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 2) Burnham Institute for Medical Research

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

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

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

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

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

1 H NMR spectroscopy of hypoxic fly muscle 0.5% O minutes supervised by Laurence Coquin MAMMALIAN TISSUE: Troy H et. al. Metabolomics 2005; 1:

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

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

Reconstructing the Drosophila metabolic network Database integration Database integration KEGG: metabolic genes, enzymes, reactions, EC numbers, pathways KEGG: metabolic genes, enzymes, reactions, EC numbers, pathways Flybase: complete genome, proteins, function, compartment, mutant stocks, references Flybase: complete genome, proteins, function, compartment, mutant stocks, references Filtered gene index Pathways109 EC numbers 437 Genes1322 Genes (mitochondrial) 125 Genes (stocks available) 507

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

Alanine Acetyl-CoA α -Oxoglutarate Glutamate 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

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

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

Hypoxia simulation: key fluxes 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

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

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

Acknowledgements Polly Huang Polly Huang Palsson lab, UCSD Bioengineering Palsson lab, UCSD Bioengineering Adam Feist Adam Feist Thuy Vo Thuy Vo Khoi Pham Khoi PhamQuestions

Flux variability