Quantitative Analysis of Mammalian Cellular Metabolism

Abstract

Metabolism is a complex process that transforms nutrients into energy, reducing power, and biosynthetic precursors, thereby enabling cellular functions such as mechanical work, signal transduction and macromolecule biosynthesis. Mammalian metabolism consists thousands of interconnected, tightly regulated biochemical reactions that take place in multiple cellular compartments. Understanding mammalian metabolism is particularly important within the context of cancer, given that in cancer cells metabolism is altered to support rapid cell growth. To gain a quantitative understanding of mammalian cell metabolism, we developed a comprehensive approach that integrates LC-MS-based isotope tracer studies with uptake/excretion measurements into metabolic flux models. We developed novel metabolic flux quantification methods in two ways: (1) We first applied oxygen uptake rate as a constraint and constructed a redox-balance model, (2) Based on traditional isotope tracers (e.g. 13C , 14C, 15N), we developed a new deuterium tracer approach that directly measures redox active hydrogen transfer, which enables quantifying reaction contribution in a cofactor specific manner. The potential pitfalls in isotope-based metabolic flux quantification that result from reaction reversibility have also been investigated using the specific example of isocitrate dehydrogenase, an enzyme of great interest in recent literature. We have further applied experimental-computational methods to quantitatively study the metabolism of cancer cells with particular emphasis on cofactor balance, which includes quantifying the contribution of various pathways in production and consumption of ATP (main currency of energy) and NADPH (main currency of reducing power). We found that glutamine-driven oxidative phosphorylation is a major means of ATP production, even in hypoxic cancer cells. And we identified and confirmed that, beyond canonical pathways, in proliferating cells, the oxidation of serine-derived one-carbon units via folate-dependent pathway is a major NADPH source. Since metabolism is a dynamic process that responds to genetic and environmental conditions, we also investigated how oncogene activation and hypoxia influence cellular metabolism. Metabolism can also in turn regulate other cellular functions, here we demonstrated that human phosphoglycerate dehydrogenase, an enzyme amplified in tumors, produces the "oncometabolite" D-2-hydroxy-glutarate and influences histone methylation, providing an example in which the moonlighting activity of a metabolic enzyme has a potentially important role in epigenetic regulation.