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.

Figure 1. Citrate isotopomer analysis

(A) Isotopomers generated during the 1^st (red), 2^nd (orange), and 3^rd (yellow) turn of the TCA cycle. Citrate isotopomers were grouped into families depending on whether the label is originated from PDH flux only (Cit_a, Cit_f, Cit_b, Cit_i, Cit_d, Cit_e and Cit_k) or from both PDH and PC fluxes (Cit_h, Cit_c, Cit_j, Cit_g, Cit_f’, and Cit_b’). (B) Enrichment time course of the deconvolved citrate isotopomers from PDH flux alone or (C) from both PDH and PC fluxes. (D) Total PDH (∑ Cit_{a, f, i, d, h, j}) and PC flux (∑ Cit_{h, c}) into citrate. (E) Measured vs. predicted glutamate C2, C3 and C4 NMR multiplets patterns from deconvolved citrate isotopomer data. All data are reported as mean ± S.E.M.

Figure 2. Tracking of ¹³C-label

(A) [1,2-¹³C₂]Acetyl-CoA (from glutamate MIDA) compared to its precursor [U-¹³C₃]PEP. (B–D) Mitochondrial M⁺², M⁺⁴ and [(1,2,3)(2,3,4)-¹³C₃] (from PC flux) isotopomer evolution in OAA, aspartate and malate. (E) Incorporation of [U-¹³C₆]glucose into the glycolytic intermediates fructose-1,6-bis phosphate (F1,6bP, M⁺⁶), 3-phosphoglycerate (3PG, M⁺³), 2-phosphoglycerate (2PG, M⁺³), PEP(M⁺³) and pyruvate (M⁺³). (F) Evolution of ¹³C-label from glycolysis into the TCA cycle through PDH flux and (G) anaplerotic PC flux. All data are reported as mean ± S.E.M.

Figure 3. Isotopic steady state analysis

(A) Graphical scheme of the 52 steady-state precursor-product relationships between the metabolic intermediates contributing to the TCA cycle. (B–D) Map and the ratio of precursor (X) to product (Y) enrichments (Φ_X→Y) of (B) segmental (C) serial, and (D) anaplerotic/cataplerotic reactions of the TCA cycle. * P < 0.001, ** P < 0.0001. All data are reported as mean ± S.E.M.

Figure 4. Comparison of steady state analyses by NMR and MIMOSA

(A) V_PDH, V_PC and V_PK fluxes relative to V_CS calculated from NMR-based tcaCALC simulations (blue) compared to equivalent MIMOSA values (Φ, red). Pyruvate cycling here is denoted as ¹³C label that cycles out of the TCA cycle into pyruvate via PEPCK and ME. In tcaCALC this is the ‘PK’ term but is distinct from glycolysis. In MIMOSA it is the transfer of oxaloacetate label to the full pyruvate pool, Φ_OP. (B) Measured and predicted glutamate NMR multiplet isotopomer distributions from (blue) direct, (red) predicted from tcaSIM using the tcaCALC flux values (PDH=1, PC=0.409, Ys=0.838, PK=0.77) and (green) predicted by tcaSIM using MIMOSA-derived constraints (PDH=1, PC=0.15, Ys=1, PK=0.26). (C) Total enrichments of ¹³C₂-pyruvate isotopomers directly measured by LC-MS/MS (blue), predicted by tcaSIM using tcaCALC fluxes (red), or using steady state MIMOSA values (green). All data are reported as mean ± S.E.M. Predicted values by tcaSIM have no uncertainty levels reported by the software.

Figure 5. Relationship of MIMOSA modeled mitochondrial rates determined by CWave to insulin secretion

(A) Overview of the rates and isotopomers considered in the CWave model: Pyruvate kinase (V_PK); pyruvate dehydrogenase (V_PDH); pyruvate carboxylase (V_PC); conversion of oxaloacetate (OAA) into pyruvate (V_{PEP/PyrCycling}); citrate/isocitrate exchange with αKG (V_ICDH); αKG exchange with glutamate(V_GlutExc); dilution of glutamate(V_GlutDil); the racemizing exchange between the malate/fumarate and (OAA V_SC). The arrows associated with V_CS have different colors indicating different turns (blue (1^st), orange (2^nd), and purple (3^rd)). The isotopomers with the greatest impact are displayed next to each pool and their carbons are color-coded to match their associated turn of the TCA cycle. (B) Insulin secretion from cells incubated with 2.5, 5, 7 and 9mM glucose. (C) [PEP] normalized to intracellular [taurine]. (D) PEP M⁺³ enrichments from glycolysis of glucose M⁺⁶. (E–H) precursor/product relationships Φ_PO, Φ_POCit, Φ_OP and Φ_PAc, respectively. (I) V_PC/V_CS calculated from MIMOSA rates (Table 1) (J) Absolute rates and (K) fold change through CS, PDH, PC, lipid oxidation (β–ox) and ICDH calculated using the model. (L) Correlation between CS and (M) PDH and PC fluxes and insulin secretion. (N) Correlation between O₂ consumption and CS. (O) O₂ consumption from ATP synthesis and mitochondrial leak. All data are reported as mean ± S.E.M. * P < 0.01, ** P < 0.001.

Figure 6. Flux analysis in C2C12 myoblast cells and primary rat hepatocytes

Evolution of ¹³C-label from glycolysis into the TCA cycle through PDH flux in C2C12 cells incubated with (A) glucose (5mM), (B) glucose and insulin (100nM) and (C) glucose and fatty acids (0.2mM palmitate:oleate (1:2)). Absolute (D) V_CS, (E) V_PDH and (F) V_βox. Relative contributions of (G) V_PDH and (H) V_βox to V_CS calculated from by CWave and (I) the equivalent with steady state measurement Φ_PAc. (J) Absolute V_PC flux and (K) Φ_PO calculated using OAA enrichments or malate and citrate as surrogate for OAA. (L) Calculated Φ_AcCit to assess the existence of reversed ICDH flux. (M) Evolution of ¹³C-label from [U-¹³C₃]lactate into the TCA cycle through PC flux in primary rat hepatocytes and (N) respective fluxes calculated by MIMOSA. Absolute flux data is reported as mean ± standard deviation of the flux distribution calculated by CWave. All other data are reported as mean ± S.E.M. * P < 0.01, ** P < 0.001.