Metabolic flux rewiring in mammalian cell cultures

Abstract

Continuous cell lines (CCLs) engage in 'wasteful' glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells.

Figure 1. Typical metabolic phenotypes of proliferating and non-proliferating CCLs

(A) Exponentially growing cultures exhibit aerobic glycolysis and rely on elevated glutamine consumption to fuel mitochondrial metabolism. This results in increased lactate and ammonium production as cells rewire their metabolism to maintain carbon, nitrogen, and redox balance. (B) Stationary phase cultures metabolize glucose mainly by oxidation in the TCA cycle, which provides much higher ATP yields and reduced byproduct accumulation. An increased proportion of incoming glucose is diverted into the oxidative pentose phosphate pathway to maintain NADPH levels.

Figure 2. Major pathways of central carbon metabolism and key regulatory proteins that control enzyme expression and activation

The enzymes PFK1 and PFK2/FBPase (discussed in the text) have been lumped under Glycolysis and are not shown explicitly. See list of abbreviations for explanation of nomenclature.

Figure 3. Alternative fates of glutamine carbon entering the TCA cycle

Glutamine carbon can be converted to lipids or other macromolecular building blocks through either (A) normal anaplerosis (in the oxidative direction) or (B) reductive carboxylation. On the other hand, glutamine can be used to supply ATP and/or NADPH without retention of carbon by either (C) glutaminolysis to form lactate + CO₂ or (D) complete oxidation to CO₂.