(566g) Comprehensive Analysis of the Gluconeogenesis Pathways through the Combined Use of Multiple Isotopic Tracers

Methods for measuring the metabolic rate of gluconeogenesis (GNG) in vivo, relative to hepatic glucose production (HGP), rely on the use of stable isotopes (13C and 2H) and assessment of glucose labeling distributions by gas chromatography mass spectrometry (GC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Here, we applied multiple 13C-, and 2H-labeled tracers and analyzed the resulting mass isotopomer distribution of multiple glucose fragments by GC/MS. From this data we estimated, for the first time, both net and exchange fluxes in the upper gluconeogenesis pathway. We identified limitations in current methods to estimate gluconeogenesis in vivo, and developed a novel multiple-tracer method for accurate, quantitative analysis of gluconeogenesis independent of the isotopic steady-state assumption.

Limitations of current methods. It has been argued that the algebraic relationships based on the [U-13C]glucose method will underestimate gluconeogenesis in vivo. A major reason is the failure to consider the exchange of labeled precursors in the TCA cycle and the contribution of glycerol to gluconeogenesis. In the early 1990s condensation polymerization methods offered a new approach to estimating biosynthesis. [13C]Glycerol tracers were proposed for quantifying gluconeogenesis using mass isotopomer distribution analysis (MIDA). However, the possibility of zonation of the tracer across the liver has led to questions about the validity of this method. In fact, several studies with hepatocyte cell cultures, perfused livers, and whole animals have shown that mass isotopomer distributions (MID) of glucose from [13C]glycerol tracers are incompatible with the assumption that these is a single constant pool of triose phosphates, presumably reflecting the presence of different cell populations with differential preference for various gluconeogenic precursors. Malloy and colleagues proposed a quantitative approach based on the fate of [U-13C]propionate and the analysis of hepatic glutamate and glucose. This method uncovered a discrepancy between the analyses of different compounds indicative of compartmentation of metabolism. This important finding further complicated the search for a clear quantitative method for estimating hepatic fluxes from carbon labeling data. Recently, we applied [2H5]glycerol to measure net and exchange fluxes in the gluconeogenesis pathway. From mass isotopomer distribution analysis of glucose fragments we estimated the extent of equilibration of phosphoglucose isomerase (PGI) and triose phosphate isomerase (TPI) reactions with precision, i.e. 80-86% equilibration for PGI and 79-81% equilibration for TPI, in isolated hepatocytes. However, our results further suggested that [2H5]glycerol will underestimate the net flux of GNG, by about 15% in our experiments. Currently, the most widely used method for measuring GNG is the 2H2O initially introduced by Landau. This method has the potential to provide an unbiased estimate of the GNG flux. However, in current use this method assumes complete equilibration of PGI and TPI reactions, which may not hold true in all situations. We have shown previously in isolated hepatocytes that lack of complete equilibration for PGI reaction resulted in 20% overestimation of GNG via this method. Thus, despite over many decades of experiments, the search for a well-accepted method for quantifying gluconeogenesis from stable isotope tracers in vivo continues.

Here, we present a novel two tracer method for accurate determination of net and reversible fluxes in the gluconeogenesis pathway in vivo. Our method builds on Landau's 2H2O method with an additional novel doubly-labeled glycerol tracer, i.e. [U-13C,2H8]glycerol. In this method two tracers (2H2O and [U-13C,2H8]glycerol) are applied simultaneously and fluxes are estimated with precision form the incorporation of 2H from 2H2O into glucose after talking into account incomplete equilibration of GPI and TPI reactions that is estimated from the incorporation of 13C and 2H isotopes from [U-13C,2H5]glycerol into glucose. In this method we make use of GC/MS method to analyze three glucose derivatives that were recently introduced, i.e. aldonitrile pentapropionate ion fragments at m/z 173, 259, 284, and 370; methyloxime pentapropionate ion fragment at m/z 145; and di-O-isopropylidene propionate ion fragment at m/z 301. From these fragments we obtained an overdetermined data set from which accurate metabolic fluxes can be estimated. To interpret the complex labeling patterns of glucose from these experiments we apply rigorous mathematical tools based on elementary metabolite units (EMU) framework that was recently introduced and shown to be the most efficient method for analyzing mass isotopomer data in labeling experiments. Thus, we estimated for the first time both net and exchange fluxes in the upper gluconeogenesis pathway independent of the isotopic steady-state assumption.