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, PhD Burnham Institute for Medical Research Andrew McCulloch, PhD Cardiac Mechanics Research Group University of California, San Diego

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 Reconstruct interaction networks 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

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

Reconstruction Overview Gene-protein-reaction associations Literature and Databases Annotated Genome Stoichiometric matrix Metabolic network reconstruction NMR metabolomics Thorax microarray

Reconstruction steps Database integration –KEGG: metabolic genes, enzymes, reactions, EC numbers, pathways –Flybase: complete genome, proteins, function, compartment, mutant stocks, references Filtered gene index Pathways109 EC numbers437 Genes1322 Genes (mitochondrial)125 Genes (stocks available)507

Reconstruction steps

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

Phase 0Phase 1 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

Constraint-based modeling Steady state assumption: dx/dt = Sv = 0 Flux constraints: v i = (C i t2 – C i t1 )/∆t Optimize for objective function: Z = cv Mass and charge balance inherent Particular solution (optimal) Null Space of S Solution space Metabolic network reconstruction S matrix

Price, et. al. (2004) Nat Rev Microbiol 2,

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

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

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) me for advanced copy: –COBRA Toolbox for Matlab Future directions –Different tissues –Applications Mechanistic understanding Multiple interventions

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

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

Alanine Acetyl-CoA α-OxoglutarateGlutamate Cytosol Mitochondria Acetate Acyl-carnitine shuttle Glucose PyruvateLactate Main Energetic Pathways in Model ATP Oxaloacetate NADH Acetyl-CoA Citrate ATP Pyruvate ATP NADH NADH/FADH 2 NADH NADH/FADH 2 O2O2 H2OH2O TCA cycle Oxidative phosphorylation Glycolysis NADH FADH α-GPDH shuttle Products seen in NMR Hypothesized pathways Known Drosophila pathways ATP CO 2 NH 4

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 lac ala ac glc

GenomeNetworksModulesFunctional Models BioinformaticsSystems Biology Multiscale Modeling cell tissue organ Experiment