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： 环境毒素2，3，7，8-四氯二苯并-对-二恶英（二恶英， TCDD）引起代谢毒性，包括抑制葡萄糖产生、能量衰竭和肝毒性。 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是重要的并且是PARP和sirtuins的催化活性所需的底物， 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 通过探索我们的发现，TCDD降低NAD+，了解AHR代谢毒性的基础 通过AHR靶基因TiPARP（TCDD诱导的聚ADP核糖基化酶，PARP 7）的水平，PARP之一 消耗NAD+而ADP-核糖基化蛋白质的酶。我们将在这里利用我们的发现， TiPARP通过消耗NAD+和抑制诱导而有助于降低肝葡萄糖产生 由PEPCK的sirtuin 1，一种植物异生的限速酶。长期接触TCDD也会增加 肝脏PARP 1水平表明PARP 1可能有助于降低NAD+水平。我们提出了两 相关具体目标（SA）：SA 1将研究NAD+耗竭对沉默调节蛋白活性的影响， 线粒体生物能量学，将建立TiPARP和PARP1在哺乳动物TCDD毒性中的作用 模型，并将寻求确定NAD-补充作为一种预防和纠正策略，对TCDD毒性。 SA2将检查TiPARP引起的ADP-核糖基化在AHR作用中的作用。具体而言，这些研究将 阐明NAD+耗竭对Sirts 1、3和6的生物和催化活性的影响，使用 新的sirtuin特异性检测（SA1a1）。在线粒体生物能量学中，TCDD消耗NAD+的作用将是 通过使用Seahorse技术检查耗氧量和糖酵解的研究解决了这一问题（SA1a2）。 鸡胚肝细胞将在整个资助过程中使用，我们将确认在人类中的主要发现。 原代肝细胞肝脏特异性TiPARP和PARP1 KO小鼠将用于评估它们在肿瘤治疗中的作用。 在哺乳动物模型中产生TCDD代谢毒性（SA1b）。SA1c将确定NAD+是否 补充烟酰胺和其他NAD+补充剂可以预防并可能纠正TCDD 毒性SA2将检查TiPARP引起的ADP-核糖基化在AHR代谢毒性中的作用， PEPCK被TiPARP腺苷二磷酸核糖基化。我们将检查（SA 2a）PEPCK的ADP-核糖基化的影响， 通过TiPARP对PEPCK稳定性和活性的影响，并将寻求鉴定其他被TiPARP ADP核糖基化的蛋白质 揭示ADP-核糖基化参与AHR作用的新途径（SA2b）。我们希望这项研究 建立一个重要的机制，其中NAD+消耗PARP活性导致TCDD肝 代谢毒性，并确定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.