将提供新的 从TCDD降低NAD的发现来理解AHR代谢毒性的基础 通过AHR靶基因TiPARP(TCDD诱导的聚合ADP核糖基酶，PARP7)表达水平，PARP是PARP之一 消耗NAD而ADP-核糖化蛋白的酶。我们将在这里利用我们的发现 TiPARP通过消耗NAD和抑制诱导而导致肝脏葡萄糖产生减少 通过糖异生中的限速酶PEPCK的sirtuin 1。长期接触TCDD也会增加 肝脏PARP1水平提示PARP1可能有助于降低NAD水平。我们建议两个 相关的特定目标(SA)：SA1将研究NAD枯竭对sirtuin活性和 线粒体生物能量学，将确定TiPARP和PARP1在哺乳动物TCDD毒性中的作用 他将努力确定NAD再补充是一种预防和纠正TCDD毒性的战略。 SA2将通过TiPARP研究ADP核糖化在AHR作用中的作用。具体地说，这些研究将 阐明NAD耗尽对Sirts1、Sirts3和Sirts6的生物和催化活性的影响 新的sirtuin特异性检测方法(SA1a1)。TCDD耗尽NAD在线粒体生物能量学中的作用 通过使用海马技术检查氧气消耗和糖酵解的研究(SA1a2)来解决。 鸡胚胎肝细胞将在整个赠款过程中使用，我们将证实人类的主要发现。 原代肝细胞。肝脏特异的TiPARP和PARP1 KO小鼠将被用来评估它们在 在哺乳动物模型中产生TCDD代谢毒性(SA1b)。SA1c将决定NAD是否 补充烟酰胺和其他NAD复制剂可以预防并可能纠正TCDD 毒物。SA2将检测TiPARP的ADP核糖化在AHR代谢毒性中的作用，我们发现 PEPCK是由TiPARP进行ADP核糖化的。我们将研究(SA2a)对PEPCK的ADP-核糖化的影响 TiPARP对PEPCK稳定性和活性的影响，并将寻求鉴定其他由TiPARP修饰的ADP-核糖化蛋白 揭示ADP核糖化参与AHR作用的新途径(SA2b)。我们期待着这项研究 建立PARP活性导致NAD耗竭导致TCDD肝损害的重要机制 代谢毒性和确定NAD补充作为预防和/或纠正AHR代谢的方法 体内毒性。

ABSTRACT This research will address a major knowledge gap in aryl hydrocarbon receptor (AHR) biology: how transcriptional activation of the AHR by the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes metabolic toxicities including suppressed glucose production, energy failure and hepatic steatosis. We will investigate the role of NAD+ depletion in AHR metabolic toxicities. NAD+ is a small molecule important in cellular redox reactions and is a required substrate for the catalytic activity of PARPs and sirtuins, enzymes that regulate energy metabolism, genome stability and aging. This research will provide new understanding of the basis of AHR metabolic toxicities by probing our finding that TCDD decreases NAD+ levels through the AHR target gene TiPARP (TCDD-inducible poly ADP-ribosylase, PARP7), one of the PARP enzymes which consume NAD+ while ADP-ribosylating proteins. We will exploit here our discoveries that TiPARP contributes to decreased hepatic glucose production by consuming NAD+ and suppressing induction by sirtuin 1 of PEPCK, a rate limiting enzyme in gluconeogenesis. Longer exposure to TCDD also increases hepatic PARP1 levels suggesting that PARP1 may contribute to lowering NAD+ levels. We propose two connected Specific Aims (SA): SA1 will study the consequences of NAD+ depletion on sirtuin activity and mitochondrial bioenergetics, will establish the role of TiPARP and PARP1 in TCDD toxicities in mammalian model and will seek to identify NAD-repletion as a preventive and corrective strategy against TCDD toxicity. SA2 will examine the role of ADP-ribosylation by TiPARP in AHR action. Specifically, these studies will elucidate the consequences of NAD+ depletion for biological and catalytic activities of Sirts1, 3 and 6, using new sirtuin specific assays (SA1a1). A role of NAD+ depletion by TCDD in mitochondrial bioenergetics will be addressed by studies using Seahorse technology to examine oxygen consumption and glycolysis (SA1a2). Chick embryo hepatocytes will be used throughout the grant, and we will confirm major findings in human primary hepatocytes. Liver-specific TiPARP and PARP1 KO mice will be used to assess their roles in the production of TCDD metabolic toxicities in a mammalian model (SA1b). SA1c will determine whether NAD+ repletion with nicotinamide, and other NAD+ repleting agents, can prevent and possibly correct TCDD toxicities. SA2 will examine the role of ADP-ribosylation by TiPARP in AHR metabolic toxicities as we found that PEPCK is ADP-ribosylated by TiPARP. We will examine (SA2a) the effects of ADP-ribosylation of PEPCK by TiPARP on PEPCK stability and activity and will seek to identify other proteins ADP-ribosylated by TiPARP to reveal new ways by which ADP-ribosylation could participate in AHR action (SA2b). We expect this research to establish a significant mechanism in which NAD+ depletion by PARP activity leads to TCDD hepatic metabolic toxicities and to identify NAD+-repletion as an approach to prevent and/or correct AHR metabolic toxicities in vivo.