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Publication
Mass spectrometry-based microassay of 2H and 13C plasma glucose labeling to
quantify liver metabolic fluxes in vivo.
Authors Hasenour CM, Wall ML, Ridley DE, Hughey CC, James FD, Wasserman DH, Young JD
Submitted By Jamey Young on 7/24/2015
Status Published
Journal American journal of physiology. Endocrinology and metabolism
Year 2015
Date Published
Volume : Pages 309 : E191 - E203
PubMed Reference 25991647
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. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes
were delivered intravenously in short- (9 h) and long-term-fasted (19 h)
C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were
performed on three 40-µl 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 glycogen 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 oxaloacetate to PEP were severalfold higher
than VCS in 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.




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