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Limitations of detection of anaplerosis and pyruvate cycling from metabolism of [1-13C] acetate

Nature Medicine volume 21, pages 108–109 (2015)Cite this article

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To the Editor:

Recently Befroy and colleagues1 measured tricarboxylic acid (TCA) cycle flux and anaplerosis in human liver by analyzing 13C enrichment in the C1 and C5 positions of glutamate during [1-13C]acetate infusion1. The authors concluded that anaplerosis was equal to ∼1.4 times TCA cycle flux, and that other methods overestimate this flux1. The entry of [1-13C]acetyl-CoA into the cycle yields [5-13C]glutamate on each turn of the cycle, but [1-13C]glutamate appears only on subsequent turns. Anaplerosis, for instance via pyruvate carboxylation, results in the influx of unlabeled oxaloacetate. The subsequent loss of 13C-oxaloacetate from the cycle to make phosphoenolpyruvate (PEP) decreases the enrichment in glutamate C1 but not glutamate C5. Hence, the steady-state ratio of [1-13C]glutamate to [5-13C]glutamate should reflect anaplerosis relative to TCA cycle activity (i.e., citrate synthase flux = 1). PEP formed from decarboxylation of oxaloacetate has at least two fates, either to contribute to gluconeogenesis (GNG) or to regenerate pyruvate via pyruvate kinase (PK). PK, and other pathways (for example, malic enzyme), contribute to a futile pyruvate cycle whereby oxaloacetate → pyruvate → oxaloacetate. The relationship among these variables is simple: GNG = anaplerosis – PK flux. This pathway has been included in virtually all models of hepatic GNG2,3,4,5,6,7,8,9. In humans, various values of PK flux relative to citrate synthase (CS) have been reported, varying from 1.5 to 5.8 when determined using a [3-14C]lactate tracer with radioisotope detection8, [3-13C]lactate tracer with mass isotope detection3 or [U-13C]propionate tracer with nuclear magnetic resonance (NMR) detection6,9. In fact, pyruvate cycling accounted for the majority of anaplerosis in these human studies3,6,8,9, yet was excluded from anaplerosis by Befroy et al.1 This exclusion complicates the analysis considerably because pyruvate cycling in the presence of H13CO3−, as observed in the in vivo 13C NMR spectra1, regenerates [1-13C]glutamate. Hence, the steady-state ratio of [1-13C]glutamate to [5-13C]glutamate no longer reflects total anaplerosis. Given the implications, it is worth examining the effect of pyruvate cycling on 13C in glutamate C1 and C5 using the conditions reported by Befroy et al.1

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Figure 1: The effect of pyruvate cycling on the steady-state ratio of [1-13C]glutamate to [5-13C]glutamate (13C1/13C5).

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Acknowledgements

We thank S.F. Previs for helpful discussion. Support for this work was provided by the US National Institutes of Health (DK078184, DK058398, DK087977, HL034557, EB015908, EB016197), the Cancer Prevention and Research Institute of Texas (RR-101243), the Portuguese Science Foundation (EXCL/DTP/PIC/0069/2012), the European Regional Development Fund (PEst-C/SAU/LA0001/2011) and the Robert A. Welch Foundation (SCB I-1804).

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Authors and Affiliations

  1. Advanced Imaging Research Center Division of Metabolic Mechanisms of Disease, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Shawn C Burgess, Mathew E Merritt, Jeffrey D Browning, A Dean Sherry & Craig R Malloy

  2. Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Shawn C Burgess

  3. Center for Neurosciences and Cell Biology University of Coimbra, Coimbra, Portugal

    John G Jones

  4. Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Jeffrey D Browning

  5. VA North Texas Health Care System, Dallas, Texas, USA

    Craig R Malloy

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All authors contributed to the design of this report. S.C.B., A.D.S., M.E.M. and C.R.M. wrote the initial draft and all authors contributed to the final draft.

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Correspondence to Shawn C Burgess.

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Burgess, S., Merritt, M., Jones, J. et al. Limitations of detection of anaplerosis and pyruvate cycling from metabolism of [1-13C] acetate. Nat Med 21, 108–109 (2015). https://doi.org/10.1038/nm.3789

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