Transcript Flexibility in energy metabolism supports hypoxia tolerance in

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
Cellular hypoxia response
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Hypoxia is the cause of cell death in many
pathologies, mechanism not known
All cells have intrinsic defenses
Hypoxia tolerant organisms have highly
orchestrated metabolic regulation
Drosophila as a model for hypoxia research
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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
human
fly
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
Systems analysis of hypoxia response
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Complex balances must be
maintained to tolerate hypoxia
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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
General hypothesis for hypoxia
tolerance
Flexible metabolic regulation is the major source
of hypoxia tolerance
Immediate (minutes)
 Global (ATP production, biosynthesis, protein
translation)
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Our systems approach to modeling ATPgenerating metabolism:
Metabolomics to find all anaerobic pathways
 Flux-balance analysis to simulate pathways under
varying oxygen
 Generate novel, specific, testable hypotheses for
hypoxia tolerance
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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
Global metabolic profile
• Concentrations measured by targeted profiling (Chenomx): peak identification, alignment, subtraction
• Lower confidence group due to spectra overlap
Significant metabolites
1H
NMR spectroscopy of flight muscle at
t=0,1,10,60,240 minutes
Reconstructing the Drosophila
metabolic network
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Database integration
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KEGG: metabolic genes, enzymes, reactions, EC numbers,
pathways
Flybase: complete genome, proteins, function,
compartment, mutant stocks, references
Filtered gene index
Pathways
109
EC numbers
437
Genes
1322
Genes (mitochondrial)
125
Genes (stocks available)
507
Reconstructing the network
Network model of central metabolism
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162 genes, 143 proteins and 158 reactions
Includes glycolysis, TCA cycle, oxidative
phosphorylation, β-oxidation, amino acids
Elementally- and chargeStoichiometric
balanced
matrix
Metabolic network reconstruction
Literature and Databases
Gene-protein-reaction
associations
Annotated Genome
Reed JL et. al., Nat Rev Genet. 2006 Feb;7(2):130-41.
Drosophila central
metabolism
Main energetic pathways in model
Glucose
NADH
Acetate
NH4
NADH
α-Oxoglutarate
Glycolysis
ATP
Glutamate
ATP
NADH
Pyruvate
Alanine
Lactate
Acetyl-CoA
α-GPDH shuttle
NADH
Cytosol
Mitochondria
Pyruvate
Acyl-carnitine
shuttle
FADH
NADH
CO2
Acetyl-CoA
Oxaloacetate
Known Drosophila pathways
ATP
Citrate
TCA cycle
NADH/FADH2
CO2
ATP
O2
H2O
Oxidative phosphorylation
Hypothesized pathways
Products seen in NMR
NADH/FADH2
Flux-balance analysis
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Steady state assumption, flux constraints
Optimize for objective function
Mass and charge balance inherent
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Null Space of S
ATP supply and demand
Redox potential
pH
S matrix
Metabolic network
reconstruction
Solution space
Particular solution
(optimal)
glc
Flux-balance analysis of hypoxia
Simulation conditions
ac
lac
ala
- Glucose (and equivalents) only carbon substrate
- Lactate, alanine, acetate constrained to NMR fluxes
- Varied O2 uptake constraint
- Objective: maximize ATP production
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
Conclusions
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‘Exotic’ anaerobic pyruvate pathways in fly may
contribute to hypoxia tolerance
New hypotheses to test: alanine and
acetate production essential under hypoxia
Systems modeling revealed emergent
behavior
Perturbation Analysis of Energy
Metabolism in Hypoxia
Model
Genetic perturbation
Experiment
Validate
Refine
NMR metabolomics
Candidate genes
Acknowledgements
Polly Huang
 Palsson lab, UCSD Bioengineering
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Adam Feist
 Thuy Vo
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Khoi Pham
Questions
Flux variability