Elucidating modulators of hepatic metabolism by quantitative flux analysis

描述（由申请人提供）：目前还不习惯测量细胞做什么，因为生物医学研究中使用的大多数分析方法测量细胞内分子，因此提供了细胞状态的相当静态的视图。拟议研究的一个中心原则是，细胞内代谢通量是细胞和组织功能的极好描述符，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，简单地通过将那些反应定位在代谢网络中，所述代谢网络响应于用调节剂的处理而表现出显著的通量变化。在先前的研究中，我们已经开发了最先进的方法，从GC-MS数据的代谢通量定量。它们在一个强大的软件工具Metran中实现，Metran还允许通量可观测性分析和通量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，证明Metran在设计和进行高分辨率测定肝细胞中心碳代谢（CCM）通量的实验中的能力。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作用。具体而言，我们最近发现，在肝细胞中的己糖胺生物合成途径（HBP）的通量调节影响其胰岛素敏感性，如通过糖原沉积或葡萄糖输出测量的。然而，由于这些重要现象的机械起源在很大程度上是未知的，我们将在不同的调制器条件下应用通量测量，以确定网络中通量扰动的主要位置。我们的项目有5个具体目标：评估亚稳原子轰击电离以提高流量测定的MS数据质量，验证Metran用于肝细胞CCM流量定量，然后应用于研究葡萄糖酸-PEP循环机制，被认为在控制肝细胞中葡萄糖输出方面具有重要意义。在此之后，我们将应用我们的方法来研究特定的激素（瘦素），药物（蛋氨酸）和HBP通量调节剂（己糖胺，aloxan，azasaline）的兴趣2型糖尿病。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。

DESCRIPTION (provided by applicant): 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 framework for 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 indispensable tool in biomedical research.