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Cell Metab. 2015 Nov 3;22(5):936-47. doi: 10.1016/j.cmet.2015.08.021. Epub 2015 Sep 24.

Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.

Author information

1
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
2
Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
3
Department of Diagnostic Radiology and Psychiatry, Yale University School of Medicine, New Haven, CT 06520, USA.
4
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address: richard.kibbey@yale.edu.

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.

PMID:
26411341
PMCID:
PMC4635072
DOI:
10.1016/j.cmet.2015.08.021
[Indexed for MEDLINE]
Free PMC Article
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