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[] Tracing Compartment-Specific Redox Pathways Using Stable Isotopes and Mass Spectrometry

University of California, San Diego, Department of Bioengineering
20 November 2017
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Abstract

Metabolism is central to virtually all cellular functions and contributes to a range of diseases.  A quantitative understanding of how biochemical pathways are dysregulated in the context of diseases such as cancer and metabolic syndrome is necessary to identify new therapeutic targets.  To this end we apply stable isotope tracers, mass spectrometry, and metabolic flux analysis (MFA) to study metabolism in mammalian cells, animal models, and human patients.  Using these approaches we have characterized how proliferating and differentiated cells regulate flux of glucose and amino acids into mitochondria for maintaining redox homeostasis and lipid biosynthesis. Recently, we have developed novel methods for studying pyridine nucleotide metabolism, employing 2H tracers and mass spectrometry to quantify how specific metabolic pathways are used to regenerate NADH and NADPH. To better understand how redox pathways are regulated in the cytosol and mitochondrial matrix we have generated compartment-specific enzyme reporters that exploit the neomorphic activity of mutant isocitrate dehydrogenases (IDHs). Specifically, R132H IDH1 and R172K IDH2 produce (D)2-hydroxyglutarate (2HG) in the cytosol and mitochondria, respectively. Quantitation of labeling from specifically labeled 2H tracers provides critical insights into NAD(P)H-producing pathways in each compartment. We have employed this approach to identify redox pathway regulation under hypoxia, where oxidative pentose phosphate pathway flux is upregulated to fuel reductive carboxylation. The application of MFA to cell and animal models greatly improves our ability to characterize intracellular metabolic processes, providing a mechanistic understanding of cellular physiology and metabolic function.

Keywords

metabolic flux analysis, NADPH, NADH, mitochondria, cytosol, IDH1, hypoxia, serine, one carbon metabolism

Cite this article as

Metallo, C. Tracing Compartment-Specific Redox Pathways Using Stable Isotopes and Mass Spectrometry. In Proceedings of the The 2nd International Electronic Conference on Metabolomics, 20–27 November 2017; Sciforum Electronic Conference Series, Vol. 2, 2017 ; doi:10.3390/iecm-2-05000

Presentation

Author biographies

Christian Metallo
Christian Metallo joined the University of California, San Diego in 2011 and is currently an associate professor in the Department of Bioengineering. He received his bachelor’s in chemical engineering from the University of Pennsylvania in 2000 before joining Merck Research Laboratories to conduct bioprocess engineering research. He received his Ph.D. from the University of Wisconsin-Madison Department of Chemical and Biological Engineering in 2008 and was an American Cancer Society Postdoctoral Fellow in Chemical Engineering at the Massachusetts Institute of Technology. Christian was the recipient of the Biomedical Engineering Society Rita Schaffer Young Investigator Award in 2012 and a Searle Scholar Award in 2013. He received a 2015 NSF CAREER Award and a Camille and Henry Dreyfus Teacher-Scholar Award in 2017.

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