Systems model of the ATP-generating metabolic network in Drosophila flight muscle

1. Systems model of the ATP-generating metabolic network in Drosophila flight muscle Jacob Feala, Laurence Coquin, Andrew McCulloch, Giovanni Paternostro, Presenting work from the labs of: Giovanni Paternostro, MD, PhDBurnham Institute for Medical ResearchAndrew McCulloch, PhD Cardiac Mechanics Research GroupUniversity of California, San Diego

2. Systems Biology • Choose well known model organism with sequenced genome and genetic tools • Use high-throughput technologies to acquire genome-scale data on biological components under the context of interest • Reconstructinteractionnetworks from the annotated genome and high-throughput data • Develop predictive,quantitative models of systems properties and integrative functions • Systematically perturb components, in-silico and experimentally. • Compare simulation with experiment to refine model and generate new experimentally testable hypotheses

3. Model context: hypoxic metabolism • Global regulation of metabolism during hypoxia • To survive, must balance: • ATP supply/demand • Acid production • Redox state • Metabolic intermediates • Use a genome-scale model to keep track of it all – how do flies maintain homeostasis? Hochachka, 1996

4. Reconstruction Overview Literature and Databases Metabolic network reconstruction Stoichiometric matrix Annotated Genome Thorax microarray Gene-protein-reactionassociations NMR metabolomics

5. Reconstruction steps • Database integration • KEGG: metabolic genes, enzymes, reactions, EC numbers, pathways • Flybase: complete genome, proteins, function, compartment, mutant stocks, references

6. Reconstruction steps

7. NMR Metabolomics 1H NMR spectroscopy of flight muscle at t=0,1,10,60,240 minutes Reconstruction steps

8. Reconstruction steps Network model of central metabolism • 211 genes, 196 reactions (74 associated with genes), 6 pathways • Glycolysis, TCA cycle, oxidative phosphorylation, β –oxidation, amino acid degradation, glutathione redox cycle, superoxide production and scavenging • Elementally- and charge-balanced Phase 0 Phase 1

9. Constraint-based modeling Steady state assumption: dx/dt = S•v = 0 Flux constraints: vi = (Cit2 – Cit1)/∆t Optimize for objective function: Z = c•v Mass and charge balance inherent Null Space of S S matrix Solution space Particular solution (optimal) Metabolic network reconstruction

10. Price, et. al. (2004) Nat Rev Microbiol2, 886-897

11. Hypoxia simulation: 3 pyruvate pathways vs 1 (Pseudo-) Mammalian Drosophila Stable pH Reduced glucose uptake Equivalent ATP Using pathways that generate alanine and acetate increases ATP/H+ and ATP/glucose ratios Abbreviations: • atp: ATP production • co2: CO2 production • glc: glucose uptake • h: proton production • ac: acetate accumulation • lac: lactate accumulation • ala: alanine accumulation

12. Aging and hypoxia tolerance • Data-driven FBA of young and old flies after 4 hours hypoxia and 5 minutes recovery •  Map of flux ratios on recovery (green:red = young:old) • Young flies recover mitochondrial respiration pathways better, old flies rely on anaerobic acetate production • Poster 854B, Laurence Coquin

13. Resources and applications • Resources for the community • Drosophila model files in SBML Feala et. al., 2007, Mol Sys Biol (Supplementary Material) • Updated model (in submission) email me for advanced copy: jfeala@ucsd.edu • COBRA Toolbox for Matlab http://systemsbiology.ucsd.edu/Downloads • Future directions • Different tissues • Applications • Mechanistic understanding • Multiple interventions

14. Acknowledgements • Matt Owen • UCSD Undergrads • Francis Le • Polly Huang • Khoi Pham • Palsson Lab • Bernhard Palsson • Adam Feist • Thuy Vo

16. 1H NMR spectroscopy of hypoxic fly muscle High-throughput technologies • 0.5% O2 • 240 minutes • NMR specialist: Laurence Coquin MAMMALIAN TISSUE: Troy H et. al. Metabolomics 2005; 1: 293-303

17. 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

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

19. organ Experiment tissue Multiscale Modeling cell Genome Networks Modules Functional Models Bioinformatics Systems Biology