Metabolic Activities and Their Control at the Mitochondria-Cytosol Interface in CHO Cells

Mitochondrial activity and its regulation are of major importance for eukaryotic cell factories as well as in many health-related aspects. Mitochondria play a central role in energy generation that is crucial for the energy-intensive biosynthesis of proteins. The respiratory chain and oxidative phosphorylation require TCA cycle activity supplying NADH, succinate and ADP. Various transporters are involved in the exchange of metabolites between cytosol and mitochondria. Interestingly, not all transport activities are clearly assigned to specific genes. For example, only in 2010, the genes coding for the central pyruvate transporter proteins were identified.
Chinese Hamster Ovary (CHO) cells are the major workhorse in biopharmaceutical production. They are also an important mammalian cell model system. To study metabolic activities at the cytosol- mitochondria interface, we used a novel systems-oriented approach combining different methods to elucidate rate limiting steps and robustness of metabolism. These methods were mainly dynamic metabolic flux analysis and the measurement of compartment-specific enzyme activities. Metabolic fluxes represent in vivo rates or fluxes that can be related to maximum possible in vitro rates. Comparing these two values provides valuable information about metabolic control in a metabolic network. We applied dynamic metabolic flux analysis using measurements of biomass formation, extracellular carbohydrates, organic acids and amino acids. Batch cultivation of CHO resulted in different distinct growth phases with significantly different metabolic flux distributions. The first phase (phase I) was characterized by high glycolytic activity with secretion of lactate and consumption of pyruvate, glutamine, asparagine, aspartate, serine and secretion of alanine and glycine. The ratio of lactate production to glucose consumption was about 1.5 mol/mol and the ratio of TCA cycle flux to glycolytic flux varied around a value of 1 mol/mol. After consumption of glutamine, metabolism switched to consumption of lactate and glutamate. The ratio of lactate consumption to glucose consumption was now around a value of 1 mol/mol while the TCA/glycolysis ratio stabilized at a value of about 3 mol/mol. Hexokinase activities were low in phase I, only slightly higher than in vivo glycolytic fluxes indicating their rate controlling function. All other glycolytic fluxes were much smaller than corresponding in vitro activities. All glycolytic ratios increased significantly in phase II. TCA cycle was clearly limited by mitochondrial isocitrate dehydrogenase with an in vitro/in vivo activity ratio of close to one. Interestingly, the fraction of in vitro phosphofructokinase (PFK), that was measured after digitonin treatment leading to selective permeabilization of the cell membrane but leaving mitochondrial membrane intact, increased with increasing cultivation time. This shows that a large fraction of PFK was initially quite strongly attached to mitochondrial proteins thus blocking its active center. Later, an increasing fraction was directly accessible after digitonin treatment. This behavior is consistent with earlier indications of glycolytic substrate channeling. Flux analysis and compartment specific in vitro enzyme activities showed that in phase I mitochondrial pyruvate is primarily derived via malate and alanine that are transported into the mitochondria and there converted to pyruvate. Glycolytic pyruvate primarily reacts to lactate. This is supported but channeling of glycolytic intermediates directly to lactate. Anaplerotic supply of C4 units is then accomplished by glutamine feeding into the alpha-ketoglutarate pool via glutamate dehydrogenase. In phase II, lactate was taken up and converted to pyruvate that was now directly transported to the mitochondrial matrix. The major anaplerotic reaction was now by transport of aspartate into mitochondria and conversion to oxaloacetate.
Our findings show that there is a subtle control of glycolytic reactions and there connection to mitochondrial activities by channeling. Such findings were possible by a combined systems-oriented approach combining dynamic metabolic flux analysis and compartment specific enzyme activities. We think that simultaneous reporter gene studies will help to further refine mechanisms of such type
of control and later to derive methods for intervention to improve energy metabolism in cells producing recombinant proteins but also to cure diseases related to such processes.