Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3‐¹³C₃]propionate

Abstract

Simple equations that relate glucose and glutamate 13 C-NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of |l,2,3-13 C3lpropionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13 C spectra of glucose collected from rats after gastric versus intravenous administration of [l,2,3-13 C 3 ]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13 C-NMR spectrum of blood collected after oral administration of enriched propionate.

Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR

“…Relative flux values through various pathways in isolated perfused rat livers have been measured using a combination of stable isotope tracers and NMR or MS [32,39]. Absolute fluxes through the tricarboxylic acid cycle and associated pathways have been measured by an elegant combination of mass-isotopomer-distribution analysis ('MIDA') and substrate balance across the perfused rat liver [40] or by using a rigorous stoichiometric analysis of metabolite uptake and production [41].…”

“…The 13 C labelling pattern in OAA (and therefore effluent glucose) reflects fluxes associated with the tricarboxylic acid cycle. Specifically, the C2 resonance in the 13 C spectrum of effluent glucose (after conversion into MAG) was used to determine anaplerosis, pyruvate cycling and GNG from PEP relative to tricarboxylic-acid-cycle flux [32]. Normalizing this latter relative flux with the absolute flux, GNG PEP , determined from glucose production and 2 H NMR results in absolute fluxes for all of the pathways shown in Scheme 1 [28].…”

“…To assess the relationship between the tricarboxylic acid cycle and GNG PEP , the perfusion media also contained [U- 13 C]propionate and the resulting glucose isotopomers were evaluated by 13 C NMR [32]. A typical 13 C spectrum of MAG derived from effluent glucose is shown in Figure 2.…”

“…The insets show expanded C2 resonances of MAG derived under three different physiological perfusion conditions. The individual C2 multiplet components (D12, D23, Q) were deconvoluted and those areas were used to perform a 13 C isotopomer analysis of biochemical fluxes into and out of the tricarboxylic acid cycle [32]. Differences between multiplets from perfusion with high lactate ( Figure 2B) versus low lactate ( Figure 2A) were relatively minor, with a slight increase noted in the D23 component relative to the central singlet (S) and a slight decrease in the D12 component relative to the quartet (Q).…”

“…Flux through PEPCK (v6/v7; also includes flux through the malic enzyme), pyruvate cycling (v5/v7; includes PK and malic enzyme) and gluconeogenic flux from PEP (v4/v7), all relative to tricarboxylic-acid-cycle flux, were determined from 13 C-NMR data [32] and values are shown in Table 1. Interestingly, these values are relatively independent of the ratio of lactate to pyruvate in the perfusate and even of the absence of lactate and pyruvate.…”