CAREER: Metabolic Determinants of Programmed Cell Death in Hepatic Lipotoxicity

本申请的总体目标是阐明代谢变化激活暴露于升高的饱和脂肪酸（SFA）的肝细胞中的凋亡信号传导的机制。中心假设是由SFA诱导的内质网（ER）应激导致中心碳途径之间的偶联改变，其随后触发活性氧物质（ROS）的线粒体积累，导致细胞凋亡。拟议研究的基本原理是，一旦知道代谢途径通量如何影响脂质诱导的细胞凋亡，这些途径可以通过靶向引起脂毒性的潜在代谢因素的治疗干预来调节。计划是通过追求以下具体目标来检验中心假设。首先，确定SFAs诱导肝细胞中ROS积聚的机制。第二，确定负责诱导SFA处理的肝细胞ER应激的脂质中间体。这些目标将使用系统方法来实现，该方法侧重于综合代谢途径与个体反应，其中代谢网络的概念至关重要。该方法涉及GC-MS代谢分析，以同时跟踪许多不同细胞内代谢物的相对浓度和同位素标记模式，以及代谢通量分析（MFA），以重建代表整个网络中物质动态流动的综合通量图。通过对肝细胞进行各种旨在干扰脂质和中心碳代谢的治疗，他们将剖析SFA诱导的脂毒性所涉及的途径。

0955251YoungThe overall objective of the current application is to elucidate the mechanism by which metabolic changes activate apoptotic signaling in hepatic cells exposed to elevated saturated fatty acids (SFAs).The central hypothesis is that endoplasmic reticulum (ER) stress induced by SFAs leads to altered coupling between central carbon pathways, which subsequently triggers mitochondrial accumulation of reactive oxygen species (ROS) resulting in apoptosis. The rationale for the proposed research is that, once it is known how metabolic pathway fluxes impact lipid-induced apoptosis, these pathways can be modulated by therapeutic interventions that target the underlying metabolic factors causing lipotoxicity. The plan is to test the central hypothesis by pursuing the following specific aims. First, to determine the mechanism by which SFAs induce ROS accumulation in hepatic cells. Second, to identify the lipid intermediate responsible for inducing ER stress in SFA-treated hepatic cells. These aims will be accomplished using a systems approach that focuses on integrated metabolic pathways versus individual reactions and where the concept of the metabolic network is of central importance. The approach involves both GC-MS metabolic profiling to track the relative concentrations and isotopic labeling patterns of many different intracellular metabolites simultaneously, as well as metabolic flux analysis (MFA) to reconstruct comprehensive flux maps that represent the dynamic flow of material throughout the network. By subjecting hepatic cells to a variety of treatments aimed at perturbing lipid and central carbon metabolism, they will dissect the pathways involved in SFA-induced lipotoxicity.