Mass spectrometry-based microassay of 2H and 13C plasma glucose labelling to quantify liver metabolic fluxes in vivo

Clinton M. Hasenour, Martha L. Wall, D. Emerson Ridley, Curtis C. Hughey, Freyja D. James, David H. Wasserman, Jamey D. Young

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [13C3]propionate, [2H2]water, and [6,6-2H2]glucose isotopes were delivered intravenously in short-(9h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-jxl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from gly-cogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short-to long-term fasting. CAC flux [i.e., citrate synthase (Vcs)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of ox-aloacetate to PEP were severalfold higher than VCSin long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.

Original languageEnglish (US)
Pages (from-to)E191-E203
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Volume309
Issue number2
DOIs
StatePublished - Jul 15 2015
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2015 the American Physiological Society.

Keywords

  • Gluconeogenesis
  • Isotopomer model
  • Liver physiology
  • Metabolic flux analysis
  • Nutrient metabolism

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