[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate

Abstract

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain metabolism of exogenous pyruvate

“…The higher 13 C enrichment of glutamate C 3 than of the C 2 from [2‐ 13 C]pyruvate agrees with previous findings that the product of pyruvate carboxylation in liver does not undergo complete equilibration with fumarate (Heath and Rose 1985). This finding may be explained if the malate or oxaloacetate formed through pyruvate carboxylation is metabolized directly to citrate without prior equilibration with the symmetric fumarate over fumarase.…”

“…This finding suggests incomplete scrambling (Fig. 7a) of the product of pyruvate carboxylation in the fumarate step of the hepatic TCA cycle (Heath and Rose 1985).…”

The effects of stress and injury on the activity of phosphoenolpyruvate carboxykinase in the liver of the rat

“…Scald injury therefore always involved stress from ether, and, when DMSO was used as a vehicle for drugs, stress from DMSO also. The effects of injury were expected to predominate, since, although it is not painful, owing to the destruction of the nerve endings, it produces most of the effects of a severe injury on glucose metabolism (Heath, 1985…”

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the 13C Labeling of Citrate Released by Perfused Rat Hearts

“…Mathematical models of increasing complexity were developed for the study of the CAC in various organs or tissues (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), including the heart (13)(14)(15)(16)(17). Solving for flux parameters in equations derived from these models requires measuring the incorporation of 14 C-or 13 C-labeled substrate(s) into various CAC metabolites (8,9) or the distribution of label between carbons of given molecules such as glutamate (1,(13)(14)(15)(17)(18)(19) or citrate (4,6). The use of 13 C-enriched labeled substrate(s) and measurements of 13 C labeling of CAC intermediates or related metabolites by nuclear magnetic resonance (NMR) or gas chromatography-mass spectrometry (GCMS) offers several advantages over classical 14 C methods.…”