Metabolic flux and enrichment studies using MIMOSA

Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is an LC-MS/MS based method which follows the step-wise flow of mass isotopomers, generated from stable isotope-labeled glucose and glutamine tracers, along intersecting metabolic nodes of central carbon metabolism (Krebs cycle [TCA cycle] and glycolysis). MIMOSA can capture both steady-state and dynamic metabolic fluxes by resolving positional isotopomers of the Krebs cycle. As a consequence, MIMOSA can determine the rates of individual intracellular fluxes as well as the relative contribution of multiple pathways converging onto the same biochemical reaction.

Glucose label (Glc)

Label introduced by 13C-glucose is used to evaluate relative metabolic flux through glycolysis into the Krebs cycle, that is, pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH). The anaplerotic flux (non-oxidative flow of carbons into the TCA cycle) through PC is expressed relative to citrate synthase (CS) and denoted as PC/CS.

Flux through PDH relative CS describes the relative contribution of glucose oxidation versus ß-oxidation into the Krebs cycle and is denoted as PDH/CS.

Following conversion of pyruvate to acetyl-CoA by PDH, CS catalyzes the condensation of acetyl-CoA with oxaloacetate to form citrate, after which the oxidative reactions of the Krebs cycle ensue. These are described by the fractional synthesis of glutamate from citrate, succinate from glutamate, and malate from succinate, which are represented in the data by 𝝋CitrateGlutamate, 𝝋 Glutamate Succinate and 𝝋SuccinateMalate (M2; M4; see below explanation). Since Mal M+2 can originate from multiple sources (PEP cycling, TCA), it is a less robust form of estimating fractional synthesis of malate by succinate, we also report 𝝋 SuccinateMalate M4, which can only be generated from succinate and therefore from the TCA cycle only. Ideally, both calculations should be very similar; however, in general, 𝝋SucMal M4 is more variable and might not be as reliable. Malate M3TCA is an estimate of malate M+3 originating within the TCA cycle as opposed to coming from PC flux. Mal M3 PC aids in calculating the PC/CS anaplerotic contribution to the Krebs
cycle.

Glutamine label (Q)

To begin with, it should be noted that due to their fast exchange, glutamate is used as a surrogate metabolite for 𝛼-ketoglutarate in our analysis. The relative contribution of glutamine label to the intramitochondrial Krebs cycle pool through glutamate via anaplerotic GDH flux is described by the 𝝋GlutamateSuccinate. Subsequent oxidative reactions through the Krebs cycle are represented by the 𝝋 SuccinateMalate (SDH), 𝝋MalateCitrate (CS), and 𝝋CitrateGlutamate (IDH). 𝝋GlutamineGlutamate represents the fractional contribution of glutamine to glutamate through glutaminase. The Q label is used to assess reductive carboxylation of 𝛼-ketoglutarate into citrate, which is mediated by reversed IDH and which provides substrate for citrate lyase flux (ACLY). This reaction is responsible for the breakdown of citrate into malate and acetyl CoA in the cytosol and it is the reaction that immediately precedes the first committed step of de novo lipogenesis (DNL). The 𝝋GlutamateCitrate describes the fractional synthesis of citrate from reversed IDH, whereas 𝝋CitrateMalate describes the fractional ACLY. This reaction is responsible for the breakdown of citrate in the cytosol and is the reaction that immediately precedes the first committed step of de novo lipogenesis. Moreover, the glutamine labeling strategy allows for assessment of cataplerosis through PEPCK (synthetic flow of carbons from the TCA cycle) into gluconeogenesis (pyruvate→OAA/malate→PEP→glucose) or into pyruvate cycling (pyruvate→OAA/malate→PEP→pyruvate). Comparing the enrichments from OAA to PEP functions as a proxy for relative contribution of PEPCK to pyruvate (𝝋MalatePEP), whereas OAA's contribution to pyruvate (𝝋MalatePyruvate) serves as a readout of malic enzyme fractional contribution (NB: malate and oxaloacetate act as one pool as far as fractional enrichments are concerned).

Metabolite concentrations

Standard curves are run for each metabolite and used to calculate metabolite concentrations. Concentration data are normalized to total protein content in the sample, which is determined by Bradford assay. Concentrations are provided for glycolytic/gluconeogenic intermediates (DHAP, G3P, 3PG, PEP, pyruvate, lactate) as well as Krebs cycle intermediates (citrate, succinate, malate) and extramitochondrial metabolites, e.g. glutamine and glutamate. In addition to normalized concentrations, also included in the analysis are data sets with the raw (not normalized) concentrations.

Results/data

All data will be provided in self-explanatory Prism files and a companion Microsoft PPT format. Data files contain processed mass spec data, including background and natural abundance correction, calculations of label enrichments, Phi value (𝜑) analysis, and key relative fluxes (PC/CS, PDH/CS). Data tables and graphs can be used by the client for their own statistical analysis and additional data interpretation.

Publications

Alves TC, Pongratz RL, Zhao X, Yarborough O, Sereda S, Shirihai O, Cline GW, Mason G, Kibbey R, (2015) Integrated, step-wise, mass-isotopomeric flux analysis of the TCA cycle. Cell Metab. 2015 Nov 3;22(5):936-47. doi: 10.1016/j.cmet.2015.08.021. Epub 2015 Sep 24. PMID: 26411341

Boutagy NE, Fowler JW, Grabinska KA, Cardone R, Sun Q, Vazquez KR, Whalen MB, Zhu X, Chakraborty R, Martin KA, Simons M, Romanoski CE, Kibbey RG, Sessa WC. TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes. Proc Natl Acad Sci U S A. 2023 Sep 19;120(38):e2218150120. doi: 10.1073/pnas.2218150120. Epub 2023 Sep 11. PMID: 37695914; PMCID: PMC10515159.