Elucidating modulators of hepatic metabolism by quantitative flux analysis

描述（由申请人提供）：目前还没有习惯测量细胞的功能，因为生物医学研究中使用的大多数分析方法都是测量细胞内分子，因此，提供了一个相当静态的细胞状态视图。提出的研究的一个核心原则是，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效果提供了一个信息框架。通量对于识别受宏观通量调节剂影响的特定酶特别有效，只需将这些反应定位在代谢网络中，这些反应对调节剂的处理反应表现出显著的通量变化。在先前的研究中，我们开发了最先进的方法，从GC-MS数据中进行代谢通量定量。它们在功能强大的软件工具Metran中实现，该工具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。本研究有两个主要目标：首先，证明Metran在设计和实施肝细胞中心碳代谢（CCM）通量高分辨率测定实验方面的能力。这意味着从GC-MS数据中计算肝细胞CCM中的每个单一反应通量。其次，利用Metran和定量通量分析阐明肝通量调节剂对胰岛素抵抗和糖尿病的重要作用。具体来说，我们最近发现肝细胞中己糖胺生物合成途径（HBP）的通量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，由于这些重要现象的机制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来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：评估亚稳态原子轰击电离以提高通量测定的MS数据质量，验证用于肝细胞CCM通量定量的Metran，然后应用于丙酮酸- pep循环机制的研究，该机制被认为对控制肝细胞的葡萄糖输出具有重要意义。之后，我们将把我们的方法应用于对2型糖尿病感兴趣的特定激素（瘦素）、药物（二甲双胍）和HBP通量调节剂（己糖胺、亚氧嘧啶、阿扎塞林）的研究。最终，我们希望将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.