to all of these metabolites via a stoichiometrically precise reaction network that includes and therefore balances NAD+ and NADH. Also important is the timescale of the experiments. Creatine and phosphocreatine are important as a buffer for short-term fluctuations in ATP/ADP ratios, and ade- nylate kinase similarly protects ATP/AMP ratios in the short term. However, we have chosen a 4-hour timescale to emphasize steady-state fluxes over short-term feedback, and so even though these reactions exist in the model, they are not used. Ten metabolites were chosen for fitting the model, based on the requirements that they were present in the NMR spectra in sufficient concentrations for all experimental conditions, were represented in our metabolic reconstruction, and changed concentration in a direction that was feasible with the model. Fluxes were approximated by dividing differences in two concentrations by the experiment time, and standard errors for fluxes were derived by adding the variances of the two concentration measurements. Glycogen availability was left unconstrained for all simulations. This assumption was supported by the large increase in free glucose for most hypoxic measurements, indicating that glycogen and trehalose breakdown supplied glucose monomers faster than the system could use them. Reactions for fatty acid catabolism are present in the model, but literature data has suggested that flight muscle metabolism in closely related insects is almost completely based on carbohydrates. The large deposits of glycogen in flight muscle of flies, the depletion of these reserves after prolonged flights, and the rapid catabolism by flight muscle in vitro, indicate that glycogen is the carbohydrate that provides the major source of energy for meeting the metabolic requirements of active flight. Instead, for each experiment we swept the oxygen consumption constraint over ranges that encompassed the qualitative features of the model. Oxygen consumption requirements in simulation Every simulation required some minimum level of oxygen to produce the organic end products observed in the NMR spectra, below which the model was infeasible. The minimum feasible oxygen uptake was less than 5 nmol O2 per minute per mg protein for all three simulations, much lower than physiological measurements of normoxic oxygen uptake on the order of 1,000 and 3,000 nmol O2 per minute per mg protein in mitochondria, and PubMed ID: per mg dry weight in whole flies, respectively. In SCH58261 cost hypoxia at 4% O2, oxygen consumption was previously measured roughly on the order of 2,200 nmol O2 per mg, deviating only a small amount between nave and adapted flies, and still two orders of magnitude higher than the minimum uptake suggested by the model. A trend of lower minimum oxygen consumption in adapted flies can be seen, possibly suggesting greater flexibility in regulating oxygen demand, but this trend was not statistically significant for the three groups tested. Key hypoxia tolerance indicators At the physiological ranges of oxygen uptake noted above, mitochondrial respiration still dominates central metabolism, masking subtler differences in fluxes. Therefore, to focus on interesting differences between groups and since we do not have measurements of true oxygen uptake “operating points” for each group, we compared ATP production across models using a common oxygen uptake that was as low as possible while still producing a feasible result for all simulations. The true flux distrib

Flux-balance analysis was used to simulate system flux distributions during acute hypoxia for each group

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