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Jacob Feala 1,2 Laurence Coquin, PhD 2 Andrew McCulloch, PhD 1 Giovanni Paternostro, PhD 1,2

Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic and computational systems analysis. Jacob Feala 1,2 Laurence Coquin, PhD 2 Andrew McCulloch, PhD 1 Giovanni Paternostro, PhD 1,2 1) UCSD Bioengineering

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Jacob Feala 1,2 Laurence Coquin, PhD 2 Andrew McCulloch, PhD 1 Giovanni Paternostro, PhD 1,2

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  1. Flexibility in energy metabolism supports hypoxia tolerance in Drosophila flight muscle: metabolomic and computational systems analysis Jacob Feala1,2 Laurence Coquin, PhD2 Andrew McCulloch, PhD1 Giovanni Paternostro, PhD1,2 1) UCSD Bioengineering 2) Burnham Institute for Medical Research

  2. Cellular hypoxia response • Hypoxia is the cause of cell death in many pathologies, mechanism not known • All cells have intrinsic defenses • Hypoxia tolerant organisms have highly orchestrated metabolicregulation

  3. human fly Drosophila as a model for hypoxia research • Flies are hypoxia tolerant • Simple system, genetic tools and libraries • Genetic screen found gene required for tolerance 1 • Hypoxia tolerance gene was successfully transferred to mammalian cells 2 1Haddad GG et. al., Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10809-12. 2Chen Q et. al., J Biol Chem. 2003 Dec 5;278(49):49113-8. Epub 2003 Sep 16. Phylogenetic tree

  4. Systems analysis of hypoxia response • Complex balances must be maintained to tolerate hypoxia • ATP supply and demand • Redox potential • Metabolic intermediates • pH • Systems biology to understand and model the complex control systems Hochachka, P. W. J Exp Biol 2003; 206:2001-2009

  5. General hypothesis for hypoxia tolerance Flexible metabolic regulation is the major source of hypoxia tolerance • Immediate (minutes) • Global (ATP production, biosynthesis, protein translation)

  6. Our systems approach to modeling ATP-generating metabolism: • Metabolomics to find all anaerobic pathways • Flux-balance analysis to simulate pathways under varying oxygen • Generate novel, specific, testable hypotheses for hypoxia tolerance

  7. 1H NMR spectroscopy of hypoxic fly muscle • 0.5% O2 • 240 minutes • supervised by Laurence Coquin • MAMMALIAN TISSUE: Troy H et. al. Metabolomics 2005; 1: 293-303

  8. Global metabolic profile • Concentrations measured by targeted profiling (Chenomx): peak identification, alignment, subtraction • Lower confidence group due to spectra overlap

  9. Significant metabolites 1H NMR spectroscopy of flight muscle at t=0,1,10,60,240 minutes

  10. Reconstructing the Drosophila metabolic network • Database integration • KEGG: metabolic genes, enzymes, reactions, EC numbers, pathways • Flybase: complete genome, proteins, function, compartment, mutant stocks, references

  11. Reconstructing the network Network model of central metabolism • 162 genes, 143 proteins and 158 reactions • Includes glycolysis, TCA cycle, oxidative phosphorylation, β-oxidation, amino acids • Elementally- and charge-balanced Metabolic network reconstruction Stoichiometric matrix Drosophila central metabolism Literature and Databases Gene-protein-reactionassociations Annotated Genome Reed JL et. al., Nat Rev Genet. 2006 Feb;7(2):130-41.

  12. Main energetic pathways in model Glucose NADH Acetate NH4 Glycolysis NADH ATP α-Oxoglutarate Glutamate ATP NADH Acetyl-CoA Pyruvate Lactate Alanine α-GPDH shuttle NADH Cytosol Mitochondria Acyl-carnitine shuttle Pyruvate FADH NADH CO2 Acetyl-CoA Oxaloacetate Citrate NADH/FADH2 ATP O2 H2O Known Drosophila pathways TCA cycle ATP Oxidative phosphorylation Hypothesized pathways NADH/FADH2 Products seen in NMR CO2

  13. Flux-balance analysis • Steady state assumption, flux constraints • Optimize for objective function • Mass and charge balance inherent • ATP supply and demand • Redox potential • pH Null Space of S S matrix Solution space Particular solution (optimal) Metabolic network reconstruction

  14. glc Flux-balance analysis of hypoxia Simulation conditions - Glucose (and equivalents) only carbon substrate - Lactate, alanine, acetate constrained to NMR fluxes - Varied O2 uptake constraint - Objective: maximize ATP production ac lac ala

  15. Hypoxia simulation: key fluxes (Pseudo-) Mammalian Drosophila Stable pH Reduced glucose uptake Equivalent ATP • Abbreviations: • atp: ATP production • co2: CO2 production • glc: glucose uptake • h: proton production • ac: acetate accumulation • lac: lactate accumulation • ala: alanine accumulation

  16. Conclusions • ‘Exotic’ anaerobic pyruvate pathways in fly maycontribute to hypoxia tolerance • New hypotheses to test: alanine and acetate production essential under hypoxia • Systems modeling revealed emergent behavior

  17. Perturbation Analysis of Energy Metabolism in Hypoxia Model Experiment Genetic perturbation Validate Refine NMR metabolomics Candidate genes

  18. Acknowledgements • Polly Huang • Palsson lab, UCSD Bioengineering • Adam Feist • Thuy Vo • Khoi Pham Questions

  19. Flux variability

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