Elementary Flux Analysis and Non-Stationary 13C Fluxomics Combine to Characterise Cytosol-Mitochondria Compartmentalisation in the CHO-K1 Cell Line

Introduction

The metabolism of mammalian cells is characterised by compartmentalisation, metabolite exchange and channelling. Mitochondria play an important part in the organisation of the eukaryotic metabolism. They are the powerhouse of the cell, hosting the TCA cycle, oxidative phosphorylation and other essential reactions from the central metabolism. In addition, sub-compartmental structures are scarcely investigated forms through which the eukaryotic metabolism is controlled by the cellular micro-structure.

Methods

In the first part of this study, CHO-K1 cells were selectively permeabilised to obtain ghost cells containing functional mitochondria. Several selected mitochondrial substrates and substrate combinations were fed. The elementary flux modes were computed for each case using the elementary modes of a reduced mitochondrial model and the measured uptake/secretion rates. In the second part, the CHO-K1 metabolism was investigated by feeding two labelled substrates, [U-¹³C₆] glucose and [U-¹³C₅] glutamine, to a batch shake flask culture. The dynamics of intra- and extracellular mass isotopomers was sampled at selected time points and used to estimate the intracellular fluxes, reversibilities and intracompartmental concentrations by applying non-stationary ¹³C metabolic flux analysis.

Results

Addition of ADP stimulated the uptake of most metabolites by the selectively permeabilised cells and favoured substrate metabolisation to CO₂ without enhancing the production of other metabolites. ADP exerted a higher control on the uptake of metabolites supplied to the first part of the TCA cycle: pyruvate, citrate, α-ketoglutarate and glutamine. In the second part of the TCA cycle, the rates were controlled by the concentrations of C4- dicarboxylates. The activity of the pyruvate carboxylase – malate dehydrogenase – malic enzyme cycle consumed the ATP produced by oxidative phosphorylation when ADP was not supplied externally, preventing its accumulation and maintaining metabolic steady state conditions. Uptake fluxes by the mitochondria in ghost cells were 4-to-50 times higher than those computed for live cells.

The metabolism of CHO-K1 cells exhibited a high pentose phosphate pathway activity in response to oxidative stress. Important features were a low TCA cycle, low cataplerotic fluxes, and simultaneous catabolism and production of amino acids. Malate and glutamate were cycled between cytosol and mitochondria via several transporters. Cytosolic NADH was regenerated partially using the malate-aspartate shuttle and partially by cytosolic lactate dehydrogenase. The different labelling patterns of lactate and pyruvate were modelled by considering channelling effects in the cytosol and mitochondria, as well as by including a mitochondrial lactate pool. Using non-stationary ¹³C metabolic flux analysis produced a comprehensive description of the metabolic fluxes and estimations for several compartment concentrations.

Conclusions

Two systems biology methods were combined to analyse compartmentalisation in CHO-K1 cells. Selectively permeabilised cells  and elementary mode analysis allowed studying the mitochondrial metabolism, transport and regulation. Non-stationary ¹³C metabolic flux analysis produced a high-resolution exploration of transport reversibility, metabolic compartmentalisation, mitochondrial transport and metabolic channelling. Combining the two methods offers a new perspective into the intricate system of metabolic fluxes in mammalian cells.

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