Mass spectrometry-based microassay of²H and¹³C plasma glucose labeling to quantify liver metabolic fluxes in vivo

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.

Hepatic mTORC1 Opposes Impaired Insulin Action to Control Mitochondrial Metabolism in Obesity

“…was increased by 50% in fed Insr LKO mice (Figure 4E) and decreased by 35% in fed Tsc1 LKO mice on a chow diet (Figure 4F). Oxidative metabolism in the TCA cycle was generally decreased by fasting (Figure 4G and H), consistent with a recent study using similar methodology (Hasenour et al, 2015). Insr LKO mice had an overall increase in hepatic TCA cycle flux but remained responsive to nutritional state (Figure 4G).…”

Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver

“…However, when a regression model including randomization was used, data from [U- 13 C]lactate/[U- 13 C]pyruvate-perfused livers gave values identical to those from [U- 13 C]propionate and simple equations ( Figure 1G). Hence, in contrast to a recent report (21), but in agreement with another (22), these findings demonstrate that tracer doses of propionate do not perturb glucose production or anaplerosis and explain why [U- 13 C]pyruvate/lactate/alanine tracers provide lower estimates of pyruvate cycling when incomplete randomization is not modeled (23).…”

“…This agrees with our previous experience, where we optimized propionate administration in mice to avoid elevated glucose or insulin levels (62). Recently, investigators from the Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University, used an identical tracer approach, but an independent mass spectrometry platform, and found fluxes that were similar to those we previously reported; they also found that tracer amounts of [U- 13 C]propionate had no effect on hepatic metabolic flux in mice when fit to a complete regression model (22). Notably, stoichiometric analysis of oxygen consumption correlated strongly with flux measurements using 13 C and 2 H isotopomer analysis.…”

Assessment of Hepatic Mitochondrial Oxidation and Pyruvate Cycling in NAFLD by 13C Magnetic Resonance Spectroscopy

“…Such high rates of hepatic pyruvate cycling would use a significant fraction of the ATP available, and place the hepatocyte in a metabolically precarious position. In a recent study we found that an intra-arterial infusion of propionate, that was lower than (Hasenour et al, 2015; Jin et al, 2005; Jin et al, 2004; Satapati et al, 2015) or similar to (Sunny et al, 2011) the total amount of sodium propionate administered in previous studies, dose-dependently increased: hepatic propionyl CoA concentrations up to 18 fold, hepatic pyruvate cycling up to 20–30 fold, hepatic TCA intermediates (malate and succinate) and aspartate by 2–3 fold, and rates of hepatic glucose production by up to 2 fold in awake rats (Perry et al, 2016). It is well established that propionyl CoA potently stimulates pyruvate carboxylase activity and flux (Perry et al, 2016; Scrutton, 1974) and this may explain at least in part the large increases in hepatic pyruvate carboxylase flux and pyruvate cycling that Sunny et al observed in their [1,2,3- 13 C 3 ]propionate labeling studies of hepatic mitochondrial metabolism in humans (Sunny et al, 2011).…”