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Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy

Abstract

Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new 13C-labeling strategy in combination with 13C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (VTCA) and anaplerotic flux (VANA) to be 0.43 ± 0.04 μmol g−1 min−1 and 0.60 ± 0.11 μmol g−1 min−1, respectively, in twelve healthy, lean individuals. We also found the VANA/VTCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for assessing the effectiveness of new therapies targeting these pathways in humans.

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Figure 1: Plasma acetate concentration (black diamonds) and 1-13C enrichment (gray diamonds) during the [1-13C]acetate infusion protocol (n = 12).
Figure 2: In vivo 13C MRS of the human liver.
Figure 3: Time courses of hepatic glutamate C5 and C1 enrichment determined by localized in vivo 13C MRS during an infusion of [1-13C]acetate (n = 12).
Figure 4: Metabolic model of liver acetate oxidative metabolism used to estimate hepatic VTCA and VANA.

References

  1. Jones, J.G., Solomon, M.A., Cole, S.M., Sherry, A.D. & Malloy, C.R. An integrated 2H and 13C NMR study of gluconeogenesis and TCA cycle flux in humans. Am. J. Physiol. Endocrinol. Metab. 281, E848–E856 (2001).

    Article  CAS  Google Scholar 

  2. Jones, J.G., Solomon, M.A., Sherry, A.D., Jeffrey, F.M. & Malloy, C.R. 13C NMR measurements of human gluconeogenic fluxes after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Am. J. Physiol. 275, E843–E852 (1998).

    PubMed  CAS  Google Scholar 

  3. Sunny, N.E., Parks, E.J., Browning, J.D. & Burgess, S.C. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 14, 804–810 (2011).

    Article  CAS  Google Scholar 

  4. Landau, B.R. et al. 14C-labeled propionate metabolism in vivo and estimates of hepatic gluconeogenesis relative to Krebs cycle flux. Am. J. Physiol. 265, E636–E647 (1993).

    PubMed  CAS  Google Scholar 

  5. Landau, B.R. et al. Use of 2H2O for estimating rates of gluconeogenesis. Application to the fasted state. J. Clin. Invest. 95, 172–178 (1995).

    Article  CAS  Google Scholar 

  6. Shulman, G.I. et al. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy. N. Engl. J. Med. 322, 223–228 (1990).

    Article  CAS  Google Scholar 

  7. Befroy, D.E. et al. Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients. Diabetes 56, 1376–1381 (2007).

    Article  CAS  Google Scholar 

  8. Lebon, V. et al. Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle. J. Clin. Invest. 108, 733–737 (2001).

    Article  CAS  Google Scholar 

  9. Mason, G.F. et al. Simultaneous determination of the rates of the TCA cycle, glucose utilization, α-ketoglutarate/glutamate exchange, and glutamine synthesis in human brain by NMR. J. Cereb. Blood Flow Metab. 15, 12–25 (1995).

    Article  CAS  Google Scholar 

  10. Mason, G.F. et al. Measurement of the tricarboxylic acid cycle rate in human grey and white matter in vivo by 1H-[13C] magnetic resonance spectroscopy at 4.1T. J. Cereb. Blood Flow Metab. 19, 1179–1188 (1999).

    Article  CAS  Google Scholar 

  11. Petersen, K.F. et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science 300, 1140–1142 (2003).

    Article  CAS  Google Scholar 

  12. Szendroedi, J. et al. Abnormal hepatic energy homeostasis in type 2 diabetes. Hepatology 50, 1079–1086 (2009).

    Article  CAS  Google Scholar 

  13. Schmid, A.I. et al. Liver ATP synthesis is lower and relates to insulin sensitivity in patients with type 2 diabetes. Diabetes Care 34, 448–453 (2011).

    Article  CAS  Google Scholar 

  14. Befroy, D.E., Rothman, D.L., Petersen, K.F. & Shulman, G.I. 31P-magnetization transfer magnetic resonance spectroscopy measurements of in vivo metabolism. Diabetes 61, 2669–2678 (2012).

    Article  CAS  Google Scholar 

  15. Mitchell, C.S. et al. Resistance to thyroid hormone is associated with raised energy expenditure, muscle mitochondrial uncoupling, and hyperphagia. J. Clin. Invest. 120, 1345–1354 (2010).

    Article  CAS  Google Scholar 

  16. Boumezbeur, F. et al. Altered brain mitochondrial metabolism in healthy aging as assessed by in vivo magnetic resonance spectroscopy. J. Cereb. Blood Flow Metab. 30, 211–221 (2010).

    Article  CAS  Google Scholar 

  17. Lebon, V. et al. Astroglial contribution to brain energy metabolism in humans revealed by 13C nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism. J. Neurosci. 22, 1523–1531 (2002).

    Article  CAS  Google Scholar 

  18. Li, S., Yang, J. & Shen, J. Novel strategy for cerebral 13C MRS using very low RF power for proton decoupling. Magn. Reson. Med. 57, 265–271 (2007).

    Article  CAS  Google Scholar 

  19. Li, S. et al. In vivo13C magnetic resonance spectroscopy of human brain on a clinical 3 T scanner using [2-13C]glucose infusion and low-power stochastic decoupling. Magn. Reson. Med. 62, 565–573 (2009).

    Article  CAS  Google Scholar 

  20. Sailasuta, N. et al. Clinical NOE 13C MRS for neuropsychiatric disorders of the frontal lobe. J. Magn. Reson. 195, 219–225 (2008).

    Article  CAS  Google Scholar 

  21. Befroy, D.E. et al. Increased substrate oxidation and mitochondrial uncoupling in skeletal muscle of endurance-trained individuals. Proc. Natl. Acad. Sci. USA 105, 16701–16706 (2008).

    Article  Google Scholar 

  22. Beylot, M., Soloviev, M.V., David, F., Landau, B.R. & Brunengraber, H. Tracing hepatic gluconeogenesis relative to citric acid cycle activity in vitro and in vivo. Comparisons in the use of [3-13C]lactate, [2-13C]acetate, and α-keto[3-13C]isocaproate. J. Biol. Chem. 270, 1509–1514 (1995).

    Article  CAS  Google Scholar 

  23. Puchowicz, M.A. et al. Zonation of acetate labeling across the liver: implications for studies of lipogenesis by MIDA. Am. J. Physiol. 277, E1022–E1027 (1999).

    PubMed  CAS  Google Scholar 

  24. Jin, E.S. et al. Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative. Anal. Biochem. 327, 149–155 (2004).

    Article  CAS  Google Scholar 

  25. Jones, J.G., Carvalho, R.A., Franco, B., Sherry, A.D. & Malloy, C.R. Measurement of hepatic glucose output, Krebs cycle, and gluconeogenic fluxes by NMR analysis of a single plasma glucose sample. Anal. Biochem. 263, 39–45 (1998).

    Article  CAS  Google Scholar 

  26. Alves, T.C. et al. Regulation of hepatic fat and glucose oxidation in rats with lipid-induced hepatic insulin resistance. Hepatology 53, 1175–1181 (2011).

    Article  CAS  Google Scholar 

  27. Jones, J.G. et al. Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3-13C3]propionate. FEBS Lett. 412, 131–137 (1997).

    Article  CAS  Google Scholar 

  28. Shen, J., Rycyna, R.E. & Rothman, D.L. Improvements on an in vivo automatic shimming method. Magn. Reson. Med. 38, 834–839 (1997).

    Article  CAS  Google Scholar 

  29. Patel, A.B., de Graaf, R.A., Rothman, D.L., Behar, K.L. & Mason, G.F. Evaluation of cerebral acetate transport and metabolic rates in the rat brain in vivo using 1H-[13C]-NMR. J. Cereb. Blood Flow Metab. 30, 1200–1213 (2010).

    Article  CAS  Google Scholar 

  30. Katz, J., Wals, P. & Lee, W.N. Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate. J. Biol. Chem. 268, 25509–25521 (1993).

    PubMed  CAS  Google Scholar 

  31. Heath, D.F. & Rose, J.G. [14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate. Biochem. J. 227, 851–865 (1985).

    Article  CAS  Google Scholar 

  32. Petersen, K.F., Blair, J.B. & Shulman, G.I. Triiodothyronine treatment increases substrate cycling between pyruvate carboxylase and malic enzyme in perfused rat liver. Metabolism 44, 1380–1383 (1995).

    Article  CAS  Google Scholar 

  33. Fernandez, C.A. & Des Rosiers, C. Modeling of liver citric acid cycle and gluconeogenesis based on 13C mass isotopomer distribution analysis of intermediates. J. Biol. Chem. 270, 10037–10042 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Impellizeri, Y. Kosover, I. Smolgovsky, M. Smolgovsky, G. Solomon, C. Parmelee and the staff of the Yale Center for Clinical Investigation Hospital Research Unit for their technical support and the volunteers for their participation in these studies. We also acknowledge the contributions of G. Mason, who assisted in the implementation and interpretation of the metabolic modeling of the rat [1-13C]acetate infusion data. This publication was supported by grants from the US Public Health Service (R24 DK-085638, R01 AG-23686, R01 DK-49230, P30 DK-45735 and UL1 RR-024139), a Distinguished Clinical Investigator Award from the American Diabetes Association (K.F.P.) and an investigator-initiated grant from Pfizer (K.F.P.). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the US National Center for Research Resources or National Institutes of Health.

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D.E.B., R.J.P., J.T., J.B., K.F.P., D.L.R. and G.I.S. designed the experimental protocols. D.E.B., R.J.P., S.D., G.W.C. and K.F.P. performed the studies. D.E.B., N.J., R.J.P., S.D., G.W.C. and D.L.R. analyzed the data. D.E.B., R.J.P., K.F.P., D.L.R. and G.I.S. contributed to the writing of the manuscript.

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Correspondence to Gerald I Shulman.

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Befroy, D., Perry, R., Jain, N. et al. Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy. Nat Med 20, 98–102 (2014). https://doi.org/10.1038/nm.3415

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