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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK075850-01
Application #
7132918
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Serrano, Jose
Project Start
2006-09-30
Project End
2011-07-31
Budget Start
2006-09-30
Budget End
2007-07-31
Support Year
1
Fiscal Year
2006
Total Cost
$298,044
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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Zhang, Jie; Ahn, Woo Suk; Gameiro, Paulo A et al. (2014) 13C isotope-assisted methods for quantifying glutamine metabolism in cancer cells. Methods Enzymol 542:369-89
Gameiro, Paulo A; Laviolette, Laura A; Kelleher, Joanne K et al. (2013) Cofactor balance by nicotinamide nucleotide transhydrogenase (NNT) coordinates reductive carboxylation and glucose catabolism in the tricarboxylic acid (TCA) cycle. J Biol Chem 288:12967-77
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Gameiro, Paulo A; Yang, Juanjuan; Metelo, Ana M et al. (2013) In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation. Cell Metab 17:372-85
Keibler, Mark A; Fendt, Sarah-Maria; Stephanopoulos, Gregory (2012) Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism. Biotechnol Prog 28:1409-18
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Noguchi, Yasushi; Young, Jamey D; Aleman, Jose O et al. (2011) Tracking cellular metabolomics in lipoapoptosis- and steatosis-developing liver cells. Mol Biosyst 7:1409-19
Gaglio, Daniela; Metallo, Christian M; Gameiro, Paulo A et al. (2011) Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth. Mol Syst Biol 7:523
Hiller, Karsten; Metallo, Christian; Stephanopoulos, Gregory (2011) Elucidation of cellular metabolism via metabolomics and stable-isotope assisted metabolomics. Curr Pharm Biotechnol 12:1075-86

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