Elucidating modulators of hepatic metabolism by quantitative flux analysis

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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.