A new mathematical approach to the metabolism of [14C]glucose, with applications to sensory ganglia of chicken embryos

Abstract— The available models of carbohydrate metabolism are not suitable for analysis of experiments on dorsal root ganglia of chicken embryos because they assume that certain products of the pentose cycle mix freely with those of glycolysis, which appears not to be true in this tissue, and because full isotopic equilibration, needed before the start of measurements, is not achieved while the excised ganglia are reasonably fresh. Therefore, new equations were developed which assume only a steady state of relevant metabolic intermediates and make use of the process of isotopic equilibration as a source of information. It is also assumed that an initially unknown but calculable fraction of the products of each pentose cycle re-enters the next cycle, the remainder leaking either to glycolysis or to the incubation medium. From measurements of the time course of output of labelled CO₂ in the presence of [1-¹⁴C]- and [6-¹⁴C]glucose and the incorporation and release in lactate of labelled C from [l-¹⁴C]glucose, the equations permit the estimation of many features of carbohydrate metabolism, such as the partitioning of material between the pentose cycle and glycolysis, the partitioning of CO₂ output between the pentose and citric acid cycles, the partitioning of the products of glycolysis between CO₂ and other destinations, such as lactate, and the degree of recycling from one pentose cycle into the next. In addition, the time course of labelled CO₂ output from [2-¹⁴C]glucose can be predicted; this, by comparison with the observed output, serves to support some variants of the basic model, while invalidating others. In dorsal root ganglia from 15-day chicken embryos, the assumption of a metabolic steady state was supported by a constant output of labelled CO² from [l-¹⁴C]glucose for 15 or more hours, except for the initial period of isotopic equilibration. By use of the new equations, it is concluded that in these ganglia (a) recycling in the pentose cycle can be 100% efficient in some incubation conditions, but not in others, (b) more CO₂ is released from the pentose cycle than from the citric acid cycle, (c) large, quantifiable differences exist between the utilization of the various carbon atoms of glucose, and (d) a pool of intermediates within the pentose cycle, with a time constant of about 1 h, explains a large delay observed in the output of C-6 of glucose into CO², which occurs with a time constant as long as 5 h under some conditions. Under conditions where recycling is complete in the pentose cycle, this cycle must operate in isolation from glycolysis, which would otherwise convert much of the output of the pentose cycle to lactate. This may explain the role of fructose-1,6-diphospha-tase in the tissue, without recourse to the oft-proposed, puzzling, and ATP-degrading‘futile cycle’between fructose-6-P and fructose-1,6-diP. It is proposed that the new equations may be suitable for similar analyses on some, but not all, other tissues.     

Time-dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1-(13) C]glucose metabolism

[1-(13) C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono- and dicarboxylic amino acids were determined from (1) H-observed/(13) C-edited and (13) C-NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate-glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re-evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.     

Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction

Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals. However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells. This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia. Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis. It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK-21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem. Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control. Moreover, a 1.4-fold-higher adenosine triphosphate (ATP) content was achieved. The flux of labeled [(14)C]-glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation. Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs.     

Long-term metabolism of [U-14C]glucose in the whole rat brain

The experiment was performed on rats to which a single injection of [U-14C]glucose had been administered. Results were observed from the 7th to the 281st day following contamination. At 280 days only the lipids in the brain contained radioactivity, the highest degree of specific activity being found in the cerebrosides.     

14C] GABA and [14C]GABA-pantoyl metabolism in mice

It is shown that more than 90% of the labelled substance D-[1-14C] calcium homopantotenate is rapidly removed from the organism with urea; 6-8% are products of its transformation, among them GABA is identified. An insignificant transformation of D-[1-14C] calcium homopantotenate up to 14CO2 is observed. After the preparation administration only unchanged D-[1-14C] calcium homopantotenate was found in the tissues, except of the liver where, as in urea, there is a nonidentified product with small Rf. [1-14C] GABA is rapidly transformed to 14CO2 and only its insignificant part is removed with urea, chiefly as products of transformation.     

Measurement of exogenous carbohydrate oxidation: a comparison of [U-14C]glucose and [U-13C]glucose tracers

The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHO(EXO)). To accomplish this, seven healthy male subjects (24 +/- 3 yr, 74.8 +/- 2.1 kg, V(O2(max)) 62 +/- 4 ml x kg(-1) x min(-1)) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHO(EXO) to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO(2) in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHO(EXO) measured with [13C]glucose ([13C]CHO(EXO)) was significantly greater than CHO(EXO) measured with [14C]glucose ([14C]CHO(EXO)) from 30 to 120 min. There was a 15 +/- 4% difference between [13C]CHO(EXO) and [14C]CHO(EXO) such that the absolute difference increased with the magnitude of CHO(EXO). Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the "strength" of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285-294, 1977). Therefore, the explanation for the discrepancy in calculated CHO(EXO) remains to be fully understood.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Str?m, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

The metabolism of [14C]nicotine in the cat

The metabolism of [2'-(14)C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [(14)C]nicotine and [(14)C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40mug. of [(14)C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [(14)C]Nicotine is metabolized extremely rapidly, [(14)C]cotinine appearing in the blood within 2.5min. of intravenous injection. [(14)C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [(14)C]nicotine. Metabolites of [(14)C]nicotine have been identified in liver and urine extracts. [(14)C]Nicotine-1'-oxide has been detected in both liver and urine.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells.

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Strøm, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.