Elucidating modulators of hepatic metabolism by quantitative flux analysis

通过定量通量分析阐明肝脏代谢调节剂

• 批准号: 7132918
• 负责人: GREGORY STEPHANOPOULOS
• 金额: $ 29.8万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7132918
• 关键词: azo compounds; biological signal transduction; carbon; data quality /integrity; genetically modified animals; glucosamine; glucose metabolism; hexosamines; hormone metabolism; insulin sensitivity /resistance; ionization; laboratory mouse; leptin; liver cells; liver metabolism; mass spectrometry; metformin; method development; nutrient bioavailability; pharmacokinetics; pyruvates; radiotracer; stable isotope; statistics /biometry

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.

描述(由申请人提供)：目前还不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测量细胞内的分子，因此提供了细胞状态的相当静态的视图。这项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，并为研究疾病和药物的效果提供了一个信息框架。对于识别受宏观通量调节剂影响的特定酶，焊剂特别有效，只需在代谢网络中定位这些反应，这些反应表现出显著的通量变化，以响应调节剂的处理。在以前的研究中，我们已经开发了从GC-MS数据中定量代谢通量的最先进的方法。它们是在一个强大的软件工具Metran中实现的，该工具还允许对通量的可观测性进行分析和计算通量的统计特性和可信区间。这项研究有两个广泛的目标：首先，展示Metran在设计和进行高分辨率测定肝细胞中心碳代谢(CCM)通量方面的实验能力。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。第二，使用Metran和定量流量分析来阐明肝脏流量调节剂在胰岛素抵抗和糖尿病中的作用。具体地说，我们最近发现，肝细胞中氨基己糖生物合成途径(HBP)的通量调节影响了它们的胰岛素敏感性，无论是通过糖原沉积还是葡萄糖输出来衡量。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，我们将应用在不同调制器条件下的磁通测量来确定磁通扰动在网络中的主要位置。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用于通量测定的MS数据的质量，验证Metran用于肝细胞CCM通量定量，然后应用于丙酮酸-PEP循环机制的研究，这被认为在控制肝细胞葡萄糖输出方面具有重要意义。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流量调节剂(氨基己糖、阿罗生、氮丝氨酸)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。