1. Metabolomic and computational systems analysis of hypoxic metabolism in Drosophila
Jacob Feala1,2
Laurence Coquin, PhD2
Andrew McCulloch, PhD1
Giovanni Paternostro, PhD1,2
1) UCSD Bioengineering
2) Burnham Institute for Medical Research

2. Cellular hypoxia response
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
Metabolic response is immediate and global
Drosophila is hypoxia tolerant model
Cellular hypoxia is root of necrosis and apoptosis in myocardial infarction and stroke.
Want to better understand cellular hypoxia because, although much work has been done, actual mechanisms of cell death and the cellular defense are not well known.
All cells have intrinsic defenses to oxygen fluctuations, but some organisms better than others.
These hypoxia-tolerant organisms generally have highly orchestrated regulation of metabolism to prevent cellular injury due to lowered ATP production in the absence of oxygen. We want to better understand this regulation in order to try to improve hypoxia sensitivity in human tissues.

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

4. Drosophila as a model for hypoxia research
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 Sep 30;94(20):
2Chen Q et. al., J Biol Chem Dec
5;278(49): Epub 2003 Sep 16.
Phylogenetic tree

5. General hypothesis for hypoxia tolerance
Flexible metabolic regulation is the major source of hypoxia tolerance
Immediate (minutes)
Global (ATP production, biosynthesis, protein translation)
One general hypothesis for the essence of hypoxia tolerance of certain organisms is that these cells have very flexible, highly orchestrated regulation of metabolism, and that this regulation occurs quickly (within minutes) and globally (affecting almost all metabolic processes in the cell)

6. Drosophila as a genetic model
No heart
yeast
human
fly
worm
Drosophila is the most genetically tractable model organism with a circulatory system.
Phylogenetic tree
Humans and flies share many cardiac genes:
tinman (Nkx2.5)
ether-a-go-go (hERG)
troponin
714 out of 929 (77%) of human disease genes had a Drosophila homolog (26 cardiovascular) (Reiter, 2001)
Metazoa  …  Arthropoda  …  Insecta  …  Diptera  …  Drosophilidae  …  Drosophila  melanogaster
Disease gene study: PMID (Reiter, 2001)
RNA and protein sequence match
Homologous disease alleles (cardiovascular?)
Sequence similarity: Flies vs. humans

7. Drosophila as a model organism
Cardiac aging
[From Giovanni’s slides]
Cardiac hypoxia
Fruit flies are highly tolerant to oxygen fluctuations
Genetic screen for hypoxia tolerance: Adar (Haddad, 1997)
Cardiac systems biology
Several high-throughput datasets (protein interactions, genetic interactions, microarrays)
Well annotated genome
Ease of manipulation and short lifespan for phenotype screens
Original haddad paper (genetic screen): PMID: Several reviews and follow-ups in recent years
Adar = pre-mRNA adenosine deaminase. Edits a sodium channel mRNA (gene name: paralytic or para)

8. Response to low O2 in flies and mammals
Standard physiological endpoints for resistance to ischemia
Infarct size: gold standard for mammals
Myocardial stunning
Recovery of mechanical function
Post-ischemic arrhythmias
Sensitivity of fly heart to oxygen fluctuations not yet characterized
Measurable in flies

9. Systems analysis of hypoxia response in Drosophila heart
Regulation of metabolism is key in hypoxia-tolerant organisms
Systems biology to understand and model the complex control systems
Hochachka PMID:
Hochachka, PMID:

10. Hypoxia Genes and Human Disease
Apolipoprotein D
Expressed in stroke, aging, alzheimers
Defense against hypoxia-reperfusion injury in flies
CD36
Fatty acid transporter
Mouse KO had reduced ischemia tolerance (41% drop in cardiac output during ischemia)
ApoD: Walker, et. al., 2006
CD36: Null mouse had same baseline CO as wild-type, but after 6 min of ischemia CO dropped 41%

11. Our systems approach to modeling ATP-generating metabolism:
Metabolomics to find all anaerobic pathways
Flux-balance analysis to simulate pathways under varying oxygen
Generate novel, specific, testable hypotheses for hypoxia tolerance
We chose to apply our systems biology approach to first understand regulation of ATP-generating central metabolism in Drosophila flight muscle. To find all anaerobic pathways used in flies, we gathered global metabolomic profiles across several hypoxic timepoints using NMR spectroscopy. We reconstructed the pathways of central metabolism to simulate these pathways using flux-balance analysis, and in the end came up with novel, specific, and testable hypotheses for hypoxia tolerance mechanisms within the cell.

13. The fly heart
heart organ
Microscope view
Normal beating

14. Automated measurement of heart function in Drosophila
Gene Disruption Project
Mail-order mutants
Breed and cross
(Bloomington, Indiana)
Computer automated
Mutants generated by individual investigators and BDGP (P-element insertion) >40% of genome (Bellen, et. al., PMID: )
Explain back-crossings. Balancer to prevent homologous recombination introduces genetic noise and is removed by crossing. Trait selection based on curly wing marker on balancer.
Heterozygous mutations against a uniform background (WT is Oregan S). Ideally would do more back-crossings against WT strain
Detect and measure heart
Anesthetize and mount on slide

15. Automated cardiac phenotyping

16. Automated anesthesia and mounting
pressurized air
anesthesia chamber
fly vial
vacuum
N2 gas
anesthesia
slide
computer-controlled valve

17. Cardiac Mechanics Research Group

18. Heart rate measurementO2
detected beat
time
microscope view
M-mode image
Heart rate is monitored at baseline, hypoxia (via N2 gas), and during recovery

21. Preliminary Research: Cardiac phenotype
Aim 1
Hypoxic cardiac phenotype
Decrease in rate and fractional shortening, fast recovery
Recovery is affected by %O2, duration
Lots of data collected, needs better analysis
120s hypoxia

22. Environmental factors
Reflexive body contraction (loss of muscle tone?) below 2%, hard to measure heart
Myocardial stunning at < 5%
Increased response at high temperature

24. 1H NMR spectroscopy of hypoxic fly muscle
240 minutes
supervised by Laurence Coquin
MAMMALIAN TISSUE:
Troy H et. al. Metabolomics 2005; 1:

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

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

28. Reconstructing the Drosophila metabolic network
Database integration
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

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

30. Acyl-carnitine shuttle
Main energetic pathways in model
Glucose
NADH
Acetate
NH4
Glycolysis
NADH
ATP
α-Oxoglutarate
Glutamate
ATP
NADH
Acetyl-CoA
Alanine
Pyruvate
Lactate
α-GPDH shuttle
NADH
Cytosol
Mitochondria
Acyl-carnitine shuttle
Pyruvate
FADH
CO2
NADH
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

31. Flux-balance analysis
Steady state assumption, flux constraints
Optimize for objective function
Mass and charge balance inherent
ATP supply and demand
Redox potential pH
Null Space of S
We then used flux balance analysis to generate network simulations.
FBA relies on steady state assumption to create a solution space of all possibilities for flux distributions created by the network from a set of constraints
A particular solution can be chosen within this solution space by optimizing for some objective function (can be one reaction or linear combination of rxns)
Nice thing about FBA is that many of the complexities of hypoxic metabolism discussed before are inherently taken care of by the mass-balance requirement of FBA. Depending on boundary conditions, these conditions can be met:
S matrix
Solution space
Particular solution
(optimal)
Metabolic network reconstruction

32. Price, et. al. (2004) Nat Rev Microbiol 2, 886-897

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

34. Hypoxia simulation: key fluxes
Drosophila
(Pseudo-) Mammalian
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

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

38. Cardiac phenotypes and single deletion analysis
WT vs mutant heart rate under hypoxia
Colors represent effect of deletion on ATP production
Here are a few example cardiac hypoxia traces overlaid on the single deletion analysis. Warm colors are enzymes whose deletions have a greater effect on ATP production in hypoxia

40. Conclusions
Multiple 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

41. Questions Acknowledgements and many thanks Polly Huang
Palsson lab, UCSD Bioengineering
Adam Feist
Thuy Vo
Khoi Pham
Questions