A model for determining cardiac mitochondrial substrate utilisation using stable 13C-labelled metabolites

Abstract

IntroductionRelative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use—yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle.ObjectivesWe aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the 13C labelled proportion of acetyl-CoA entering the Krebs cycle.MethodsA model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with 13C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture).ResultsThe contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-13C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively.ConclusionThese results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.Electronic supplementary materialThe online version of this article (10.1007/s11306-019-1618-y) contains supplementary material, which is available to authorized users.

High Throughput Procedure for Comparative Analysis of In Vivo Cardiac Glucose or Amino Acids Use in Cardiovascular Pathologies and Pharmacological Treatments

“…The proportion of each intermediate (which is a few units heavier as a result of 13 C labeling) is directly detectable, and thus, differences in the ratios of 13 C-labeled metabolic substrate oxidation are detected in the labeling patterns. Stable isotope labeling is commonly used for the study of metabolic fluxes, mainly to investigate the proportional contribution of different substrates to the Krebs cycle under varying conditions and across different tissues [ 24 , 25 , 26 ]. Nevertheless, analysis of cardiac substrate preference requires specific method conditions.…”