Flexibility in energy Flexibility in energy metabolism supports metabolism supports hypoxia tolerance in hypoxia tolerance in
Drosophila flight muscle: Drosophila flight muscle: metabolomic and metabolomic and
computational systems computational systems analysisanalysis
Jacob FealaJacob Feala1,21,2
Laurence Coquin, PhDLaurence Coquin, PhD22
Andrew McCulloch, PhDAndrew McCulloch, PhD11
Giovanni Paternostro, PhDGiovanni Paternostro, PhD1,21,2
1) UCSD Bioengineering 1) UCSD Bioengineering 2) Burnham Institute for Medical Research2) Burnham Institute for Medical Research
Cellular hypoxia responseCellular hypoxia response Hypoxia is the cause of cell death in many Hypoxia is the cause of cell death in many
pathologies, mechanism not knownpathologies, mechanism not known All cells have intrinsic defenses All cells have intrinsic defenses Hypoxia tolerant organisms have highly orHypoxia tolerant organisms have highly or
chestrated chestrated metabolicmetabolic regulationregulation
Drosophila as a model for Drosophila as a model for hypoxia researchhypoxia research
Flies are hypoxia tolerantFlies are hypoxia tolerant Simple system, genetic tools and librariesSimple system, genetic tools and libraries Genetic screen found gene required for tolerance Genetic screen found gene required for tolerance
11
Hypoxia tolerance gene was successfully Hypoxia tolerance gene was successfully transferred to mammalian cells transferred to mammalian cells 22
humanfly
Phylogenetic tree
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.
Systems analysis of hypoxia Systems analysis of hypoxia responseresponse
Complex balances must be maiComplex balances must be maintained to tolerate hypoxiantained to tolerate hypoxia ATP supply and demandATP supply and demand Redox potentialRedox potential Metabolic intermediatesMetabolic intermediates pHpH
Systems biology to understand Systems biology to understand and model the complex control and model the complex control systemssystems
Hochachka, P. W. J Exp Biol 2003; 206:2001-2009
General hypothesis for hypoxia tolerGeneral hypothesis for hypoxia toleranceance
Flexible metabolic regulation is the major soFlexible metabolic regulation is the major source of hypoxia toleranceurce of hypoxia tolerance Immediate (minutes)Immediate (minutes) Global (ATP production, biosynthesis, protein trGlobal (ATP production, biosynthesis, protein tr
anslation)anslation)
Our systems approach to modeling Our systems approach to modeling ATP-generating metabolism:ATP-generating metabolism:
Metabolomics to find all anaerobic Metabolomics to find all anaerobic pathwayspathways
Flux-balance analysis to simulate Flux-balance analysis to simulate pathways under varying oxygenpathways under varying oxygen
Generate novel, specific, testable Generate novel, specific, testable hypotheses for hypoxia tolerancehypotheses for hypoxia tolerance
11H NMR H NMR spectroscospectrosco
py of py of hypoxic fly hypoxic fly
musclemuscle• 0.5% O2
• 240 minutes• supervised by Laurence Coquin
MAMMALIAN TISSUE:
Troy H et. al. Metabolomics 2005; 1: 293-303
• Concentrations measured by targeted profiling (Chenomx): peak identification, alignment, subtraction• Lower confidence group due to spectra overlap
Global metabolic profileGlobal metabolic profile
11H NMR spectroscopy of flight H NMR spectroscopy of flight muscle at t=0,1,10,60,240 muscle at t=0,1,10,60,240 minutesminutes
Significant Significant metabolitesmetabolites
Reconstructing the Reconstructing the Drosophila Drosophila metabolic metabolic
networknetwork Database integrationDatabase integration
KEGG: metabolic genes, enzymes, reactions, KEGG: metabolic genes, enzymes, reactions, EC numbers, pathwaysEC numbers, pathways
Flybase: complete genome, proteins, function, Flybase: complete genome, proteins, function, compartment, mutant stocks, referencescompartment, mutant stocks, references
Filtered gene indexFiltered gene indexPathwaysPathways 109109EC numbersEC numbers 437437GenesGenes 13221322Genes Genes (mitochondrial)(mitochondrial)
125125
Genes (stocks Genes (stocks available)available)
507507
Network model of central Network model of central metabolismmetabolism 162 genes, 143 proteins and 158 reactions162 genes, 143 proteins and 158 reactions Includes glycolysis, TCA cycle, oxidative Includes glycolysis, TCA cycle, oxidative
phosphorylation, phosphorylation, β-oxidation, amino acids-oxidation, amino acids Elementally- and charge-Elementally- and charge-
balancedbalanced
Reconstructing the network Reconstructing the network
Gene-protein-reactionassociations
Literature and Databases
Annotated Genome
Stoichiometric matrix
Metabolic network reconstruction
Drosophila central metabolism
Reed JL et. al., Nat Rev Genet. 2006 Feb;7(2):130-41.
AlanineAcetyl-CoA
α-Oxoglutarate Glutamate
Cytosol
Mitochondria
Acetate
Acyl-carnitine shuttle
Glucose
Pyruvate Lactate
Main energetic pathways in model
ATP
Oxaloacetate
NADH
Acetyl-CoA
Citrate
ATP
Pyruvate
ATP
NADH
NADH
NADH/FADH2
NADH
NADH/FADH2
O2 H2OTCA cycle
Oxidative phosphorylation
Glycolysis
NADH
FADH
α-GPDH shuttle
Products seen in NMR
Hypothesized pathways
Known Drosophila pathwaysATP
CO2
CO2
NH4
Flux-balance analysisFlux-balance analysis Steady state assumption, flux Steady state assumption, flux
constraintsconstraints Optimize for objective functionOptimize for objective function Mass and charge balance inherentMass and charge balance inherent
ATP supply and demandATP supply and demand Redox potentialRedox potential pHpH
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 Flux-balance analysis of hypoxiahypoxia
lac
ala
ac
glc
Hypoxia simulation: key fluxesHypoxia 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
ConclusionsConclusions ‘‘Exotic’ anaerobic pyruvate Exotic’ anaerobic pyruvate
pathways in fly maypathways in fly may contribute to contribute to hypoxia tolerancehypoxia tolerance
New hypotheses to test: alanine New hypotheses to test: alanine and acetate production and acetate production essential under hypoxiaessential under hypoxia
Systems modeling revealed Systems modeling revealed emergent behavioremergent behavior
Candidate genes
Genetic perturbation
ModelExperiment
NMR metabolomics
Validate
Perturbation Analysis of Perturbation Analysis of Energy Metabolism in Energy Metabolism in
HypoxiaHypoxia
Refine
AcknowledgementsAcknowledgements Polly HuangPolly Huang Palsson lab, UCSD BioengineeringPalsson lab, UCSD Bioengineering
Adam FeistAdam Feist Thuy VoThuy Vo
Khoi PhamKhoi Pham
QuestionsQuestions
Flux variabilityFlux variability