Elucidating modulators of hepatic metabolism by quantitative flux analysis

目前还不习惯于测量细胞的功能，因为生物医学领域使用的大多数分析方法都是 研究测量细胞内的分子，并且同样地，提供细胞状态的相当静态的视图。一 本研究的中心原则是细胞内代谢通量是细胞代谢的极好描述符， 和组织功能，并为研究疾病和药物作用提供了一个信息框架。 通量对于鉴定受宏观通量调节剂影响的特定酶特别有效 简单地通过在代谢网络中定位那些反应， 用调节剂治疗。在先前的研究中，我们已经开发了最先进的代谢方法， 来自GC-MS数据的通量定量。它们在一个强大的软件工具Metran中实现， 允许通量可观测性分析和通量的统计特性和置信区间的计算。 这项研究有两个广泛的目标：第一，展示Metran在设计和 进行高分辨率测定中央碳代谢（CCM）通量的实验， 肝细胞这意味着从GC-MS数据计算肝细胞CCM中的每个单个反应通量。 第二，使用Metran和定量流量分析来阐明肝流量调节剂对 胰岛素抵抗和糖尿病的重要性。具体来说，我们最近发现， 肝细胞中己糖胺生物合成途径（HBP）的调节影响其胰岛素敏感性， 通过糖原沉积或葡萄糖输出来测量。然而，作为这些的机械起源， 重要的现象在很大程度上是未知的，我们将在不同的调制器下应用通量测量 条件，以确定网络中通量扰动的主要位置。我们的项目有5个具体的 目的：评价亚稳原子轰击电离，以提高通量MS数据的质量 测定、验证Metran用于肝细胞CCM通量定量，然后应用于研究 在控制葡萄糖输出方面被认为是非常重要的 在肝细胞中。之后我们将把我们的方法应用到特定激素（瘦素）、药物 （二甲双胍）和HBP通量调节剂（己糖胺、aloxan、阿扎胞苷）。 最终，我们渴望建立通量量化作为生物医学研究中不可或缺的工具。

It is not presently customary to measure what a cell does, as most analytical methods used in biomedical research measure intracellular molecules and, as such, provide a rather static view of the cellular state. A central tenet of the proposed research is that intracellular metabolic fluxes are excellent descriptors of cell and tissue function and provide an informative frameworkfor studying disease and the effect of drugs. Fluxes are particularly effective for identifying specific enzymes impacted by macroscopic flux modulators simply by locating those reactions in a metabolic network exhibiting significant flux changes in response to treatments with the modulators. In prior research we have developed state-of-the-art methods for metabolic flux quantification from GC-MS data. They are implemented in a powerful software tool, Metran, that also allows flux observability analysis and calculation of statistical properties and confidence intervals of fluxes. This research has two broad objectives: First, to demonstrate the power of Metran in designing and conducting experiments for the high-resolution determination of central carbon metabolic (CCM) fluxes in hepatocytes. This means the calculation of every single reaction flux in hepatocyte CCM from GC-MS data. Second, to use Metran and Quantitative Flux Analysis in elucidating the effect of hepatic flux modulators of importance to insulin resistance and diabetes. Specifically, we have recently discovered that the flux modulation of the hexosamine biosynthetic pathway (HBP) in hepatocytes affects their insulin sensitivity as measured by either glycogen deposition or glucose output. However, as the mechanistic origin of these important phenomena is largely unknown, we will apply flux measurements under varying modulator conditions to identify the primary locations of flux perturbations in the network. Our project has 5 specific aims: Evaluation of Metastable Atom Bombardment ionization to improve the quality of MS data for flux determination, validation of Metran for hepatocyte CCM flux quantification, and then application to the study of the pyruvate-PEP cycling mechanism, recognized to be of major importance in controlling glucose output in hepatocytes. After that we will apply our method to the study of specific hormones (leptin), drugs (Metformin) and HBP flux modulators (hexosamine, aloxan, azaserine) of interest to Type-2-Diabetes. Ultimately, we aspire to establish flux quantification as an indispenable tool in biomedical research.