title: Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.
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.
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Tiago C
authors.initials: TC
authors.lastname: Alves
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Pongratz
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Orlando
authors.initials: O
authors.lastname: Yarborough
authors.affiliation: Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Sam
authors.initials: S
authors.lastname: Sereda
authors.affiliation: Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Orian
authors.initials: O
authors.lastname: Shirihai
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Gary W
authors.initials: GW
authors.lastname: Cline
authors.affiliation: Department of Diagnostic Radiology and Psychiatry, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Graeme
authors.initials: G
authors.lastname: Mason
authors.affiliation: 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.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Kibbey
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes; D002951:Citrates / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism*; D002952:Citric Acid Cycle / Q000235:genetics*; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats
chemical_list: D002247:Carbon Isotopes; D002951:Citrates; D007328:Insulin; D011768:Pyruvate Dehydrogenase Complex; D019343:Citric Acid; D062907:Oxaloacetic Acid; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D007521:Isocitrate Dehydrogenase; D011766:Pyruvate Carboxylase