Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle
Tiago C Alves, Rebecca L Pongratz, Xiaojian Zhao, Orlando Yarborough, Sam Sereda, Orian Shirihai, Gary W Cline, Graeme Mason, Richard G Kibbey, Tiago C Alves, Rebecca L Pongratz, Xiaojian Zhao, Orlando Yarborough, Sam Sereda, Orian Shirihai, Gary W Cline, Graeme Mason, Richard G Kibbey
Abstract
Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.
Copyright © 2015 Elsevier Inc. All rights reserved.
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Source: PubMed