A model for determining cardiac mitochondrial substrate utilisation using stable 13C-labelled metabolites
Tóm tắt
Relative 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. We 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. A 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). The 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. These 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.
Tài liệu tham khảo
Aubert, G., Martin, O. J., Horton, J. L., Lai, L., Vega, R. B., Leone, T. C., et al. (2016). The failing heart relies on ketone bodies as a fuel. Circulation, 115, 698–705.
Bell, R. M., Mocanu, M. M., & Yellon, D. M. (2011). Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion. Journal of Molecular and Cellular Cardiology, 50(6), 940–950.
Chatham, J. C., Forder, J. R., Glickson, J. D., & Chance, E. M. (1995). Calculation of absolute metabolic flux and the elucidation of the pathways of glutamate labeling in perfused rat heart by 13C NMR spectroscopy and nonlinear least squares analysis. Journal of Biological Chemistry, 270, 7999–8008.
Crown, S. B., & Antoniewicz, M. R. (2013). Publishing 13C metabolic flux analysis studies: A review and future perspectives. Metabolic Engineering, 20, 42–48.
Crown, S. B., Kelleher, J. K., Rouf, R., Muoio, D. M., & Antoniewicz, M. R. (2016). Comprehensive metabolic modeling of multiple 13C-isotopomer data sets to study metabolism in perfused working hearts. American Journal of Physiology-Heart and Circulatory Physiology, 311(4), H881–H891.
Dargie, H. (2005). Heart failure post-myocardial infarction: A review of the issues. Heart, 91, 3–6.
Hue, L., Taegtmeyer, H., Randle, P., Garland, P., & Hales, N. (2009). The Randle cycle revisited: A new head for an old hat. American Journal of Physiology. Endocrinology and Metabolism, 297, 578–591.
Katzs, J., Walss, P., & Lee, W. N. (1993). Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate. The Journal of Biological Chemistry, 268(34), 25509–25521.
Khairallah, M., Labarthe, F., Bouchard, B., Danialou, G., Petrof, B. J., & Des Rosiers, C. (2004). Profiling substrate fluxes in the isolated working mouse heart using 13 C-labeled substrates: Focusing on the origin and fate of pyruvate and citrate carbons. American Journal of Physiology-Heart and Circulatory Physiology, 286(4), H1461–H1470.
Krebs, H. A., Salvin, E., & Johnson, W. (1938). The formation of citric acid and α-ketoglutaric acids in the mammalian body. The Biochemical Journal, 32(1), 113–117.
Le Belle, J. E., Harris, N. G., Williams, S. R., & Bhakoo, K. K. (2002). A comparison of cell and tissue extraction techniques using high-resolution 1H-NMR spectroscopy. NMR in Biomedicine, 15(1), 37–44.
Lewandowski, E. D., & Hulbert, C. (1991). Dynamic changes in 13C NMR spectra of intact hearts under conditions of varied metabolite enrichment. Magnetic Resonance in Medicine, 19(1), 186–190.
Lewandowski, E. D., White, L. T., Damico, L. A., Yu, X., Doumen, C., & LaNoue, K. F. (1996). Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates. Magnetic Resonance in Medicine, 35(2), 149–154.
Liu, J., Wang, P., Douglas, S. L., Tate, J. M., Sham, S., Lloyd, S. G., et al. (2016). Impact of high-fat, low-carbohydrate diet on myocardial substrate oxidation, insulin sensitivity, and cardiac function after ischemia-reperfusion. American Journal of Physiology-Heart and Circulatory Physiology, 44, 1–10.
Lloyd, S., Brocks, C., & Chatham, J. C. (2003). Differential modulation of glucose, lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart. American Journal of Physiology-Heart and Circulatory Physiology, 285, 163–172.
Lloyd, S. G., Wang, P., Zeng, H., Chatham, J. C., Steven, G., Wang, P., et al. (2004). Impact of low-flow ischemia on substrate oxidation and glycolysis in the isolated perfused rat heart. American Journal of Physiology-Heart and Circulatory Physiology, 287, H351–H362.
Melo, T. M., Haberg, A. K., Risa, Ø., Kondziella, D., Pierre-gilles, H., & Sonnewald, U. (2011). Tricarboxylic acid cycle activity measured by 13C magnetic resonance spectroscopy in rats subjected to the Kaolin model of obstructed hydrocephalus. Neurochemical Research, 36, 1801–1808.
Neubauer, S. (2007). The failing heart—An engine out of fuel. The New England Journal of Medicine, 356, 1140–1151.
Randle, P., Garland, M., Hales, C., & Newsholme, E. (1963). The glucose fatty-acid cycle: Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. The Lancet, 281(7285), 785–789.
Saddik, M., Gamble, J., Witters, L. A., & Lopaschuk, G. D. (1993). Acetyl-CoA carboxylase regulation of fatty acid oxidation in the heart. Journal of Biological Chemistry, 268(34), 25836–25845.
Saddik, M., & Lopaschuk, G. D. (1991). Myocardial triglyceride turnover and contribution to energy. The Journal of Biological Chemistry, 266(13), 8162–8170.
Taegtmeyer, H., Young, M. E., Lopaschuk, G. D., Abel, E. D., Brunengraber, H., Darley-Usmar, V., et al. (2016). Assessing cardiac metabolism—A scientific statement from the American Heart Association. Circulation Research, 118(10), 1659–1701.
Yang, L., Kasumov, T., Kombu, R. S., Zhu, S., Cendrowski, A. V., David, F., et al. (2008). Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. The Journal of Biological Chemistry, 283(32), 21988–21996.
Yu, X., White, L. T., Lewandowski, E. D., Damico, L. A., Kathryn, F., & Alpert, N. M. (1995). Kinetic analysis of dynamic 13C NMR spectra: Metabolic flux, regulation, and compartmentation in hearts. Biophysical Journal, 69(5), 2090–2102.