2.4. Metabolic flux analysis

Steady state metabolic fluxes were calculated by combining extracellular flux measurements (glucose/glutamine consumption, lactate/glutamate/alanine/aspartate secretion) and mass isotopomer distributions using the isotopomer network compartmental analysis (INCA) software (Young, 2014), which simulates mass isotopomer distributions (MIDs) using the elementary metabolite unit (EMU) framework (Young et al., 2008; Antoniewicz et al., 2007). The metabolic flux analysis assumptions are:

Cells are at metabolic and isotopic steady state (Figs. S2A and B).

Produced CO₂ is not reincorporated via carboxylation reactions.

Succinate and fumarate are symmetrical, and their metabolism does not favor a particular orientation.

Pyruvate and aspartate are present in both the cytosol and mitochondria; malate, citrate and oxaloacetate freely exchange between the cytosol and mitochondria.

During metabolite extraction, mitochondrial and cytosolic pools are mixed and GC-MS analysis of isotopic enrichments reflects the mixture of different pools.

Biomass requirements (v29) were assumed to be similar to CHO cells (Ahn and Antoniewicz, 2011).

Glucose, lactate, glutamine and glutamate concentrations were measured in fresh and spent culture medium using a BioProfile Basic-4 analyzer (NOVA). Alanine and aspartate concentrations in media were quantitated using GC-MS by spiking a known concentration of labeled standard into samples prior to extraction. Secretion and consumption rates for metabolites were calculated by normalizing to total protein amount and time. Reaction networks describing the stoichiometry and atom transitions of central carbon metabolism are listed in Table S1. Extracellular flux reactions (v1, v8, v10, v23, v24, v26) were constrained based on measured values and uncertainties. Cytosolic and mitochondrial pools are assumed to mix during metabolite extraction, and mixing reactions are incorporated into the reaction network (v31–v36). Steady state (12hr labeling) mass isotopomer distributions from [U–¹³C]-glucose and [U–¹³C]-glutamine experiments were used to fit the model and estimate flux values. Flux estimations were initiated from random values at least 50 times to ensure a global minimum was reached. Goodness-of-fit was assessed by a chi-squared test. 95% confidence intervals (Table S1) were calculated by assessing the sensitivity of the sum of squared residuals to individual variations of each flux parameter. Fluxes within the malate-aspartate shuttle were found to be unconstrained, and the network model was therefore simplified to ignore compartmentalization of malate, aspartate, and oxaloacetate. (Table S1).