utions in the organism are a superimposition of the hypoxic pathways shown, plus some large flux through glycolysis and mitochondrial respiration. Since respiration is neutral in terms of protons and produces no end products besides CO2, and also since small changes in ATP production rates can have major effects in concentration over the long term, hypoxic flux patterns shown here are likely to be important for hypoxia tolerance even though their magnitudes are small relative to the concurrent high levels of respiration seen in physiological conditions. gest that this holds for any given O2 uptake rate. As a corollary, O2 consumption is also lower in adapted flies for any given ATP output. Therefore, although we did not have measurements of oxygen consumption for each group, simulations suggest that the hypoxia-adapted metabolism is more efficient in terms of ATP/O2, regardless of where the O2 “operating point” may lie. Key ratios of hypoxia tolerance were compared across groups at this common oxygen uptake rate. As shown in Comparison of active pathways We inspected differences in enzyme fluxes at this simulated oxygen uptake. Each experimental group likely operated at a different O2 uptake, but for the reasons argued above, simulations were again compared at minimum feasible O2 for all groups. As with the previous two figures, In adapted flies ATP production is higher at a common O2 uptake than both groups of nave flies, and experiments sweeping the oxygen constraint sug- Page 6 of 15 BMC Systems Biology 2009, 3:91 http://www.biomedcentral.com/1752-0509/3/91 30 ATP production at equivalent oxygen uptake ATP production 25 20 15 olytic flux in adapted flies. Pyruvate fermentation to alanine by alanine transaminase is active in controls but shut down almost completely in adapted flies, but lactate production from lactate dehydrogenase, shown for comparison, is similar among the groups. Pyruvate carboxylase, an anaplerotic reaction producing purchase AZ-6102 oxalacetate from pyruvate, is less by nearly half in adapted flies, while pyruvate dehydrogenase and acetate production from acetyl-CoA synthase are greater in the adapted population. The electron transport chain also shows important differences among the groups. During hypoxia, adapted flies utilize Complex I at a higher rate, while nave flies rely more on Complex II activity. The Complex II flux in nave flies is driven 
by the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19799341 glycerol phosphate shuttle, which transports accumulated cytosolic reducing equivalents in the form of NADH to the mitochondria in the form of FADH2. A reducing equivalent entering the electron transport chain via Complex I generates more ATP and consumes an additional proton, compared with one entering via Complex II. Experiments in isolated mitochondria have also demonstrated that activation of Complex II produced a lower P/O ratio than Complex I. 10 5 0 20% -> 4% 20% -> 0.5% Nave controls 4% -> 0.5% Hypoxia adapted Discussion Previously, Zhou et. al. used experimental selection over several generations to adapt a Drosophila population to be able survive chronic hypoxia. These flies are also able to recover more quickly after acute hypoxia than “nave” control flies. Adaptation to hypoxia is a remarkable feat for directed evolution over a relatively small number of generations, considering the complexity and scale of cellular mechanisms involved in oxygen regulation. We studied metabolic aspects of this adaptation, first measuring metabolic concentration profiles using 1H NMR
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